Contributing Authors
Massimo Avoli, Department of Neurology and Neurosurgery, McGill University, Montreal, Canada
mental, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Roy A. Bakay, Department of Neurological Surgery, Chicago Institute of Neurosurgery and Neuroresearch, Rush Presbyterian-St. Luke’s Medical Center, Chicago, IL
Wei-Ping Chen, Department of Physiology and Pharmacology, SUNY Health Science Center, Brooklyn, NY Suzanne Clark, Department of Biomedical Science, Colorado State University, Fort Collins, CO
Scott C. Baraban, Department of Neurological Surgery, University of California San Francisco, San Francisco, CA
Miguel A. Cortez, Department of Neurology, Hospital for Sick Children, Toronto, Ontario, Canada
Tallie Z. Baram, Departments of Pediatrics and Anatomy and Neurobiology, University of California Irvine, Irvine, CA
Marco de Curtis, Department of Experimental Neurophysiology, Instituto Nazionale Neurologico Carlo Besta, Milan, Italy
Stefania Bassanini, National Neurological Institute Carlo Besta, Milan, Italy
Antoine Depaulis, Université Joseph Fourier, Grenoble, France
Giorgio Battaglia, National Neurological Institute Carlo Besta, Milan, Italy Christophe Bernard, INMED-INSERM U29, Marseilles, France
Marc A. Dichter, Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA
Edward H. Bertram, Department of Neurology, University of Virginia, Charlottesville, VA
Céline Dinocourt, Department of Physiology, University of Maryland, Baltimore, MD
Ronald A. Browning, Department of Physiology, Southern Illinois University School of Medicine, Springfield, IL
Céliné M. Dubé, Department of Anatomy and Neurobiology, University of California Irvine, Irvine, CA
Paul S. Buckmaster, Department of Comparative Medicine, Stanford University, Stanford, CA
F. Edward Dudek, Department of Physiology, University of Utah, Salt Lake City, UT
Daniel L. Burgess, Department of Neurology, Baylor College of Medicine, Houston, TX
Jerome Engel, Jr., Reed Neurological Research Center, Department of Neurology, UCLA School of Medicine, Los Angeles, CA
Thomas Budde, Institut fur Physiologie, Otto-vonGuericke-Universitat, Universitatsklinikum, Magdeburg, Germany
Aristea S. Galanopoulou, Departments of Neurology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY
Xiang Cai, Department of Physiology, University of Maryland, Baltimore, MD
Mary E. Gilbert, Neurotoxicology Division, US Environmental Protection Agency, Research Triangle Park, NC
Esper A. Cavalheiro, Escola Paulista de Medicina (UNIFESP/EPM), Laboratorio de Neurologia Experi-
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Contributing Authors
Jeffrey H. Goodman, Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, West Haverstraw, NY Ali Gorji, Institut fur Physiologie, Universitat Munster, Munster, Germany Heidi Grabenstatter, Department of Biomedical Science, Colorado State University, Fort Collins, CO Kevin D. Graber, Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA Uwe Heinemann, Institute of Neurophysiology, Charite, Humboldt University, Berlin, Germany Gregory L. Holmes, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, NH
Joseph J. LoTurco, Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT Heiko J. Luhmann, Institut fur Physiologie und Pathophysiologie, Johannes-Gutenberg-Mainz Universitat, Mainz, Germany Gilles van Luijtelaar, Department of Biological Psychology, Radboud University of Nijmegen, Nijmegen, The Netherlands Pavel Maresˇ, Institute of Physiology, Academy of Sciences, Videnska, Prague, Czech Republic Gary W. Mathern, Department of Neurological Surgery, University of California Los Angeles, Los Angeles, CA
John R. Huguenard, Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA
Andrey M. Mazarati, University of California Los Angeles, West Los Angeles VA Medical Center, Los Angeles, CA
John G.R. Jefferys, Department of Neurophysiology, University of Birmingham Medical School, Birmingham, United Kingdom
Tracy K. McIntosh, Traumatic Brain Injury Laboratory, Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA
Frances E. Jensen, Department of Neurology, Children’s Hospital, Harvard Medical School, Boston, MA
Dan C. McIntyre, Department of Psychology, Carleton University, Ottawa, Ontario, Canada
Phillip C. Jobe, Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, IL
James O. McNamara, Department of Neurobiology, Duke University Medical Center, Durham, NC
Oliver Kann, Institute of Neurophysiology, Charite, Humboldt University, Berlin, Germany
Luiz E. Mello, Department of Physiology, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Kevin M. Kelly, Department of Neurology, AlleghenySinger Research Institute, Allegheny General Hospital, Pittsburgh, PA
Solomon L. Moshé, Departments of Neurology, Neuroscience and Pediatrics, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY
Irina Kharatishvilli, Department of Neurobiology, AI Virtanen Institute for Molecular Science, University of Kuopio, Kuopio, Finland
Maria G. Naffah-Mazzacoratti, Escola Paulista de Medicina (UNIFESP/EPM), Laboratorio de Neurologia Experimental, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Rüdiger Köhling, University of Rostock, Institute of Physiology, Rostock, Germany Hana Kubová, Institute of Physiology, Academy of Sciences, Videnska, Prague, Czech Republic Sanjay S. Kumar, Department of Comparative Medicine, Stanford University, Stanford, CA
Astrid Nehlig, Faculty of Medicine, University of Strasbourg, Strasbourg, France Prosper N’Gouemo, Department of Pharmacology, Georgetown University Medical Center, Washington, DC
João P. Leite, Department of Neurology, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
Jari Nissinen, Department of Neurobiology, AI Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
Laura Librizzi, Department of Experimental Neurophysiology, Instituto Nazionale Neurologico Carlo Besta, Milan, Italy
Jeffrey L. Noebels, Developmental Neurogenetics Laboratory, Department of Neurology, Baylor College of Medicine, Houston, TX
Dean D. Lin, Brain Institute, Department of Neurological Surgery, University of Florida, Gainesville, FL
Michael W. Nestor, Department of Physiology, University of Maryland, Baltimore, MD
Wolfgang Löscher, Department of Pharmacology, Toxicology & Pharmacy, School of Veterinary Medicine, Hannover, Germany
Andre Obenaus, Department of Radiation Medicine, Radiobiology Program, Loma Linda University, Loma Linda, CA
Contributing Authors
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Jeffrey Ockuly, Department of Neurology, University of Wisconsin, Madison, WI
O. Carter Snead III, Department of Neurology, The Hospital for Sick Children, Toronto, Ontario, Canada
Hans-Christian Pape, Institut fur Physiologie, Otto-vonGuericke-Universitat, Universitatsklinikum, Magdeburg, Germany
Erwin-Josef Speckmann, Institut fur Physiologie, Universitat Munster, Munster, Germany
Asla Pitkänen, Department of Neurobiology, AI Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland John Pollard, Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA David A. Prince, Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA Dominick P. Purpura, Dean, Albert Einstein College of Medicine, Bronx, NY Raddy L. Ramos, Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT Charles E. Ribak, Department of Anatomy and Neurobiology, University of California Irvine, Irvine, CA Michael A. Rogawski, Epilepsy Research Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD Steven N. Roper, Brain Institute, Neurological Surgery, University of Florida, Gainesville, FL Russell M. Sanchez, Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX Raman Sankar, Departments of Neurology and Pediatrics, Brain Research Institute, University of California Los Angeles, Los Angeles, CA Sebastian Schuchmann, Institute of Neurophysiology, Charite, Humboldt University, Berlin, Germany Philip A. Schwartzkroin, Department of Neurological Surgery, University of California Davis, Davis, CA Laszlo Seress, Central Electron Microscopic Laboratory, Faculty of Medicine, University of Pecs, Pecs, Szigeti, Hungary Lee A. Shapiro, Department of Anatomy and Neurobiology, University of California Irvine, Irvine, CA Yukiyoshi Shirasaka, University of California Los Angeles, West Los Angeles VA Medical Center, Los Angeles, CA
Ajay Srivastava, Department of Pharmacology & Toxicology, Anticonvulsant Drug Development Program, University of Utah, Salt Lake City, UT Carl E. Stafstrom, Department of Neurology, University of Wisconsin, Madison, WI Mark Stewart, Department of Physiology and Pharmacology, SUNY Health Science Center, Brooklyn, NY Janet L. Stringer, Department of Pharmacology, Baylor College of Medicine, Houston, TX Lucie Suchomelova, University of California Los Angeles, West Los Angeles VA Medical Center, Los Angeles, CA Thomas P. Sutula, Departments of Neurology and Anatomy, University of Wisconsin Medical School, Madison, WI Kerry W. Thompson, University of California Los Angeles, West Los Angeles VA Medical Center, Los Angeles, CA Scott M. Thompson, Department of Physiology, University of Maryland, Baltimore, MD William J. Triggs, Department of Neurology, College of Medicine, University of Florida, Gainesville, FL Yuto Ueda, Department of Psychiatry, Miyazaki Medical College, Miyazaki, Japan Laura Uva, Department of Experimental Neurophysiology, Instituto Nazionale Neurologico Carlo Besta, Milan, Italy Libor Velísˇek, Departments of Neurology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY Jana Velísˇková, Departments of Neurology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY Matthew C. Walker, Institute of Neurology, University College London, London, UK Claude Wasterlain, Brain Research Institute, University of California Los Angeles, Los Angeles, CA
Margaret N. Shouse, Department of Neurobiology, University of California Los Angeles, Los Angeles, CA
H. Jürgen Wenzel, Department of Neurological Surgery, University of California Davis, Davis, CA
Misty Smith-Yockman, Department of Pharmacology and Toxicology, Anticonvulsant Drug Development Program, University of Utah, Salt Lake City, UT
H. Steve White, Department of Pharmacology & Toxicology, Anticonvulsant Drug Development Program, University of Utah, Salt Lake City, UT
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Contributing Authors
Karen S. Wilcox, Department of Pharmacology & Toxicology, Anticonvulsant Drug Development Program, University of Utah, Salt Lake City, UT
Robert K.S. Wong, Department of Physiology and Pharmacology, SUNY Health Science Center, Brooklyn, NY
Philip A. Williams, Department of Biomedical Science, Colorado State University, Fort Collins, CO
Qian Zhao, Department of Medicine, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, NH
L. James Willmore, Department of Neurology, Saint Louis University School of Medicine, St. Louis, MO
Foreword
In 1972, J. Kiffin Penry and I co-edited a volume entitled “Experimental Models of Epilepsy.” The field of epilepsy research had advanced to such a level by that date to warrant, we thought, a manual that would assist laboratory workers to identify and apply optimal model systems for their investigations. The intervening years have borne witness to advances in our understanding of seizures, of epilepsy, and of epileptogenic mechanisms that could not have been envisioned thirty years ago. This progress has come about largely through the study of animal model systems that have allowed us to reconstruct many of the intimate details—e.g., of what controls excitation and inhibition—that give rise to seizures in cortical and sub-cortical brain regions. Fundamental to these studies has been the growing appreciation of the genetic factors influencing neuronal activity, and of the specific role of developmental events in the emergence of seizure disorders. Our increasing sophistication has led, in turn, to a growing complexity of features—systems, cellular, molecular, and genetic—that need to be factored into our vision of seizure mechanisms and epileptic syndromes. To meet that challenge, the number of “model systems” has grown exponentially. The use of animal models has a critical role for all of modern biomedical research. Developing such models, that reproduce critical features of clinical syndromes and phenotypes, has long been a major aspect of epilepsy research. The number of different directions and testable hypotheses offered through these models is sometimes intimidating. The models themselves—ranging from “simple systems” to intact models of complex epilepsy syndromes—provide access to questions about seizures, epileptogenesis, and various forms of the epilepsies, some with important agespecific features. Research using these systems has yielded important insights into human epileptic syndromes, with respect to mechanisms of disease processes and potential
therapeutic interventions—including antiepileptogenic and antiepileptic treatments. The current volume provides an invaluable resource for the modern investigator who hopes to make an intelligent choice of subjects for future research studies. There has been no other attempt to gather together major model possibilities since the 1972 publication of “Experimental Models of Epilepsy.” The current volume is therefore not only useful, but timely—especially given the major advances of recent research efforts, and the sense that modern epilepsy research is on the threshold of still more important and dramatic advances. There is much being said today for the value of “translational research.” While not specifically identified as such thirty years ago, research in epilepsy has always recognized the links between experimental and clinical studies. Indeed, some of the most significant advances in understanding epileptogenic processes have come from investigators with a keen awareness of the problems of detection and management of epilepsy at the clinical level. Conversely, some of the important improvements in treatment have been made possible by laboratory bench research. This complementary approach to the field is intrinsic to the history of research in epilepsy—one of the earliest recognized disorders in medicine, mistakenly considered a “Sacred Disease.” We now stand at the cusp of great advances still to come—in basic understanding and clinical treatments. The present volume highlights one of the most critical considerations that most investigators face—the choice of models—in their determination of how to go about “epilepsy research.” These choices, and the related experimental strategies, will determine if, how, and when we reach our ultimate goal—seizure elimination and epilepsy cures. Dominick P. Purpura, M.D. December 2004
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Preface
new emphasis on “developmental” issues—to which only one chapter was devoted in the 1972 book, but which is an important discussion point in many of the current chapters. This renewed overview also offers some hints about what we might learn in studying certain types of model systems. Such guidance is certainly useful for the young epilepsy researcher. We hope it is also helpful to those investigators studying related disorders and/or phenomena, to clinicians interested in the current state of the research art, and to the experienced laboratory investigator who is looking for fresh ways of addressing experimental questions. To help us in this effort, we’ve enlisted the assistance of a large group of epilepsy researchers, each of whom has particular expertise in the application of a given model or model type. We’ve asked the author(s) of each chapter to describe the model of interest within the context of the following outline: What does the model model? How is it generated? What are the characteristic and defining features of the model? What are its limitations? And what insights have been developed (through research on this model) into human epileptic disorders? While the ease with which these points are covered varies considerably from chapter to chapter (model to model), the contributing authors have done a superb job in presenting experimentally-useful information as well as conceptually challenging discussions. We have also charged a set of investigators with the task of describing some of the most generally applied technical approaches for investigating epilepsy-relevant models. Again, these chapters not only offer technical overviews, but also provide important discussions about the requirements, advantages, and limitations of these experimental approaches. We hope that the reader will find that these descriptions are useful in thinking about how to do productive epilepsy research.
In 1972, Purpura and colleagues (Purpura, Penry, Tower, Woodbury, and Walter) published a “definitive” volume describing the available models for epilepsy research (Experimental Models of Epilepsy—A Manual for the Laboratory Worker, Raven Press, New York). At the time, epilepsy research was still in its early stages—perhaps not its infancy, but certainly its childhood. The authors could have hardly imagined either the long-reaching influence of their publication, or the incredible maturation of this field. Research on the basic mechanisms of, and treatments for, the epilepsies has become a significant part of many clinical and basic neuroscience programs. Technical advances in cellular and molecular biology have provided a driving force for much of this research, making feasible previously onlydreamed-of experiments. There has been a rush of excitement with the realization that we can study almost any experimental subject—from gene to intact human subject. This excitement has been tempered with a perhaps not-sorapid development of our conceptual sophistication. What questions are important to ask? And in what systems can these questions be best addressed? The response to the latter of the questions is reflected in an explosion of potentially useful “models”—models of epileptiform cellular activity, models of seizure generation, models of epilepsy and epileptogenesis. On the one hand, these models constitute the life-blood of our research efforts. They allow us to examine—in ways that are not available in clinical studies—hypotheses about basic mechanisms. They provide us with opportunities to test the efficacy of new treatments and novel therapies. On the other hand, the plethora of such models confronts us with a need for significant choices. To be effective in our quest for answers (and cures), we need to be intelligent about how best to use the model systems that are available. The current volume represents, in part, an attempt to provide an updated “list” of epilepsy models. While some of the models described in Purpura et al. are still in general use, others have fallen out of favor. One noteworthy change is the
Asla Pitkänen (Kuopio, Finland) Philip A. Schwartzkroin (Davis, California) Solomon L. Moshé (Bronx, New York)
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1 What Should Be Modeled? JEROME ENGEL, JR. AND PHILIP A. SCHWARTZKROIN
Epilepsy accounts for a significant portion of the disease burden worldwide (Murray and Lopez, 1994). The economic, social, and personal costs of this disorder are due largely to uncontrolled seizures (Begley et al., 2000), which underscores the need for more research into new approaches for the diagnosis, treatment, and prevention of epilepsy and its consequences. Although one could argue that research on human epilepsy ideally should be carried out on humans with epilepsy, this approach is not always possible or practical (Engel, 1998). Obvious ethical constraints exist, particularly those associated with the invasive techniques often needed to pursue important investigative questions. It is difficult to control for clinical variables, and control data can be impossible to obtain. Statistical analysis frequently requires larger populations than can be obtained from most clinical practices. Finally, the cost of carrying out research projects on patients would be prohibitive. Consequently, despite a tremendous increase in the opportunities for noninvasive research on the human brain provided by modern neuroimaging and growing access to direct investigations in the setting of epilepsy surgery, animal models of epilepsy and epileptic seizures are—and most likely will remain for the foreseeable future—essential to epilepsy research. This book provides an update on the large variety of animal models available to neuroscientists interested in carrying out research on epilepsy. This introductory chapter reviews the various reasons why animal models might be used, what specifically can and should be modeled, and how these models might provide insight into questions of interest and concern (i.e., what can be measured).
(usually epilepsy related) and normal brain function. Animal models of epilepsy are also important, however, for research designed specifically to devise new diagnostic approaches or to test the efficacy of new antiepileptic drugs or other novel therapeutic interventions. It is likely that in the future animal models will be needed to test preventive (i.e., antiepileptogenic) measures as well.
Modeling to Understand Basic Mechanisms Elucidation of the fundamental mechanisms of epilepsy and epilepsy-related phenomena is essential for devising new diagnostic, therapeutic, and preventative approaches to human epilepsy and its consequences. Until relatively recently, almost everything we knew, or thought we knew, about neuronal events underlying epileptic phenomena derived from research using animals. From the outset, however, the most pertinent questions that needed to be answered about epilepsy derived from observations of patients. Answers obtained in the animal laboratory required validation of clinical relevance, again by observing patients. Although the use of animal models to understand the fundamental mechanisms of epilepsy and its consequences is the major theme of this volume, it is important to recognize that animal models of epilepsy, and of epileptic seizures, also have been used to elucidate neuronal mechanisms of normal brain function (Engel et al., 2001). For instance, the discrete perturbations induced by various epileptogenic insults have served as a valuable tool for mapping pathways and synaptically related regions in the brain, both anatomically (e.g., using histological 2-deoxyglucose [2DG] autoradiography and immediate early gene methodologies) and physiologically (e.g., with strychnine neuronography). Epileptic seizures have also been commonly used by physiological psychologists for a variety of investigational
WHY MODEL? Animal models of epilepsy most often are used to investigate fundamental neuronal mechanisms of both abnormal
Models of Seizures and Epilepsy
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Copyright © 2006, Elsevier Inc. All rights of reproduction in any form reserved.
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Chapter 1/What Should Be Modeled?
paradigms that require controlled interruption of behavior. Studies of oscillatory brain waves—a prominent feature of normal cerebral function—have been approached through the window of epileptiform phenomena. Our understanding of inhibitory control of brain excitability has been based largely on studies of epilepsy models. Perhaps most important is that modern investigations into brain “plasticity” mechanisms have a significant basis in studies of epileptogenesis. Research using models of different aspects of epilepsyand seizure-related phenomena provides different types of insights into basic mechanistic questions: Acute seizure models: Studies of acute seizures in otherwise normal animals have made us aware of a host of potentially important cellular and molecular processes that might be involved in both the generation and the termination of epileptic seizure, and these possibilities remain the focus of much of today’s research (Avanzini et al., 1998; Engel, 1992; Purpura et al., 1972; Schwartzkroin, 1993). Early workers produced acute generalized epileptic seizures in animals using maximal electroshock and various chemoconvulsants (laboratory procedures that are still commonly used today) as well as a variety of other insults, such as insulin shock and trauma. Localized acute epileptic seizures were created by focal electrical stimulation and by local application of convulsant drugs such as strychnine and penicillin. Such techniques provoked acute ictal (i.e., seizure) activity in otherwise normal brains, permitting investigations of the fundamental neuronal basis of ictal discharge and seizure termination. These studies led to a number of hypotheses regarding the cellular bases of seizure activity, including alterations in intrinsic properties of neurons, loss of inhibition or increased excitatory synaptic activity, and changes in the extracellular milieu. This research also led to theories about seizure termination, such as energy depletion, desynchronization, depolarization block, and release of antiseizure substances like adenosine. Although it was often possible to determine why experimental interventions produced seizures, these manipulations were artificial and there was little or no way to relate these mechanisms of seizure initiation to those of spontaneous generation of seizures in patients. Acute seizures have also been useful, as indicated previously, as a perturbation for those interested in investigating normal brain function. Chronic epilepsy models: Neuroscientists interested in understanding mechanisms of epilepsy have developed a number of chronic animal models with which to investigate persistent epileptogenic abnormalities present between seizures, or interictally. Chronic animal models have offered opportunities to investigate a large set of potentially important and clinically relevant mechanisms,
including excitotoxicity and synaptic reorganization, altered voltage-gated channel functions, sprouting with synaptic reorganization, the development of novel receptors and receptor complements, and astrocyte activation. Many chronic epilepsy models were created specifically to reproduce particular types of human epilepsy, particularly the most common form, mesial temporal lobe epilepsy (MTLE) (Wieser et al., 2004). Seizures in MTLE originate in mesial temporal limbic structures, particularly the hippocampus and adjacent parahippocampal cortex. Most patients with MTLE have a unique epileptogenic structural disturbance— hippocampal sclerosis—but the cause of this disturbance is unknown. Many other lesions in this area, such as tumors, malformations, and infections, can also cause MTLE. A popular laboratory model of MTLE is amygdala kindling, which brings the process of limbic epileptogenesis under experimental control but, as usually performed, does not result in spontaneous seizures. Nevertheless, kindling does induce an enduring epileptogenic process that can be studied profitably. Chronic models of MTLE that give rise to spontaneous seizures can be produced by a variety of interventions, including systemic or intrahippocampal application of excitotoxic agents, such as kainic acid or pilocarpine, as well as focal electrical stimulation that gives rise to self-sustained status epilepticus. Chronic neocortical models include freeze lesions; the partially isolated cortical slab; application of metals such as alumina, cobalt, tungstic acid, and ferric chloride; tetanus toxin; and antimonosialogangioslide (GM1) antibodies. With the advent of high-resolution magnetic resonance imaging (MRI), it has become apparent that malformations of cortical development, particularly cortical dysplasia, account for a large percentage of intractable epilepsies in patients (Schwartzkroin and Walsh, 2000). These conditions have been modeled by in utero chemical exposures and early irradiation or by localized freeze lesions in the neonate. The genetic basis of epilepsy is receiving increasing attention (Noebels, 2003), and a large number of genetic epilepsies also occur in animals. Some of these resemble human genetic epilepsies, such as the Genetic Absence Epilepsy Rat from Strasbourg (GAERS) model of human absence epilepsy. Research with these models, too, have revealed a large number of potential epileptogenic mechanisms, such as voltage-gated channel abnormalities or deletions, aberrant neuronal–glial interactions, and abnormalities in energy metabolism. For most of these genetic models, however, the relevance to human epilepsy remains to be demonstrated. Multifactorial models: It is becoming increasingly apparent that the etiologies of many human epileptic conditions, particularly those that are refractory to medication, are
Why Model?
likely to be multifactorial. Most commonly this would involve both a genetic predisposition and a specific insult. For instance, patients with MTLE are more likely than the general population to have a family history of epilepsy and either a complex febrile seizure or some other initial precipitating insult within the first 5 years of life, insults that are now believed to be important contributors to the cell loss and neuronal reorganization characteristic of hippocampal sclerosis (Wieser et al., 2004). Such pathophysiologic disturbances, when superimposed on a genetic predisposition for seizure activity, may greatly increase the likelihood that a sclerotic hippocampus will ultimately become epileptogenic. Just as there was a major paradigm shift for epilepsy researchers to realize that seizures in a normal brain are not the same as chronic epilepsy, we are now facing the realization that chronic epilepsy induced in a normal brain is not the same as chronic epilepsy induced in a brain that is genetically predisposed to specific epileptogenic disturbances (Engel and Bertram, 2004). There is a great need to identify epilepsy “susceptibility genes” in patients so that multifactorial animal models can be devised and the interactions of multiple etiologic contributions studied.
Modeling to Devise New Approaches for Diagnosis Electrophysiologic Diagnosis The first laboratory test capable of measuring human cerebral function was the electroencephalogram (EEG), which rapidly became the premier diagnostic tool for epilepsy and provided a key element for the classification of epileptic seizures (Commission on Classification, 1981). Interictal and ictal epileptiform discharges on EEG are not only useful for making a general diagnosis of epilepsy, but they also provide a basis for determining what type of epilepsy is present and for localizing the epileptogenic abnormality when surgical treatment is being considered. Animal models of epilepsy played an important role in understanding the neuronal mechanisms responsible for the interictal EEG spike and wave as well as for the various ictal EEG patterns that characterize specific seizure types. Much still can be done using model systems to improve the diagnostic value of EEG: 1. Surrogate markers of epileptogenicity and epileptogenesis. Interictal spikes are too often falsely localizing because it is difficult (at least at times) to distinguish among spikes generated by the primary epileptogenic region, those that are propagated into the area under the recording electrode,
3
and those that occur in epileptogenic areas incapable of generating spontaneous seizures. Furthermore, interictal epileptiform EEG discharges can be seen in some people who do not have epileptic seizures. In patients who do have epileptic seizures, interictal spikes can be recorded from areas distant from the primary epileptogenic regions. Finally, for most patients with epilepsy, the characteristics of the interictal epileptiform discharges provide no information about the frequency or severity of seizure occurrence. Consequently, these EEG events have limited value as markers of epileptogenicity and epileptogenesis. There is a great need, therefore, for more accurate surrogate or biological markers of epilepsy to (1) localize the epileptogenic region for surgical resection, (2) predict whether someone will develop epilepsy following a potentially epileptogenic insult, and (3) determine the efficacy of a therapeutic intervention (for instance, an antiepileptic drug) without the need to wait for another seizure to occur. One possible surrogate marker under investigation is a very high frequency (250–600 Hz) EEG oscillation, termed fast ripples (FR) (Bragin et al., 1999). FR are uniquely seen in association with interictal EEG spikes recorded from areas capable of generating spontaneous seizures. Although FR are easily identified with depth electrode recordings from patients with MTLE, they cannot yet be recorded noninvasively. Therefore ascertaining the diagnostic value of FR will require investigations in animal models. 2. Seizure prediction. There is much current interest in developing methods for anticipating the onset of seizure activity with sufficient latency to allow automated interventions to abort the seizure. Development of computational paradigms, based on EEG signals, is the basis for current approaches to this goal (Lehnertz and Litt, 2005). Most of this research has been carried out directly on patients with epilepsy; however, animal models in which seizures occur unpredictably, but with reasonable frequency, could become crucial for developing practical analysis paradigms and eventually for devising and testing seizure-aborting interventions that can be triggered from alarm signals. Neuroimaging Technical progress in the neuroimaging field has led to a revolution in diagnostic procedures for epilepsy. Modern neuroimaging now allows us to assess structural, functional, and metabolic features of suspect brain areas. Although this revolution has often proceeded at the level of clinical investigation, the possibility of addressing specific imagingrelated questions lies primarily in the application of these techniques to animal models. In particular we still do not understand the relationship of abnormalities revealed by
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Chapter 1/What Should Be Modeled?
neuroimaging to epileptogenesis, to seizure initiation, and to seizure propagation. These clinical diagnostic approaches will help us to define more clearly what features of brain abnormality are strongly coupled to epilepsy and epileptogenesis for further investigations in animal models. The following are examples of such features: 1. Structural abnormalities. Magnetic resonance imaging (MRI) is the most important diagnostic test for identifying potentially epileptogenic structural lesions in patients with epilepsy, and the clinical value of this diagnostic approach was developed through extensive clinical application. It has also been used in clinical studies to document the progressive nature of some epileptogenic lesions. Small-animal MRI has become available at many centers throughout the world. Application of this structural imaging tool in animal models should lead to improved efficacy of clinical applications as well as a clearer definition of epilepsyrelated structural abnormalities. Putative structural surrogate markers of epileptogenicity and epileptogenesis, for example, axon sprouting in MTLE or specific patterns of cortical dysplasia, could be investigated in animals using high-resolution MRI. Whereas clinical studies are normally cross-sectional, much more valuable longitudinal paradigms can be constructed in small animal models to investigate early epileptogenesis as well as epilepsy-induced progressive disturbances (still a controversial issue). 2. Functional abnormalities. Functional neuroimaging is playing an increasingly important role in the diagnosis of epilepsy (Henry et al., 2000). The most frequently used functional neuroimaging approach, positron emission tomography with fluorodeoxyglucose (FDG-PET), was based on animal experiments with 2DG autoradiography. FDG-PET studies in human temporal lobe epilepsy, for instance, were preceded by 2DG autoradiography studies of amygdala kindling. Now a number of other tracers are being used clinically for diagnostic purposes in epilepsy, not only with PET, but with single-photon emission computed tomography (SPECT). One tracer, alphamethyl-tryptophan (AMT), may also be a surrogate marker of epileptogenicity (Chugani et al., 1998). Development of these diagnostic tools in animal models can help to focus their capabilities. A unique capacity of functional MRI (fMRI) as a functional neuroimaging tool is its high temporal resolution. This capability makes it possible to image the evolution of ictal activity throughout the brain, in three dimensions, during the course of a seizure and then to examine the postictal consequences of this activity. Furthermore, this temporal resolution permits imaging of the anatomic substrates of brief EEG transients, such as
interictal spikes. Consequently, the use of EEG together with fMRI has great potential for localizing the epileptogenic region in surgical candidates without requiring expensive long-term monitoring to capture ictal events. Whereas interictal spikes on the EEG can be falsely localizing, EEG-fMRI eventually may be able to discriminate between interictal spikes from the primary epileptogenic region and those that are propagated or occur in areas not capable of generating spontaneous seizures. Such EEG-fMRI events could be surrogate markers for predicting which patients are likely to develop epilepsy following insult or for determining which antiepileptic intervention is most likely to be effective without waiting for another seizure to occur. At present EEG-fMRI research is being carried out almost exclusively in patients; however, realization of the full potential of this technique will require much more extensive research with experimental animal models. Animal models are also likely to play an important role in the development and effective clinical application of newer diagnostic techniques, such as magnetoencephalography (MEG), magnetic resonance spectroscopy (MRS), microdialysis, optical imaging, and transcranial magnetic stimulation (TMS).
Modeling to Test New Therapies Pharmacotherapy Beginning with studies of phenytoin, investigations in animal models have been used to determine the efficacy and safety of new antiepileptic compounds before they are tried in patients. Animal models of epileptic seizures have been indispensable for this purpose. With one exception (levetiracetam), however, all potential new antiepileptic compounds have been screened against only two mouse models of epileptic seizures: maximal electroshock and subcutaneous Metrazol (Levy et al., 2002). Undoubtedly testing drugs in these models of generalized tonic-clonic seizures and absence seizures, respectively, has resulted in identification of many drugs effective against epilepsies associated with these two seizure types. However, this approach has been less useful in the discovery of drugs for treating other seizure types, particularly focal seizures and atonic seizures. Consequently epilepsy associated with focal and atonic seizures are often medically refractory. It is likely that many compounds that might have been excellent for preventing focal and atonic ictal events were discarded because they did not have anticonvulsant or antiabsence properties. These two acute seizure mouse models continue to be used for drug screening because large numbers of compounds can be screened inexpensively. In contrast it is very expensive to
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What to Model
screen large numbers of compounds against chronic models (e.g., the amygdala kindling model of MTLE). There is therefore a great need to develop new animal models of those seizure types that are particularly refractory to current pharmacotherapy that could be cost-effectively used for drug screening. Development of model systems based on surrogate markers might possibly fill this role.
Alternative Therapies Alternative treatments for epilepsy also benefit from animal research. Vagus nerve stimulation (VNS), for instance, was first investigated with acute seizures in dogs and then with chronic epilepsy in monkeys before it was tried in humans (Schachter and Wheless, 2002). Animal research is also playing an important role in developing the techniques of deep brain stimulation as a treatment for epilepsy. Epilepsy surgery, gamma knife surgery, and the ketogenic diet (KD) have been used effectively in patients without preliminary studies in animals. However, animal research continues to be of value in our efforts to understand how these therapeutic interventions work, what might be done to improve them, and which patients are most likely to benefit from their use. Identifying appropriate animal models for testing therapeutic interventions is a prime concern. For example, because KD is principally used in children, understanding and improving KD-based approaches require comparable immature brain experimental animal models (Stafstrom and Rho, 2004).
Antiepileptogenesis Currently no treatments are available that focus explicitly on the issue of preventing the development of chronic epilepsy (antiepileptogenesis) or on ameliorating the biological consequences of chronic seizure activity. Although there has been much discussion about the need for such approaches, the few existent clinical studies (e.g., prophylactic treatments for posttraumatic epilepsy) have been disappointing. None of our current antiepileptic drugs is known to be antiepileptogenic. Whereas clinical studies on this important theme cannot be cost-effectively pursued without better prognostic indications, current animal model systems provide an important opportunity to begin to assess novel treatments that could prevent the development of chronic seizures following a known epileptogenic manipulation (Loescher, 2002). Animal models can also be used to help identify treatments that might decrease the morbidity associated with seizures, including the progression of the epileptogenic process, interictal disturbances in behavior (e.g., learning
and memory deficits in MTLE), the occurrence of depression, and developmental delay in infants and small children. Although these disturbances represent a major cause of disability in patients, their causal relationship to seizures remains controversial because this association is difficult to document clinically, let alone investigate in a controlled fashion (Sutula and Pitkänen, 2002). Animal studies, however, have shown that kindling causes seizures to become more severe, that rats exposed to repeated seizures when they are young are deficient in some learning and memory behaviors (even though the initial seizure exposure does not result in spontaneous seizures in the adult or in demonstrable neuropathologic changes), and that seizures can alter behavior in ways that suggest psychiatric disturbances (Engel et al., 1991). Such animal models present opportunities for assessing therapies that might treat or prevent these progressive disturbances.
WHAT TO MODEL The International League against Epilepsy (ILAE) agreed on the following definitions for epileptic seizures and epilepsy (Fisher et al., 2005). “An epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain.” “Epilepsy is a chronic disorder of the brain characterized by an enduring predisposition to generate epileptic seizures, and by the neurobiological, cognitive, psychological, and social consequences of this condition. The definition of epilepsy requires the occurrence of at least one epileptic seizure.” When devising models of human epileptic seizures and epilepsy, therefore, it is essential to distinguish between (1) models of acute epileptic seizures that occur in a normal brain and do not necessarily indicate the presence of an epileptic condition and (2) models of epilepsy that are associated with permanent “epileptogenic” disturbances. The latter is present whether or not seizures are occurring and can be associated not only with seizure phenomena but also with enduring or progressive nonepileptic consequences of these disturbances. There are many different types of epileptic seizures, and each may be associated with different epileptogenic mechanisms (Commission on Classification, 1981; Engel and Pedley, 1997); such likely differences need to be considered when creating animal models. There are also many different epilepsy syndromes that are characterized by the occurrence of one or more specific seizure type(s) as well as by other clinical features such as age of onset, response to antiepileptic drugs, family history, interictal disturbances, pathophysiologic mechanisms and anatomic substrates (Commission on Classification, 1989; Engel and Pedley, 1997). The variety of these
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syndromes also needs to be considered when animal models are created. It is debatable whether animal models exist—or can be created—that faithfully reproduce any given human epilepsy syndrome. Consequently, one approach to modeling human epilepsy is to define the component parts of epileptic disorders and to model each part individually. Even a single epileptic seizure (with a few exceptions) consists of an evolution of events, each event with different mechanisms and anatomic substrates that can be individually modeled and independently studied. Laboratory phenomena that either represent a basic component part of an epileptic disturbance or provide information about such a disturbance have been referred to as epilepsy equivalents (Engel, 1992); some surrogate markers can serve as epilepsy equivalents.
Epileptic Seizures The ILAE classified epileptic seizures most recently in 1981 (Commission on Classification, 1981) (Table 1), and this classification is undergoing revision (Engel, 2001). The 1981 classification is based solely on clinical and EEG descriptive phenomena. It defines seizures that begin in a part of one hemisphere as partial seizures and those that begin in both hemispheres at the same time as generalized seizures. Generalized seizures are often divided into
TABLE 1 International Classification of Epileptic Seizures I.
Partial (focal, local) seizures A. Simple partial seizures 1. With motor signs 2. With somatosensory or special sensory symptoms 3. With autonomic symptoms or signs 4. With psychic symptoms B. Complex partial seizures 1. Simple partial onset followed by impairment of consciousness 2. With impairment of consciousness at onset C. Partial seizures evolving to secondarily generalized seizures 1. Simple partial seizures evolving to generalized seizures 2. Complex partial seizures evolving to generalized seizures 3. Simple partial seizures evolving to complex partial seizures evolving to generalized seizures
II. Generalized seizures (convulsive or nonconvulsive) A. Absence seizures 1. Typical absences 2. Atypical absences B. Myoclonic seizures C. Clonic seizures D. Tonic seizures E. Tonic-clonic seizures F. Atonic seizures (astatic seizures) III. Unclassified epileptic seizures From Commission on Classification, 1981, with permission.
convulsive and nonconvulsive types. The convulsive types include generalized tonic-clonic seizures (previously called grand mal seizures) as well as seizures that are purely clonic, seizures that are purely tonic, and various combinations of these ictal manifestations. Nonconvulsive generalized seizures include typical absence seizures (previously called petit mal seizures) as well as atypical absence seizures. Typical absences involve brief losses of consciousness, without warning or postictal symptoms, associated with a regular three-per-second generalized spike-and-wave EEG discharge. Atypical absences last longer, there can be postictal symptoms, and the EEG patterns are more irregular. Other nonconvulsive generalized seizures are generalized myoclonic seizures, which consist of sudden bilateral myoclonic jerks, and atonic seizures, which involve sudden losses of muscle tone. Myoclonic, atonic, and brief clonic seizures can all result in falls, referred to as astatic seizures or “drop attacks.” Generalized convulsive seizures, typical absences, and myoclonic seizures occur in the benign genetic epileptic disorders that are unassociated with brain damage; in contrast generalized convulsive seizures, atypical absences, and atonic seizures occur in conditions in which brain damage is so diffuse that seizures begin bilaterally. The 1981 ILAE classification divides partial seizures into simple if there is no alteration of consciousness or complex if consciousness is impaired. Simple partial seizures can have motor, sensory, autonomic, or psychic signs or symptoms, depending on the function of the cortex where the seizures arise. The term complex partial seizures has been erroneously used synonymously with temporal lobe seizures because most often these events begin in mesial temporal limbic structures. It is important to understand, however, that consciousness can be impaired as a result of ictal activity in other brain areas so that not all complex partial seizures are of mesial temporal origin. Furthermore, mesial temporal seizures often do not propagate to produce impaired consciousness; they can give rise to characteristic simple partial signs and symptoms that occur with clear consciousness, typically sensations of epigastric rising, emotional experiences such as fear, and autonomic changes. Partial seizures commonly evolve. Simple partial seizures can progress to complex partial seizures, and both simple and complex partial seizures can progress to secondarily generalized seizures. Simple partial seizures without motor signs that proceed to complex partial or secondarily generalized seizures are commonly referred to as auras. Clearly the pathophysiologic mechanisms and anatomic substrates of ictal events change during this evolution. Similarly, different mechanisms and different brain areas (e.g., forebrain and brainstem) are involved at different times during progression of a generalized tonic seizure to the clonic phase.
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What to Model
Because partial seizures often involve widespread disturbances, sometimes involving both hemispheres, the term partial was preferred over focal when the 1981 classification was devised. The often-used term epileptic focus gives the misleading impression that epileptogenic abnormalities are limited to a small, discrete area of the brain. This impression resulted in part from early research on the experimental penicillin focus, which did involve seizures generated from activation of a small cluster of neurons. The more accurate replacement term, partial, however, has also caused confusion because it can be misinterpreted to mean part of a seizure rather than an entire seizure. Consequently the ILAE recommends returning to the earlier term, focal seizure (Engel, 2001), but with the explicit understanding that most “focal” epilepsies involve a widely distributed epileptogenic substrate; indeed, in chronic focal epilepsies, the entire brain may be abnormal. The ILAE is also planning to eliminate the distinction between simple and complex events based on impaired consciousness (Engel, 2001). Although impairment of consciousness is clinically important, it is difficult to assess in many patients and is no longer considered an appropriate criterion for classification of ictal phenomena. The concept of complex is, in any event, suspect with respect to animal models; indeed it is usually difficult or impossible to determine the state of consciousness of a rat or mouse. It will be much easier to justify a claim that seizures in an animal model reproduce human seizure types when these clinical ictal events are better defined based on pathophysiologic mechanisms and anatomic substrates. There is also concern about the designation of generalized seizures because there is a question as to whether any seizures are truly generalized at their onset (Engel, 2001). Although advanced EEG and functional neuroimaging techniques can help to determine the true site of ictal onset for various types of human “generalized” seizures, animal models of these ictal events will greatly enhance our ability to answer this important question. Within the context of this current volume on animal models, it is noteworthy that clinicians have had such difficulty in defining epileptic phenomena. Certainly it would be much easier to justify a claim that seizures in an animal model reproduce human seizure types if these clinical ictal events were better defined. To eliminate confusion and misconceptions caused by the 1981 seizure classification, the ILAE is currently attempting to identify ictal events that represent discrete pathophysiologic mechanisms and anatomic substrates that can be used as diagnostic entities rather than to rely only on phenomenologic designations developed for descriptive purposes (Engel, 2001). This attempt at redefinition is important and welcome news for animal research in areas where human phenomenology may be difficult to reproduce but pathophysiologic mechanisms and anatomic
substrates can be effectively modeled and verified. There was insufficient information to base seizure classification on mechanistic and anatomic criteria when the current ILAE classification was accepted in 1981, and concern remains as to whether our knowledge of basic mechanisms of epileptic seizures today is adequate to permit a more “diagnostic” classification. Nevertheless, a tentative list of discrete seizure types, based on this new classification scheme, has been published (Engel, 2001) (Table 2). These seizure types are being individually evaluated to determine whether there is sufficient justification to consider them discrete diagnostic entities. This evaluation will take into account the following criteria: pathophysiologic mechanisms (e.g., electrophysiology, neural networks, and neurotransmitter actions), anatomic substrates, response to antiepileptic drugs, ictal EEG patterns, propagation patterns and postictal features, and associated epilepsy syndromes. The list in Table 2 includes a number of seizure types that are characteristic of specific epilepsy syndromes but that are not part of the 1981 classification, such as myoclonic atonic seizures, myoclonic absences, negative myoclonus, gelastic seizures, and epileptic spasms. Whereas epileptic spasms were previously referred to as infantile spasms and considered to be purely an age-related ictal phenomenon characteristic of West syndrome, it is now accepted that epileptic spasms can also occur in older children and adults. No valid animal model of this often intractable and devastating seizure type exists. Some of these epileptic seizure types are easily treated by antiepileptic drugs, and others are highly refractory to pharmacotherapy. The development of animal models of the more refractory seizure types is obviously valuable to understanding their fundamental neuronal mechanisms and to devising better treatments. There is also a great need to identify surrogate markers or epilepsy equivalents of these phenomena that could serve to develop costeffective screening procedures for potential antiepileptic compounds.
Epilepsies and Epilepsy Syndromes The current ILAE classification of epilepsies and epilepsy syndromes (Table 3) was approved in 1989 (Commission on Classification, 1989) and is also now undergoing revision (Engel, 2001). The 1989 classification divides epilepsies into generalized and localization-related categories, depending on whether the characteristic seizures begin simultaneously on both sides of the brain or in a part of one hemisphere. Generalized epilepsies are never associated with partial seizures, but localization-related epilepsies can be associated with generalized seizures if they are secondarily generalized. Epilepsy syndromes are further divided by this classification into idiopathic and symptomatic categories. The term
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Chapter 1/What Should Be Modeled?
TABLE 2 Epileptic seizure types and precipitating stimuli for reflex seizures Self-limited seizure types Generalized seizures Tonic-clonic seizures (includes variations beginning with a clonic or myoclonic phase) Clonic seizures Without tonic features With tonic features Typical absence seizures Atypical absence seizures Myoclonic absence seizures Tonic seizures Spasms Myoclonic seizures Eyelid myoclonia Without absences With absences Myoclonic atonic seizures Negative myoclonus Atonic seizures Focal seizures Focal sensory seizures With elementary sensory symptoms (e.g., occipital and parietal lobe seizures) With experiential sensory symptoms (e.g., temporoparietooccipital junction seizures) Focal motor seizures With elementary clonic motor signs With asymmetric tonic motor seizures (e.g., supplementary motor seizures) With typical (temporal lobe) automatisms (e.g., mesial temporal lobe seizures) With hyperkinetic automatisms With focal negative myoclonus With inhibitory motor seizures Gelastic seizures Hemiclonic seizures Secondarily generalized seizures Continuous seizure types Generalized status epilepticus Generalized tonic-clonic status epilepticus Clonic status epilepticus Absence status epilepticus Tonic status epilepticus Myoclonic status epilepticus Focal status epilepticus Epilepsia partialis continua of Kojevnikov Aura continua Limbic status epilepticus (psychomotor status) Hemiconvulsive status Precipitating stimuli for reflex seizures Visual stimuli Flickering light: color to be specified when possible Patterns Other visual stimuli Thinking Music Eating Praxis Somatosensory Proprioceptive Reading Hot water Startle Modified from Engel 2001, with permission.
TABLE 3 International Classification of Epilepsies, Epileptic Syndromes, and Related Seizure Disorders 1. Localization-related (focal, local, partial) 1.1. Idiopathic (primary) Benign childhood epilepsy with centrotemporal spikes Childhood epilepsy with occipital paroxysms Primary reading epilepsy 1.2. Symptomatic (secondary) Temporal lobe epilepsies Frontal lobe epilepsies Parietal lobe epilepsies Occipital lobe epilepsies Chronic progressive epilepsia partialis continua of childhood Syndromes characterized by seizures with specific modes of precipitation 1.3. Cryptogenic, defined by: Seizure type Clinical features Etiology Anatomical localization 2. Generalized 2.1. Idiopathic (primary) Benign neonatal familial convulsions Benign neonatal convulsions Benign myoclonic epilepsy in infancy Childhood absence epilepsy (pyknolepsy) Juvenile absence epilepsy Juvenile myoclonic epilepsy (impulsive petit mal) Epilepsies with grand mal seizures (GTCS) on awakening Other generalized idiopathic epilepsies Epilepsies with seizures precipitated by specific modes of activation 2.2. Cryptogenic or symptomatic West syndrome (infantile spasms, Blitz-Nick-Salaam Krämpfe) Lennox-Gastaut syndrome Epilepsy with myoclonic-astatic seizures Epilepsy with myoclonic absences 2.3. Symptomatic (secondary) 2.3.1. Nonspecific etiology Early myoclonic encephalopathy Early infantile epileptic encephalopathy with suppression bursts Other symptomatic generalized epilepsies 2.3.2. Specific syndromes Epileptic seizures may complicate many disease states 3. Undetermined epilepsies 3.1. With both generalized and focal seizures Neonatal seizures Severe myoclonic epilepsy in infancy Epilepsy with continuous spike-waves during slow wave sleep Acquired epileptic aphasia (Landau-Kleffner syndrome) Other undetermined epilepsies 3.2. Without unequivocal generalized or focal features 4. Special syndromes 4.1. Situation-related seizures (Gelegenheitsanfälle) Febrile convulsions Isolated seizures or isolated status epilepticus Seizures occurring only when there is an acute or toxic event due to factors such as alcohol, drugs, eclampsia, nonketotic hyperglycemia From Commission on Classification, 1989, with permission.
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What to Model
idiopathic derives from the Greek idio, meaning “self,” and it refers to conditions that are only epilepsy (and nothing else). The term idiopathic does not mean “cause unknown” (epilepsies of unknown cause are called cryptogenic). Idiopathic epilepsies are genetic conditions without structural abnormalities and usually are without interictal clinical signs or symptoms. They are age-related and almost always benign in that seizures are highly responsive to pharmacotherapy and, for many syndromes, remit spontaneously as the brain matures. Symptomatic epilepsies are due to structural or metabolic disturbances, which can be acquired (e.g., from infections, trauma, or other insults), endogenous (e.g., tumors or congenital malformations), or genetic disturbances associated with structural or metabolic disturbances (e.g., tuberous sclerosis and phenylkentonuria). More than half of epilepsies are symptomatic, and these represent essentially all the conditions that are typically refractory to pharmacotherapy. The prognosis for symptomatic localizationrelated epilepsies depends on their etiology. The most common of these, and also the most common form of human epilepsy, is MTLE with hippocampal sclerosis (Wieser et al., 2004). MTLE is among the most difficult of the epilepsies to treat medically, although it is usually amenable to surgical therapy. Symptomatic generalized epilepsies are due to diffuse brain damage and manifest differently at different ages (e.g., severe myoclonic epilepsy shortly after birth, West syndrome in infants, and the Lennox-Gastaut syndrome in older children). These conditions are extremely difficult to treat and are usually associated with mental retardation and other neurologic deficits that contribute greatly to the disability. The ILAE also established a revised list of epilepsy syndromes (Engel, 2001) (Table 4) and is attempting to validate their existence as discrete diagnostic entities according to the following criteria: epileptic seizure type(s), age of onset, progressive nature, interictal EEG pattern, associated interictal signs and symptoms, pathophysiologic mechanisms, anatomic substrates, and etiologies. Justifications for the generalized versus localization-related dichotomy and for the idiopathic versus symptomatic dichotomy are being reevaluated; these dichotomies are not likely to be carried into any new classification scheme (Engel, 2001). Epilepsy experts now believe that none of these conditions is likely to be truly generalized and that both genetic and structural factors play a role in many (if not most) of these syndromes. Research with appropriate animal models could help to confirm this point of view. Several familial syndromes are listed in Table 4 that were not included in the 1989 classification, such as autosomaldominant nocturnal frontal lobe epilepsy and familial lateral and mesial temporal lobe epilepsies; these syndromes were recognized after the 1989 classification was completed. Further, some syndromes, such as familial focal epilepsy
TABLE 4 Epilepsy syndromes and related conditions Benign familial neonatal seizures Early myoclonic encephalopathy Ohtahara syndrome * Migrating partial seizures of infancy West syndrome Benign myoclonic epilepsy in infancy Benign familial infantile seizures Benign infantile seizures (nonfamilial) Dravet’s syndrome HHE syndrome * Myoclonic status in nonprogressive encephalopathies Benign childhood epilepsy with centrotemporal spikes Early onset benign childhood occipital epilepsy (Panayiotopoulos type) Late onset childhood occipital epilepsy (Gastaut type) Epilepsy with myoclonic absences Epilepsy with myoclonic-astatic seizures Lennox-Gastaut syndrome Landau-Kleffner syndrome (LKS) Epilepsy with continuous spike-and-waves during slow-wave sleep (other than LKS) Childhood absence epilepsy Progressive myoclonus epilepsies Idiopathic generalized epilepsies with variable phenotypes Juvenile absence epilepsy Juvenile myoclonic epilepsy Epilepsy with generalized tonic-clonic seizures only Reflex epilepsies Idiopathic photosensitive occipital lobe epilepsy Other visual sensitive epilepsies Primary reading epilepsy Startle epilepsy Autosomal dominant nocturnal frontal lobe epilepsy Familial mesial temporal lobe epilepsy Familial lateral temporal lobe epilepsy * Generalized epilepsies with febrile seizures plus * Familial focal epilepsy with variable foci Symptomatic (or probably symptomatic) focal epilepsies Limbic epilepsies Mesial temporal lobe epilepsy with hippocampal sclerosis Mesial temporal lobe epilepsy defined by specific etiologies Other types defined by location and etiology Neocortical epilepsies Rasmussen syndrome Other types defined by location and etiology Conditions with epileptic seizures that do not require a diagnosis of epilepsy Benign neonatal seizures Febrile seizures Reflex seizures Alcohol withdrawal seizures Drug or other chemically induced seizures Immediate and early post cerebral insult seizures Single seizures or isolated clusters of seizures Rarely repeated seizures (oligoepilepsy) * Syndromes in development Modified from Engel 2001, with permission.
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Chapter 1/What Should Be Modeled?
with variable foci and generalized epilepsy with febrile seizures plus (GEFS+), deviate from previous concepts of syndromes because they are syndromes of families rather than of individuals; that is, the diagnosis can be made only if the appropriate family history is available. There is nothing characteristic about the proband otherwise. Genetic discovery is now a major area of investigation in epilepsy. The search for new genetic syndromes, and for epilepsy genes, will undoubtedly yield information that will help to elucidate mechanisms of epilepsy and to provide a more objective basis for classification. At the same time, this work has greatly complicated efforts to classify and categorize specific epilepsy syndromes because we now recognize that “clearly defined” syndromes can be caused by several different gene abnormalities and that a given identified epilepsy gene abnormality can cause different epilepsy syndromes in different members of the same family. Animal models based on gene discovery will play an important role in sorting through this extremely important, highly complicated field of inquiry. Along with the reevaluation of epilepsy syndromes, a new concept of epileptic encephalopathy was introduced (Engel, 2001). This refers to epilepsy syndromes in which progressive behavioral and epileptic features are believed to be due to repeated ictal events rather than to the underlying etiology. A number of such syndromes are now recognized, including the Landau–Kleffner syndrome, epilepsy with continuous spike-and-wave during slow sleep, early myoclonic encephalopathy, Ohtahara syndrome, Dravet syndrome, myoclonic status in nonprogressive encephalopathies, West syndrome, Lennox-Gastaut syndrome, and perhaps even MTLE with hippocampal sclerosis. For these syndromes seizures appear to cause progressive deterioration, suggesting that early effective therapeutic intervention can prevent severe long-term disability resulting from nonepileptic deficits. Here, too, animal models will be essential in efforts to elucidate mechanisms that cause progression in the various epileptic encephalopathies, to devise diagnostic techniques that will determine when to intervene, and to develop more effective therapeutic interventions. Another interesting addition to the new list of syndromes is hypothalamic hamartoma with gelastic seizures, which may be an epileptic encephalopathy. Although this condition has been recognized for many years, it has only recently been demonstrated that the gelastic seizures appear to arise from the hamartoma itself and that in some cases the seizures can be abolished by surgical removal of the hamartoma (Berkovic et al., 2003). How a hypothalamic hamartoma can give rise to seizures is an interesting and still unanswered question; at present no animal model of this condition is available to help investigators unravel the mystery.
Component Parts of Epilepsy It should be clear from this review of epileptic seizure types and epilepsy syndromes that it may not be possible to develop accurate animal models in which the complex features of a seizure or syndrome are faithfully reproduced. Therefore animal modeling must adopt strategies that will help define and address the critical questions associated with the varied and complex seizure and syndrome phenotypes that have been clinically described. Perhaps the most widely accepted (explicit or implicit) strategy has been to focus on key components of seizures and syndromes (Engel, 1998). Examples of component parts that can be individually modeled are described in the following sections. This list is undoubtedly incomplete but will give the reader a sense of how such a strategy can be applied in the animal laboratory. Epileptogenesis Acquired epilepsies that begin with an epileptogenic insult require time to develop. This is likely to be the case even if the insult occurs on a background of preexisting seizure susceptibility. The period during which the insultinitiated processes give rise to a condition of spontaneous seizure discharge has been referred to as the latent period. For instance, for MTLE with hippocampal sclerosis (HS), there is thought to be an initial precipitating insult (within the first 5 years of life) that presumably results in the death of selective hippocampal neurons, with subsequent axonal spratting, synaptic reorganization and gliosis (Wieser et al., 2004). Other changes can also be associated with this epileptogenic process, including alterations in receptors and channels on key hippocampal cell populations as well as changes in the relative rates of neurogenesis and apoptosis. These changes appear to promote spontaneous hypersynchronous discharges that over time will recruit other limbic and distant structures into the epileptogenic process. At some later time—perhaps several years later—spontaneous seizures will emerge. This hypothesized mechanism of epileptogenesis has been modeled in animals using agents or techniques that cause similar damage to the hippocampus, such as kainic acid, pilocarpine, and stimulation-induced selfsustained status epilepticus. The subsequent recruitment of other limbic and distant structures into the epileptogenic process has been brought under laboratory control with amygdala and hippocampal kindling. For other types of epilepsy, it is likely that the epileptogenic process is very different. For instance, in agedependent idiopathic epilepsies, there may be no epileptogenic process per se (certainly no identifiable discrete initiating insult) but rather a substrate that is unmasked during a particular period of brain maturation. Such epilepsies often appear to be expressed during that period of brain development in which synaptogenesis reaches a peak, particularly
What to Model
when N-methyl-d-aspartate (NMDA)-type glutamate receptors are highly expressed. Even for the acquired epilepsies, such developmental processes influence epileptogenesis, determine an age-related expression of seizure activity, and must be considered when animal models are created. The Interictal State One of the most intriguing—and puzzling—features of epilepsy is that seizures occur sporadically, intermittently. That is, most of the time, there are no seizures, but the brain is still characterized by an epileptic condition. Given this seizure capability, the chronic interictal period is perhaps most interesting for the natural homeostatic mechanisms that prevent seizure generation. What factors are responsible for maintaining an interictal state and assuring that seizures do not occur continuously? What are the bases for the breakdown of such protective mechanisms when seizures do occur? Given that mechanisms of ictal onset differ among the various epileptic seizure types and epilepsy syndromes, it is possible that the protective seizuresuppressing mechanisms also differ. This area of investigation, which can be most easily pursued in the animal laboratory, should provide insights into novel clinical approaches to treat or prevent epilepsy. Ictal Onset Much remains to be learned regarding the transition from the interictal to the ictal state. This transition clearly differs markedly from one seizure type to another. In some conditions the transition can take considerable time, opening the potential for the application of electrophysiologic techniques to predict seizure onset minutes to hours before they occur (Lehnertz and Litt, 2004). In some cases, the preictal EEG findings merely reflect changes in normal brain function that decrease the threshold for seizure occurrence; in other cases they could reflect the accrual of pathological changes that slowly build up to the generation of a clinical ictal event. Animal models of these electrophysiologic phenomena could help to improve technologies for seizure prediction and to reveal “reversible” mechanisms that could be targeted by new strategies for seizure prevention. Ictus and Seizure Termination The ictal event for typical absence seizures is stereotypically repetitive and most likely represents a single pathophysiologic mechanism. However, for the vast majority of ictal events, there is a pattern of evolution that reflects a sequence of pathophysiologic disturbances at the neuronal level and in the extracellular space that results in recruitment of adjacent and distant anatomic structures. Depending on the seizure type, various ictal phases can be further broken
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down and studied independently using appropriate animal models. In addition, the mechanisms of synchrony—the feature that defines most seizure states—can be analyzed, potentially yielding insight into how to interfere with ongoing seizure activity. Desynchronization could represent a naturally occurring process that terminates seizure events. Unfortunately little information about the mechanisms of seizure termination is available. It is perhaps reasonable to assume that there are as many different neuronal mechanisms for terminating seizures as there are seizures types. Using animal models of different seizure types, we should be able to determine why different types of seizures stop and also why this process fails in status epilepticus. Epilepsia partialis continua, or focal seizures that can continue for hours or years, is an interesting condition because it indicates that the mechanisms that prevent seizure spread are not necessarily the same as the mechanisms that terminate seizures. Much could be learned from an animal model of this phenomenon. The Postictal Period Most seizures are followed by a period of focal or generalized neurologic deficit, ranging from generalized “postictal depression” to focal signs and symptoms, such as aphasia and Todd paralysis. The features of these postictal disturbances depend on the seizure type. For some patients, these disturbances can be more disabling than the seizures themselves. Often postictal deficits are a consequence of the natural mechanisms that act to terminate the seizure, suggesting that interventions designed to exploit these homeostatic events to stop seizures could exacerbate postictal dysfunction. More animal research is needed to elucidate the neuronal events responsible for these disturbances. Long-Term Consequences It is widely hypothesized that “seizures beget seizures” and that a significant consequence of a seizure (or series of seizures) is alteration in subsequent seizure manifestations, such as increased frequency and severity. In addition, clinical investigators have documented the development of interictal behavioral disturbances that are thought to be direct consequences of the seizures (Sutula and Pitkänen, 2002). Similar behavioral changes, such as kindling, have been shown in animal models. Alternatively such behavioral changes could reflect the effects of homeostatic seizuresuppressing mechanisms that act to maintain the interictal state. Long-term consequences of seizures could be potentially reversible (functional) or permanent (structural). Many patients are more disabled by interictal behavioral disturbances than they are by their seizures. These behavioral consequences are particularly crippling for the epileptic encephalopathies. There is an urgent need to understand the
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Chapter 1/What Should Be Modeled?
mechanisms of these consequences of epilepsy to prevent and treat them more effectively.
WHAT TO MEASURE Research on epilepsy with animal models, as with experimental animal research in all areas of biomedicine, begins with the identification of clinically relevant questions— questions that cannot easily be answered by studying patients but might be answered by studies in “simpler” systems in which the key variables can be brought under experimental control. The selection of the model depends on the question of interest as well as on the technical expertise of the investigators and available facilities. Regardless of whether the question can be addressed using models of epileptic seizures induced in a normal brain, of chronic epilepsy, or of epileptic equivalents, the investigator has a choice of experimental subjects (ranging from primates to flies), preparations (from oocytes to intact behaving animals), and technical approaches. Animal models can be studied electrophysiologically in vivo, either acutely (in which the animal is sacrificed at the end of the experiment) or chronically (where the same animal can be studied repeatedly over long periods). In vitro electrophysiologic investigations can be carried out on normal tissue that is made epileptic “in the dish” or on tissue removed from animal models of chronic epilepsy. In vitro preparations can be acute (e.g., slices, dissociated cells) or long-term (e.g., tissue culture). Live tissue used for electrophysiologic, imaging, and metabolic studies can be fixed later and studied in detail for structural elements. Whereas morphologic investigations in the past have required that animals be sacrificed and brains removed, it is now possible to perform both functional and structural in vivo neuroimaging in chronic animal models. Ictal and interictal behavioral investigations typically require in vivo experimental paradigms, but ictal and interictal electrophysiologic equivalents can be investigated in vitro. Neurochemical investigations are usually performed in vitro; however, they can now also be carried out in vivo using techniques such as microdialysis, PET, and MRS. Pharmacologic investigations can be pursued in awake behaving animals, with tissue preparations, and with PET and SPECT. Genetic investigations are now feasible in complex animal models by using new microarray techniques; surveys of thousands of genes (from excised cell or tissue samples) that may be altered by specific epileptogenic interventions can be assessed and quantified. Such powerful technology complements ongoing studies of population genetics carried out in mice or simpler organisms (such as flies). Gene discovery in turn leads to identification of gene products. Such discoveries raise new questions about mechanisms that must be addressed using in vitro molecular neurobiological techniques or in vivo gene
manipulations (e.g., knockouts, conditional transgenics, vector gene manipulations). Finally data derived from animal research can be used to create mathematical and computational models where variables are maximally controlled. All these approaches, however, provide information that is of clinical value only if it is subsequently validated as relevant to the human condition. All answers obtained from questions asked of animal models therefore ultimately require follow-up questions that must be pursued with patients. The number of animal models for epilepsy investigations is quite remarkable, and the techniques available for probing these models are extremely powerful. The experimenter is therefore faced with a set of difficult choices. 1. Which model should I use? As indicated, the choice of model depends critically on what questions are to be addressed. There is no easy answer. The following chapters provide some alternatives and, it is hoped, some guidance. 2. What should I measure, particularly with respect to “validating” the model for epilepsy relevance? The issues at this level are realistically dictated (as suggested previously) by the investigator’s expertise, colleagues, and facilities. There is, however, a standard set of possible measures that will be invaluable for characterizing any epilepsy model and for comparing the model to the relevant human condition. Validation, therefore, can be based on one or more of these measures. Electrophysiologic Characterization Clinical epilepsies are defined in part by typical interictal and ictal EEG patterns (whether in vivo or in vitro). Models of human absence epilepsy, for instance, are validated by ictal EEG discharges that resemble the threeper-second spike-and-wave discharges seen in humans (Avoli et al., 1990). Local circuit populations can provide important information about epilepsy surrogate markers and equivalents such as FR or long-term potentiation. At the single cell level, it is critical to describe the electrical features of neurons (or glia) that participate in the epileptic phenomena, such as burst discharge and details of current flux. Behavioral Manifestations The defining feature of epileptic seizures is the clinical behavioral nature of the ictal phenotype. Careful description of the behaviors characterizing a rat or a mouse seizure, although perhaps difficult, is extremely important, perhaps not so much as a means of validating the seizures with respect to human epilepsies (it may well be that a mouse does not reproduce human behavioral phenotypes) but for providing other investigators with a useful set of measures to replicate and to extend studies on a given model. An
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What to Measure
example is the Racine scale, which is always used to define the various stages of kindled seizures (Racine, 1972). Similarly, a description of the behavioral deficits associated with a given seizure or epilepsy model provides information necessary to assess the consequences of seizures that characterize particular epilepsy syndromes (Engel et al., 1991). Structural Measures At least some epilepsy syndromes are characterized by specific abnormalities in structural organization. An example is the hippocampal sclerosis of MTLE which is reproduced in the chronic animal models of this condition (Wieser et al., 2004). Detailed histologic and immunocytochemical measures provide important data about cell organization, structural plasticity, and changes in receptor complements. Ultrastructural analysis can help to identify more subtle changes (e.g., in synapse morphology, number, and targets). These traditional measures are now complemented by information obtained with sophisticated imaging approaches, which in noninvasive paradigms can provide longitudinal information about progressive structural changes in the same animal. Age (Development), Gender, Species, and Strain Specificity Increasingly laboratory studies are discovering differences between adult and immature animals with respect to seizure sensitivity, seizure types, and seizure mechanisms. It is clear that the immature brain is not simply a small version of the adult brain (Schwartzkroin et al., 1995). These results parallel the growing awareness of such differences in patients. For instance, the immature brain is more susceptible to acute seizures than the adult brain, but it is less susceptible to the development of chronic epilepsy in both animal models and humans. Similarly, it is clear that there are seizure phenomena that appear to be “sex-linked,” whether because of sex-linked genes attributable to specific hormonal issues or related to specific differences in brain structure. These powerful influences must be acknowledged, controlled, and preferably measured in animal model studies. In addition, differences between animal species, and even between strains of the same species, must be taken into consideration when designing studies and interpreting results. Genetic Background and Predisposition As indicated previously, it is clear that there are not only epilepsy genes (i.e., genes that, when mutated, result in a seizure phenotype) but also genetic predispositions in both humans and animals. Many epilepsy genes are now well documented. We continue to struggle, however, with the
issue of genetic background as a basis for seizure predisposition. Explicit reporting of genetic background (particularly in mouse models) has become an important feature of all epilepsy studies. Identification of “susceptibility” genes will constitute a major focus for modern epilepsy research. Genetic abnormalities identified in humans can then be reproduced in animals, and genetic disturbances identified as causing or influencing epileptic manifestations in animals can then be sought in patients. Response to Therapy Our ultimate goal in studying animal models of epilepsy is to develop more effective treatments and to design preventive measures for seizures and epilepsies in the human population. Characterization of animal models on the basis of their responsiveness to antiepileptic (or antiepileptogenic) treatment is therefore a key element in their validation. The number of therapeutic strategies available to the laboratory investigator precludes any complete characterization for a given model. However, as one nears the point of establishing a model as clinically relevant and offers hypotheses regarding underlying mechanisms that might give rise to specific treatments, assessing the model with respect to responses to conventional pharmacologic therapy is both useful and necessary.
Acknowledgments Original research reported by the authors was supported in part by grants NS-02808, NS-15654, NS-33310 (JE), and NS-18895 (PAS) from the National Institutes of Health.
References Avanzini, G., Moshé, S.L., Schwartzkroin, P.A., and Engel, J. Jr. 1998. Animal models of partial epilepsy. In Epilepsy: A Comprehensive Textbook Ed. J. Engel, Jr., and T.A. Pedley. pp. 427–442. Philadelphia: Lippincott–Raven. Avoli, M., Gloor, P., Kostopoulos, G., and Naquet, R. (Eds.) 1990. Generalized Epilepsy. Boston: Birkhäusen. Begley, C.E., Famulari, M., Annegers, J.F., Lairson, D.R., Reynolds, T.F., Coan, S., Dubinsky, S., et al. 2000. The cost of epilepsy in the United States: An estimate from population-based clinical and survey data. Epilepsia 41: 342–351. Berkovic, S.F., Arzimanoglou, A., Kuzniecky, R., Harvey, A.S., Palmini, A., and Andermann, F. 2003. Hypothalamic hamartoma and seizures: A treatable epileptic encephalopathy. Epilepsia 44: 969–973. Bragin, A., Engel, J. Jr., Wilson, C.L., Fried, I., and Mathern, G.W. 1999. Hippocampal and entorhinal cortex high frequency oscillations (100–500 Hz) in kainic acid-treated rats with chronic seizures and human epileptic brain. Epilepsia 40: 127–137. Chugani, D.C., Chugani, H.T., Muzik, O., Shah, J.R., Shah, A.K., Canady, A., Mangner, T.J., et al. 1998. Imaging epileptogenic tubers in children with tuberous sclerosis complex using alpha-[11C]methyl-L-tryptophan positron emission tomography. Ann Neurol 44: 858–866. Commission on Classification and Terminology of the International League Against Epilepsy. 1981. Proposal for revised clinical and
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electroencephalographic classification of epileptic seizures. Epilepsia 22: 489–501. Commission on Classification and Terminology of the International League Against Epilepsy. 1989. Proposal for revised classification of epilepsies and epileptic syndromes. Epilepsia 30: 389–399. Engel, J. Jr. 1992. Experimental animal models of epilepsy: Classification and relevance to human epileptic phenomena. Epilepsy Res (Suppl 8): 9–20. Engel, J. Jr. 1998. Research on the human brain in an epilepsy surgery setting. Epilepsy Res 32: 1–11. Engel, J. Jr. 2001. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: Report of the ILAE Task Force on Classification and Terminology. Epilepsia 42: 796–803. Engel, J. Jr., Bandler, R., Griffith, N.C., and Caldecott-Hazard, S. 1991. Neurobiological evidence for epilepsy-induced interictal disturbances. In Advances in Neurology, vol. 55 Ed. D. Smith, D. Treiman, and M. Trimble. pp. 97–111. New York: Raven Press. Engel, J. Jr., and Bertram, E.H. 2004. The search for pharmacological and non-pharmacological targets for curing epilepsy. Epilepsy Res 60: 125–131. Engel, J. Jr., and Pedley, T.A. (Eds.) 1997. Epilepsy: A Comprehensive Textbook, vol 1, 2, and 3. Philadelphia: Lippincott–Raven. Engel, J. Jr., Schwartzkroin, P.A., Moshé, S.L., and Lowenstein, D.H. (Eds.) 2001. Brain Plasticity and Epilepsy: A Tribute to Frank Morrell. San Diego: Academic Press. Fisher, R.S., van Emde Boas, W., Blume, W., Elger, C., Genton, P., Lee, P., and Engel, J. Jr. 2005. Epileptic seizures and epilepsy (ILAE) and the International Bureau for Epilepsy (IBE). Epilepsia 46: 470–472. Henry, T.R., Duncan, J.S., and Berkovic, S.F. (Eds.) 2000. Functional Imaging in the Epilepsies. Philadelphia: Lippincott Williams & Wilkins. Lehnertz, K., and Litt, B. (Eds.) 2005. The First International Collaborative Workshop on Seizure Prediction. Clin Neurophysiol 116: 493–505.
Levy, R.H., Mattson, R.H., Meldrum, B.S., and Perucca, E. (Eds.) 2002. Antiepileptic Drugs, 5th ed. Philadelphia: Lippincott Williams & Wilkins. Loescher, W. 2002. Animal models of epilepsy for the development of antiepielptogenic and disease-modifying drugs: A comparison of the pharmacology of kindling and post status epilepticus models of temporal lobe epilepsy. Epilepsy Res 50: 105–123. Murray, C.J.L., and Lopez, A.D. (Eds.) 1994. Global Comparative Assessment in the Health Sector; Disease Burden, Expenditures, and Intervention Packages. Geneva: World Health Organization. Noebels, J.L. 2003. The biology of epilepsy genes. Ann Rev Neurosci 26: 599–625. Purpura, D.P., Penry, J.K., Tower, D.B., Woodbury, D.M., and Walter R.D. (Eds.) 1972. Experimental Models of Epilepsy—A Manual for the Laboratory Worker. New York: Raven Press. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32: 281–294. Schachter, S.C., and Wheless, J.W. (Guest Eds.) 2002. Vagus nerve stimulation therapy 5 years after approval: A comprehensive update. Neurology 59(Suppl 4): S1–S61. Schwartzkroin, P.A. (Ed.) 1993. Epilepsy: Models, Mechanisms and Concepts. Cambridge, UK: Cambridge University Press. Schwartzkroin, P.A., Moshe, S.L., Noebels, J.L., and Swann, J.W. (Eds.) 1995. Brain Development and Epilepsy. New York: Oxford University Press. Schwartzkroin, P.A., and Walsh, C.A. 2000. Cortical malformations and epilepsy. Ment Retard Dev Disabilities Res Rev 6: 268–280. Stafstrom, C.E., and Rho, J.M. (Eds.) 2004. Epilepsy and the Ketogenic Diet. Totowa, NJ: Humana Press. Sutula, T., and Pitkänen, A. (Eds.) 2002. Do Seizures Damage the Brain? Progress in Brain Research, vol 135. Amsterdam: Elsevier. Wieser, H.-G. 2004. Mesial temporal lobe epilepsy with hippocampal sclerosis: Report of the Commission on Neurosurgery. Epilepsia 45: 695–714.
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2 Single Nerve Cells Acutely Dissociated from Animal and Human Brains for Studies of Epilepsy MARK STEWART, WEI-PING CHEN, AND ROBERT K. S. WONG
generation and patterning. One of the most significant advantages of the acutely dissociated cell preparation is that different stages of neuron development or different pathological conditions can be studied by harvesting cells from animals of different ages or from brains with existing pathology. This procedure can be carried out not only on animal models of interest but also on tissues obtained from human surgery (e.g., resections for medically refractory epilepsy). Important technical advantages of the preparation make possible some approaches to studying electrogenesis that are difficult or impossible with other preparations. First, acutely dissociated cells have reduced dendritic appendages, and as such they are more suitable for voltage clamp studies because space clamping is considerably improved. Second, ionic currents are more amenable to pharmacologic isolation. Thus it is possible to study the currents underlying action potential and firing-pattern generation, as contributed by both the soma and proximal dendrites. Studies of ionic currents in acutely dissociated cells have been used to define stereotypical and distinct firing patterns from different populations of acutely dissociated primary cells (e.g., hippocampal CA3 and CA1 and subicular pyramidal cells) as well as the features of other cell populations (e.g., subtypes of interneurons in CA1 (Fan and Wong, 1996). Similar studies have been used to compare developmental changes in firing properties and to contrast “normal” and “abnormal” cells from the same region.
GENERAL DESCRIPTION OF THE MODEL The study of the basic mechanisms of epilepsy has benefited from the use of many different in vitro experimental preparations, including the intact whole-brain model and progressively more “reduced” preparations, such as brain slices, acutely dissociated neurons, neuron fragments (e.g., dendritic fragments), and single-channel patch preparations. The acutely dissociated neuron remains sufficiently intact to generate many kinds of cellular activity, including action potentials, voltagegated currents, and currents associated with activation of specific receptor-transmitter systems. The acutely dissociated neuron preparation is particularly well suited for some types of investigations involving intrinsic properties of neurons; in addition, it has several advantages when recordings from dissociated cells are compared with recordings from single neurons embedded within intact brains or brain slices or with recordings from single neurons in culture. In this chapter we discuss the usefulness of the acutely dissociated cell preparation for the study of epilepsy and offer our own procedures for preparing and studying isolated neurons.
Advantages of the Acutely Dissociated Cell Preparation A critical feature of the acutely dissociated cell is that action potential electrogenesis is preserved. The action potential is not only a sensitive indicator of channel activity, but it is also the output mechanism of the neuron. Most studies of the acutely dissociated cell aim toward understanding the roles of ion channels in action potential
Models of Seizures and Epilepsy
Disadvantages of the Acutely Dissociated Cell Preparation There are obvious disadvantages to studying the acutely dissociated cell. It has been literally torn from the brain
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Chapter 2/Acutely Dissociated Cell Preparation
during the isolation process. Perhaps surprisingly the main disadvantage is not poor cell condition. Indeed acutely dissociated cells routinely remain viable during recordings for tens of minutes to hours. The main disadvantage is that the partial preservation of the cell produces a distorted view of the intact cell’s properties. For example, complex firing properties that depend on distributed conductances along the somatodendritic axis are no longer available for analysis. In this context, however, we note that different firing patterns for the soma and dendrites of CA1 pyramidal cells in guinea pig brain slices have been demonstrated (Wong and Stewart, 1992). Another important disadvantage is the absence of synaptic connectivity, a feature that disallows analysis of cell-to-cell interactions. This feature of the acutely dissociated cell preparation does preclude using this system to model epileptiform activity per se; that is, it is not possible for an isolated, acutely dissociated cell to generate seizurelike discharge. However, as discussed later, this limitation does not mean that dissociated cells are unsuitable for epilepsy research. On the contrary, we believe that the preparation offers important advantages (see later) that, when used in combination with other animal models (or human tissue), provide powerful insights into epilepsy-related mechanisms.
Overview To illustrate the usefulness of the acutely dissociated cell preparation for the study of epilepsy, we offer a number of examples of how this preparation has been or can be used. In the following sections we describe (1) our method for preparing acutely dissociated cells, (2) studies of intrinsic membrane properties, and (3) studies of receptor properties of acutely dissociated cells. The examples we highlight here are by no means a complete review of the studies that have used acutely dissociated cells to elucidate the basic mechanisms of epilepsy. Our examples are meant to demonstrate the range of applications of the acutely dissociated cell preparation for epilepsy research.
METHODS OF GENERATION Animal Issues Acutely dissociated cells from many different animal species have been studied. Cells from all the most common animal models (e.g., rat, mouse, guinea pig), and even humans (surgical resections) have been studied. In fact the acutely dissociated cell preparation is particularly appropriate for studies of human tissue that has been removed during a surgical biopsy or resection where individual tissue samples can be quite small. An important aspect of the acutely dissociated cell preparation is that cells can be harvested from animals of any age
(Mody et al., 1989; Oh et al., 1995; Thompson and Wong, 1991) or from animals that are at different stages in a disease process, for example, at some particular time after a convulsive treatment. This feature of the preparation gives the investigator the ability to study neuronal maturation processes or to contrast “normal” and “abnormal” cell function as part of a disease process.
Dissociation There are many variations on the isolation protocol. Indeed we have varied our own protocol at times (Chen et al., 1998; Fan et al., 1994; Kay and Wong, 1986). We describe our current protocol in this chapter and provide references for many other variations in the bibliography. Sprague-Dawley rats (of essentially any age; we have been using animals that are 21 to 35 days old) are deeply anesthetized with 2-bromo-2-chloro-1,1,-trifluoethane (halothane) and decapitated. The brain is quickly removed from the skull by first cutting the scalp along the dorsal surface of the head and then splitting the skull and carefully removing bone and dura mater from over the dorsal surface of the brain. A small spatula is used to lift the brain from the skull as it cuts through the cranial nerves. The whole brain is placed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in millimolars [mM]) 124 NaCl, 26 NaHCO3, 3 KCl, 2 CaCl2, 2 MgCl2, and 10 glucose, which is bubbled with 95% O2/5% CO2 to maintain a pH of 7.4. Blocks of tissue (several millimeters thick) are cut from the regions of interest (e.g., ventral hippocampal regions cut in the horizontal plane) from each hemisphere. These blocks are sectioned using a motorized sectioning system to make brain slices. Slices are cut at 400 to 500 mm (slightly thicker than slices used for electrophysiologic studies in a slice chamber) using a Vibratome sectioning system (Pelco, Redding, CA) and transferred to ACSF solution at room temperature. Each slice is dissected to isolate a particular brain region (e.g., CA3 or subiculum). The resulting pieces of tissue are approximately 1 mm square and the thickness of the slice. Microdissection of brain slices is a useful way to refine the isolation of a specific brain region and cell population. Microdissection of slices is relatively straightforward in hippocampal-limbic system slices because the boundaries for many subregions are clearly visible using a dissecting microscope. At least one (sometimes more) enzyme treatment is used to facilitate cell dissociation. The specific enzyme, its concentration, and the duration of exposure vary considerably among the investigators using these techniques (Oyama et al., 1990). Our current enzyme protocol for acutely dissociating cells starts as we first transfer tissue pieces (<1 mm3) to a small chamber containing a piperazine diethanesulfonic acid (PIPES) dissociation solution (in mM) as follows: 120 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 PIPES, 25 glucose, and
Methods of Generation
1 mg/ml protease type XIV (pH 7.0 with NaOH) at a temperature of 31° C, where the tissue remains for 30 to 40 minutes. After enzymatic treatment, the tissue is washed three times with enzyme-free PIPES solution, and pieces remain in this solution until they are ready for dissociation. For the final step, a single piece of the enzyme-treated tissue is triturated by passing it through progressively smaller tip sizes of fire-polished pipettes (approximately, 0.8-, 0.4-, 0.2mm tip diameters). The dissociated neurons are placed in an uncoated 35-mm petri dish (Corning, NY) containing ACSF solution bubbled with 95% O2 / 5% CO2. Cells are allowed to settle for 3 to 5 minutes by stopping the gas supply to the dish. Cellular debris can be reduced by gently replacing ACSF and allowing the cells to resettle in the petri dish. The petri dish is moved to the microscope stage for cell selection and recording.
Characterization of Intrinsic Neuronal Behavior The acutely dissociated cell is an intermediate preparation—between single cells from neurons in situ (brain slice, whole brain, or tissue culture) and subcellular preparations (all forms of frank patch recording: inside-out, outside-out, etc.). Enough of the cell remains intact to permit the investigator to study macroscopic (whole-cell) current behavior, including action potential generation. Also the isolated cell soma maintains many of its signaling cascades. Action potential discharge properties are studied using current clamp techniques. Voltage-clamping methods can be used to study currents and take advantage of the dissociated cell’s relatively compact electrical structure (for effective space clamping). Pharmacologic isolation of currents is particularly effective in this preparation because the cell’s membrane is entirely exposed to the extracellular environment. Because there are no synaptic connections in this preparation, dissociated cell studies focus on intrinsic cellular properties. Although the absence of synaptic connections can be seen as a limitation of the preparation, the relative ease of characterizing intrinsic cell properties in the complete absence of synaptic activity may also be viewed as a significant advantage of this preparation. The most commonly used recording methods (we will not discuss any of these in detail here) employ low-resistance pipettes for whole-cell recording techniques (various configurations: whole-cell mode, perforated patch mode, cell-attached patch mode). The selection of recording configuration and pipette solution depend on the specific goals of the study. The ionic composition of the lowresistance pipettes can itself be a significant variable in such studies because ingredients of the pipette solution can block some channels or substantially alter the kinetic properties of these channels. For example, Kay and colleagues (1986) showed that intracellular fluoride (com-
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monly used in patch pipettes) accelerated the inactivation kinetics of the L-type calcium current in hippocampal neurons. Thus, as in all studies of current kinetics, such studies on dissociated cells must be interpreted with attention to these details. In general, to assess the firing properties of neurons, the pipette solution should be “physiologic.” Alternatively the investigator chooses a recording configuration (such as perforated patch (Kanemoto et al., 2002)) that will minimally alter the intracellular contents of a cell. To study particular currents, one can also choose pipette solutions that will be helpful in isolating currents of interest (e.g., QX-314 to block sodium channels or cesium ions to block potassium channels). Any study of how a particular cellular “pathology” (such as pacemaker bursting) or a genetic mutation of ionic channels (channelopathy) contributes to epileptogenic mechanisms requires an understanding of the baseline characteristics of intrinsic currents of the neuron. Such measurements can be made on cells from “normal” and “experimental” tissue to compare cells on properties of interest. The following is a general plan for characterizing the acutely dissociated cell: 1. Determine the basic properties of a cell type by recording the cell’s behavior in current-clamp mode to define the action potential firing capabilities and patterns of the neurons. This behavior can be compared with the firing patterns of comparable cells in slices or in “intact” preparations. What discharge features characterize the dissociated cell? Injected current (steady direct current or brief pulses) can be used as the stimulus to evoke action potential firing. The action potentials (burst versus single spike after potentials) and the firing patterns (e.g., burst conversion to single spikes and spike frequency as a function of stimulus amplitude) are sensitive indicators of channel function. 2. Make a qualitative assessment of currents that are activated in response to hyperpolarizing or depolarizing stimuli. Determine the channels that are involved in suprathreshold activity (e.g., burst firing versus regular spiking, firing rates) and subthreshold changes (e.g., membrane oscillations) by testing the effects of specific channel blockers (one at a time). 3. Make a quantitative assessment of currents by using voltage-clamp techniques in the presence of drugs that will block all channels except the one you wish to study. Given the ease of obtaining an accurate image of the cell itself, it will be possible to relate the sizes of currents (from a well space-clamped cell) to the actual size of the neuron. It will also be possible to relate the sizes of different currents (recorded separately) to one another. Such quantitative details will allow the investigator to draw specific inferences about channel distribution and channel density.
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Chapter 2/Acutely Dissociated Cell Preparation
4. Finally, use the computer to reconstruct the neuron (electrically) using the quantitative data you have collected on ionic currents. Will the computer “predict” the suprathreshold and subthreshold properties you have recorded from your acutely dissociated cells? Obviously, data on as many ionic currents as possible are likely to result in “better” computer simulations. However, even relatively simple computer models can help to identify “critical” currents for particular kinds of suprathreshold and subthreshold behavior. From a practical standpoint, experimental emphasis on a small subset of these guidelines (e.g., studies of a single current) can be used to compare cells from animals of different ages or to compare cells from normal animals with cells from a disease model (e.g., following kainic acid treatment or kindling). In studies of neurons removed from human patients (tissue resection as part of a surgical treatment), it is difficult to establish the suprathreshold and subthreshold parameters for “normal” cells. As such, conclusions about disease-related features of human cells are risky.
used to apply drug to a regionally restricted portion of a single cell while comparing properties of somatic and dendritic compartments (Huguenard et al., 1989). A final interesting example is the “concentration-jump” technique (Akaike et al., 1989), which employs rapid flow changes in tubing to create a “push” or “pull” of drug from the tubing. In general, because functional transmitter receptors are present on the acutely dissociated cell, studies of receptor function can be carried out that exploit the flexibility of the preparation for drug application.
STUDIES OF IONIC CURRENTS In the previous section we discussed some of the techniques used to study acutely dissociated cells. In the next two sections we describe some of the experiments that have employed the acutely dissociated cell approach specifically for epilepsy research. These examples are meant to illustrate the preparation “in action” and to suggest other possible applications.
Endogenous Bursting and Pacemaker Cells Characterization of Receptor Properties As much as the acutely dissociated cell cannot, by definition, be used as a model to study seizures per se (abnormal neuronal synchronization—involving multiple neurons—is an essential component of the definition of a seizure), the preparation can be used to study abnormalities in cellular properties that contribute to seizure generation. Indeed the investigator is not completely limited to the study of voltage- and calcium-dependent currents. It is also possible to study receptor properties in the dissociated cell preparation. Here again, the dissociated cell has some important advantages as an experimental preparation. First, drugs can be applied to a recorded cell using any of a variety of methods; pressure ejection, iontophoresis, and bath application are all appropriate methods. The special advantage of the dissociated cell preparation is that a drug delivery device can be placed anywhere in relation to the recorded cell and can be moved within the recording chamber without substantially disturbing the recording. This feature permits the rapid application not only of single drugs but also of multiple drugs to a given cell. As one example, g-aminobutyric acid (GABA) receptor rundown has been studied in acutely dissociated hippocampal neurons by pressure application of GABA (Stelzer et al., 1988). As another example, the effect of N-methyl-d-aspartate (NMDA) receptor activation on GABA-receptor-mediated currents was studied with a piezo-electric switching device that allows the experimenter to change pipette solutions rapidly (Chen and Wong, 1995). A pair of pipettes, both streaming solutions (one drug-containing and one control solution), has been
We propose that a basic definition for a seizure generator is a brain region containing “pacemaker” neurons and connectivity to cause synchrony (Miles and Wong, 1983; Wong et al., 1986). Given such a starting point, the acutely dissociated cell preparation is ideal for the study of pacemaker properties—that is, those intrinsic neuronal properties that lead to a burst firing pattern—of neurons in a generator region. These properties may be observed as properties of normal cells (physiologic pacemakers), such as hippocampal CA3 pyramidal cells (Wong et al., 1986) and subicular pyramidal cells (Harris and Stewart, 2001; Harris et al., 2001; Stewart and Wong, 1993), which have endogenous burst firing properties and recurrent connections. We have compared properties of acutely dissociated CA1 pyramidal cells in hippocampus with subicular pyramidal cells (Figure 1) and found that the acutely dissociated subicular cells generate a pattern of inward currents (when the cells are stimulated with depolarizing current) that is consistent with action potential burst generation. Burst electrogenesis is a complex phenomenon that appears to vary across cell types (even among closely related areas such as CA1, CA3, and subiculum) and is a key determinant of a pacemaker region (Funahashi et al., 1999; Jung et al., 2001; Sah et al., 1988; Staff et al., 2000; Stewart and Wong, 1993; Wong and Prince, 1978; Wong and Stewart, 1992; Wong et al., 1979).
T-Current and Absence Seizures The acutely dissociated cell, with its advantages for voltage-clamp studies of ionic currents, has been useful for
Studies of Receptor Properties
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FIGURE 1 Examples of acutely dissociated cells and their electrophysiologic discharge patterns. Top row shows images of pyramidal neurons from rat subiculum (left) and hippocampal CA1 (right). Below the cell images are examples of current-clamp responses to small hyperpolarizing and depolarizing current pulses to illustrate differences in the intrinsic firing properties of these cells. Note that the subicular cell (left) discharge consists of an initially complex action potential, which is composed of prominent sodium- and calcium-dependent components (pharmacologic data to dissect out these currents, using tetrodotoxin and cadmium salts, are not shown). The initial response is followed by simpler action potentials that more closely resemble the action potentials generated by the CA1 cell (right). Size, voltage, and time calibrations are given in the figure.
evaluating the role of the transient, low-threshold (T-type), voltage-gated calcium current in absence seizures (Coulter et al., 1989; Huguenard et al., 1991). “Rebound” bursting in thalamic relay neurons was suppressed by ethosuximide (a drug commonly used to treat absence seizures), and the acutely dissociated cell preparation was used to demonstrate that ethosuximide selectively blocks the T-current in these thalamic cells. A similar study of the T-type calcium current showed that this current is prominent in hippocampal pyramidal cells before postnatal day 30, but it disappears after this age. It is noteworthy that ethosuximide did not appear to affect the Tcurrents in hippocampal CA1 pyramidal cells (Thompson and Wong, 1991), suggesting that different subtypes of Tchannels are present in thalamic versus hippocampal neurons. These studies illustrate how the acutely dissociated cell preparation can be used for the study of specific drug actions.
Other Specific Currents Action potential generation, and more specifically burst electrogenesis, depends on voltage-gated sodium and calcium currents. Accordingly, these currents have been
studied in various animal models of epilepsy and in cells from human patients with epilepsy. Calcium and potassium currents were altered in acutely dissociated neurons from rat dentate gyrus (Mody et al., 1992) and hippocampal CA1 (Vreugdenhil and Wadman, 1992, 1995) after kindling. Changes in calcium currents from neurons of human dentate gyrus (Beck et al., 1999; Nagerl and Mody, 1998), potassium currents from human neocortical neurons (Ruschenschmidt et al., 2004), and sodium currents from human entorhinal (Agrawal et al., 2003; Cummins et al., 1994) and subicular (Vreugdenhil et al., 2004) neurons have all been hypothesized as possibly contributing to seizure generation (or at least characterizing an epileptic brain region). Currents in isolated dendritic segments have also been studied (Kavalali et al., 1997). Thus alterations in dendritic currents during epileptogenesis can be studied in isolation using this dissociated cell fragment preparation.
STUDIES OF RECEPTOR PROPERTIES The hypersynchronized activity that defines a seizure is largely dependent on glutamate receptor activation and is prevented (at least to some extent) by synaptic inhibition.
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Chapter 2/Acutely Dissociated Cell Preparation
Because many synaptic receptors are well preserved in acutely dissociated cells, the preparation offers the investigator the possibility of studying postsynaptic receptor properties in normal and pathological tissue.
Ionotropic Receptors Many dissociated cell studies have aimed at defining the properties of ionotropic receptor types, such as GABA and NMDA, as well as defining the pharmacologic modulation of receptor-ion channel properties (Chen and Wong, 1995; ffrench-Mullen et al., 1993; Hussy et al., 1997; Numann and Wong, 1984; Oh et al., 1995; Stelzer et al., 1988). An important aspect of these studies has been the comparison of the receptor properties of normal versus abnormal neurons. For example, larger-amplitude NMDA receptor-mediated currents were found in dentate granule cells isolated from kindled rat brains (Kohr et al., 1993) compared with currents in normal control granule cells. As with the experiments specifically to antagonize voltage-gated channels with anticonvulsant drug application, receptor-gated channels have also been studied in the acutely dissociated cell preparation. For example, the sedative and anticonvulsant properties of drugs such as pentobarbital and phenobarbital were examined in acutely dissociated CA1 neurons in studies that contrasted the differential drug actions on calcium currents and GABA receptor–mediated currents (ffrench-Mullen et al., 1993).
Metabotropic Receptors The preservation of an intact cell soma in acutely dissociated cells suggests that many intracellular signaling pathways—for example, involving g-protein–coupled receptors (“metabotropic” receptors)—remain intact. Previously it was thought that such structurally complex receptors would not survive the enzymatic action used for the cell dissociation. However, through careful monitoring of enzymatic concentration and duration of treatment, robust gprotein–coupled receptor activity can be preserved. This development, together with the accessibility of the intracellular space in the acutely dissociated cell, made it possible to characterize intracellular signaling cascades activated by these g-protein—coupled receptors (Penington, 1996; Penington and Kelly, 1990; Penington et al., 1991). However, one prominent metabotropic receptor-mediated current, the M-current, is not seen in acutely dissociated cells. This absence may be explained by the recent finding that these channels are localized near the axon hillock (Devaux et al., 2004). The cholinergic receptors coupled to the M-current may be located on the membrane of acutely dissociated cells, but the coupled potassium channels (KCNQ) required for the M-current appear to be eliminated by the dissociation process.
Currents recorded as a consequence of receptor activation in the acutely dissociated cell preparation (as well as other preparations that employ “bulk” drug application) will necessarily include an unknown combination of synaptic and extrasynaptic receptor-mediated channel activation. To study synaptically mediated channel activation specifically, it is possible to include vesicle-containing boutons with the isolation of neuronal somata during the dissociation process (Drewe et al., 1988). The dissociation process is similar to the one described in this chapter but with critical differences in the concentrations of calcium (0 to 0.2 mM) and magnesium (5 mM) salts used during the isolation and incubation steps. This procedure permits the observation of miniature synaptic currents in a preparation that is well suited for highresolution voltage-clamp recordings.
STUDY OF GENETIC MODELS The genetically altered animal is an increasingly useful tool for the neuroscientist. Channels, pumps, or signaling cascades that determine channel behavior can be studied in acutely dissociated cells to determine the consequences of genetic manipulations at the level of simple cell properties. The acutely dissociated cell preparation can be useful for testing the physiologic consequences of altered channels in genetically engineered animals or in search of altered channel behavior as a possible basis for the seizures in a diseased animal or human. Specific channelopathies have been associated with some seizure disorders such as severe myoclonic epilepsy of infancy (Rhodes et al., 2004; Sugawara et al., 2002; Wallace et al., 2003). As an adjunct to physiologic studies on tissue with potential genetic abnormalities, a potentially useful manipulation is to employ single-cell polymerase chain reaction (PCR) techniques to amplify and identify specific genetic material (or its absence or modification in the case of knockout or transgenic animals). This process for identifying a cell’s genetic makeup is facilitated in the acutely dissociated cell preparation. With the dissociated cell preparation, one is spared the task of aspirating the contents of a cell (the required procedure when single-cell PCR is applied to neurons, e.g., in slices) because the whole cell can be processed directly and with minimal risk of contamination from other cells.
CONCLUSION This chapter highlights some examples of how the acutely dissociated cell preparation can be useful for studying the properties of neurons (or, in theory, even glia) that might contribute to epilepsy (e.g., burst electrogenesis), for studying the changes in cells that could provide the basis for
References
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Harris, E., Witter, M.P., Weinstein, G., and Stewart, M. 2001. Intrinsic connectivity of the rat subiculum: I. Dendritic morphology and patterns of axonal arborization by pyramidal neurons. J Comp Neurol 435(4): 490–505. Huguenard, J.R., Coulter, D.A., and D. A. Prince, D.A. 1991. A fast transient potassium current in thalamic relay neurons: kinetics of activation and inactivation. J Neurophysiol 66(4): 1304–1315. Huguenard, J.R., Hamill, O.P., and Prince, D.A. 1989. Sodium channels in dendrites of rat cortical pyramidal neurons. Proc Natl Acad Sci U S A 86(7): 2473–2477. Hussy, N., Deleuze, C., Pantaloni, A., Desarmenien, M.G., and Moos, F. 1997. Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation. J Physiol 502 (Pt 3): 609–621. Jung, H.Y., Staff, N.P., and Spruston, N. 2001. Action potential bursting in subicular pyramidal neurons is driven by a calcium tail current. J Neurosci 21(10): 3312–3321. Kanemoto, Y., Ishibashi, H., Doi, A., Akaike, N., and Ito, Y. 2002. An electrophysiological study of muscarinic and nicotinic receptors of rat paratracheal ganglion neurons and their inhibition by Z-338. Br J Pharmacol 135(6): 1403–1414. Kavalali, E.T., Zhuo, M., Bito, H., and Tsien, R.W. 1997. Dendritic Ca2+ channels characterized by recordings from isolated hippocampal dendritic segments. Neuron 18(4): 651–663. Kay, A.R., Miles, R., and Wong, R.K. 1986. Intracellular fluoride alters the kinetic properties of calcium currents facilitating the investigation of synaptic events in hippocampal neurons. J Neurosci 6(10): 2915–2920. Kay, A.R., and Wong, R.K. 1986. Isolation of neurons suitable for patchclamping from adult mammalian central nervous systems. J Neurosci Methods 16(3): 227–238. Kohr, G., De Koninck, Y., and Mody, I. 1993. Properties of NMDA receptor channels in neurons acutely isolated from epileptic (kindled) rats. J Neurosci 13(8): 3612–3627. Miles, R., and Wong, R.K. 1983. Single neurones can initiate synchronized population discharge in the hippocampus. Nature 306(5941): 371–373. Mody, I., Kohr, G., Otis, T.S., and Staley, K.J. 1992. The electrophysiology of dentate gyrus granule cells in whole-cell recordings. Epilepsy Res Suppl 7: 159–168. Mody, I., Salter, M.W., and MacDonald, J.F. 1989. Whole-cell voltageclamp recordings in granule cells acutely isolated from hippocampal slices of adult or aged rats. Neurosci Lett 96(1): 70–75. Nagerl, U.V., and Mody, I. 1998. Calcium-dependent inactivation of highthreshold calcium currents in human dentate gyrus granule cells. J Physiol 509(Pt 1): 39–45. Numann, R.E., and Wong, R.K. 1984. Voltage-clamp study on GABA response desensitization in single pyramidal cells dissociated from the hippocampus of adult guinea pigs. Neurosci Lett 47(3): 289–294. Oh, K.S., Lee, C.J., Gibbs, J.W., and Coulter, D.A. 1995. Postnatal development of GABAA receptor function in somatosensory thalamus and cortex: whole-cell voltage-clamp recordings in acutely isolated rat neurons. J Neurosci 15(2): 1341–1351. Oyama, Y., Hori, N., Allen, C.N., and Carpenter, D.O. 1990. Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons of Aplysia californica. Cell Mol Neurobiol 10(2): 193–205. Penington, N.J. 1996. Actions of methoxylated amphetamine hallucinogens on serotonergic neurons of the brain. Prog Neuropsychopharmacol Biol Psychiatry 20(6): 951–965. Penington, N.J., and Kelly, J.S. 1990. Serotonin receptor activation reduces calcium current in an acutely dissociated adult central neuron. Neuron 4(5): 751–758. Penington, N.J., Kelly, J.S., and Fox, A.P. 1991. A study of the mechanism of Ca2+ current inhibition produced by serotonin in rat dorsal raphe neurons. J Neurosci 11(11): 3594–3609.
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Rhodes, T.H., Lossin, C., Vanoye, C.G., Wang, D.W., and George A.L. Jr. 2004. Noninactivating voltage-gated sodium channels in severe myoclonic epilepsy of infancy. Proc Natl Acad Sci U S A 101(30): 11147–11152. Ruschenschmidt, C., Kohling, R., Schwarz, M., Straub, H., Gorji, A., Siep, E., Ebner, A. et al. 2004. Characterization of a fast transient outward current in neocortical neurons from epilepsy patients. J Neurosci Res 75(6): 807–816. Sah, P., Gibb, A.J., and Gage, P.W. 1988. The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons. J Gen Physiol 91(3): 373–398. Staff, N. P., Jung, H.Y., Thiagarajan, T., Yao, M., and Spruston, N. 2000. Resting and active properties of pyramidal neurons in subiculum and CA1 of rat hippocampus. J Neurophysiol 84(5): 2398–2408. Stelzer, A., Kay, A.R., and Wong, R.K. 1988. GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors. Science 241(4863): 339–341. Stewart, M., and Wong, R.K. 1993. Intrinsic properties and evoked responses of guinea pig subicular neurons in vitro. J Neurophysiol 70(1): 232–245. Sugawara, T., Mazaki-Miyazaki, E., Fukushima, K., Shimomura, J., Fujiwara, T., Hamano, S., Inoue, Y. et al. 2002. Frequent mutations of SCN1A in severe myoclonic epilepsy in infancy. Neurology 58(7): 1122–1124. Thompson, S.M., and Wong, R.K. 1991. Development of calcium current subtypes in isolated rat hippocampal pyramidal cells. J Physiol 439: 671–689.
Vreugdenhil, M., Hoogland, G., van Veelen, C.W., and Wadman, W.J. 2004. Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy. Eur J Neurosci 19(10): 2769–2678. Vreugdenhil, M., and W.J. 1992. Enhancement of calcium currents in rat hippocampal CA1 neurons induced by kindling epileptogenesis. Neuroscience 49(2): 373–381. Vreugdenhil, M., and Wadman, W.J. 1995. Potassium currents in isolated CA1 neurons of the rat after kindling epileptogenesis. Neuroscience 66(4): 805–813. Wallace, R.H., Hodgson, B.L., Grinton, B.E., Gardiner, R.M., Robinson, R., V. Rodriguez-Casero, V. et al. 2003. Sodium channel alpha1-subunit mutations in severe myoclonic epilepsy of infancy and infantile spasms. Neurology 61(6): 765–769. Wong, R.K., and Prince, D.A. 1978. Participation of calcium spikes during intrinsic burst firing in hippocampal neurons. Brain Res 159(2): 385–390. Wong, R.K., Prince, D.A., and Basbaum, A.I. 1979. Intradendritic recordings from hippocampal neurons. Proc Natl Acad Sci U S A 76(2): 986–990. Wong, R.K., and Stewart, M. 1992. Different firing patterns generated in dendrites and somata of CA1 pyramidal neurones in guinea-pig hippocampus. J Physiol 457: 675–687. Wong, R.K., Traub, R.D., and Miles, R. 1986. Cellular basis of neuronal synchrony in epilepsy. Adv Neurol 44: 583–592.
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3 Cell Culture Models for Studying Epilepsy MARC A. DICHTER AND JOHN POLLARD
In the early 1970s, it became clear to a number of investigators that to begin to understand neurobiology at a cellular and ultimately molecular level, in vitro preparations of mammalian CNS were needed. Almost simultaneously a small number of investigators began to grow and maintain embryonic mammalian CNS in dissociated cell cultures (Dichter, 1975, 1978; Fischbach and Dichter, 1974) while others learned how to maintain viable sections of more mature CNS in slice chambers (Schwartzkroin, 1975; Schwartzkroin and Prince, 1976, 1977; Schwartzkroin and Wester, 1975). These discoveries led to a virtual explosion of new information about the physiology and pharmacology of mammalian CNS and, in parallel, a great expansion of our knowledge of epilepsy mechanisms and the mechanisms of action of antiepileptic drugs. Thus it is impossible to separate “pure epilepsy research” using cell culture models from basic “neurobiological research” that led to major insights into epilepsy mechanisms. For example, once the physiology of glutamate-mediated excitatory synaptic function was analyzed in cell cultures, and AMPA and N-methyld-aspartate (NMDA) receptors were identified, our understanding of the role of glutamate receptor-mediated events in seizures could develop. Similarly, once GABA and its receptors were identified and characterized in cell cultures, it was possible to understand the role of GABAmediated inhibition in epileptiform events as well as the mechanisms of action of several of our most important antiepileptic drugs. In this chapter, we briefly review techniques for establishing and maintaining dissociated cell cultures of those parts of mammalian CNS that are most relevant to epilepsy studies, referring to primary sources for more detailed methodology. We then discuss the kinds of questions about epilepsy mechanisms and antiepileptic drugs that can best be answered by studies employing this model, and offer
INTRODUCTION AND HISTORICAL REVIEW To understand the role of cell culture models in epilepsy research, one must start at the time cell culture models were first “invented” for the study of mammalian central nervous system (CNS). Much work was performed in the 1960s and early 1970s to investigate the cellular phenomena occurring in the mammalian brain during interictal discharges and seizures (Ayala et al., 1970a, b; Dichter and Spencer, 1969a, b; Matsumoto and Ajmone Marsan, 1964; Matsumoto et al., 1969; Prince, 1968a, b, 1969; Prince and Futamachi, 1968, 1970; Prince and Gutnick, 1971; Prince and Wilder, 1967). This research developed in parallel with neurophysiologic studies of neuronal and synaptic function in general. At that time there was a basic understanding of neuronal physiology, still based on the Hodgkin and Huxley model of the squid giant axon, although even such elementary discoveries as the presence of Ca-dependent action potentials in mammalian neurons had not yet been made. In addition, there was a rudimentary understanding of synaptic physiology and pharmacology. The identities of the inhibitory neurotransmitters were just being discovered, and the identities of the excitatory transmitters were still somewhat a mystery, as was even the basic physiology of excitatory synaptic potentials. Almost nothing was known about the pharmacology of g-aminobutyric acid (GABA) or glutamate receptors. All the epilepsy research until then was performed in intact animals that had been made to exhibit seizures by either chemical or electrical stimulation. Mechanisms underlying the seizures had to be inferred from these in vivo experiments and were based on the then-current understanding of neurophysiology. At the same time, relatively little was known about the mechanisms of action of any of the then-available antiseizure drugs.
Models of Seizures and Epilepsy
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Copyright © 2006, Elsevier Inc. All rights of reproduction in any form reserved.
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Chapter 3/Cell Culture Models for Studying Epilepsy
examples from the literature to illustrate how these strategies can produce important information. We focus on how these cell culture models can be used to approach some of the unanswered questions about epilepsy and epileptogenesis. Finally, we discuss limitations of the model.
CELL CULTURE METHODS Species Used Dissociated cell cultures of CNS tissue can be prepared from essentially any mammalian species. Most cultures used to date, however, are derived from rat and mouse embryos or early postnatal animals. Rats and mice are small, abundant, and relatively inexpensive. Animal supply facilities can produce timed pregnant dams so that embryos or pups can be obtained at the appropriate age. Data derived from the cultured tissue can be compared with results from analysis of intact tissue (although questions remain about what constitutes comparable age) because rat and mouse brain slices are also commonly employed as experimental preparations. In addition, there are substantial numbers of studies on intact rat (or mouse) models with seizures or epilepsy with which one can compare both slice and culture in vitro preparations. Because so much of the original in vivo work and culture work developed in rats, this species was the preferred laboratory subject for many years. Later, as research on transgenic mice become widespread, more cell culture work has focused on mouse tissue. It is noteworthy that there is little literature on potential differences between rat and mouse CNS neurons. Comparisons in culture would be extremely useful, given that clear differences between species have been identified in intact systems. Most forms of epilepsy occur in cortex-archicortex or neocortex. As such, studies of cellular mechanisms most relevant to epilepsy or antiepileptic drugs have been performed on cultures developed from embryonic cortex or hippocampus (Banker and Cowan, 1977; Dichter, 1978). Other brain regions can also be studied as dictated by specific research questions. These regions include thalamus, cerebellum, and brainstem. In addition, many studies involving mechanisms of action of antiepileptic drugs (AEDs) have utilized dissociated cultures of spinal cord (Fischbach, 1972; Hosli et al., 1972) or dorsal root ganglion (DRG) (Dichter and Fischbach, 1977); these regions produce large, relatively easily studied neurons with reproducible characteristics (Dichter and Fischbach, 1977).
Specific Culture Methods CNS neurons can be cultured in a variety of formats, depending on the desired experimental paradigm. For most physiologic experiments, neurons are cocultured with glial
cells in a relatively enriched medium (Dichter, 1978). For other experiments neurons can be grown in an almost glialfree environment with carefully controlled and characterized media (Brewer, 1995; Brewer et al., 1993; Evans et al., 1998). Cells can be grown at varying densities, depending on the nature of the experiment to be performed. For example, for routine physiology and pharmacology, neurons are often grown at relatively high density on top of proliferating astrocytes. These neurons are best studied at a density that allows identification of individual cells (soma and dendrites) in a microscopic field filled with many neurons. Under these conditions the neurons are healthy, synapse formation is abundant, and small networks develop (Dichter, 1978). For biochemical experiments, cultures such as these can be grown on the surface of large plates or in flasks. One drawback of the usual “high-density” cultures is that finding pairs of synaptically connected neurons is difficult. In these cultures, all the neurons receive abundant excitatory and inhibitory synaptic inputs, but when one records from pairs of neurons near one another, connections are rarely found. By growing cultures at very low densities, such that only two or three neurons are visible in a microscopic field, coupled pairs are easily found (Wilcox and Dichter, 1994; Wilcox et al., 1994). An extreme example of this arrangement is found when neurons are cultured in isolation, often seeded at very low densities onto astrocytes or microdots of adherent substrate (Segal, 1991; Segal and Furshpan, 1990). Under these conditions, neurons form abundant autapses onto themselves such that each neuron is both presynaptic and postsynaptic to itself. When the neuron is glutamatergic, receptor antagonists must be included in the medium to prevent excitotoxic autostimulation. There are many subtle variations on the main methodologic scheme employed to establish CNS cell cultures. These can best be obtained from either original papers or from texts devoted to CNS cell cultures (Buchhalter and Dichter, 1991; Dichter, 1978; Wilcox et al., 1994). In general, pregnant rats (or mice) are anesthetized, fetuses are removed, and hippocampus or neocortex is dissected (hippocampus at 18 to 21 days of gestational age, neocortex at 15 to 21 days). The brain tissue is dissociated by incubation in trypsin, minced into smaller pieces, and triturated by drawing the tissue back and forth through a pipette tip. Some investigators prefer to avoid enzymes, and dissociate the cells by mechanical methods alone. This latter procedure tends to produce a much larger number of cells, but with a much lower viability. Particulate matter settles out of the mixture; remaining single cells are suspended in media, counted, and plated onto special glass coverslips or plastic tissue culture plates at specific densities. Both the coverslips and plates are coated with an adherent substrate, most often polylysine or polyornithine. Collagen can also be utilized, but neurons grown on collagen have more of a tendency to develop in clumps and remain less dispersed. For routine
Cell Culture Methods
cultures, cells are plated at 400 to 600/mm2 in media containing 5% or 10% serum. A variety of enriched growth media can be employed (Banker, 2002). For very-lowdensity cultures, cells are plated at 100 to 110/mm2 in medium containing high (20 mM) potassium concentration. Cultures remain in CO2 incubators at 37° C until they are used. Figure 1 illustrates sample microscopic fields from hippocampal cultures grown under three conditions: high density (400,000 cells/35-mm plate) with serum; low density (60,000 cells/35-mm plate) with serum and high K; and intermediate density (100,000 cells/35-mm plate) in neurobasal medium without serum. In the phase-contrast images of the top row, one can see astrocytes underlying the neurons at 2 and 3 weeks. These cells are absent or very sparse in the neurobasal cultures. Within hours of plating, most viable cells settle on the substrate, adhere, and begin sending out processes. Even at this early point, some neurons appear pyramidal, some stellate, and some bipolar. Whether these morphologies correspond to what the cells were destined to become in vivo has never been determined. By 3 to 5 days in vitro (DIV), the neurons have clearly differentiated and have developed extensive processes. Depending on the brain region being cultured and the age at which it was dissected, synapses begin to appear at about 4 to 7 DIV and increase in density and complexity for days thereafter. Given the nature of the
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connectivity in the high density cultures, it is likely that axons extend fairly significant distances before synapses are made. Cultures of this kind can be maintained in a robust state for 4 to 6 weeks or longer. As the cultures age, there appears to be a gradual decline in the number of viable neurons, although those that remain often are quite large and have extensive dendritic trees and abundant spines. When cultures are grown in high density with astrocytes, it is often necessary to use mitotic inhibitors (for example cytosine arabinoside at 10 mM) at about 4 to 7 DIV to prevent overgrowth of glia and loss of neurons. This procedure does not appear to affect the neurons adversely. Highdensity cultures grown in defined media have minimal astrocytes and do not require mitotic inhibition. Despite the lack of astrocytes in low-density cultures, the neurons appear quite normal and form abundant excitatory and inhibitory synapses. When synaptic interactions are to be studied using paired recordings, cultures are often grown at very low density. These cultures are maintained in lower volumes of media containing higher than usual amounts of potassium (e.g., 20 mM) (Mattson and Kater, 1988, 1989). Under these conditions, glial cells grow very slowly and do not overgrow the cultures (Wilcox et al., 1994). The basic properties of these neurons appear similar or identical to those grown at higher density, although this similarity has not been rigorously studied.
FIGURE 1 Three sets of hippocampal dissociated cell cultures: at 1 day (left column), 2 weeks (middle column), and 3 weeks (right column) in vitro. For each field, there is a photomicrograph of the same field (40¥ magnification) in phase contrast (left) and after neuronal staining with antibodies to MAP2 (right). The top row shows cultures grown at high density (400,000 cells/35-mm plate) with serum; the middle row shows cultures grown at low density (60,000 cells/35-mm plate) with serum and high K; the bottom row shows cultures grown at intermediate density (100,000 cells/35-mm plate) in neurobasal medium without serum. In the phase-contrast images of the top and middle row, one can see astrocytes at 2 and 3 weeks. These cells are absent or very sparse in the neurobasal cultures.
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Chapter 3/Cell Culture Models for Studying Epilepsy
UTILITY OF CNS CELL CULTURES AS A MODEL TO HELP UNDERSTAND THE PATHOPHYSIOLOGY OF EPILEPSY AND THE MECHANISMS OF ACTION OF ANTIEPILEPTIC DRUGS Neuronal and Synaptic Physiology and Pharmacology Ionic Currents Underlying Action Potentials in Cortical Neurons Our understanding of the physiologic basis of the action potential (AP) was derived from pioneering studies of the squid giant axon and other similar preparations. In these cells and axons, APs are generated by the opening of voltage-dependent Na channels followed by a rapid voltagesensitive inactivation. In addition, voltage-dependent K channels open to facilitate repolarization in many neurons. In a few invertebrate neurons, investigators also demonstrated that voltage-dependent Ca currents could sustain APs. Early studies in cell culture demonstrated that some vertebrate, including mammalian, neurons had Cadependent APs as well. These were first demonstrated in chick DRG neurons (Dichter and Fischbach, 1977), a preparation that then became very useful for studying mechanisms by which neuromodulators were able to affect Ca currents (Dunlap and Fischbach, 1978). Voltage-dependent Ca APs were also demonstrated in mammalian cortical neurons, both in slice preparations (Schwartzkroin and Slawsky, 1977) and in culture. Under baseline conditions, these currents were not apparent because of the rapid depolarizationrepolarization sequence initiated by Na and K currents, but when Na currents were inhibited, the cortical neurons developed slower, powerful Ca-dependent APs (Dichter and Zona, 1989; Dichter et al., 1983). Once the Ca APs were identified, it was noted that some groups of neurons in cortex, hippocampus, and thalamus had quite prominent Ca APs and tended to fire bursts of action potentials when depolarized. These neurons appeared capable of acting as “pacemaker” neurons for seizure-like discharges (Wong and Prince, 1978), and hypotheses about the origin of rhythmic discharges in cortex, hippocampus, and thalamocortical circuits were developed based on these findings. Similar observations were made on various K currents that were characterized in cortical and hippocampal neurons, some of which contributed to a neuron’s having a propensity for endogenous bursting. Physiology and Pharmacology of Inhibitory Synapses Earlyin vivo studies demonstrated that inhibitory postsynaptic potentials (IPSPs) were generated by opening Cl channels and were associated with an increase in membrane conductance. GABA had been tentatively identified as the
likely inhibitory neurotransmitter in mammalian forebrain, but glycine was also thought to be significant as a neurotransmitter. Studies in mammalian CNS cell culture demonstrated that GABA was the main, if not exclusive, neurotransmitter responsible for postsynaptic inhibition (Dichter, 1980; White et al., 1980) and that glycine likely played no, or very little, role in forebrain inhibition. These studies also showed that GABAergic inhibition was very labile at most synapses and declined rapidly with repetitive stimulation, mostly as a result of presynaptic factors (Wilcox and Dichter, 1994) (i.e., not due to receptor desensitization or changes in Cl gradient). These discoveries in cell culture led to extensive examinations of the pharmacology of GABAergic synapses, including the elucidation of the mechanisms of action of some of the most important AEDs available. From both biochemical (e.g., receptor binding) studies on processed brain tissue and from studies at the cellular level in cell culture, the GABAA receptor was recognized as a GABA receptor complex (GRC). Studies utilizing cell cultures first described the allosteric modulation of the GRC by benzodiazepines (Choi et al., 1977), a group of drugs used extensively to treat status epilepticus and other forms of seizures but whose mechanism of action had been unknown. Similar studies demonstrated that barbiturates, another class of antiepileptic drug, could allosterically modulate the GRC at a different site (Macdonald and Barker, 1977, 1978; Macdonald and McLean, 1982). Further, unlike the benzodiazepines, barbiturates at high concentrations could directly activate the GRC. Studies of membrane patches removed from cultured neurons also demonstrated how these drugs worked at the single channel level (Macdonald et al., 1988). Other work focused on a number of new AEDs that were developed based on animal models and whose mechanisms of action are unknown. At least one of these drugs, topiramate, appears to work partially by potentiating GABAmediated inhibition as demonstrated in hippocampal cultures (White et al., 1997, 2000). GABA also activates other classes of receptors, most notably GABAB receptors which are found on both somas and axon terminals, and GABAC receptors which are most prominent in retina. Much of what we know of the physiology of GABAB receptors is derived from studies in cell culture. These receptors are G-protein coupled to Ca channels in axon terminals, where they block Ca entry and thereby downregulate neurotransmitter release. They are also G-protein coupled to K channels on somas and dendrites, where they mediate slow and prolonged inhibition (Bowery et al., 1983, 1984, 1989; Bowery et al., 1984). Both these sets of events have important implications for epileptiform mechanisms. For example, it is thought that activation of GABAB-mediated inhibition plays a role in the termination of interictal discharges and possibly in the termination of seizures (Sperk et al., 2004). In addition, increased activation of GABAB receptors is thought to
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Utility of CNS Cell Cultures
contribute to enhanced bursting in thalamocortical relay neurons and the generation of spike-wave discharges in primary generalized epilepsy (Hosford et al., 1992, 1995). Inhibition of GABA release by activation of GABAB receptors at GABAergic terminals has also been implicated in the reduction of inhibition seen with repetitive activation of inhibitory synapses (Deisz and Prince, 1989). In addition, activation of GABAB receptors is likely to be responsible for the complex effects of two new AEDs that were designed to increase GABA levels at inhibitory synapses: tiagabine (by blocking GABA uptake) and vigabatrin (by blocking GABA metabolism). Both agents are antiseizure drugs effective against partial seizures. However, both can also enhance primary generalized absence seizures and possibly status epilepticus, presumably by allowing released GABA to act on presynaptic GABA terminals and paradoxically decreasing inhibition (Oh and Dichter, 1994) or by enhancing hyperpolarization on thalamocortical neurons and thereby enhancing synchronized spike-wave activity. Physiology and Pharmacology of Excitatory Synapses The physiology of excitatory synapses was much less well understood than even inhibitory synapses before the introduction of neuronal cell cultures. Unlike IPSPs, excitatory PSPs (EPSPs) were often not associated with measurable conductance changes and had unusual current-voltage (I-V) characteristics. The main hypothesis to explain these observations was that EPSPs occurred far out on dendrites and the characteristics that one could measure in the soma were distorted by the cable properties of the neurons. In addition, although glutamate and aspartate were hypothesized to be the excitatory neurotransmitters in the brain, their identities had not been confirmed and the cellular mechanisms by which these acidic amino acids depolarized neurons were not understood. This picture changed relatively rapidly once these properties could be analyzed in vitro. The response of neurons to applied glutamate was characterized (Ransom et al., 1977); the odd I-V curve was attributed to a voltage-dependent block by Mg of a subpopulation of glutamate receptors (Ascher and Nowak, 1988; Ascher et al., 1988); different glutamate receptor subtypes (e.g., AMPA, NMDA, kainate) were identified and characterized (Dingledine et al., 1990; McLennan, 1983; Westbrook, 1994), and the physiology of excitatory synapses was much better understood. Moreover, a virtual “industry” of glutamate pharmacology was born. Each of the advances in our understanding of the mechanisms of excitatory transmission had direct impact on epilepsy research. The nature of the large depolarizing events seen both during interictal discharges and ictal events was characterized within the framework of different glutamate receptor events. The role of NMDA receptors especially was emphasized as an important component of
epileptiform activity, and the autoregenerative nature of NMDA-mediated synaptic events (with depolarization reducing the Mg block of NMDA receptors, which in turn promoted further depolarization by glutamate) became a critical part of the explanation for the all-or-nothing development of some forms of epileptic activity. The characterization of excitatory synaptic transmission was also determined at the single-channel level. AMPA receptors were demonstrated to have very rapid and unusual desensitization properties such that they opened once in response to agonist and then desensitized until agonist was removed (Tang et al., 1989; Trussell et al., 1988). NMDA receptor-coupled channels, however, continued to open and close for as long as glutamate remained present. In addition, it was demonstrated that NMDA receptors were very sensitive to changes in extracellular pH, becoming inhibited at acidic values and enhanced by alkalosis, within pH ranges that were found in the brain during physiologic or pathological stimuli (Tang et al., 1990). Investigators also demonstrated that most AMPA receptors were permeable to only monovalent cations, whereas NMDA receptors were permeable to Ca as well. As the molecular biology of the receptors was further understood, AMPA receptors that lacked a properly edited version of the GluR2 subunit were found to be permeable to Ca. These channels occurred naturally on some inhibitory interneurons (Hollmann et al., 1991) and were proposed as a contributing component of some disease states (Bennett et al., 1996; Pellegrini-Giampietro et al., 1997). Each of these discoveries had great implications for our understanding of epileptic processes and for current theories of epileptogenesis. For example, the use of hyperventilation to stimulate epileptic changes in the electroencephalogram (EEG) may be due to the brief metabolic alkalosis induced by this procedure. In addition, the vulnerability of subsets of inhibitory interneurons to hypoxia may be related to their Ca-permeable AMPA receptors. In addition to the fast depolarization mediated by ionotrophic glutamate receptors, it was also noted that glutamate could produce a slow and long-lasting change in neuronal properties as a result of activation of “metabotropic” glutamate receptors. The discovery and characterization of these receptors also added significantly to our understanding of how epileptiform events could become established and propagate throughout the brain (Ghauri et al., 1996; Lee et al., 2002).
Synaptic Plasticity Most studies of synaptic plasticity in the vertebrate CNS were performed using the preparation of in vitro brain slices, most commonly hippocampal slices. A vast literature on long-term potentiation and long-term depression is derived from these studies. In general, with a few exceptions, such studies rely on pathway stimulation and on single-cell or
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Chapter 3/Cell Culture Models for Studying Epilepsy
field recordings to infer synaptic mechanisms. Parallel studies in cell cultures have allowed direct investigation of synaptic plasticity using intracellular recordings from both the presynaptic and postsynaptic elements of the synaptic pair. This approach provides more precise control over the relevant synaptic events. However, this preparation also has significant drawbacks for these studies. The cell culture tissue appears to be more immature than the tissue usually studied in slices; the nature of the synaptic interactions may be different from those that develop in the more complex CNS; and it may be very difficult or impossible to activate multiple inputs to the same postsynaptic neuron in culture preparations. In addition, some cellular mechanisms “wash out” during the intracellular dialysis that occurs with wholecell patch-clamp recordings (although this problem is not unique to cell culture preparations). The latter issue is particularly important when considering longer-term changes in synaptic efficacy. Nevertheless, important observations about short-term plasticity have been made utilizing cell culture preparations. In general, when studied with paired recordings, inhibitory synapses almost always decrement when repetitively activated. After even a single action potential, the probability of release of GABA declines for as long as several seconds. With brief trains of action potentials, inhibitory synaptic efficacy may decline by more than 50%, all because of presynaptic factors independent of GABAB autoreceptor feedback (Wilcox and Dichter, 1994). Such decreases in inhibitory efficacy may have profound implications for ictogenesis. Repetitive firing of excitatory principal cells may excite inhibitory interneurons, but the resulting firing of the inhibitory interneurons may become less and less effective in inhibiting principal cells. By contrast, excitatory neurons have different response characteristics to repetitive activation. They also often demonstrate a decrease in release of synaptic transmitter after single action potentials, but this decrease is much more variable than at inhibitory synapses (Kaplan et al., 2003; Wilcox and Dichter, 1994). With trains of high-frequency stimuli, some excitatory synaptic responses potentiate, some appear to remain stable, and some decrement (Kaplan et al., 2003). The net result is that if both excitatory and inhibitory synapses are part of a recurrent circuit, and if they are simultaneously activated repetitively, net inhibition will decline with respect to net excitation and the circuit will become unstable. This developing instability is likely one of the critical mechanisms underlying the development and spread of seizures through the brain.
Single-Cell Molecular Profiling and Physiology One of the most widely employed techniques in molecular biology is expression profiling of various tissues under
various physiologic and pathological conditions. Changes in gene expression, as measured at the mRNA level, are often analyzed to determine how a specific group of cells reacts to a change in physiology. For the complicated CNS, such expression profiling is most informatively performed on single identified neurons (or at least on small groups of identified neurons) to allow an appropriate understanding of how changes in gene expression produce changes in network behavior. Some of the earliest applications of single-cell gene-expression profiling were performed on identified neurons in cell culture (Eberwine et al., 1995). These studies were then extended to neurons identified in live brain slices. Subsequently it was determined that RNA could be relatively faithfully amplified and analyzed even from fixed tissues (Crino et al., 1996). Initial studies focused on molecular differences between identified inhibitory and excitatory neurons. Surprisingly little difference was found in the relative expressions of most channels and receptors. Even more surprising, however, was the finding of relatively large amounts of mRNA for glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of the inhibitory neurotransmitter, GABA, in all excitatory neurons in culture (Cao et al., 1996). High levels of GAD mRNA were found, despite the fact that these neurons were not synthesizing GAD protein or GABA. These observations were then extended to more intact CNS tissues, where it was also found that excitatory pyramidal cells, granule cells in hippocampus, and granule cells in cerebellum also contained relatively large amounts of GAD mRNA (Cao et al., 1996). A new hypothesis was proposed, the “standby neurotransmitter hypothesis,” which suggested that under some circumstances normally excitatory neurons could become inhibitory (Cao et al., 1996). Important from the point of view of epilepsy research, it was subsequently demonstrated that just such a “switch” to GABA synthesis could occur during prolonged seizures. Dentate gyrus granule cells, which are normally excitatory, after seizures exhibit dense staining for GAD and GABA all the way out into axon terminals; these cells can produce inhibition on downstream neurons (Sloviter et al., 1996). Subsequent to this demonstration, investigators have shown that the number of GAD-positive neurons in particular brain nuclei varies significantly (and reversibly) under other physiologic circumstances, such as diurnal and estrus cycles. Obviously control of inhibitory elements in critical brain circuits can have a profound effect on excitability and seizure development and spread. The application of single-cell molecular profiling has been extended well beyond the early studies in cell culture. Much work focuses on changes in gene expression in different cells within the hippocampus after status epilepticusinduced epileptogenesis (Brooks-Kayal et al., 1998; Becker et al., 2002; Chen et al., 2001; Crino et al., 2002; Zhang et al., 2004). Changes in receptor numbers and subtypes, and
Utility of CNS Cell Cultures
alterations in ion channels, have been implicated in the development of the hyperexcitable state (Beck et al., 1996; 1997a, b; Bernard et al., 2004; Brooks-Kayal et al., 1998; Coulter, 1999; Reckziegel et al., 1998).
Excitotoxicity The concept of excitotoxicity is so common at present that it is perhaps surprising to recognize that this hypothesis was only developed in the 1970s and 1980s. Although initial observations by Olney and others were based on in vivo work (Coyle et al., 1981; Olney, 1969, 1976; Olney and Ho, 1970; Olney et al., 1974; Rothman and Olney, 1986), much of the subsequent progress in understanding underlying mechanisms proceeded in studies of CNS cell cultures. These developments included the role played by the various glutamate receptor subtypes (Choi, 1993), the cellular mechanisms involved in both the necrotic and apoptotic components of cell death (Choi, 1993), and the effects of drugs on these processes. It would be unrealistic to attempt to summarize all these activities in this chapter, but the reader is referred to a number of excellent recent reviews (Mattson, 2003; Sattler and Tymianski, 2001). This literature makes it clear that excitotoxicity plays in an important role in epileptogenesis and in seizure-induced brain damage.
Mechanisms of Action of Antiepileptic Drugs Cultures of mammalian CNS have played a critical role in our modern understanding of the cellular and molecular mechanisms of antiepileptic drugs. Almost all the currently available AEDs were developed based on animal screening models. The mechanisms of action of these drugs were completely unknown when the drugs were introduced, and to some extent these mechanisms remain unknown for several important “old” and “new” AEDs. However, we have a sophisticated understanding of the mechanisms by which many of our currently used AEDs work. Sodium Channel Blockers Several very potent AEDs have as a primary mechanism of action the ability to block voltage-dependent Na channels in a voltage- or use-dependent fashion. Using preparations of mammalian spinal cord or cortical cell cultures, several groups demonstrated that phenytoin, carbamazepine, lamotrigine, and zonisamide could block sustained repetitive firing (SRS) in neurons as induced by prolonged depolarizing current pulses (Macdonald and McLean, 1982; Macdonald et al., 1985). Early action potentials in the train were generally minimally affected, but as the neurons remained depolarized, the drugs were able increasingly to block more and more channels. Subsequent voltage-clamp experiments demonstrated the voltage-dependent nature of the block.
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GABA Receptor Complex Allosteric Modulators Several of our more commonly employed AEDs work as allosteric modulators of the GABA receptor complex (GRC), mechanisms first elucidated by studies of cell cultures. Benzodiazepines enhance the affinity of GABA at the receptor and allow enhanced channel openings (Macdonald et al., 1986); barbiturates prolong channel openings and can produce channel openings even in the absence of GABA (Macdonald et al., 1988). Topirimate also appears to enhance GABA-mediated inhibition (White et al., 1997, 2000). GABA Uptake Inhibitors Tiagabine was developed as an inhibitor of GABA uptake. It was hypothesized that such inhibition would result in more GABA extracellularly, and thus there would be enhanced inhibition. Studies in cell culture have produced evidence that this assumption may reflect an oversimplification of the processes involved. In the cell culture system, enhanced GABA at the synapse may not produce stronger inhibition, either at GABAA or GABAB receptors. Paradoxically the increased GABA primarily affects presynaptic GABAB receptors on interneurons and therefore downregulates GABA release, thereby producing less inhibition (Oh and Dichter, 1994). This surprising result may account for the complex response of some seizure types to tiagabine. This drug appears to exacerbate absence-like seizures and possibly some forms of status epilepticus, but it reduces partial seizures (Eckardt and Steinhoff, 1998; Ettinger et al., 1999; Fitzek et al., 2001; Genton, 2000; Knake et al., 1999; Piccinelli et al., 2000; Schapel and Chadwick, 1996; Shinnar et al., 2001; Skardoutsou et al., 2003; Skodda et al., 2001; Solomon and Labar, 1998; Steinhoff and Eckardt, 1999). Similarly complex results were obtained when analyzing the effects of glutamate transport inhibitors on excitatory synaptic physiology. Instead of simply enhancing excitation, these agents often dampened excitatory synapses by an action of the excess synaptic glutamate on presynaptic metabotropic glutamate receptors (mGluRs), which decreased transmitter release from excitatory terminals (Maki et al., 1994). Glutamate Receptor Antagonists It has been hypothesized that antagonists of either the AMPA or NMDA receptor subtype could be effective AEDs—if such antagonism were not too toxic. Many of the drugs that work as antagonists at these receptors were developed and characterized using a CNS cell culture system. So far, however, drugs that target these receptors have not been as useful as hoped, partly because of toxicity and partly because of limited efficacy. One of the new AEDs,
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Chapter 3/Cell Culture Models for Studying Epilepsy
topiramate, has been shown to act by blocking kainate receptors (Gibbs et al., 2000; Gryder and Rogawski, 2003) in addition to its effect on GABA receptors. This type of multiple targeting, with drugs that affect multiple receptor (or channel) types, may prove to be a new avenue for AED development. Other drugs that act as weak noncompetitive antagonists at AMPA or NMDA receptors are being developed, and these may prove to be more effective than the earlier compounds tested (Rogawski, 2000; Donevan and Rogawski, 2000). Potassium Channel Modulators Another new class of possible AEDs is being developed based on a mechanism that was first demonstrated in CNS cell cultures. These agents enhance the opening of subclasses of voltage-dependent K channels or in some cases open the channels directly. The net effect is to dampen neuronal excitability.
Circuit Behavior and Ictogenesis: Do Cell Cultures Exhibit “Seizure-like” Phenomena? Models of Epilepsy in Cell Culture When neurons from cortex or hippocampus are grown in dissociated cell culture, they form extensive synaptic interactions and local circuits. The nature of the circuitry has not been extensively examined, but a few principles have emerged. Neurons in routine high-density culture do not tend to form synapses with neighbors in culture, despite extensive overlap of processes. Instead synapses are formed with more distant targets. When neurons are monitored by intracellular recordings, they tend to fire in bursts at either irregular, or occasionally, regular intervals. These bursts, which resemble those seen in epileptic tissue in vivo, occur despite an inability of these neurons to generate endogenous bursts in response to depolarizing pulses (Buchhalter and Dichter, 1991; Dichter, 1978) (as opposed to some endogenous bursting neurons in intact cortex and hippocampus). When pairs of neurons in culture are recorded, it is common for the neurons to exhibit synchronous bursting or synchronous activation, presumably driven by synaptic events originating elsewhere in the cultures. Thus the circuit organization and synaptic interactions among neocortical and hippocampal neurons maintained in dissociated cell cultures spontaneously re-create the kinds of circuits needed to sustain epileptiform types of activity. Synchronous bursting in the cultures can be enhanced by many of the same stimuli that provoke epileptic activity in intact cortex (e.g., GABA receptor antagonists and NMDA receptor activators). Such epileptogenicity occurs in the absence of endogenous bursting cells, electrical interactions between the neurons, or ephaptic interactions among the neurons (as a result of large extracellular currents flowing in
the media). This synchronous discharge can also occur when neurons are grown in the virtual absence of astrocytes. Surprisingly this “epileptic-type activity” and epileptogenic circuitry have been largely ignored by epilepsy researchers using cell cultures to study other aspects of cellular biophysics, physiology and pharmacology, or mechanisms of action of AEDs. One prominent exception is the work of DeLorenzo and colleagues (Gibbs et al., 1997; Pal et al., 2001; Sombati and DeLorenzo, 1995), who have exploited the enhanced synchrony produced by low magnesium exposure to create a cell culture model of epilepsy. Reducing Mg in the media in which neurons are bathed enhances activity of NMDA receptors and produces an increase in excitability of the cultures. When cells are maintained in this condition for longer than 3 hours, the cultures remain hyperexcitable for the life of the cells, even when normal Mg is restored. The DeLorenzo laboratory uses hippocampal cultures that are plated on a 2-week-old astrocyte feeder layer. After about 2 weeks, the culture media is replaced with media containing no magnesium and a small amount of glycine, sufficient for activation of NMDA receptors. During exposure to this lowmagnesium media, the neurons exhibit a continuous synchronous firing pattern reminiscent of a status epilepticus (Sombati and DeLorenzo, 1995). After 3 hours of exposure to this activating media, the cell cultures are switched back to the original media. Following this treatment, the neurons exhibit activity that has been dubbed spontaneous recurrent epileptiform discharges (SREDs) (DeLorenzo et al., 1998). In contrast to control tissue, the low Mg-exposed neurons have a much higher likelihood of synchronous depolarizations; this tendency persists for the life of the cells, typically more than 2 weeks. DeLorenzo’s group has carried out a number of experiments to support their view that this model simulates focal epileptogenesis, dependent on aberrant activation of NMDA receptors. They showed that blockade of the NMDA receptors during the low-Mg treatment of these cultures prevents the development of hyperexcitability (as evidenced by SREDs) (DeLorenzo et al., 1998). Prolonged direct activation of the NMDA receptors, using the physiologic agonist glutamate instead of low magnesium, resulted in a similar long-term hyperexcitability and disruption of calcium homeostasis, likely through alteration in calcium/calmodulin kinase II activity (Sun et al., 2001). These alterations in calcium homeostasis may also lead to increased GABAA receptor endocytosis or altered GABAA receptor composition. Low-magnesium-treated cells exhibit a lower current density for a given concentration of GABA and show a lower sensitivity to the benzodiazepine, clonazepam. Blocking clathrin-mediated endocytosis in postsynaptic cells reverses hyperexcitability caused by the low-magnesium treatment (Blair et al., 2004). The proposed mechanism of this clathrin action is by blockade of GABAA receptor
Summary: Insights Into Human Epilepsy Derived from Studies of Neuronal Cell Cultures
endocytosis, which in turn causes the levels of GABAA receptor to return to baseline; concommitantly, the hyperexcitability of the cell cultures returns to baseline (Blair et al., 2004). Chronic Cultures of Dissociated Neurons Maintained on a Chip Another recent approach to analyzing epileptic-like events in cell culture has developed around an interesting new way of culturing the neurons. Cells can be grown on microchips with multiple exposed electrodes (Potter and DeMarse, 2001). When a neuron lands on one of these electrodes, an extracellular recording from that cell can be maintained, often for weeks at a time. Because the microchip contains many electrodes, many individual cells or small groups of neurons can be monitored simultaneously. Under these conditions, the neurons tend to fire in bursts, and there are varying degrees of synchrony occurring in the cultures (Potter, personal communication). Because the neurons can be monitored for weeks, this preparation has the potential for important studies of dynamic circuit behaviors, including the response of these synchronously discharging cells to AEDs or other possible antiseizure therapies.
LIMITATIONS Despite the extensive studies performed on dissociated cell cultures since they were first “invented” in the early 1970s, there are a number of limitations that must be mentioned about “models” of epilepsy developed with this approach. Many different laboratories now maintain cell cultures, and there is undoubtedly significant variability in cultures (methods of generation and maintenance, cell types, etc.) from one laboratory to the next. For some types of experiments, this variability may be unimportant. However, for some studies that focus on epileptic-like events and conditions, such differences could be quite important; for example, a variation in the percent of inhibitory neurons, from 30 to 40% of the culture (Cao et al., 1996; White et al., 1980) to less than 10% (Benson et al., 1994). Similarly, the presence or absence of astrocytes may be important for some kinds of studies. Another issue that always arises with respect to cell cultures is how faithfully they mimic their in vivo counterparts. Surprisingly, for most properties studied, the resemblance is remarkable. However, for other properties, there may be very significant differences between cultured neurons and neurons in the intact brain. For example, one group of GABAB receptors (K-channel–coupled GABAB receptors on cell bodies and dendrites) are absent from cultured neurons. Similarly, as we understand more about developmental expression of different ion channels and transmitter receptors, it will be necessary to validate culture studies with in
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vivo data. It is now clear that different mechanisms of epileptogenesis, seizure generation, and response to AEDs are characteristic of CNS neurons at different stages of maturity. It will therefore be important to determine what maturational level is reproduced in cell cultures before drawing general conclusions from culture data for the intact brain. Perhaps the most interesting aspect of the comparison between cell cultures and the intact brain relates to the epileptiform activity expressed by neurons in culture. Do the circuits that form after cells are completely dissociated and then plated onto a two dimensional surface (covered with an artificial substrate) re-create the kinds of circuits that are capable of developing and sustaining epileptiform activity? Depending on how one defines epileptic, it appears that cell cultures can do just that. What insights will this preparation provide about the developing brain and the response to injury? How will culture-determined properties of cells and circuits be useful for designing strategies to prevent seizures or the hyperexcitability that underlies human epilepsy?
SUMMARY: INSIGHTS INTO HUMAN EPILEPSY DERIVED FROM STUDIES OF NEURONAL CELL CULTURES Studies utilizing the preparation of dissociated cell cultures have already provided significant insights into the basic physiology, pharmacology, and molecular biology of neocortical and hippocampal neurons and synapses and have led to many advances in our understanding of epileptiform events. Such studies have contributed greatly to our current understanding of the mechanisms of action of AEDs. Further work on receptor (or channel) subtype-specific pharmacology will likely lead to new classes of AEDs—drugs that have more focused targets and better side-effect profiles. Other studies focusing on mechanisms underlying shortterm synaptic plasticity and on the local circuitry involved in synchronous neuronal activation may also lead to new strategies for suppressing seizures. Each of these areas may result in new targets for AED screening. One of the main areas of neglect in epilepsy research is that of epileptogenesis. Studies in cell culture on such issues as axonal sprouting, new synapse formation and altered molecular phenotype in response to activity or injury may provide new insights into these processes and may lead to new hypotheses about how to control them. It is clear that we need multiple models in our struggle to understand the mechanisms underlying seizures, the changes in brain structure and function that underlie epilepsy, and the best methods for developing new antiseizure and antiepileptogenic strategies. This volume provides an introduction to many of these models, both in the intact animals and in vitro. It is important to remember, however, that it is “the question” to be answered that
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remains the most important focus of our research, not the model per se. As critical questions are posed, experimenters must make use of—and develop—those models that can best answer those questions.
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Lee, A.C., Wong, R.K., Chuang, S.C., Shin, H.S., and Bianchi, R. 2002. Role of synaptic metabotropic glutamate receptors in epileptiform discharges in hippocampal slices. J Neurophysiol 88: 1625–1633. Macdonald, R.L., and Barker, J.L. 1977. Phenobarbital enhances GABAmediated postsynaptic inhibition in cultured mammalian neurons. Trans Am Neurol Assoc 102: 139–140. Macdonald, R.L., and Barker, J.L. 1978. Different actions of anticonvulsant and anesthetic barbiturates revealed by use of cultured mammalian neurons. Science 200: 775–777. Macdonald, R.L., McLean, M.J. 1982. Cellular bases of barbiturate and phenytoin anticonvulsant drug action. Epilepsia 23(Suppl 1): S7–18. Macdonald, R.L., McLean, M.J., and Skerritt, J.H. 1985. Anticonvulsant drug mechanisms of action. Fed Proc 44: 2634–2639. Macdonald, R.L., Weddle, M.G., and Gross, R.A. 1986. Benzodiazepine, beta-carboline, and barbiturate actions on GABA responses. Adv Biochem Psychopharmacol 41: 67–78. Macdonald, R.L., Twyman, R.E., Rogers, C.J., and Weddle, M.G. 1988. Pentobarbital regulation of the kinetic properties of GABA receptor chloride channels. Adv Biochem Psychopharmacol 45: 61–71. Maki, R., Robinson, M.B., and Dichter, M.A. 1994. The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate depresses excitatory synaptic transmission via a presynaptic mechanism in cultured hippocampal neurons. J Neurosci 14: 6754–6762. Matsumoto, H., and Ajmone Marsan, C. 1964. Cellular mechanisms in experimental epileptic seizures. Science 144: 193–194. Matsumoto, H., Ayala, G.F., and Gumnit, R.J. 1969. Neuronal behavior and triggering mechanism in cortical epileptic focus. J Neurophysiol 32: 688–703. Mattson, M.P. 2003. Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromolecular Med 3: 65–94. Mattson, M.P., and Kater, S.B. 1988. Isolated hippocampal neurons in cryopreserved long-term cultures: development of neuroarchitecture and sensitivity to NMDA. Int J Dev Neurosci 6: 439–452. Mattson, M.P., and Kater, S.B. 1989. Development and selective neurodegeneration in cell cultures from different hippocampal regions. Brain Res 490: 110–125. McLennan, H. 1983. Receptors for the excitatory amino acids in the mammalian central nervous system. Prog Neurobiol 20: 251–271. Oh, D.J., and Dichter, M.A. 1994. Effect of a gamma-aminobutyric acid uptake inhibitor, NNC-711, on spontaneous postsynaptic currents in cultured rat hippocampal neurons: implications for antiepileptic drug development. Epilepsia 35: 426–430. Olney, J.W. 1969. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science 164: 719–721. Olney, J.W. 1976. Brain damage and oral intake of certain amino acids. Adv Exp Med Biol 69: 497–506. Olney, J.W., and Ho, O.L. 1970. Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature 227: 609–611. Olney, J.W., Rhee, V., and Ho, O.L. 1974. Kainic acid: a powerful neurotoxic analogue of glutamate. Brain Res 77: 507–512. Pal, S., Sun, D., Limbrick, D., Rafiq, A., and DeLorenzo, R.J. 2001. Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy. Cell Calcium 30: 285–296. Pellegrini-Giampietro, D.E., Gorter, J.A., Bennett M.V., and Zukin, R.S. 1997. The GluR2 (GluR-B) hypothesis: Ca(2+)-permeable AMPA receptors in neurological disorders. Trends Neurosci 20:464–470. Piccinelli, P., Borgatti, R., Perucca, E., Tofani, A., Donati, G., and Balottin, U. 2000. Frontal nonconvulsive status epilepticus associated with high-dose tiagabine therapy in a child with familial bilateral perisylvian polymicrogyria. Epilepsia 41: 1485–1488. Potter, S.M., and DeMarse, T.B. 2001. A new approach to neural cell culture for long-term studies. J Neurosci Methods 110: 17–24.
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4 An Overview of In Vitro Seizure Models in Acute and Organotypic Slices UWE HEINEMANN, OLIVER KANN, AND SEBASTIAN SCHUCHMANN
Following a seizure-like event, there is usually a postictal depression period. Interictal discharges then reappear, eventually leading to the next seizure-like event. Interictal discharges can be “proictogenic,” but there are also examples where the frequent occurrence of interictal discharges prevents seizure generation. Criteria to discriminate between proepileptic and antiepileptic interictal discharges in the clinical environment have not yet been worked out and can only be experimentally determined. For example, reduction of magnesium concentration in the artificial cerebrospinal fluid (ACSF) bathing rat hippocampal slices (the low-Mg2+ model) induces recurrent short discharges that fulfill the criteria of paroxysmal depolarization shifts. When baclofen, a g-aminobutyric acid (GABA)B agonist, is added to the bathing medium, these short, recurrent discharges are reduced in frequency and seizure-like events emerge (Anderson et al., 1986). Seizure-like events are typically accompanied by rises in extracellular potassium concentration ([K+]o) to levels near 12 mM (with an undershoot to below baseline following the seizure-like event), decreases in extracellular calcium concentration ([Ca2+]o) by up to 0.6 mM, decreases in sodium and chloride concentrations ([Na+]o and [Cl-]o), and changes in the size of the extracellular space (Lux et al., 1986). Changes in pH are also characteristic, with an initial alkalotic shift followed by acidosis. Intracellular recordings from neurons reveal transient depolarizations superimposed by bursts of action potentials during the initial phase; a sustained depolarization by about 30 mV during the tonic phase; and depolarization shifts, which again evoke bursts of action potentials superimposed on a slowly declining membrane potential (Schmitz et al., 1997). During the tonic phase, intrasomatic recordings show frequently aborted spikes or
INTRODUCTION Characteristic Features of Seizures to Be Modeled In Vitro In vitro preparations offer a variety of options for studying the mechanisms of the generation, spread, and termination of seizures using methods that are difficult to employ under in vivo conditions. Particularly, in vitro preparations permit precise control of temperature and extracellular environment. However, in vitro seizure models lack the behavioral and motor components of clinical seizures. They must therefore rely on “equivalents” of seizures that have been observed in vivo. Such equivalents are characteristic changes of electrical activity and in the ionic environment as measured using either intracellular or extracellular recording techniques. Seizure models—in vivo or in vitro— should also share with human patients a similar sensitivity to antiepileptic drugs. Because about 30% of epileptic patients do not become seizure-free with classic anticonvulsants, there is a need for models that mimic drugresistant epilepsy. In vitro criteria for seizure models include electrographic features and changes in ionic concentrations that are comparable to features observed in human and animal studies. A seizure-like event (SLE) in most cortical structures lasts more than 10 seconds. In extracellular field potential recordings, a negative shift accompanies the SLE; superimposed on the negative potential shift are tonic-like or clonic-like electrographic features. Tonic-like discharges are characterized by relatively fast, small transients with a frequency range above 8 Hz; clonic-like discharges consist of relatively large-field potential transients of 50- to 200-ms duration.
Models of Seizures and Epilepsy
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Chapter 4/An Overview of In Vitro Seizure Models in Acute and Organotypic Slices
no action potentials at all as a result of depolarizing inactivation of sodium currents. However, as the depolarization propagates electrotonically into the axon, axonal action potentials will occur where there is insufficient depolarization to cause inactivation of sodium currents; hence, high-frequency axonal firing can be observed during the tonic phase of seizure-like events. In some cases ectopic action potential generation in presynaptic terminals, resulting from changes in the ionic environment and heterosynaptic or homosynaptic effects of released transmitters, can also be involved in ictogenesis (Gutnick and Prince, 1972). Following a SLE, a long-lasting (~30 seconds) afterhyperpolarization is often observed, with reduced synaptic noise and initial suppression of interictal discharges. Occurrence and spread of SLEs can be monitored using extracellular recording techniques with multiple electrodes, with voltage-sensitive dyes (Demir et al., 1998), or by using intrinsic optical signals (Buchheim et al., 1999).
raise the possibility that adaptive homeostatic plastic changes develop in chronically epileptic tissue, which prevents frequent occurrence of seizures. Understanding such mechanisms might open new strategies for the cure of epilepsies.
IN VITRO PREPARATIONS We address two types of in vitro preparations that can be used for studies on seizure generation: acute slices and organotypic slice cultures. More intact preparations of early postnatal neocortex or hippocampus offer advantages for studying epilepsy features in preparations that correspond to perinatal human tissue (Khalilov et al., 1999) (see Chapter 6). An isolated brain preparation offers additional research options (Paré et al., 1992) (see Chapter 9). More “reduced” in vitro preparations also offer advantages for specific applications (e.g., see Chapters 2 and 3).
SPECIAL FEATURES OF IN VITRO MODELS
EPILEPTIFORM ACTIVITY IN ACUTE SLICES
Treatments that induce seizures in animals and humans often induce only relatively short recurrent discharges in slice preparations. This important “discrepancy” is most notably the case with agents that interfere with GABAergic or glycinergic synaptic transmission, such as strychnine, bicuculline, penicillin, etc. By contrast, agents that block potassium currents, such as 4-aminopyridine (4-AP), barium, and cesium, are more likely to induce seizure-like events, as is the case in vivo (Avoli and Perreault, 1987). Conditions where changes in Mg and Ca bathing medium concentrations are used to induce seizure-like events bear some relationship to eclamptic seizures. In such cases, opening the blood-brain barrier as a consequence of sudden surges in blood pressure leads to a hypomagnesaemia and a hypocalcaemia in the interstitial space of the central nervous system (CNS), which in turn may contribute to ictogenesis (Thomas, 1998). Delayed inactivation of sodium currents, as observed with veratridine, can induce ictal events both in vivo and in vitro (Alkadhi and Tian, 1996). Based on these observations, it appears that interference with intrinsic neuronal excitability is more likely to result in seizure-like generation in vitro than is interference with synaptic transmission. It should also be noted that most seizure models are used in otherwise healthy tissue. Little information is yet available about seizure characteristics in chronically epileptic tissue. For example, it remains unclear why we were unable to induce seizure-like events in slices from human epileptic tissue with low-Mg2+ bathing medium, bicuculline, 4-AP, and other agents, but potassium-evoked seizure-like events were generated (Gabriel et al., 2004). Such observations
The most popular in vitro preparations are acute brain slices, consisting of 200- to 600-mm thin sections of living brain tissue, which can be obtained from any species (including nonmammalian species) with a complex brain. Although there is much literature on burst discharges from invertebrate preparations, there is a current bias that invertebrate systems show too many differences from mammalian brain to make them useful tools to study epileptic activity (however, see Chapter 15). Many areas of the brain have been used for acute slices. Complex slice preparations, which partially preserve inter-area connectivity, for example, between the entorhinal cortex and the hippocampus (Dreier and Heinemann, 1991; Walther et al., 1986), between the amygdala and the entorhinal cortex-hippocampal formation (von Bohlen et al., 1998), or between the neocortex and the thalamus (Coulter and Lee, 1993), have been used for studies in experimental epilepsy. Acute slice preparations exploiting brainstem tissue, involving regions often involved in generation of myoclonic seizures and in secondary generalization of seizures, have been much less studied. Numerous problems must be taken into account by investigators using acute slice preparations. Inevitably such preparations have undergone a period of ischemia. Further, many fiber projections entering and leaving the brain slice are severed. Therefore all studies on ictogenesis in such brain slices are performed in tissue which has, per se, undergone an acute lesion. Moreover the thickness of the preparation requires a supply of nutrients that is dependent on exchange by diffusion from the ACSF. The average distance between capillaries is only 50 mm in brain tissue, providing
Epileptiform Activity in Organotypic Slice Cultures
efficient exchange of oxygen and nutrients to the neural tissue. In acute slices, diffusion processes are much less efficient. Exchange of the extracellular solution in acute brain slices takes about 60 minutes when investigations are performed under interface conditions and about 20 minutes when investigations are performed under submerged conditions. As a result of the acute lesion, and in analogy to in vitro observations after acute transection of the spinal cord, there is a drastic reduction in spontaneous synaptic activity. This reduction is particularly important in neocortical preparations where synaptic input from extrinsic sources provides a major source of excitatory drive, and is less important where physiologic synaptic activity can be maintained by intraslice circuitry (e.g., in the hippocampal formation). Because of limitations of diffusion and to provide a sufficient oxygen and glucose supply, solutions saturated with 95% of oxygen and high glucose content are typically used for maintaining acute slices. This approach results in cells on the slice surface being exposed to nonphysiologic, highoxygen tensions (which might cause cell alterations), whereas cells in the center of the slice see oxygen tensions that might be too low. It is worth noting that all early measurements on oxygen tension in acute brain slice preparations were performed with electrodes that, by themselves, consumed significant amounts of oxygen; other, as yet unpublished, observations with less oxygen-consuming electrodes suggest that this potential problem is overstated (Jiang et al., 1991). Although slice preparations permit investigators to study the mechanisms underlying different forms of epileptiform activity, they are not suited for studying mechanisms of cell death (for example, during status epilepticus). Even given the fact that diffusion and equilibration are faster in thinner slices, no attempts have been made to adjust oxygen tension and glucose concentrations to the real needs of neurons in such preparations. Another diffusion-related issue arises when considering the use of patch-clamp recording techniques. Patch-clamp recordings have become a very valuable tool in neuroscience research. In brain slice preparations, visual identification of individual neurons to be patched is facilitated using submerged bathing conditions. Although diffusional exchange is faster under submerged conditions than under interface conditions, the objective that is inserted into the fluid covering the slice might cause a zone of slowed perfusion. Indeed we have found that, for submerged conditions, care must be taken to prevent acidosis in the tissue. A high perfusion rate of 4 ml/min solves this problem in submerged chambers (compared with a perfusion rate of 1.3 to 2 ml/min in interface chambers to keep the slice preparation in good condition). Unfortunately many groups using patch-clamp recordings work at room temperature. Because seizure generation is usually blocked when the temperature in the preparation falls below 32° C (Schuchmann et al., 2002),
37
conclusions based on room temperature recordings must be interpreted with care. Finally, in using slices from different species, it is important to consider that different brain regions, particularly limbic regions, serve different functions in different animals. Thus in mice the hippocampus is the predominant association cortex, and many more functions are likely to involve the hippocampal formation for neuronal computation than in humans. Because of the small size of the mouse brain, a slice from a mouse may contain more of the functional connectivity of the structure of interest; in larger brains, less connectivity is likely to be preserved. Indeed it is much easier to induce seizure-like events in hippocampal slices from mice than from rats or cats.
EPILEPTIFORM ACTIVITY IN ORGANOTYPIC SLICE CULTURES Potentially the best suited preparations for studies on seizure generation and seizure-induced cell death are organotypic slice cultures. Organotypic cultures maintain some of the intrinsic properties of the tissue, including the most important aspects of connectivity. However, the preparations are usually made from pups (P6–P10) and then studied after 7 to 30 days in vitro; therefore these preparations do not necessarily represent adult tissue. The preparations are usually maintained in an artificial growth medium, which may include serum, so at least some of their properties are probably not comparable to those of adult tissue. Finally, as the preparations are more or less deafferentate and deefferented, abnormal connectivity may develop (Gutierrez and Heinemann, 1999). The degree of abnormal connectivity depends on the age of the animal at preparation, the brain area that is prepared (with or without adjacent tissue), and the culture medium. Unfortunately, no systematic studies have yet been performed to control for such alterations. In isolated organotypic hippocampal slice cultures in which the entorhinal cortex has been removed, the development of the dentate gyrus (DG) is usually incomplete. There is evidence for some mossy fiber sprouting, even when the entorhinal cortex is left attached to the hippocampus (Routbort et al., 1999). Aberrant connectivity between area CA1 and the DG has also been reported (Gutierrez and Heinemann, 1999). Because some of this abnormal connectivity mimics aberrant connectivity in epileptic brain, organotypic slice cultures may be viewed as good models of chronically epileptic tissue, where abnormal connectivity is one of the hallmarks of tissue reorganization. In spite of the limitations mentioned here, organotypic slice cultures offer some advantages over dissociated cultures and acute slices. First, they possess a limited extracellular space and contain many, if not all, of the neuron types present in the comparable area in vivo (i.e., they are
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Chapter 4/An Overview of In Vitro Seizure Models in Acute and Organotypic Slices
“organotypic”). Second, they are not acutely damaged. Third, they are thinner (150–200 mm) than acute slice preparations (350–500 mm) and therefore do not require abnormally high oxygen and glucose levels for maintenance of excitability. In fact, high oxygen levels may damage the slices (Pomper et al., 2000; 2001). Finally, they can be put back in the incubator after a series of seizures, a manipulation that gives the investigator an opportunity to explore long-term effects of seizure-like events.
PREPARATIONS AND MAINTENANCE OF SLICES Acute Slices There are multiple variations of preparation techniques for acute slices. The precise method should depend on the purpose of the study and the area of interest. Initially most laboratories used a tissue chopper for cutting the tissue into thin slices; later, vibratomes were adopted in many laboratories because they appeared to make slices with less mechanical damage. Efforts have been made to determine which cutting parameters (with the vibratome) are most important for preservation of fine tissue details (preservation is essential, for example, for carrying out recordings from subcellular compartments such as presynaptic terminals (Geiger et al., 2002)). Likewise the storage of slices is an important issue. In our laboratory, for experiments under submerged conditions, slices are placed on a nylon grid, which is fixed at the surface of ACSF in a homemade Perspex incubation chamber (at room temperature). The incubation chamber contains 50 ml of gassed (95% O2/5% CO2) ACSF (static bath). For recordings, slices are individually transferred to a submersion-style recording chamber. For interface chamber experiments, we have found the best tissue preservation when slices are maintained in an interface chamber on a piece of lens paper, which facilitates transfer of the slice to the recording chamber. A period of 2 hours allows recovery of swollen neurons and mitochondria. Slice recordings can be performed in an interface chamber or in a submerged chamber. Alternatively a grease gap chamber can be used in which slices set over a separation sealed with Vaseline (Avsar and Empson, 2004), and recordings can be performed by measuring the potential difference between the two chambers. This procedure permits recording of different types of epileptiform activity and easy analysis of drug effects on such activity. Interface chambers are available in two basic variations: (1) the Oslotype chamber, developed in Per Andersen’s laboratory from the original design of McIllwain, who used slices to study brain metabolism (Andersen, 1975); and (2) The Haas-type chamber (Haas et al., 1979). We prefer the Haas chamber, where the slices set on the floor of the chamber, providing
mechanical stability. An always-important issue is the temperature at which the tissue is maintained because seizurelike events are difficult to induce at temperatures below 33° C. Another important issue is the composition of the ACSF, which is used to perfuse the in vitro preparations. Our laboratory uses ACSF containing (in mM): NaCl 124, KCl 3, MgSO2 1.8, CaCl2 1.6, NaH2PO4 1.25, NaHCO3 26, and glucose 10. The solution is equilibrated with carbogen gas (95% O2/5% CO2) to maintain a pH of 7.4; the osmolarity of the solution should be about 290 mOsm. Ca2+, Mg2, and K+ concentrations must be carefully adjusted because small variations in concentrations of these ions have marked effects on neuronal activity. The extracellular Ca2+ concentration ([Ca2+]o) in vivo is about 1.2 mM which is mimicked in slices by 1.6 mM. The reason for this difference is the chelating effect of HCO3- on Ca2+, which precipitates about 25% of the free [Ca2+]o. The potassium concentration in the brain is 3 mM (Lux et al., 1986), but many researchers use higher baseline concentrations following the original recipes of McIllwain. The Mg2+ concentration in CSF, as measured from the ventricles, is somewhere between 0.9 and 1.2 mM; it may be higher in the interstitial space. We use a baseline Mg2+ concentration of 1.6 mM in our experiments. Reduction of extracellular Mg2+ concentration from 1.6 to 1.2 mM has facilitating effects on synaptic transmission (Hamon et al., 1987), and further lowering to 0.9 mM is sufficient to induce epileptiform activity in slices from rat pups (Gloveli et al., 1995). An issue in preparing slices is the choice of anesthesia (and depth of anesthesia chosen before the brain is removed). Pups are frequently anesthetized by putting them on dry ice. This procedure exploits the anesthetic effect of high carbon dioxide levels combined with hypothermia. Many laboratories use gas anesthetics, such as ether or halothane. This choice gives reliable anesthesia with a reasonable therapeutic gap between deep and irreversible levels. It should be noted that very deep anesthesia leads to circulation arrest and hypoxia and causes cessation of protein synthesis (slices prepared in this way often do not exhibit long-term plasticity). Gas anesthesia works well in both sexes. Barbiturate anesthesia has the drawback that females often require more anesthetic to obtain an adequately deep state, but frequently they are put down so deeply that they die before decapitation. Ketamine anesthesia interferes with N-methyl-d-aspartate (NMDA) receptors, an effect not always desirable in studies on seizures. Some laboratories perfuse the animal with a low calcium medium in which sucrose is substituted for sodium to reduce cell death. We have not found that this modification normally enhances tissue viability. However, in cases where preparation time may be prolonged, as in chronically epileptic animals and human tissue, we have found that immersing the slices in sucrose ACSF together with alpha-tocopherol improves tissue preservation (Gabriel et al., 2004).
Imaging In Vitro Preparations
The following procedures are employed to prepare entorhinal cortex–hippocampal slices (Dreier and Heinemann, 1991). Rats (150–200 g) are anesthetized with ether and decapitated. The skull is opened, the dura cut, and the whole brain rapidly (within less than a minute) removed. The brain is submerged in cold (4° C) ACSF. The cerebellum is removed, and the brain is divided into two hemispheres. Each hemisphere is placed on its medial surface and divided into rostral and caudal sections by a transverse cut in a plane approximately parallel to the main axis of the hippocampus. The cut plane of the caudal piece is fixed to the plate of a vibratome (using cyanoacrylate glue), and five to eight horizontal slices are cut at a thickness of 400 mm. These slices contain the temporal cortex area, the perirhinal cortex, the entorhinal cortex, the subiculum, the DG, and the ventral hippocampus. Slices are placed separately on lens paper and stored in oxygenated ACSF in either an incubation chamber (experiments under submerged conditions) at room temperature or in an interface chamber (experiments under interface conditions) at 35° C. Slices are allowed to recover under these conditions for at least 60 to 90 minutes before starting the experiments. To test the viability of our slices, we monitor the extracellular field potential in the medial entorhinal cortex layer III/IV or hippocampal area CA1 following electrical stimulation (single pulse duration of 100 ms, pulse amplitude of 5–15 V) using bipolar platinum wires placed in the lateral entorhinal cortex or in stratum radiatum of area CA1, respectively. Slices are accepted for investigation if the extracellular field potential is at least 2 mV in amplitude. To validate epileptiform activity, we use standard ion-sensitive microelectrodes to measure changes in the extracellular field potential and the extracellular potassium concentration simultaneously. Ion-selective microelectrodes are prepared using double-barreled theta glass (Lux and Neher, 1973). One barrel is filled with 154 mM NaCl and serves as the reference. The other barrel is silanized (5% trimethyl-1chlorosilane in 95% CCl4) and filled with a potassium ionophore cocktail (A60031, Fluka). To compute the molar extracellular potassium concentrations from the recorded potential values, a modified Nernst equation is utilized (Heinemann and Arens, 1992).
Organotypic Slice Cultures We have found the Stoppini method for preparing slice cultures (Stoppini et al., 1991) to be the most simple and reliable. Animal age at time of preparation is an important variable. In both rats and mice, slice cultures can be prepared from tissue starting at embryonic states up to postnatal day 16. Attempts have been made to prepare cultures also from older animals, with some success up to an age of P28 (Leutgeb et al., 2003). Published protocols yield reproducible organotypic cultures in only about 20% of
39
experiments. Hence further methodologic developments are required before such organotypic slice cultures can be used for long-term investigations. The advantage of slice cultures for epilepsy research is that the effects of seizure activity (e.g., status epilepticus)—with respect to cell death, changes in neuronal excitability, neuronal interaction, and morphologic rearrangement of networks—can be followed for extended periods. However, because such changes occur on a background of developmental alterations in the tissue (and under nonphysiologic conditions), a large number of controls are required before drawing conclusions about seizurespecific effects. For maintaining slice cultures in vitro for the purpose of electrophysiologic recordings, it is important that the cultures are superfused with an ACSF that contains physiologic oxygen because ACSF with 95% oxygen can induce hyperoxic cell damage (Pomper et al., 2001; 2004). Ionic composition of bathing medium for these recordings is similar to that used for acute slices. At the time when organotypic hippocampal slice cultures are used for experimental studies (>7 days in vitro), there has been considerable reorganization which depends on two parameters: whether the entorhinal cortex is included in the preparation and which medium is used for maintaining the slices in culture. Some of the reorganizational processes observed in tissue from chronic epileptic rats and humans are also seen in slice cultures; examples include: mossy fiber sprouting; reorganization in area CA1 with extended interconnectivity between CA1 hippocampal pyramidal cells; and interconnections between CA1, subiculum and DG. Such reorganizational changes are less pronounced, or almost absent, in acute slices. As a result of such changes in slice cultures, application of bicuculline and other GABA antagonists can induce seizure-like events in this preparation (Gutierrez and Heinemann, 1999). Our procedures for preparing hippocampal slice cultures follow published protocols (Kann et al., 2003a, b; Stoppini et al., 1991). In brief, hippocampal slices (400 mm) are cut using a tissue chopper from 7- to 9-day-old Wistar rats under sterile conditions. Slices are immediately transferred to icecold, oxygenated (95%) minimal essential medium (MEM) at pH 7.3. Slices with intact anatomic structure are maintained on a biomembrane surface (0.4 mm, Millicell-CM, Millipore, Eschborn, Germany) at an interface between culture medium (1 ml, containing: 50% MEM, 25% Hank’s balanced salt solution, 25% horse serum (Gibco, Invitrogen, Karlsruhe, Germany), and 2 mM l-glutamine (pH 7.4)) and humidified atmosphere (5% CO2, 36.5° C) in an incubator. Half the culture medium is replaced every second day. Slices cultures are used after 7 days in vitro.
IMAGING IN VITRO PREPARATIONS An increasing number of fluorescent dyes have become available that permit measurements of voltage changes and
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Chapter 4/An Overview of In Vitro Seizure Models in Acute and Organotypic Slices
intracellular ionic concentration changes, permit insight into second-messenger cascades, and allow specific studies on mitochondrial function. Generally both acute slices and slice cultures (as well as dissociated cultures) can be bulk stained by exposing the preparation to a given dye. The dye must be membrane permeable, which is often achieved by using an ester form of the dye that makes the substances lipophilic. One complicating factor is that drug transporters expressed in astrocytes can take up such dyes specifically into glial compartments; unless the staining procedure is prolonged or the transport activity is blocked by inhibitors of drug transporters, this glial uptake may make visualization of neuron changes quite difficult. Hence, for any dye, a specific protocol must be developed that permits neuronal loading. Dyes can also be applied focally or via the perfusate. Because these dyes are often expensive, many groups stain the tissue in stagnant chambers and then transfer slices or cultures to the recording chamber. This procedure may result in tissue damage from hypoxia, so dye application via perfusion is preferable. To reduce costs the perfusate can be recycled. Staining in organotypic slice cultures is simpler because the dye can simply be added to the culture medium. Electrophysiologic measurements can then be combined with microfluorimetric (photomultiplier) and imaging techniques (charge-coupled device (CCD) camera, confocal laser scanning microscopy, two-photon confocal microscopy) to monitor cytosolic or mitochondrial calcium concentrations (e.g., with indicators such as calcium green and Rhod-2), mitochondrial membrane potential (rhodamine123 and JC-1) as well as the energy status of the tissue (as reflected in the fluorescence of nicotinamide adenine dinucleotides [NADH and NADPH] and flavin adenine dinucleotide [FAD]). Measurements can be done in the interface mode or under submerged conditions. Some intracellular metabolites, such as NADH, NADPH, and FAD, are fluorescent. NADH and NADPH produce an overlapping fluorescence signal and therefore cannot be readily discriminated. Moreover, cytosolic processes influence the level of NADPH and NADH (Kann, et al., 2003; Kovacs et al., 2002). By contrast, FAD is dependent only on mitochondrial metabolic activity and therefore provides a direct indicator of mitochondrial function. Fluorescent dyes can also be used to obtain insight into such processes as mitochondrial production of radical oxygen species and nitrous oxide (NO) synthesis during seizure-like activities. Finally, dyes that indicate cell loss (e.g., propidium iodide, ethidium bromide) and cell viability (e.g., acridin orange) can also be used for studying consequences of seizures (Pomper et al., 2004). Monitoring NAD(P)H autofluorescence on-line in brain slices is an easy and stable method. Electrical and chemical stimuli elicit NAD(P)H autofluorescence signals with high reproducibility (Kann et al., 2003 a, b; Schuchmann et al., 2001). This stability allows the identification of mitochon-
drial metabolism or activity-induced changes of the NAD(P)H signal. A further advantage of the method is the ubiquity of NAD(P)H in biological tissue; therefore, NAD(P)H autofluorescence can be excited in nearly every region without any pretreatment of the slices. The main disadvantage of using NAD(P)H autofluorescence results from its excitation wavelength (350 ± 20 nm, near ultraviolet). Light with such a short wavelength involves the transfer of relatively high amounts of energy to tissue (photon energy 3.55 ± 0.2 eV) and may therefore induce phototoxicity. To minimize phototoxic effects, excitation time and intensity should be optimized with respect to the experimental conditions. Generally, continuous excitation requires reduced excitation intensity, whereas highexcitation intensity requires short excitation periods. Phototoxic effects are indicated by a rapid rundown in the NAD(P)H autofluorescence signal as well as by electrophysiologic (reduction in extracellular field potential amplitude) or morphologic (light-colored “burn” spots on the slice) changes. When slices are damaged by hypoxia, ischemia, or a mechanical lesion, the NAD(P)H autofluorescence signal is reduced especially in regions of densely packed neurons (CA1, CA3, area dentata). Thin slices (<200 mm) exhibit a significantly lower NAD(P)H autofluorescence baseline signal (compared with standard 400-mm-thick slices) under identical optical conditions, again because of the reduced number of cells in these slices. Additionally, thin slices and organotypic slice cultures (and cellular monolayers) undergo significantly increased rates of phototoxic effects compared with 400 mm slices.
IN VITRO SEIZURE MODELS Generally, before studying seizure induction, the viability of the tissue should be tested; experiments should be carried out only on preparations with “normal” physiologic responses, that is, those that show no signs of epileptiform alterations such as multiple population spikes. A good test of viability (in addition to that described previously) is whether long-term potential (LTP) or long-term depression (LTD) can be maintained beyond 20 minutes. Slices exposed to prolonged ischemia lose the capability for protein synthesis and therefore cannot maintain long-lasting changes in synaptic plasticity. Induction of seizure-like events in such preparations yields atypical discharge patterns, disturbances in ionic homeostasis, and rapid decline of the preparation once SLEs are induced. Often it is impossible to induce spontaneous activity.
Low Calcium-lnduced Epileptiform Activity Recordings in vivo have shown that extracellular Ca2+ concentration can drop to levels as low as 0.2 mM (Pumain et al., 1985). Studies initiated in vitro (Jefferys and Haas,
In Vitro Seizure Models
1982; Yaari et al., 1983) showed that lowering extracellular Ca2+ (i.e., in the bathing medium) could induce regular seizure-like events that lasted for about 30 seconds and recurred in a clock-like fashion. Similar events can also be induced when the hippocampus is treated with Ca2+chelating agents in vivo (Feng and Durand, 2003). This pattern of activity depends on low (zero mM) Ca2+ treatment and simultaneous elevation of [K+]o to 5.5 mM. The induction of seizure-like events coincides with the blockade of evoked synaptic transmission. It is a result of reduced surface charge screening (which increases neuronal excitability) and reduced Ca2+-dependent K2+ currents. Electrographically the events appear to mimic tonic-like discharges with slow negative shifts on which a series of population spikes are superimposed. The appearance of population spike activity is somewhat delayed with respect to onset of the negative field potential shift. Clonic-like afterdischarges are not apparent. Intracellular measurements reveal a membrane depolarization of about 30 mV (corresponding to the negative shift in the field), with superimposed action potentials. When normal Ca2+ levels are restored in the bathing medium, the tissue behaves normally, with little sign of neuronal deterioration. Interestingly, the low Ca2+ activity spreads with a low speed (Konnerth et al., 1984), similar to that observed for seizure-like events in the entorhinal cortex induced by low Mg and 4-AP (Weissinger et al., 2000) in which synaptic transmission is maintained, suggesting that nonsynaptic mechanisms—likely potassium diffusion—are involved in seizure spread (Shuai et al., 2003). As evoked transmitter and peptide release is blocked under conditions of low Ca2+, this model is particularly well suited for studies on mechanisms of nonsynaptic seizure spread and also for studies of whether and how anticonvulsants directly modify neuronal excitability (Heinemann et al., 1985). The low-Ca2+ model works well in rat hippocampal area CA1 where neurons are densely packed. The threshold for induction is lower in tissue from juvenile animals. The low-Ca2+ model does not work in human tissue or in other brain areas with low cell packing density, unless the size of the extracellular space (ES) is reduced by treatment with hypoosmolar solutions (which, however, also affect transmembrane Cl- concentration gradients). For example, low-Ca2+-induced seizure-like events in the DG have required simultaneous application of augmented potassium concentration (ª10 mM) or modification of extracellular osmolarity (Schweitzer et al., 1992). Under submerged conditions, induction of low-Ca2+ seizure-like events appears to be facilitated by lowering the recorded temperature (for still little-understood reasons), but in interface chambers lowering of temperature blocks this activity (Schuchmann et al., 2002). Pharmacologically, low-Ca2+ seizure-like activity responds to carbamazepine (CBZ), phenytoin (PHT), and valproate (VPA) in clinically relevant concentrations. The activity can also be blocked by application of DC fields.
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Low Magnesium Induced Seizure-Like Events Epileptiform activity can be induced in virtually all epilepsy-sensitive regions of the brain by omitting Mg2+ from the ACSF. Induction of this activity depends on increased neuronal excitability resulting from reduced surface charge screening, facilitated transmitter release, and activation of NMDA receptors due to removal of the Mg2+ block of NMDA receptor-operated ion channels. Initially these events are always sensitive to application of NMDA receptor antagonists, which is consistent with the idea that activation of NMDA receptors is critical for induction of such events (Stanton et al., 1987). However, NMDA receptor antagonists may lose their efficacy with increasing time after application of low Mg2+. In such cases, a form of long-lasting synaptic potentiation is observed. In the isolated rat hippocampal slice, lowering Mg2+ induces recurrent, short-duration discharges that share the properties of interictal discharges. They are initiated in area CA3 and propagate to area CA1. The DG does normally not participate in this activity, although these recurrent epileptiform discharges do involve the hilus. These interictal-like discharges last for 60 to 150 ms, are characterized by positive-going field potential transients in stratum pyramidale of area CA1 and CA3, and are superimposed by bursts of population spikes (Mody et al., 1987). Low-Mg2+ seizure-like events can be induced in rat hippocampus by simultaneously applying baclofen and elevating potassium (Anderson et al., 1986). In mice, omitting Mg2+ from the ACSF can cause seizure-like events (Velisek et al., 1996) which are similar to those observed with low Mg2+ in rat limbic structures such as temporal neocortex, entorhinal cortex, and subiculum (Avoli et al., 1991; Dreier and Heinemann, 1991). Each of these regions can produce seizure-like events when isolated from the remaining structures of the hippocampal formation. The seizurelike events in subiculum, EC, and temporal neocortex are characterized by negative potential shifts superimposed by high-frequency, small-amplitude oscillations, followed by clonic-like afterdischarges. Intracellular recordings reveal depolarization shifts of up to 30 mV, on which are superimposed prolonged bursts of action potentials. During cloniclike after discharges, depolarization shifts that trigger series of action potential discharges are observed. In combined hippocampal–entorhinal cortex slices, the seizure-like events most frequently start in medial entorhinal cortex but can also begin in temporal neocortex, lateral entorhinal cortex, or the subiculum (Dreier and Heinemann, 1991). The seizures spread from site of origin slowly to neighboring areas. Speed of spread is slightly slower than the speed of spread for lowCa2+-induced seizure-like events (Buchheim et al., 2000). Spread of activity can be monitored by field recordings at multiple sites or by use of intrinsic optical signals. Low-Mg2+ seizure-like events possess the same pharmacologic properties as seizures induced in vivo, that is, they are sensitive to
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Chapter 4/An Overview of In Vitro Seizure Models in Acute and Organotypic Slices
CBZ, PHT, and VPA and also to benzodiazepines and barbiturates in clinically relevant concentrations (Zhang et al., 1995). Interestingly, seizure-like events induced by low Mg2+ show different properties in frontal cortex slices. In this brain region, the epileptiform pattern does not include afterdischarges. Further, the activity spreads more rapidly than in temporal and limbic structures (Armand et al., 1998), and the episodes are much shorter than in the entorhinal cortex, amygdale, and hippocampus. This feature of low Mg2+ activity suggests that the relatively short, frequent, seizure episodes often observed in clinical frontal lobe epilepsies are dependent on intrinsic properties of frontal cortical tissue. Low-Mg2+-induced seizure-like events show different characteristics of spread when induced in chronic epileptic tissue. In both pilocarpine-treated animals and kindled animals, seizure spread is facilitated (Behr et al., 1998). Two routes of low-Mg2+ seizure spread have been described in pilocarpine treated animals: one through the subiculum and one through the DG. In contrast, seizure spread in kindled animals is preferentially through the DG, suggesting that the DG has lost some of its filtering properties (Wozny et al., unpublished observations). Low-Mg2+ seizure spread is also facilitated in preparations from rat pups, where the hippocampus is variably involved (Weissinger et al., 1998). Seizure-like events disappear sometime after induction by low Mg2+ and are replaced by shorter events, which most frequently represent tonic-like discharges. These events can be mistaken for interictal discharges. However, because the ionic change and the intracellularly recorded signals accompanying these activities differ from the properties of interictal discharges in duration and amplitude, we believe these events correspond to activity patterns observed during prolonged status epilepticus. Because application of GABA and GABA-uptake inhibitors reverse these events to seizurelike patterns, a loss of GABAergic functions seems to be involved in this activity. Muscimol blocks this activity at low concentrations; the change is likely not due to alterations in GABA-receptor affinity, but more likely to consumption of GABA in the GABA shunt of the tricarboxic acid cycle (Pfeiffer et al., 1996).These late recurrent discharges are of particular interest because they are not blocked by standard anticonvulsants even when high concentrations of these agents are applied (Dreier et al., 1998). Induction of low Mg2+ activity involves omission of magnesium sulfate from the perfusate. The first epileptiform discharges appear after 40 minutes under interface recording conditions and after 20 minutes under submerged conditions. Transition to the late recurrent, pharmacoresistant discharges occurs between 30 and 90 minutes after onset of epileptiform activity.
4-Aminopyridine-Induced Seizure-like Events 4-aminopyridne (4-AP) is a blocker of transient potassium currents. At low concentrations it affects transient currents containing Kv1.3 and 1.4 subunits in low concentrations and affects other transient potassium currents only in the millimolar range. In rat hippocampus, 4-AP usually induces recurrent discharges. In parahippocampal structures such as the entorhinal cortex, subiculum, and temporal neocortex (Avoli et al., 1996) and also in the amygdala (Klueva et al., 2003), it elicits seizure-like events. The seizure-like events start with an initial bursting period and give rise to a tonic-like episode and clonic afterdischarges superimposed on slow negative-potential shifts; these electrographic events are associated with ionic changes characteristic of seizure-like events. In interface recordings, the activity commences about 40 minutes after application of 50 or 100 mm 4-AP (Brückner and Heinemann, 2000). Transition to late recurrent discharges may or may not occur after more than 1.5 hours of ongoing seizure-like activity. These seizure-like events respond to all standard anticonvulsants (Brückner and Heinemann, 2000). Late pharmacoresistant activity can be evoked by combining bicuculline with 4-AP (Brückner, et al., 1999).
Seizure-like Events Induced by Elevating Potassium Concentration Early in vivo studies in different species suggested that elevating potassium concentration can induce seizure-like events in hippocampus. This effect is readily mimicked in vitro (Jensen and Yaari, 1988) by raising extracellular potassium in ACSF from 3/3.5 mM (standard in most laboratories) to 7.5 mM. This activity also responds to standard anticonvulsant drugs. When [K+]o is elevated to 11 mM and Ca2+ is lowered at the same time, seizure-like events can be induced in the DG of Sprague-Dawley rats (Schweitzer et al., 1992). In Wistar rats, the induction of seizure-like events with high K+ and moderately lowered Ca2+ and Mg2+ is difficult to reproduce.
Stimulation-Induced Seizures Short trains of repeated high-frequency electrical stimuli have also been used to induce ictaform activity in the form of afterdischarges. In combined hippocampal–entorhinal cortex slices from young Sprague-Dawley rats (P21–P30), repeated trains (100 Hz, 0.1-ms pulses, 1- or 2-second train duration) in the presence of 0.5 mM magnesium eventually results in self-sustained status epilepticus (Rafiq et al., 1995). In slices from older Sprague-Dawley rats, ictaform afterdischarges have been induced by stimulus trains when
References
slices were bathed in low Mg and low Ca concentrations and elevated K (Stelzer et al., 1987) or with 1.3 Mg and 5 mm 4AP bathing media (Higashima et al., 2000; Ohno and Higashima, 2002). Trains of repetitive stimulation in organotypic slice cultures also, in some cases, induce afterdischarges and spreading depressions.
Focal Induction of Seizure-like Events Although most in vitro models for seizures are thought to mimic aspects of focal or partial epilepsies, few attempts have been made to induce seizure-like events by focal manipulations. Seizure-like events in the models described herein usually have focal onset, most frequently in the entorhinal cortex, sometimes in area CA3, and more rarely in area CA1. An interesting alternative approach involves focal application of bicuculline (50 mm) and high K (10 mM) to the EC (Behr et al., 1998). These investigators studied spread patterns of epileptiform activity from the EC to the hippocampus in normal and kindled rats and later in pilocarpine-treated rats. Another method of focal induction involves discrete application of carbachol (10 mM) from a pipette with short pressure pulses (10–200 ms) (Gloveli, et al., 1998).
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Brückner, C., Stenkamp, K., Meierkord, H., and Heinemann, U. 1999. Epileptiform discharges induced by combined application of bicucculline and 4-aminopyridine are resistant to standard anticonvulsants in slices of rats. Neurosci Lett 268: 163–165. Buchheim, K., Schuchmann, S., Siegmund, H., Gabriel, H.-J., Heinemann, U., and Meierkord, H. 1999. Intrinsic optical signal measurement reveal characteristic features during different forms of spontaneous neuronal hyperactivity associated with ECS shrinkage in vitro. Eur J Neurosci 11: 1877–1882. Buchheim, K., Schuchmann, S., Siegmund, H., Weissinger, F., Heinemann, U., and Meierkord, H. 2000. Comparison of intrinsic optical signals associated with low Mg2+ and 4-aminopyridine induced seizure-like events reveals characteristic features in adult rat limbic system. Epilepsia 41: 635–641. Coulter, D.A., and Lee, C.-J. 1993. Thalamocortical rhythm generation in vitro: Extra- and intracellular recordings in mouse thalamocortical slices perfused with low Mg2+ medium. Brain Res 631: 137– 142. Demir, R., Haberly, L.B., and Jackson, M.B. 1998. Voltage imaging of epileptiform activity in slices from rat piriform cortex: onset and propagation. J Neurophysiol 80: 2727–2742. Dreier, J.P., and Heinemann, U. 1991. Regional and time dependent variations of low magnesium induced epileptiform activity in rat temporal cortex. Exp Brain Res 87: 581–596. Dreier, J.P., Zhang, C.L., and Heinemann, U. 1998. Phenytoin, phenobarbital, and midazolam fail to stop status epilepticus-like activity induced by low magnesium in rat entorhinal slices, but can prevent its development. Acta Neurol Scand 98: 154–160. Feng, Z., and Durand, D.M. 2003. Low-calcium epileptiform activity in the hippocampus in vivo. J Neurophysiol 90: 2253–2260. Gabriel, S., Njunting, M., Pomper, J.K., Merschhemke, M., Sanabria, E.R., Eilers, A., Kivi, A. et al. 2004. Stimulus and potassiuminduced epileptiform activity in the human dentate gyrus from patients with and without hippocampal sclerosis. J Neurosci 24: 10416–10430. Geiger, J.R., Bischofberger, J., Vida, I., Frobe, U., Pfitzinger, S., Weber, H.J., Haverkampf K. et al. 2002. Patch-clamp recording in brain slices with improved slicer technology. Pflugers Arch 443: 491–501. Gloveli, T., Albrecht, D., and Heinemann, U. 1995. Properties of low Mg2+ induced epileptiform activity in rat hippocampal and entorhinal cortex slices during adolescence. Dev Brain Res 87: 145–152. Gloveli, T., Egorov, A.V., Schmitz, D., Heinemann, U., and Müller, W. 1998. Muscarinic control of excitability and [Ca2+]i signaling in layer II and III projection neurons of the medial entorhinal cortex. Soc Neurosci Abstr 24(2): 1583. Gutierrez, R., and Heinemann, U. 1999. Synaptic reorganization in explanted cultures of rat hippocampus. Brain Res 815: 304–316. Gutnick, M.J., and Prince, D.A. 1972. Thalamocortical relay neurons: antidromic invasion of spikes from a cortical epileptogenic focus. Science 176: 424–426. Haas, H.L., Schaerer, B., and Vosmansky, M. 1979. A simple perfusion chamber for the study of nervous tissue slices in vitro. J Neurosci Methods 1: 323–325. Hamon, B., Stanton, P.K., and Heinemann, U. 1987. An N-methyl-d-aspartate receptor-independent excitatory action of partial reduction of extracellular [Mg2+] in CA1-region of rat hippocampal slices. Neurosci Lett 75: 240–245. Heinemann, U., and Arens, J. 1992. Production and calibration of ionsensitive microelectrodes. In Practical Electrophysiological Methods: A Guide for In Vitro Studies in Vertebrate Neurobiology. Ed. R. Grantyn and H. Kettenmann. pp. 206–212. New York: Wiley-Liss. Heinemann, U., Franceschetti, S., Hamon, B., Konnerth, A., and Yaari, Y. 1985. Effects of anticonvulsants on spontaneous epileptiform activity which develops in the absence of chemical synaptic transmission in hippocampal slices. Brain Res 325: 349–352.
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Chapter 4/An Overview of In Vitro Seizure Models in Acute and Organotypic Slices
Higashima, M., Ohno, K., Kinoshita, H., and Koshino, Y. 2000. Involvement of GABAA and GABAB receptors in afterdischarge generation in rat hippocampal slices. Brain Res 865: 186–193. Jefferys, J.G.R., and Haas, H.L. 1982. Synchronized bursting of CA1 hippocampal pyramidal cells in the absence of synaptic transmission. Nature 300: 448–450. Jensen, M.S., and Yaari, Y. 1988. The relationship between interictal and ictal paroxysms in an in vitro model of focal hippocampal epilepsy. Ann Neurol 24: 591–598. Jiang, C., Agulian, S., and Haddad, G.G. 1991. O2 tension in adult and neonatal brain slices under several experimental conditions. Brain Res 568: 159–164. Kann, O., Kovacs, R., and Heinemann, U. 2003a. Metabotropic receptormediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in rat hippocampal slice cultures. J Neurophysiol 90: 613–622. Kann, O., Schuchmann, S., Buchheim, K., and Heinemann, U. 2003b. Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat. Neuroscience 119: 87–100. Klueva, J., Munsch, T., Albrecht, D., and Pape, H.C. 2003. Synaptic and non-synaptic mechanisms of amygdala recruitment into temporolimbic epileptiform activities. Eur J Neurosci 18: 2779–2791. Konnerth, A., Heinemann, U., and Yaari, Y. 1984. Slow transmission of neural activity in hippocampal area CA1 in the absence of active chemical synapses. Nature 307: 69–71. Kovacs, R., Schuchmann, S., Gabriel, S., Kann, O., Kardos, J., and Heinemann, U. 2002. Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures. J Neurophysiol 88: 2909–2918. Leutgeb, J.K., Frey, J.U., and Behnisch, T. 2003. LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25–30) rats. J Neurosci Methods 130: 19–32. Lux, H.D., Heinemann, U., and Dietzel, I. 1986. Ionic changes and alterations in the size of the extracellular space during epileptic activity. In Advances in Neurology, vol 44. Eds. A.V. Delgado-Escueta, A.A. Ward, D.M. Woodbury, and R.J. Porter. Basic Mechanisms of the Epilepsies: Molecular and Cellular Approaches. pp. 619–639. New York: Raven Press. Lux, H.D., and Neher, E. 1973. The equilibration time course of [K+]oin cat cortex. Exp Brain Res 17: 190–205. Mody, I., Lambert, J.D.C., and Heinemann, U. 1987. Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. J Neurophysiol 57(3): 869–888. Ohno, K., and Higashima, M. 2002. Effects of antiepileptic drugs on afterdischarge generation in rat hippocampal slices. Brain Res 924: 39–45. Paré, D., DeCurtis, M., and Llinás, R.R. 1992. Role of the hippocampalentorhinal loop in temporal lobe epilepsy: Extra- and intracellular study in the isolated guinea pig brain in vitro. J Neurosci 12: 1867–1881. Pfeiffer, M., Draguhn, A., Meierkord, H., and Heinemann, U. 1996. Effects of t-aminobutyric acid (GABA) agonists and GABA uptake inhibitors on pharmacosensitive and pharmacoresistant epileptiform activity in vitro. Br J Pharmacol 119: 569–577. Pomper, J.K., Graulich, J., Kovács, R., Gabriel, S., Hoffmann, U., and Heinemann, U. Increased oxygen tension leads to cell damage in organotypic hippocampal slice cultures. 2000. International Conference Neuroprotection and Neurorepair. Cellular and molecular mechanisms in stroke ischemia and trauma. Technical Workshop, 01.03.-02.03.2000, Magdeburg, Germany. Pomper, J.K., Graulich, J., Kovacs, R, Hoffmann, U., Gabriel, S., and Heinemann, U. 2001. High oxygen tension leads to acute cell death in organotypic hippocampal slice cultures. Brain Res Dev Brain Res 126: 109–116. Pomper, J.K., Hoffmann, U., Kovacs, R., Gabriel, S., and Heinemann, U. 2004. Hyperoxia is not an essential condition for status epilepticus
induced cell death in organotypic hippocampal slice cultures. Epilepsy Res 59: 61–65. Pumain, R., Menini, C., Heinemann, U., Silva-Barrat, C, and Louvel, J. 1985. Chemical synaptic transmission is not necessary for epileptic activity to persist in the neocortex of the photosensitive baboon. Exp Neurol 89: 250–258. Rafiq, A., Zhang, Y.F., DeLorenzo, R.J., and Coulter, D.A. 1995. Long-duration self-sustained epileptiform activity in the hippocampal-parahippocampal slice: a model of status epilepticus. J Neurophysiol 74: 2028–2042. Routbort, M.J., Bausch, S.B., and McNamara, J.O. 1999. Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures. Neuroscience 94: 755–765. Schmitz, D., Empson, R.M., Gloveli, T., and Heinemann, U. 1997. Serotonin blocks different pattern of low Mg2+-induced epileptiform activity in rat entorhinal cortex, but not hippocampus. Neuroscience 76: 449–458. Schuchmann, S., Kovacs, R., Kann, O., Heinemann, U., and Buchheim, K. 2001. Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal-entorhinal cortex slices. Brain Res Brain Res Protoc 7: 267–276. Schuchmann, S., Meierkord, H., Stenkamp, K., Breustedt, J., Windmuller, O., Heinemann, U., and Buchheim, K. 2002. Synaptic and nonsynaptic ictogenesis occurs at different temperatures in submerged and interface rat brain slices. J Neurophysiol 87: 2929–2935. Schweitzer, J.S., Patrylo, P.R., and Dudek, F.E. 1992. Prolonged field bursts in the dentate gyrus: Dependence on low calcium, high potassium, and nonsynaptic mechanisms. J Neurophysiol 68: 2016–2025. Shuai, J., Bikson, M., Hahn, P.J., Lian, J., and Durand, D.M. 2003. Ionic mechanisms underlying spontaneous CA1 neuronal firing in Ca2+-free solution. Biophys J 84: 2099–2111. Stanton, P.K., Jones, R.S.G., Mody, I., and Heinemann, U. 1987. Epileptiform activity induced by lowering extracellular [Mg2+] in combined hippocampal-entorhinal cortex slices: modulation by receptors for norepinephrine and N-methyl-d-aspartate. Epilepsy Res 1: 53–62. Stelzer, A., Slater, N.T., and ten Bruggencate, G. 1987. Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy. Nature 326: 698–701. Stoppini, L., Buchs, P.-A., and Muller, D. 1991. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37: 173–182. Thomas, S.V. 1998. Neurological aspects of eclampsia. J Neurol Sci 155: 37–43. Velisek, L., Moshe S.L., and Stanton, P.K. 1996. Increased susceptibility of brain slices from carbonic anhydrase II-deficient mice to low [Mg2+]O-induced seizures. Neurosci Lett 207: 143–146. von Bohlen und Halbach, O., and Albrecht, D. (1998). Tracing of axonal connectivities in a combined slice preparation of rat brains—a study by rhodamine-dextran-amine-application in the lateral nucleus of the amygdala. J Neurosci Methods 81: 169–175. Walther, H., Lambert, J.D.C., Jones, R.S.G., Heinemann, U., and Hamon, B. 1986. Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium. Neurosci Lett 69: 156–161. Weissinger, F., Buchheim, K., Schuchmann, S., Siegmund, H., Gabriel, H.-J., Heinemann, U., and Meierkord, H. 1998. Optical imaging of epileptiform activity demonstrates age specific features in entorhinal cortex-hippocampus slices. Eur J Neurosci 10(Suppl 10): 44. Weissinger, F., Buchheim, K., Siegmund, H., Heinemann, U., and Meierkord, H. 2000. Optical imaging reveals characteristic seizure onsets, spread patterns and propagation velocities in hippocampalentorhinal cortex slices of juvenile rats. Neurobiol Dis 7: 286–298. Yaari, Y., Konnerth, A., and Heinemann, U. 1983. Spontaneous epileptiform activity of CA1 hippocampal neurons in low extracellular calcium solutions. Exp Brain Res 51: 153–156. Zhang, C.L., Dreier, J.P., and Heinemann, U. 1995. Paroxysmal epileptiform discharges in temporal lobe slices after prolonged exposure to low magnesium are resistant to clinically used anticonvulsants. Epilepsy Res 20: 105–111.
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5 The Use of Brain Slice Cultures for the Study of Epilepsy SCOTT M. THOMPSON, XIANG CAI, CÉLINE DINOCOURT, AND MICHAEL W. NESTOR
ability to induce conventional long-term potentiation (e.g., Debanne et al., 1994; Stoppini et al., 1991).
Organotypic brain slices are becoming increasingly useful and advantageous for a variety of neurobiological questions, including the study of epilepsy. In this chapter, we provide a brief review of some of the defining characteristics of this technique. We also review some recent studies in which these cultures have been used to examine various issues pertaining to the causes and consequences of epilepsy to illustrate the kinds of experimental questions that are amenable to study with this methodology. Thin sections of brain tissue can be prepared from neonatal animals and cultured as intact slices of tissue rather than as dissociated cells. These brain slice cultures offer most of the same advantages as conventional cell cultures, including (1) long-term survival in vitro; (2) precise control of experimental conditions; (3) excellent accessibility for viral vectors, biolistic transfection, and other means of gene transfection; (4) survival of tissue from neonatal–lethal transgenic animals; and (5) excellent visibility of cells and subcellular structures for morphologic and electrophysiologic studies. In addition, slice cultures offer several advantages compared with conventional dissociated cell cultures. Most notably, they retain many features of their native organotypic organization, as described extensively elsewhere (e.g., Caeser et al., 1989; Gähwiler, 1984; Gähwiler et al., 1997; Zimmer and Gähwiler, 1984). This permits the identification of defined cell groups, stimulation or lesioning of specific axonal pathways, and study of synaptic connections that more closely approximate their normal in situ patterns (Debanne et al., 1995). Finally, the slice culture technique offers the investigator the opportunity to coculture slices from different brain regions, thus facilitating the experimental manipulation and study of long-distance connections in vitro (e.g., Gähwiler et al., 1987) as well as the
Models of Seizures and Epilepsy
ORGANOTYPIC BRAIN SLICE CULTURES Preparation Two methods are currently used to prepare slice cultures. In the roller-tube technique, pioneered by Gähwiler (1981), the slices are attached to glass coverslips and placed in sealed test tubes on a roller drum in a dry-air incubator. In the membrane or interface technique, pioneered by Stoppini et al. (1991), the slices are placed on semipermeable membranes and grown statically in CO2 incubators. The primary differences between the two techniques are that the rollertube cultures generally become thinner than the membrane cultures but are somewhat more demanding and time consuming to prepare. Detailed culturing procedures can be found elsewhere (e.g., Gähwiler et al., 1998; Thompson and Mason, 2004). In both methods (Figure 1), brain slices are cut from neonatal animals. The exact age depends on the region of the brain to be cultured. The hippocampus and cortex are typically taken from 5- to 7-day-old pups, whereas the thalamus is reported to survive in culture only when taken from embryonic animals (Molnar and Blakemore, 1991). Slices are cut at a thickness of about 350 mm, as one would for an ordinary acute brain slice experiment. Because of the need to maintain sterility, it is typically easier to use a tissue chopper for the preparation of the slices; however, a vibratome preserves tissue health and integrity better for larger brain sections.
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Chapter 5/Use of Brain Slice Cultures for Study of Epilepsy
rostral tiss ue c
septum
hop per
Multiwell dish and membrane inserts
hippocampus
B
hippocampal fissure
caudal dorsal surface down
A Incubator
coverslip and fibrin clot
culture medium
semipermeable membrane
dry air
Incubator CO2 culture medium
roller drum
e e tub r u t l cu
FIGURE 1 Preparation of organotypic brain slice cultures. The tissue of interest is first dissected free from the neonatal brain, as illustrated for the hippocampus, and thin sections are cut under aseptic conditions using either a tissue chopper or a vibratome. A: In the roller-tube method, the slices are attached to cleaned glass coverslips in a film of clotted fibrin or chicken plasma; the slices are then placed in sealed test tubes and placed on a roller drum in a dry-air incubator. B: In the membrane method, the slices are placed on semipermeable membranes in multiwell plates so that they are at the gas–air interface and then maintained in a CO2 incubator.
In the roller-tube method, brain slices can be attached to glass coverslips using various substances, including chicken plasma clotted with thrombin, collagen, or synthetic fibrin solutions (e.g., Tisseal, Baxter International, Deerfield, IL), either alone or in combination. These substances serve as effective, tissue-friendly “glues” for holding the tissue down firmly in the roller drum. Coverslips containing the slices are placed in sealed test tubes containing culture medium, and the tubes are placed on a roller drum in an incubator at 36°C. The drum rotates at about 10 revolutions per hour. For the membrane cultures, the slices are placed directly on semipermeable membrane inserts (Millicell, Millipore, Billerica, MA) and need no adhesive substance because they remain static in the CO2 incubator. The inserts are placed in culture wells filled with medium so that the slices are at the interface between the medium and the air; this interface allows oxygen to diffuse into the slice at a sufficiently high concentration to maintain tissue viability. Similarly, in the
roller-drum method, the slices are only submerged in medium for part of each cycle so that adequate oxygenation is maintained. In both methods, the slices are typically grown in a semisynthetic medium containing bovine or horse serum. Serum-free medium can also be used (Annis et al., 1990), most effectively after the cultures have become established.
Properties The defining feature of slice cultures is that they retain the characteristic anatomic organization of the tissue of origin. Because slice cultures are derived from neonatal brain slices, the tissue has not yet achieved its final degree of maturation and development at the time it is explanted. In neonatal brain tissue, neurons are still elaborating their dendritic trees, axons are growing, synaptogenesis is under way, cells may be migrating, and (in some brain regions) cells may still be undergoing mitosis. The degree to which
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Organotypic Brain Slice Cultures
the culture retains its organotypic organization is therefore determined primarily by the age and maturity of the tissue at the time of explantation; older slices generally attain a more “organotypic” state. In the hippocampus, for example, many granule cells are born postnatally, and the granule cell layer is thus sometimes less well-formed than the pyramidal cell layer in slice cultures. Nevertheless, the relative proportions, transmitter phenotypes, and dendritic and axonal morphologies of most neurons in slice cultures are well preserved, even though many of these properties develop after explantation. Because of the inevitable severing of axons during the preparation of acutely prepared ex vivo brain slices, many projection pathways may be more intact in cultured slices than in ex vivo slices. It is nevertheless important to consider which extrinsic inputs are likely to be lost as the result of the tissue isolation. For example, loss of perforant path inputs to the dentate gyrus from the entorhinal cortex is likely to lead to some reorganization of mossy fibers (Laurberg and Zimmer, 1981). Likewise, there are likely to be reorganizational consequences of the loss of contralateral inputs in neocortical cultures, but this feature of cortical slice cultures has not been examined rigorously. Even removal of all extrinsic cholinergic afferents to the hippocampus does not prevent pyramidal cells in slice cultures from responding appropriately to cholinergic agonists (Gähwiler and Brown, 1985). It must be noted, however, that cultures from relatively unstudied brain regions will need to be characterized in detail to establish the degree to which they remain organotypic. One particular advantage of using cultured brain slices is that they can be maintained in vitro for prolonged periods. With hippocampal roller-tube cultures, for example, survival in vitro for 4 weeks is routine, and cultures can often survive for 6 weeks or longer. This property is particularly useful for studying the effects produced by chronic drug or toxin treatment; modifications of activity levels; or lesions, where the changes might be expected to occur slowly as gene expression is modified and proteins turn over. We have examined several aspects of synaptic structure, such as dendritic spine density, the distribution of spine morphologies, and axonal growth associated protein expression, to assess the maturity of the cultures (e.g., McKinney et al., 1999b). Our results with roller-tube hippocampal slice cultures indicate that synaptogenesis goes on during the first 2 weeks in vitro but remains stable in healthy cultures thereafter. Another defining feature of the slice culture technique is that the tissue becomes flattened during its development in vitro. This flattening, together with disappearance of the tissue that is inevitably damaged during the slicing procedure, endows cultured tissue slices with far superior optical properties than acutely prepared brain slices. The final thickness of the culture depends on the thickness of the original slice, the brain region cultured, the age of the neonatal animals used, and the type of culturing method. Hippocam-
pal brain slices prepared with the roller-tube method attain a final thickness of about 50 mm at the cell body layer (i.e., roughly three to five cells thick) and are somewhat thinner in the dendritic layers. Cerebellar cultures prepared from 1day-old rat pups, in contrast, become a true monolayer of cells (Gähwiler, 1981). In practice, this thinness allows investigators to attain high spatial resolution using standard wide-field fluorescence microscopy for many experimental applications without resorting to confocal or multiphoton microscopy. For example, we routinely perform fluorescence-based analysis of dendritic spine morphology and intracellular Ca2+ levels in single dye-loaded pyramidal cells in hippocampal slice cultures or carry out live-cell imaging of axonal growth and growth cones using wide-field fluorescence excitation and charge-coupled device (CCD) video imaging (Figure 2). Electrophysiologic recordings made over the last two decades from cultured brain slices have established that many types of neurons display the same repertoire of intrinsic ionic currents as neurons in acutely isolated ex vivo brain slices and that they express receptors for many of the same neurotransmitters. There are exceptions (Perrier et al., 2000), however, and investigators should be aware of the possibility that channel expression has changed as a result of changes in the level of ongoing synaptic activity. The synaptic physiology of cultured hippocampal slices has also been shown to closely resemble that described in ex vivo slices, indicating that much of the neuronal circuitry develops in vitro as it would in situ. Connections between CA1 pyramidal cells, for example, are rare in both ex vivo and cultured slices (Debanne et al., 1995; Thomson and Radpour, 1991). Furthermore, unitary excitatory postsynaptic responses, obtained via recordings from monosynaptically coupled pairs of CA3 pyramidal cells in hippocampal slice cultures, have amplitudes of about 1 mV or 15 pA (Debanne et al., 1995), values that are essentially identical to unitary excitatory postsynaptic potential/current (EPSP/ EPSC) amplitudes in ex vivo hippocampal slices (Miles and Wong, 1986) and quite unlike the amplitudes (of several nanoamperes) reported for dissociated cell cultures (Rothman and Samaie, 1985). In our experience, overall network excitability is also normal in healthy cultures. In designing electrophysiologic experiments in slice cultures, however, it is important to keep in mind that the circuitry is “more intact” than in ex vivo slices. The extracellular saline should therefore contain a more physiological K+ concentration (2–3 mM) and Ca2+ : Mg2+ ratio (1 : 1) than the salines typically used in ex vivo slice experiments (5 mM and 2 : 1, respectively).
Cocultures The physiology of long-range neural pathways in the brain is not amenable to study under well-controlled in vitro
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Chapter 5/Use of Brain Slice Cultures for Study of Epilepsy
FIGURE 2 Fluorescence imaging in hippocampal slice cultures. A: Low-power charge-coupled device (CCD) video image of two CA1 cells biolistically transfected with DNA for green fluorescent protein in a hippocampal slice culture prepared using the roller tube method. Higher-power CCD images of dendritic spines along apical dendritic segments are shown below. Scale bars: 50 mm upper image, 2 mm lower images. B: CCD images of an axonal growth cone of a sprouting CA3 cell axon 5 days after Schaffer collateral transection. The pair of images was captured 15 minutes apart and illustrate the dynamic processes occurring at the growth cone during axonal growth. Scale bars: 1 mm. C: CCD images of an apical terminal dendrite injected intracellularly with a red fluorescent dye (Alexa 568)(left) and an image of the fluorescence emission of the Ca2+-sensitive dye fluo-4 in the same dendrite as the result of a glutamate-induced dendritic plateau potential (see Cai et al., 2004). Scale bars: 2 mm. These results illustrate the excellent optical properties of hippocampal slice cultures prepared with the roller-tube method.
conditions because it is difficult or impossible to maintain these contacts during the preparation of ex vivo slices. In slice cultures, however, slices from two brain regions that are normally connected in vivo can be cocultured adjacent to one another, and they will form connections in vitro. This approach has been taken for septo-hippocampal (Gähwiler and Brown, 1985), thalamocortical (Molnar and Blakemore, 1991), corticostriatal (Ostergaard et al., 1991), entorhinal cortex-hippocampal (Li et al., 1994), and locus coeruleus–hippocampal connections (Knöpfel et al., 1989). Some specificity is also maintained in the growth of these
fiber pathways, as illustrated by the observation that cholinergic fibers from medial septal cells will not grow into cerebellar tissue slices, just as they do not normally innervate the cerebellum in vivo unless they are “tricked” by the addition of nerve growth factor (Gähwiler and Hefti, 1987; Gähwiler et al., 1991).
Transgenics and Gene Transfection Many transgenic animals, as a consequence of their expression of a transgene or lack of an essential gene, fail
What Aspects of Epilepsy Can be Modeled with Cultured Brain Slices?
to survive more than a few hours or days after birth due to developmental defects (often not involving the nervous system) or to behavioral defects (such as failure to suckle). This problem obviously renders it difficult or impossible to characterize the transgene-mediated central nervous system (CNS) phenotype. Obtaining brain slices from these animals at a very young age and growing them as slice cultures provide a convenient means for circumventing this problem. We have used this approach with mice lacking the growth associated protein of 43 kD (GAP-43), for example (Capogna et al., 1999), which die within 24 to 48 hours of birth as a result of axonal growth defects (Strittmater et al., 1995). Organotypic brain slices also lend themselves well to acute transfection with recombinant DNA using a variety of methods, including viral vectors, biolistic transfection, and electroporation. Viral vectors used successfully to transfect cells in slice cultures include Semiliki Forest virus, Sindbis virus (Ehrengruber et al., 1999; Jeromin et al., 2003), adenovirus (Keir et al., 1999; Ridoux et al., 1995), herpes simplex virus (Bergold et al., 1993), vaccinia virus (Pettit et al., 1995), and lentivirus (Ehrengruber et al., 2001). A particular advantage of this approach is the possibility of injecting virus into subpopulations of neurons or into one part of a coculture so that the virally delivered gene product affects only presynaptic axonal or postsynaptic dendritic compartments (e.g., Wilkemeyer and Angelides, 1995). Biolistic transfection, in which gold microparticles coated with plasmid DNA are propelled into the tissue and cells by a brief blast of N2 gas from a “gene gun” (McAllister, 2000), is a particularly simple and efficacious method of transfection. Even though the transfection efficiency is exceedingly low (<10 neurons per culture), the level of expression is high, making it suitable for morphologic and electrophysiologic studies (e.g., Cai et al., 2004; Kolleker et al., 2003). Finally, electroporation, in which a strong pulsed electrical field is used to increase membrane permeability and to allow extracellular plasmid DNA to enter cells, has been reported to produce high levels of transfection. Two methods have been described: Cells in freshly cut slices can be electroporated by bathing them in a cDNA containing saline and then applying the field across the slice (Murphy and Messer, 2001; Stühmer et al., 2002). Alternatively electroporation can be performed in neonatal animals in vivo, followed by the explantation of the slices hours or days later (Miyasaka et al., 1999). For all methods, cell type–specific promoters can lead to expression in defined cell types, even when the cDNA cannot be targeted to those cells only (Stühmer et al., 2002). Finally, slices derived from knockout animals and acute transfection can be combined to allow powerful rescue and acute “knock-in” approaches, which are considerably simpler and quicker than the generation of transgenic animals (e.g., Kolleker et al., 2003).
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Plasticity All types of cultured neurons seem to be useful for studying long-term depression (LTD) of excitatory synaptic responses. For reasons that remain unknown, however, conventional dissociated cell cultures are almost universally found not to support the induction of conventional long-term potentiation (LTP) (but see Fitzsimonds et al., 1997). In contrast, LTP is reliably induced in slice cultures in response to either tetanization of afferent inputs or pairing of lowfrequency stimulation and directly induced postsynaptic depolarization (Debanne et al., 1994; Stoppini et al., 1991). Further, the high probability of obtaining pairs of monosynaptically coupled cells in these cultures has allowed the plasticity of unitary synaptic responses to be studied in great detail (Debanne et al., 1996; Montgomery et al., 2001). Likewise, the excellent optical properties of slice cultures has facilitated the study of the morphologic correlates of synaptic plasticity (Engert and Bonhoeffer, 1999; Toni et al., 1999). Finally, the ease of transfection and the use of slices from transgenic animals have made slice cultures an accessible preparation in which to study the molecular basis of long-term potentiation expression in great detail (Kolleker et al., 2003).
WHAT ASPECTS OF EPILEPSY CAN BE MODELED WITH CULTURED BRAIN SLICES? The following are the key questions for any model system: What patterns of epileptiform activity can it generate? What epilepsy syndromes can be mimicked in the model? As with any preparation made from normal animals, no epileptiform activity is apparent in healthy cultures under control conditions. Applications of convulsants, including bicuculline, picrotoxin, tetraethylammonium (TEA), or low Mg2+- or high K+-containing saline, can trigger both interictaland ictal-like burst discharges that are synchronized throughout large cell populations in hippocampal slice cultures (e.g., Scanziani et al., 1994). These bursts appear essentially identical to those evoked in vivo or in acutely prepared hippocampal slices. Activity-dependent depression of g-aminobutyric acid (GABA)ergic synaptic inhibition, triggered by tetanic stimulation, also results in self-sustaining epileptiform discharge (Thompson and Gähwiler, 1989). It thus appears that sufficient circuitry is present within these cultures to support synchronized network activity and that the cellular network develops in such a way that synaptic inhibition and excitation are balanced appropriately. In the following sections, we describe experiments in which application of convulsants to slice cultures has been used to study the cellular basis of the neuropathology
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associated with human epilepsy, including cell death and loss of dendritic spines as well as other consequences of chronic epileptiform activity. We also describe studies in which slice cultures have been used to study the consequences of brain injury, including changes in excitability resulting from axonal injury and from denervation. Slice cultures are a valuable preparation for such studies because of the excellent experimental control afforded by the in vitro conditions. For example, inadequate blood supply during intense epileptiform discharge may result in local hypoxia, rendering it difficult to dissect the relative contribution of excitotoxicity to the neuropathology of epilepsy from that of metabolic insufficiency. Similarly, brain injuries in vivo include not only neuronal damage but also blood extravasation and clotting, inflammation and infiltration of cells from the immune system, and interruption of blood vessels. In vitro experimentation provides the means to distinguish among these possibilities. Of course it is essential to keep the potential limitations of such reduced preparations clearly in mind. The complicating factors mentioned above do, in fact, occur in the clinical setting and are undoubtedly important. Furthermore, cultured cells and tissue slices have been isolated from some of their normal inputs and have developed under in vitro conditions that are less than physiologic, but to an unknown degree. Investigators must remain aware that cell and tissue cultures may therefore have experienced some unknown plastic changes that alter the starting conditions and confound comparisons with other preparations. It is thus important that results from all in vitro preparations, including slice cultures, be replicated and compared with results from other “more intact” preparations and models.
Epilepsy-Associated Neuropathology Cell Death Ammon’s horn (or hippocampal) sclerosis, consisting of neuronal loss, gliosis, and atrophy of neuronal dendrites, is a common feature of the human epilepsies involving the temporal lobe (Meldrum and Corsellis, 1984; Scheibel et al., 1974). It is widely assumed that the death and injury of neurons is a direct consequence of the excessive glutamatergic excitation that characterizes epileptiform discharge. The mechanisms of excitotoxic neuronal death have been extensively studied in hippocampal slice cultures. Glutamate and virtually all of its receptor subtype-specific analogues can cause cell death in all principal cell groups in hippocampal slice cultures (Vornov et al., 1991). Nevertheless, because of their Ca2+ permeability, N-methyl-d-aspartate (NMDA) receptor-gated channels are prime suspects in excitotoxicity. Indeed there is evidence that NMDA receptor antagonists offer considerable neuroprotection against the consequences of ischemic/anoxic insults in slice cultures (Pringle et al., 1997; Tasker et al., 1992; Vornov et al., 1994).
NMDA receptors are also strongly activated during epileptiform activity, in both ex vivo and cultured slices (Scanziani et al., 1994). Not surprisingly, chronic epileptiform activity induced with a variety of convulsants leads to considerable cell death in hippocampal slice cultures (Kovacs et al., 1999; Thompson et al., 1996). The neuropathological changes associated with 3 days of convulsant treatment were eliminated by simultaneous application of NMDA and non-NMDA receptor antagonists, whereas neither MK-801 nor 6-Cyano-7-nitroquihoxaline-2,3-dione (CNQX) alone reduced convulsant-induced neurodegeneration significantly (Thompson et al., 1996). We concluded that only a small part of the convulsant-induced neuropathology in hippocampal slice cultures occurs as a result of the NMDA receptor–mediated excitotoxicity that has been described in response to exogenous glutamate. Presumably other mechanisms contribute to cell death associated with ongoing epileptiform discharge in slice cultures, such as free radical generation (Kovacs et al., 2002), excessive release of Zn2+ from the mossy fiber axons of dentate granule cells, Ca2+ influx via voltage-dependent Ca2+ channels, deficits in glutamate uptake (Simantov et al., 1999), metabolic insufficiency (Kovacs et al., 2001), or non-NMDA receptor– mediated excitotoxicity. Loss of Dendritic Spines The highly branched dendrites of hippocampal pyramidal cells are covered with dendritic spines, the small protrusions that form the postsynaptic side of excitatory synapses. Healthy pyramidal cells in situ, like those in slice cultures, have about one spine per micrometer of dendrite, for a total of 5000 to 10,000 spines per neuron. In addition to hippocampal sclerosis, dendritic deformities are also prominent in human hippocampal tissue taken at autopsy (Scheibel et al., 1974). These deformities include swollen and beaded dendrites and complete or partial loss of dendritic spines. Experiments in which hippocampal slice cultures were treated with convulsants have established that these changes are a direct result of epileptiform activity (Müller et al., 1993). After allowing cultures to mature for 14 days in vitro, 3 days of treatment with the convulsants bicuculline or picrotoxin caused pathological changes in dendritic morphology in cells that were not grossly swollen or vacuolated (i.e., they did not appear to be dying). Many cells had dendrites with numerous nodules that appeared to be swollen dendritic spines, resembling the “string-ofbeads” deformity described in pyramidal cells from sclerotic human hippocampi. In addition, the dendrites of most labeled cells in treated cultures displayed a significant reduction in the density of their dendritic spines. The reduction in spine density could vary between cells, even in the same culture, but was generally correlated with the number of degenerating cells. Similar results have been obtained in models of chronic focal epilepsy in situ (e.g., Jiang et al.,
What Aspects of Epilepsy Can Be Modeled with Cultured Brain Slices?
1998). Quantitative light and electron microscopic analysis (Drakew et al., 1996) indicated that the surviving spines undergo a significant decrease in their area. It was also observed that when cultures are returned to control saline for one week (after 3 days of treatment with convulsants), there is a significant recovery of dendritic spine density (Müller et al., 1993), providing a remarkable example of their capacity for structural plasticity. Many changes in the morphology of dendritic spines have been ascribed to Ca2+dependent changes in intracellular enzymatic activity, but NMDA receptor antagonists offered only partial protection of dendritic spines in convulsant-treated cultures (Thompson et al., 1996).
Consequences of Chronic Epileptiform Activity The severity of hippocampal sclerosis in human autopsy specimens is positively correlated with the number and severity of the patient’s seizures. This relationship has engendered considerable discussion about whether hippocampal sclerosis is a cause or consequence of epilepsy. We have used cultured hippocampal slices to test the hypothesis that seizure-induced neuronal injury is itself epileptogenic. We established that chronic epileptiform activity, produced by applying the convulsants picrotoxin or bicuculline for 3 days, mimics many of the pathological changes associated with epilepsy in humans, including neuronal degeneration (Müller et al., 1993). This neuropathology did not result in an increase in neuronal excitability, as might be expected if such pathological changes were causing epilepsy but was rather accompanied by a reduction in excitability and a reduction in evoked EPSP amplitudes. We concluded that hippocampal sclerosis does not necessarily cause epilepsy. Several hypotheses can be formulated to account for the decrease in EPSP amplitude in convulsant-treated cultures, including a decreased number of excitatory synapses, a decreased release of glutamate from each synapse, or a decreased number of glutamate receptors at each synapse. The loss of dendritic spines discussed previously suggests that there may be fewer excitatory synapses present in the treated cultures, although synaptic contacts between axon terminals and dendritic shafts may persist in these cultures after retraction of the spines. Evidence was obtained using in situ hybridization to support the hypothesis of a downregulation of postsynaptic glutamate receptors (GerfinMoser et al., 1995). Cultures were examined after only 2 days of convulsant treatment to avoid the neuronal degeneration observed after 3 days of epileptic activity. Significant reductions in mRNA levels for two non-NMDA, (alpha)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-preferring glutamate receptor subunits (GluR-1 and GluR-2) were observed. These changes were found to
51
be specific, not the result of a general decrease in gene expression; the mRNA levels for other receptor subunits, such as several subtypes of GABAA or NMDA receptors, were unchanged (Gerfin-Moser et al., 1995). This observation also rules out a general decrease in gene expression attributable to cell death. Despite the lack of hyperexcitability immediately after convulsant treatment, it is possible that chronic epileptiform activity might, with longer survival times, become epileptogenic. One hypothesis suggests that axonal sprouting and a reorganization of excitatory synaptic circuitry occurs with some delay after a loss of neurons. A second hypothesis is that periods of epileptiform activity lead to a potentiation of excitatory synaptic transmission. There is excellent evidence that seizures can lead to axonal sprouting in vivo and in-slice cultures. Mossy fiber sprouting has been observed by Bausch and colleagues in hippocampal slice cultures treated with kaininc acid for 48 hours (Bausch and McNamara, 2004; Routbort et al., 1999). As in human temporal lobe epilepsy (Houser, 1999; Sutula et al., 1988), large numbers of mossy fibers were detected in the supragranular dendritic regions of the dentate gyrus, where there are normally very few mossy fibers. As in ex vivo slices taken from animals with supragranular mossy fibers, dentate granule cells in cultures with supragranular mossy fibers were hyperexcitable, responding to afferent and antidromic activation with prolonged excitatory postsynaptic currents and epileptiform bursts (Bausch and McNamara, 2004). At least some of this enhanced excitability was due to growth of CA1 pyramidal cell axons into the dentate gyrus (a lesion between the two regions reduced the abnormal activity). Whether axonal sprouting by CA1 cells into the dentate gyrus also occurs in vivo is not yet known. What triggers the mossy fibers to sprout in this model? Because deafferentation of granule cells or loss of hilar mossy cells are also associated with mossy fiber sprouting, it remains unclear whether this sprouting is compensatory and due to the induction of cell death (e.g., Benedikz et al., 1993) or rather whether sprouting is induced specifically by the increased activity. For example, the sprouting of mossy fibers and CA1 cell axons may be driven by the well-documented up-regulation of neurotrophins and neurotrophin receptors that is seen after seizures in vivo and in slice cultures (Poulsen et al., 2002, 2004). Although brain-derived neurotrophic factor (BDNF) is not essential for mossy fibers to sprout in slice cultures (Bender et al., 1998), it appears likely that some tyrosine receptor kinase B (trkB) neurotrophin receptor ligand may play a critical role. First, there is biochemical and immunocytochemical evidence of trkB activation at the time when the mossy fibers are sprouting (Binder et al., 1999a, b). Second, seizure-induced mossy fiber sprouting in hippocampal slice cultures is reduced or eliminated by functionally blocking anti-BDNF antibodies or pharmacological antagonists of trkB tyrosine kinase activity (Koyama et al., 2004).
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In addition to a delayed sprouting response, there is excellent evidence of a potentiation of excitatory synaptic transmission after a period epileptiform activity (Abegg et al., 2004), potentiation that could contribute to persistent hyperexcitability (Figure 3). After a 12-hour exposure of hippocampal slice cultures to convulsants, the frequency of spontaneous miniature EPSCs was increased, and there was an increase in the ratio of the AMPA- to NMDA-receptor mediated components of the evoked EPSC. This latter effect could be attributed to the insertion of postsynaptic AMPA receptors into previously “silent” synapses, as demonstrated directly using viral transfection with fluorescently tagged AMPA receptors. Such compelling data offer a biological basis at last for Gowers’ famous dictum: seizures beget seizures.
Miniature excitatory postsynaptic currents control
20 pA 500 ms
after epileptiform discharge
Evoked excitatory postsynaptic currents control
after epileptiform discharge NMDA
AMPA
FIGURE 3 Potentiation of excitatory synaptic transmission after chronic epileptiform discharge. Epileptiform activity was induced by convulsant application in hippocampal slice cultures maintained in vitro for more than 14 days. After 12 hours, electrophysiologic recordings were made from CA1 pyramidal cells. Cultures experiencing chronic epileptiform discharge displayed higher amplitudes and frequencies of miniature excitatory postsynaptic currents than untreated sister cultures (upper panels). In addition, the ratio of the AMPA- and N-methyl-d-aspartate (NMDA)-receptor mediated components of evoked excitatory postsynaptic currents, as determined at negative and positive holding potentials, respectively, was considerably higher after epileptiform activity. This can be seen most clearly by scaling the currents so that the NMDA components were of equal amplitude (lower panels), thus controlling for differences in stimulation intensity in the two sets of cultures. These data are consistent with an epilepsy-induced potentiation of excitatory synaptic transmission mediated via the insertion of postsynaptic AMPA receptors. Data taken and adapted from Abegg et al. (2004).
Consequences of Brain Injury Head trauma and brain injury are significant factors in the etiology of some epilepsies, but the underlying mechanisms have received little attention. The percentage of all victims of a serious head injury that go on to develop posttraumatic epilepsy has been estimated at 10 to 34%, depending on the severity of the injury. One of the strongest predictors of the likelihood of posttraumatic epilepsy is a penetration of the dura (Willmore, 1990). A striking characteristic of posttraumatic epilepsy is the variable delay between the trauma itself and the development of seizures, which can last from weeks to years. Following head trauma, some relatively slow processes must be triggered, ultimately building to a permanently epileptic state. We have used organotypic hippocampal slice cultures to develop an in vitro model of posttraumatic epilepsy that allows the mechanisms underlying the development of hyperexcitability to be investigated under carefully controlled in vitro conditions (McKinney et al., 1997). Lesions of the Schaffer collateral axons of CA3 pyramidal cells in hippocampal slice cultures were made using a razor blade shard after the cultures had been allowed to develop in vitro for more than 14 days. Although the hippocampus is almost never involved in human posttraumatic epilepsy, its beautiful organization allows the presynaptic and postsynaptic consequences of injury to be studied in isolation: CA3 cells undergo a selective axonal injury, whereas CA1 cells undergo a selective partial denervation. Presynaptic Changes after Injury An important clue about the nature of the lasting changes underlying the genesis of posttraumatic epilepsy was the discovery that considerable axonal reorganization can occur in the adult brain in response to neuronal injury. For CNS neurons, this process has been studied almost exclusively for the mossy fiber axons of dentate granule cells because of the ease and specificity with which they are labeled using the Timm’s stain (Danscher and Zimmer, 1978). The mossy fiber axons of dentate granule cells have been shown to develop supragranular collaterals in the dendritic region of the dentate gyrus in various rodent models of epilepsy as well as in the hippocampi removed from patients undergoing anterior lobectomy for drug refractory, partial complex epilepsy (Houser, 1999; Sutula et al., 1988, 1989). Physiologic studies indicate that the net effect of this sprouting is to increase the excitability of the tissue (e.g., Buckmaster and Dudek, 1999). Our study of injury after Schaffer collateral transection in slice cultures indicates that pyramidal cells may also regenerate after axonal injury (McKinney et al., 1997). We first filled living CA3 pyramidal cells with biocytin to visualize the morphology of individual cells. Axons extending away from the lesion toward the dentate gyrus were largely
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What Aspects of Epilepsy Can Be Modeled with Cultured Brain Slices?
straight and unbranched, as were axons in unlesioned cultures. Collaterals of the axons growing toward the lesion, in contrast, were highly branched, had clear growth cones, and extended in circuitous, meandering trajectories. In addition, the number of presynaptic boutons per unit length along the meandering axons near the lesion was increased twofold compared with axons extending away from the lesion or axons in control cultures. We demonstrated that these collaterals were not merely preexisting axons that had become rearranged because of the proximity to the lesion but were in fact sprouted de novo by using an antibody directed against the growth-associated protein GAP-43. This protein is highly expressed early in development in vivo, when axons elongate, but is down-regulated to very low levels after synaptogenesis is complete (Skene, 1989). Consistent with these observations, GAP-43 immunoreactive fibers are numerous in hippocampal slice cultures after 3 days in vitro but are never seen by 14 days in vitro. In cultures in which the Schaffer collaterals were transected after 14 days in vitro, in contrast, there were numerous immunoreactive fibers with readily identifiable growth cones, beginning 3 days post lesion. By 14 days post lesion, large numbers of immunoreactive fibers crossed the lesion cavity itself and entered area CA1 (Figure 4). We showed that these regenerated axons form functional synapses with CA1 pyramidal cells and restore synaptic transmission across the lesion (McKinney et al., 1999a). By 21 days post lesion, there were no more GAP-43 immunoreactive fibers, suggesting that sprouting had ceased. However, transmission across the lesion persisted, indicating that the newly generated axons remained and were functional. These data thus provided direct evidence that hippocampal pyramidal cells can sprout new axon collaterals after injury (see also Perez et al., 1996). The slow time course of this growth is interesting in the context of delayed development of posttraumatic epilepsy. The functional consequences of this injury-induced axonal sprouting were examined electrophysiologically (McKinney et al., 1997). High frequencies of spontaneous EPSPs were observed in most intracellular recordings made from CA3 pyramidal cells 14 days post lesion. In addition, local stimulation within area CA3 elicited unusual polysynaptic EPSPs that were often suprathreshold for action potential initiation. Similar pathological synaptic responses have been observed in ex vivo slices taken from the vicinity of neocortical lesions (Prince and Tseng, 1993). There was a strong positive correlation between the levels of GAP-43 immunoreactivity and the frequency of spontaneous EPSPs in individual lesioned cultures. Lesioned and unlesioned cultures were indistinguishable in the amount of GABA immunoreactivity and in the amplitude of pharmacologically isolated GABAergic inhibitory postsynaptic potentials (IPSPs). Finally, most lesioned cultures responded to a challenge with a subconvulsive concentration of the GABAA
A
Five days after transection CA3 1
lesion
2
CA1
1
2 CA3
B Unlesioned CA3 CA1
FIGURE 4 Axonal sprouting after Schaffer collateral transection. Images of hippocampal slice cultures stained with an antibody against the growth associated protein, GAP-43, a marker for growing axons. A: Low-power charge-coupled device (CCD) image of the lesion site in a culture in which the Schaffer collateral pathway was transected 5 days earlier. Two higherpower confocal images from the labeled regions are shown below. Note the large number of immunoreactive fibers leaving the CA3 side of the lesion and growing toward area CA1. Numerous growth cones are visible at the tips of the growing axons. B: Confocal image of area CA3 in an unlesioned culture maintained in vitro for 14 days. Scale bars = 100 mm in upper panel of A, 25 mm in A1, A2, and B. These results illustrate the lack of growing axons in cultures at the time the lesion is made, and the strong upregulation of GAP-43 in newly sprouted axons originating in area CA3 and growing toward area CA1 after axonal injury.
receptor antagonist, bicuculline, by producing pronounced epileptiform discharges, whereas unlesioned cultures never did. Dual intracellular recordings made 14 to 21 days after the lesion revealed that the probability that any two CA3 pyramidal cells were connected by a monosynaptic excitatory synapse was increased by 50% (compared with unlesioned sister cultures). In addition, complex polysynaptic unitary EPSPs were readily elicited in lesioned cultures in response to pairs of presynaptic action potentials. This hyperexcitability outlasted the presence of immunoreactive sprouting axons, suggesting that the newly generated axons remain
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after GAP-43 expression has returned to undetectable levels. We concluded that the connectivity of the CA3 pyramidal cell population had increased after the axonal injury as a result of the sprouting of new axonal collaterals. Excitation was able to spread more readily among the population of CA3 pyramidal cells along these numerous, newly sprouted connections. Is this axonal sprouting a “culture artifact,” or is it relevant to “real” brains in situ? There are two reasons to believe that hippocampal slice cultures do indeed provide a useful model of hyperexcitability after brain injury. First, the low level of GAP-43 expression at 14 days in vitro indicates that the normal developmental phase of axonal elongation and synaptogenesis is complete at the time we make the lesion; the induced axonal sprouting thus represents plasticity of a mature system. Second, there is electron microscopic evidence of a reinnervation of area CA1 following Schaffer collateral transection in vivo (Goldowitz et al., 1979). Although the source of the regenerated presynaptic terminals in this study was not identified, it is reasonable to suspect that the presynaptic terminals were formed by regenerated Schaffer collaterals of ipsilateral CA3 pyramidal cells. We are currently taking advantage of the ability to control the extracellular environment of these cultures to explore the role of injury-induced neurotrophin secretion as trigger of axonal sprouting. We have shown that exogenous neurotrophins acting at trkB neurotrophin receptors promote axonal sprouting by pyramidal cells (Schwyzer et al., 2002) and are now examining injury-induced axonal sprouting in slice cultures from transgenic mice with reduced levels of trkB and in injured wild-type cultures exposed to immunoadhesion molecules that bind to and inactivate endogenously secreted neurotrophins. Postsynaptic Changes after Injury Although much of the current focus is on changes in synaptic circuitry and function as the cause of epilepsy, changes in intrinsic postsynaptic excitability or receptor expression are attracting increased attention (Bernard et al., 2004; Chen et al., 2001; Shah et al., 2004). There are several examples of injury-induced hyperexcitability in which amplification of intrinsic excitability has been implicated. First, axotomy increases the excitability of several cell types through alterations in the number and properties of intrinsic voltage-dependent ion channels (e.g., Abdullah and Smith, 2001). Second, denervation supersensitivity to acetylcholine is observed in denervated muscle as the result of an increased expression of receptors at both extrajunctional (Axelsson and Thesleff, 1959) and junctional sites (Davis and Goodman, 1998). A similar phenomenon also occurs in neurons. Decreasing postsynaptic activity in cultured cortical neurons with tetrodotoxin (TTX) results in increased postsynaptic receptor expression (Rutherford et al., 1998).
Finally, intrinsic excitability can also be increased following periods of low levels of cell discharge. Desai and colleagues (1999a) demonstrated that inactivation of cortical cell cultures with TTX leads to a very selective downregulation of voltage-dependent Na+ and K+ currents, perhaps including Ca2+-activated K+ current. It is noteworthy that the effects of TTX on the expression of both glutamate receptors and intrinsic conductances have been attributed to decreases in the activation of postsynaptic trkB receptors by endogenously released BDNF (Desai et al., 1996b; Rutherford et al., 1998). The possibility that injuries to brain regions susceptible to epilepsy induce similar changes in postsynaptic excitability via axonal damage, partial denervation, and decreased neuronal discharge has not yet been tested. How is the sensitivity of postsynaptic cells to synaptically released glutamate affected by axonal transection? Is there “denervation suprasensitivity”? We have taken advantage of the excellent optical properties of slice cultures to use laser microphotolysis, in which a brief pulse of ultraviolet light is focused within the preparation to cause release of chemically “caged” neurotransmitters, to study postsynaptic changes in cells in injured tissue. These experiments have yielded two exciting observations. First, we found that distal CA1 cell dendrites in control hippocampal cultures display all-or-none spike-like responses, called plateau potentials (Wei et al., 2001). Distal, terminal dendritic branches of CA1 cells responded to weak photolysis of glutamate with passive subthreshold depolarizations. Stronger glutamate applications, however, triggered all-or-none Cd2+sensitive responses. Calcium imaging revealed that the subthreshold responses generated only small local Ca2+ transients, whereas all-or-none responses resulted in Ca2+ signals throughout the stimulated dendritic segment. Intracellular 1,2-bis(o-amino-5-bromophenoxy) ethane-N,N. N’, N’-tetraacetic acid (BAPTA), apamin, and high concentrations of TEA prolonged the responses twofold to fourfold, suggesting that Ca2+-activated K+ conductance, mediated by the small conductance (SK) channel subtype, is responsible for their termination (Cai et al., 2004). We concluded that terminal dendrites respond to strong stimuli with an active spike, mediated by voltage-gated Ca2+ channels, that is confined to a single dendritic compartment. Such binary behavior could enable distal dendritic segments to perform parallel nonlinear processing of synaptic inputs. Second, we discovered that 7 days after Schaffer collateral transaction, CA1 cells display pathologically prolonged plateau potentials and an abnormal ability to produce action potentials in response to dendritic glutamate photolysis or synaptic stimulation. When we compared the effectiveness of photolysis of caged glutamate in the distal dendrites of CA1 cells in control unlesioned cultures with CA1 cells that had been deafferented 7 days earlier, we found no change in the amplitude or kinetics of the responses to the smallest
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Conclusion
Control
Denervated
+ TTX 5 mV 200 ms
Glutamate photolysis
no TTX 20 mV 200 ms
FIGURE 5 Dendritic hyperexcitability in denervated CA1 cells. Membrane potential changes elicited with microphotolysis of caged glutamate to single distal apical dendrites of control cells (left column) and cells that were denervated 7 days earlier as the result of a Schaffer collateral transaction (right column). The responses in the upper row were obtained in the presence of tetrodotoxin (TTX) to block fast-action potentials, whereas the responses in the bottom row were obtained in the absence of TTX. In each panel a series of responses was elicited using a series of photolysis steps of increasing duration (see Wei et al., 2001). Note that dendritic plateau potentials are greatly prolonged after denervation and that this facilitates the triggering of fast-action potentials.
glutamate pulses, indicating that transmitter supersensitivity does not occur in this model. We did observe a striking prolongation of the responses in the deafferented cells, however. In deafferented cells, the duration of the plateau potentials was increased sevenfold compared with control cultures (Figure 5). We also observed a marked increase in the ability of dendritic excitation to trigger action potential discharge after Schaffer collateral transaction; whereas photolysis of caged glutamate on terminal dendrites never elicited action potential discharge in control cells, the prolonged plateau potentials always elicited prolonged trains of action potentials in denervated cells. We also asked whether this prolongation of plateau potentials might contribute to the hyperexcitability seen with synaptic stimulation after Schaffer collateral transection. Indeed, unusual evoked synaptic responses were recorded from CA1 cells in the vicinity of a lesion made 7 to 21 days earlier. These data suggest that increased postsynaptic excitation may contribute to lesion-induced hyperexcitability.
CONCLUSION In summary, cultured brain slices are an advantageous experimental preparation. Their use has facilitated important contributions to our understanding of the causes and consequences of epilepsy. These studies have provided support for
the hypothesis that epileptiform activity can damage brain tissue, both in terms of inducing irreversible cell death and also triggering the retraction of dendritic spines. These results are in good agreement with results from other in vitro and in vivo studies. Current advances in neuroimaging on human subjects are facilitating clinical investigations of the relationship between the onset of epilepsy and the detection of hippocampal sclerosis; such studies will provide the ultimate test of this hypothesis. Investigation of the consequences of injury in hippocampal slice cultures has led to the novel hypothesis that deafferented cells “downstream” from a traumatic CNS injury have an increased sensitivity to glutamate and exhibit increased excitability. Together with other studies that also point to the association of altered dendritic integration and epilepsy (e.g., Bernard et al., 2004), these findings suggest novel therapeutic targets for antiepileptic drugs and offer new explanations for the anticonvulsant actions of others (Poolos et al., 2002). In addition, the description of axonal sprouting by CA3 pyramidal cells in slice cultures after Schaffer collateral transection is consistent with in vitro and in vivo observations of mossy fiber sprouting in various epilepsy models and thus supports the hypothesis that reorganization of synaptic networks is an important causative factor in some acquired epilepsies. These findings indicate further that this sprouting phenomenon is not limited to dentate granule cells but may be more widespread than has previously been appreciated. Taken together, these studies further suggest that newly sprouted axons may be forming synaptic contacts with intrinsically hyperexcitable cells, a potentially explosive situation strongly favoring the occurrence of epileptiform activity. It is our hope that the descriptions provided here will stimulate other investigators to take advantage of the unique features of these organotypic slice cultures to advance our understanding of epilepsy. The bulk of the work done to date has been on hippocampal slice cultures. We hope that future studies will also focus on other brain regions, particularly the neocortex. Similarly, most studies have been performed on tissue from normal wild-type animals. The use of slice cultures from genetically modified animals with seizure phenotypes might aid in the understanding of how single gene mutations affect network properties. Finally, models of simplified injuries in cultured slices have been used for gene array screens (Long et al., 2003; Matzilevich et al., 2002; Morrison et al., 2000), and this approach is likely to become increasingly useful to help identify genes and gene pathways that contribute to the genesis of posttraumatic and other acquired epilepsies.
Acknowledgments Supported by a grant from the National Institutes of Neurological Disorders and Stroke to SMT.
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References Abdulla, F.A., and Smith, P.A. 2001. Axotomy- and autotomy-induced changes in the excitability of rat dorsal root ganglion neurons. J Neurophysiol 85: 630–643. Abegg, M.H., Savic, N., Ehrengruber, M.U., McKinney, R.A., and Gähwiler, B.H. 2004. Epileptiform activity in rat hippocampus strengthens excitatory synapses. J Physiol 554: 439–448. Annis, C.M., Edmond, J., and Robertson, R.T. 1990. A chemically-defined medium for organotypic slice cultures. J Neurosci Methods 32: 63–70. Axelsson, J., and Thesleff, S. 1959. A study of supersensitivity in denervated mammalian skeletal muscle. J Physiol 147: 178–193. Bausch, S.B., and McNamara, J.O. 2004. Contributions of mossy fiber and CA1 pyramidal cell sprouting to dentate granule cell hyperexcitability in kainic acid-treated hippocampal slice cultures. J Neurophysiol 92: 3575–3588. Bender, R., Heimrich, B., Meyer, M., and Frotscher, M. 1998. Hippocampal mossy fiber sprouting is not impaired in brain-derived neurotrophic factor-deficient mice. Exp Brain Res 120: 399–402. Benedikz, E., Casaccia-Bonnefil, P., Stelzer, A., and Bergold, P.J. 1993. Hyperexcitability and cell loss in kainate-treated hippocampal slice cultures. Neuroreport 5: 90–92. Bergold, P.J., Casaccia-Bonnefil, P., Zeng, X.L., and Federoff, H.J. 1993. Transsynaptic neuronal loss induced in hippocampal slice cultures by a herpes simplex virus vector expressing the GluR6 subunit of the kainate receptor. Proc Natl Acad Sci U S A 90: 6165–6169. Bernard, C., Anderson, A., Becker, A., Poolos, N.P., Beck, H., and Johnston, D. 2004. Acquired dendritic channelopathy in temporal lobe epilepsy. Science 305: 532–535. Binder, D.K., Routbort, M.J., and McNamara, J.O. 1999a. Immunohistochemical evidence of seizure-induced activation of trk receptors in the mossy fiber pathway of adult rat hippocampus. J Neurosci 19: 4616–4626. Binder, D.K., Routbort, M.J., Ryan, T.E., Yancopoulos, G.D., and McNamara, J.O. 1999b. Selective inhibition of kindling development by intraventricular administration of TrkB receptor body. J Neurosci 19: 1424–1436. Buckmaster, P.S., and Dudek, F.E. 1999. In vivo intracellular analysis of granule cell axon reorganization in epileptic rats. J Neurophysiol 81: 712–721. Caeser, M., Bonhoeffer, T., and Bolz, J. 1989. Cellular organization and development of slice cultures from rat visual cortex. Exp Brain Res 77: 234–244. Cai, X., Liang, C.W., Muralidharan, S., Kao, J.P., Tang, C.M., and Thompson, S.M. 2004. Unique roles of SK and Kv4.2 potassium channels in dendritic integration. Neuron 44: 351–364. Capogna, M., Fankhauser, C., Gagliardini, V., Gähwiler, B.H., and Thompson, S.M. 1999. Excitatory synaptic transmission and its modulation by PKC is unchanged in the hippocampus of GAP-43-deficient mice. Eur J Neurosci 11: 433–440. Chen, K., Aradi, I., Thon, N., Eghbal-Ahmadi, M., Baram, T.Z., and Soltesz, I. 2001. Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability. Nat Med 7: 331–337. Danscher, G., and Zimmer, J. 1978. An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues. Histochemistry 55: 27–40. Davis, G.W., and Goodman, C.S. 1998. Synapse-specific control of synaptic efficacy at the terminals of a single neuron. Nature 392: 82–86. Debanne, D., Gähwiler, B.H., and Thompson, S.M. 1994. Asynchronous pre- and postsynaptic activity induces associative long-term depression in area CA1 of the rat hippocampus in vitro. Proc Natl Acad Sci U S A 91: 1148–1152.
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6 Hippocampal Slices: Designing and Interpreting Studies in Epilepsy Research CHRISTOPHE BERNARD
possible to identify it and correlate its morphologic properties (e.g., dendritic and axonal arbors) with its electrophysiologic characteristics. The intracellular constituents of the cell can also be retrieved and its mRNA contents determined. The development of imaging techniques, in particular multiphoton microscopy, now allows investigators to assess cellular and network function using such measures as [Ca2+]i variations in specific cell compartments (dendrites, spines) or the behavior of large populations of cells (and even neuronal networks) with voltage-sensitive fluorescent dyes. The slice preparation is thus versatile and well suited for multidisciplinary research strategies.
INTRODUCTION: DESCRIPTION OF THE HIPPOCAMPAL SLICE PREPARATION General Features of Acute Slice Preparations The development of the brain slice preparation by Yamamoto and McIlwain (Yamamoto and McIlwain, 1966) allowed major breakthroughs in the understanding of basic neurobiological processes. All fields of neuroscience benefited from this development, including anatomy, physiology, pharmacology, molecular biology, biochemistry, biophysics, and computer modeling. Brain slices, usually thin sections about 400 mm thick, can be maintained in viable condition for extended periods (>12 hours) in vitro. The slice preparation allows for easy access to many features of the tissue that would be very difficult to measure otherwise (i.e., in vivo), but at the same time it preserves much of the complex organization of the tissue. Most in vitro studies involve electrophysiologic recordings that may take different forms. Extracellular recordings are used to study population properties, including synaptic responses and neuronal firing. These parameters can be measured before and after an experimental manipulation. Within the slice, the investigator can apply a variety of stimuli, such as electrical activation of axons (e.g., to trigger synaptic plasticity) or pharmacologic treatment. In addition to the direct consequences of such stimuli on the electrophysiologic responses of the tissue, a variety of other correlated parameters can be measured (e.g., changes in gene expressions, phosphorylation levels etc.). Intracellular recordings (with sharp or patch electrodes) allow a more microscopic approach to the characterization of synaptic responses, properties of ionic channels, etc. If the recorded cell is filled with a dye, it is
Models of Seizures and Epilepsy
Specific Features of Hippocampal Slices: Comparison with Intact Preparations The advantages of the slice preparation can be summarized as follows: (1) Slices are thick enough to preserve some circuitry of a given brain area. This advantage is particularly marked in hippocampal slices, where the regular orientation and approximately lamellar organization of the structure is amenable to slices within which multisynaptic connections are maintained. (2) At the same time, the slice and its in vitro character significantly simplify much of the complexity associated with experimental protocols on intact animal preparations. This feature is also striking in hippocampal slices, which offer a window to a complex cortical structure that is integrated in widespread networks with the rest of the brain and has been implicated in complex cognitive function. (3) Stimulating and recording electrodes can be visually guided to desired positions. Because of the regular hippocampal structure, major cell types (composing densely packed cell body-containing layers) are easily
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Chapter 6/Hippocampal Slices: Designing and Interpreting Studies in Epilepsy Research
identified. Different interneuron cell bodies reside in wellstudied locations relative to these primary cell bands, and dendritic trees and axon arbors can be targeted for both stimulation and recording, based on detailed knowledge of their trajectories, features that are maintained in the slice. Thus the investigator can use a simple binocular microscope to position electrodes for population or “blind” cellular recordings (or stimulation). In addition, electrodes can be very precisely manipulated using infrared/differential interference contrast (DIC) or epifluorescence video microscopy to target specific cell types. (4) Intracellular recordings are more stable in slices than in vivo because there is no mechanical movement resulting from heartbeat or respiration (a recurrent problem of in vivo recordings). (5) Other technical approaches can be easily combined with slice electrophysiology. Despite these technical advantages, several drawbacks must be taken into account, especially when extrapolating the results of in vitro studies to the “real” in vivo situation. Brain function relies on oxygen and energy delivery, which normally occurs via exchanges at the level of blood capillaries. The relationships between oxygen delivery and energy supply and demand are complex. Indeed, studies have shown that there is a direct spatiotemporal coupling between neuronal activity and cerebral blood flow in microcircuits (Chaigneau et al., 2003). These relationships are not reproduced in hippocampal slices. Similarly, the slice “ignores” the immune signaling system; this system cannot be separated from neuronal function because immune proteins play an important role in neuronal development, synaptic plasticity, and neurologic disorders (Boulanger and Shatz, 2004). Hormones also influence neuron behavior and the construction of networks (Gahr, 2004). These reciprocal “blood-immuno-endocrino-neuronal” interactions are lost in the slice preparation. Glucose is provided at a fixed level, O2 is saturated, and no hormone is present in the artificial cerebrospinal fluid (aCSF). The composition of aCSF itself is an important issue. The CSF contains many metabolites, including amino acids, sometimes in large concentrations (such as glutamate, g-aminobutyric acid [GABA], glutamine, taurine, glutathione, etc.) These metabolites are not usually included in aCSF, and the functional consequences of such omission remain to be determined. Finally, when one is investigating a given brain structure in vitro, many of its intrinsic and most of its extrinsic connections are severed. The hippocampus is a typical example. In adult rats, a 400-mm-thick transverse slice represents less than one tenth the whole hippocampal structure. Because axonal arbors of hippocampal neurons usually have a large extension along the longitudinal axis (Freund and Buzsáki, 1996), many intrinsic connections are lost. Extrinsic connections from the contralateral hippocampus and extrahippocampal structures (septum, entorhinal cortex, parahippocampus, thalamus, etc.) are also severed. Although
many of the primary glutamatergic and GABAergic synaptic pathways are preserved within a slice, inputs that provide important modulatory function (dopaminergic, serotonergic, cholinergic, etc.) are cut. Even at the single-cell level, the slicing procedure leads to significant losses. For example, the neurons that are contained within a given slice usually have a significant portion of their own axonal and dendritic arbors severed. As a result many cells die (e.g., at least 50% of somatostatinergic interneurons in the CA1 region of the hippocampus, unpublished observation). The surviving circuitry is thus highly truncated and only approximately represents the original hippocampal network. Despite these limitations, the hippocampal slice preparation remains a useful system in which to investigate numerous neurobiological issues, such as basic properties of synapses, receptors, etc. Indeed, the hippocampal slice preparation has provided the basis for much of today’s insight into the cellular and molecular biology of neurons. The investigator simply must be cautious in interpretation and extrapolation of results, particularly those dealing with network properties. For example, the hippocampus in vivo is known to spend most of its time oscillating at various frequencies (Buzsaki, 2002; Klausberger et al., 2003). Spontaneous oscillations do occur in vitro, in the in toto (i.e., the “intact” hippocampus) preparation (Khalilov et al., 1997) prepared from animals at early developmental stages. Apart from slices from the ventral hippocampus (Colgin et al., 2004; Kubota et al., 2003), they do not occur spontaneously in adult structures in vitro, and they must be induced by pharmacologic manipulations or electrical stimulation (Whittington and Traub, 2003). How these in vitro oscillations relate to the in vivo oscillatory behavior is not known. In summary, hippocampal slices are particularly suited to studying the details of microcircuitry. Caution should be used, however, when interpreting network properties.
WHAT TYPES OF EPILEPTIC ACTIVITY CAN BE MODELLED IN HIPPOCAMPAL SLICES? The organization, generation, and propagation of seizures in temporal lobe epilepsy (TLE) can be understood via the concept of the epileptic zone (Talairach et al., 1974). TLE is indeed a disease (or perhaps a set of disorders) involving neuronal networks within the temporal lobe. In TLE, seizures engage different structures, not only at their onset (ictogenesis) but also during their course of propagation. Most past studies of TLE have concentrated on the hippocampus as the key structure in seizure generation and spread, and thus studies of hippocampal slices may provide insight into seizure activity associated with TLE. It should be noted, however, that several reports have proposed that various cortical structures participate in ictogenesis (Bartolomei et al.,
Methods of Generation (Preparing Good Slices)
1999; Munari et al., 1994; Spencer and Spencer, 1994; Talairach et al., 1974; Wieser, 1983), with particular attention focused on the critical involvement of the entorhinal cortex (EC) in ictogenesis. The EC has extensive reciprocal connections with the hippocampus and neocortical and paralimbic regions (perirhinal cortex, temporal pole). Neuropathological (Du et al., 1995) and neuroradiologic (Bernasconi et al., 1999, 2000, 2001) data have revealed anomalies within the rhinal cortex of patients with mesial TLE, but the relationship between those observations and electrophysiologic data remains hypothetical. If extrahippocampal structures are involved in the type of epilepsy under consideration, the isolated hippocampal slice might not be the best preparation for elucidating more complex seizure networks associated with TLE (e.g., see chapter 4 for consideration of the combined hippocampal-entorhinal slice). Nevertheless, hippocampal slices are useful for studying the propagation of seizures. With this preparation, one can examine some of the relationships associated with TLElike discharges (e.g., the relationships between ictal and interictal activity). Most of the extrinsic—and many of the intrinsic—connections are severed in the hippocampal slice, so it is hardly surprising that ictal-like discharges do not occur spontaneously in slices obtained from human resections or animal models. The same connectivity issues may be relevant to the paucity of spontaneous interictal-like activities in these preparations, although they can occasionally be recorded in human tissue (Cohen et al., 2002). As a consequence, ictalor interictal-like discharges must, in most hippocampal slice experiments, be artificially induced. Several different protocols can be used, as described in the section Studying Epilepsy Using the Slice Preparation. One major drawback with artificially induced epileptiform activity in vitro is the absence of behavioral and clinical seizure correlates. To address this obvious problem, experimenters have generally tried to produce electrographic seizure equivalents in vitro. An in vitro equivalent of an in vivo TLE-like seizure should satisfy several conditions. Before its occurrence, interictal activity can (should?) be present; that is, large-amplitude field potential “spikes” (in the clinical electroencephalographic [EEG] sense). Individual spikes last up to 100 ms and may occur in bursts. Just before seizure onset, these spikes may increase in amplitude, become more rhythmic (~1 Hz), and display striking synchrony when recorded across different hippocampal subregions and limbic structures. There is then a flattening of the EEG (field potential) signal, and the seizure starts with a high frequency (~20 Hz) discharge. The electrographic seizure then typically consists of tonic rhythmic discharge and clonic bursting phases (rhythmic spikes that increase in amplitude and then decrease in frequency). Alternatively TLE-like seizures may start independently of interictal activity; such seizures usually have the same electrographic signature with seizures
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lasting 30 to 60 seconds. These general descriptions, based on EEG recordings from intact animals (or humans) with TLE-associated seizures, provide the general target for experiments using slices that focus on seizure activity involving hippocampus. Can we match that description in hippocampal slices?
METHODS OF GENERATION (PREPARING GOOD SLICES) The method and care of slice preparation will determine the tissue’s viability and in large part the validity and applicability of the experimental results obtained in the preparation. Every laboratory uses its own “magical” tricks to prepare good slices. Variations include the type of anesthetic used before decapitating the animal. Some laboratories find that slice quality is improved when animals (before decapitation) are intracardially perfused cold (~ 4° C) oxygenated aCSF in which NaCl is replaced by an equimolar concentration of sucrose or choline (Hirsch et al., 1996; Hoffman and Johnston, 1998). Other tricks include the use of glutamatergic antagonists or low Ca2+/high Mg2+ (0.5/6.0 mM) concentrations in the modified aCSF to prevent glutamate excitotoxicity and to reduce synaptic transmission. The use of prior brain perfusion becomes a critical issue when juvenile or adult brains are used (>21 days postnatal). A pump can be used for these perfusions, although a simple gravity system is sufficient. The temperature should be controlled, as a too-cold (0° C) aCSF is deleterious. The next step is to remove the brain from the skull as quickly as possible. The detail of this manipulation depends somewhat on the animal species. In rats the difficulty of this process increases with age because the bone becomes thicker and harder. Once the bone covering the cerebellum is removed, it is possible to make a cut along the midline with sharp scissors. Rongeurs or pliers can be used to remove the top part of the skull in one rotating movement. The dura should then be carefully cut so that the brain can be safely extracted using a scoop. The intact brain is then placed into a beaker of ice-cold oxygenated, modified aCSF. This procedure should not take more than 15 to 20 seconds (from time of decapitation). Although it will be more cumbersome, the whole extraction procedure can be performed with the animal’s head submerged in ice-cold oxygenated, modified aCSF. The extracted brain is then prepared for the slicing procedure. Standard procedures for cutting hippocampal slices in rats include removing the cerebellum and the most frontal regions of cortex. The hemispheres are then typically separated. If a “chopper” is used, the hippocampus is removed from the brain and placed on a cutting stage. Caution must
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be used to limit the mechanical stresses and stretches. Most hippocampal slice studies use transverse sections, that is, slices cut perpendicular to the longitudinal axis of the hippocampus. Because of the curvature of the hippocampus (Figure 1A), it is not possible to obtain transverse slices from the whole hippocampus without artificially stretching the structure. When a vibroslicer is used to cut slices (Figure 1B), the investigator may leave the hippocampus embedded within the whole brain hemisphere and glue the hemisphere onto a support block. Although leaving the hippocampus protected inside the hemisphere reduces the number of slices that can be made in the “transverse” orientation, this procedure offers the advantage of lesser manipulation of the hippocampal structure before slicing. Further, by cutting the brain surface (to be glued to the support block) at an appropriate angle, the experimenter can manipulate the orientation of the slice with respect to the longitudinal axis of the hippocampus and thus “expose” hippocampal substructures of particular interest (e.g., for recording from pyramidal cell dendrites) (Hoffman et al., 1997).
A
The slicing procedure itself is a critical step. A vibroslicer with minimal Z-deflection and a glass or sapphire blade should be used for best results. Optimal slicing parameters (e.g., cut speed, amplitude, frequency of oscillation of the blade) depend on the preparation. Tissue from adult animals cut differently from tissue from immature animals because of the presence of choroid tissue, myelin, etc. Tissue from rats and mice cut somewhat differently from, for example, primate hippocampus, not least of all because of the different size of the structure in these different species. The issue of cutting parameters becomes particularly important when preparing slices from the human brain (following extraction of an epileptic hippocampus during surgery for medically intractable TLE), as the density and rigidity of the tissue may be very different according to the underlying reorganizations (sclerosis, gliosis, etc.). Cutting slices from immature brain presents the investigator with other types of challenges. Because the immature rat hippocampus easily “falls apart” when sliced, it is preferable to keep it embedded within the rest of the hemisphere during cutting, and to
B
C Longitudinal axis
FIGURE 1 A: Schematic showing the three-dimensional organization of the hippocampus within a rodent brain. B: Schematic showing key features of the slice-cutting procedure. The cortex (not shown) containing the hippocampus is glued on an agar block. Transverse slices are limited to the ventral part of the hippocampus. C: Typical transverse hippocampal slice showing the major cell regions within a slice. The hippocampal slices typically consist of the dentate gyrus (with granule cells), the CA3 and CA1 pyramidal cell regions, and the subiculum. Thick lines represent the distribution of the somata of the principal neurons (granule cells in the dentate gyrus and pyramidal cells in the CA3 and CA1 regions).
Studying Epilepsy Using the Slice Preparation
use a very slow forward speed and a high-frequency oscillation. Whatever the type of tissue, a general “rule of thumb” is to use a slow forward speed and large lateral amplitude and high frequency oscillation. However, it is important to invest the necessary time in establishing the appropriate set of parameters for a given preparation (and slicing equipment). As stressed already, all the experimental procedures and data depend on the quality of the slices. Slice thickness is another critical parameter, and it usually represents a compromise between the desired amount of intrinsic connectivity and the experimenter’s ability to provide adequate oxygenation to the core of the slice. Slices from adult tissue are rarely thicker than 400 mm. In the immature brain, this limit is less critical; the whole hippocampus can survive for 48 hours in vitro (Khalilov et al., 1997). Not all the slices made from a given hippocampus are useful for a given set of experiments. Which slices are chosen depends on the goals of the study. For example, the connectivity patterns in the ventral, dorsal, and midhippocampus are very different (Moser and Moser, 1998; Witter and Groenewegen, 1984). Physiologic features may also vary according to the slice location (along the longitudinal axis) and orientation (Ferbinteanu and McDonald, 2001). Slices are normally cut into cold aCSF and then transferred (for “storage”) into a holding chamber at room temperature. Slices needed for study are transferred to the recording chamber. The choice of interface versus submerged chamber system is another critical issue, and each offers its own advantages. For example, extracellular space (ES) is reduced when an interface chamber is used, thus increasing the ephaptic interactions, a potentially important issue in epilepsy studies (Schuchmann et al., 2002). Ictallike discharges are more easily obtained in interface versus submerged chambers. All perfusion systems should allow for a continuous flow of aCSF into the recording chamber. The perfusion speed appears to be a critical parameter because biologically relevant hippocampal rhythms can be recorded only when high flow rates (i.e., >4 ml/min vs. the usual 1.5 ml/min) are used (Hajos et al., 2004; Wu et al., 2002). Differences in such apparently subtle parameters may explain interlaboratory discrepancies, for example, when using interface versus submerged chambers (Bracci et al., 1999). A higher flow rate (with O2-saturated aCSF) is usually needed for submerged chambers in which all oxygenation is derived from the medium. In contrast, much of the oxygen supply to slices in interface chambers is derived from the O2-saturated gas environment above the tissue. A typical aCSF formulation consists of (in millimolar): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 2, NaH2PO4 1.25, NaHCO3 25, and dextrose 10. The aCSF is equilibrated with 5% CO2/95% O2 gas which is bubbled through the solution. The concentrations of K+, Ca2+, and Mg2+ are particularly variable (within 1 mM or so) from laboratory to laboratory.
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These concentrations are important because even minor changes in these ionic concentrations will alter cell and network excitability, synaptic transmission, and intracellular processes. As pointed out earlier, typical slice aCSF does not contain many metabolites that are usually contained in the CSF of intact animals. To be “more relevant” to physiologic conditions, slice experiments should be performed close to physiologic temperature (~35° C). Below 30° C, transporters, ionic channels, etc., have different biophysical properties. Working close to physiologic temperature, however, raises a number of challenges. First, there is often a “dead space” between the system that warms aCSF (e.g., a temperature-regulated water bath) and the recording chamber. Therefore, aCSF will have to be warmed to a higher temperature than the target, raising the risk of precipitation (e.g., Ca2+) and driving out needed oxygen. One can reduce the dead space by using a system close to the recording chamber. However, such a system is normally of smaller capacity and must also be set to a high level because the time that the aCSF spends in the warming apparatus is so short. The best technical solution is to use two warming systems: a temperature-regulated bath to prewarm aCSF and a small system close to the chamber for final adjustment. These temperature control issues are particularly difficult when high flow rates are used. There are two good tests to check the condition of a slice. The first one involves a visual inspection with infrared microscopy. The surface of the slice should be planar, and the neurons at the surface should be healthy, that is, having no “balloon-like” somata (with a clearly visible nucleus) and no dying neurons (with a strong black-and-white contrast soma contour). The second test involves determining whether oscillations can be induced (Hajos et al., 2004). This latter assessment can be performed even when “blind” cellular or field recording techniques are used.
STUDYING EPILEPSY USING THE SLICE PREPARATION Many different types of epilepsy exist. Hence multiple models have been developed, for example, partial or generalized epilepsies and convulsive or nonconvulsive epilepsies. A similar diversity of “models” has been reported using the hippocampal slice preparation (and variations thereon), most focusing on generating activity relevant to TLE. Many different issues can be addressed in the slice preparation. A major area of investigation has been the issue of ictogenesis, including where ictal discharges are generated, how they propagate, how they stop (or how we can stop them), and what underlying mechanisms might be involved. Another important theme involves studies of epileptogenesis, that is, what processes lead to an epileptic state following an initial insult. In pursuit of these and related questions, two general
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types of preparations have been used for the past three decades: (1) brain slices obtained from “normal” animals, in which in vitro manipulations have been used to generate epileptiform activities; and (2) brain slices obtained from human patients or chronically epileptic animals. Slices from human patients are difficult to obtain and study (see chapter 8), and generating epileptic animals is time consuming (as described elsewhere in this volume). Many investigators thus turned to normal brain slices to study ictogenesis. In addition to easy accessibility, the extensive (but far from complete) knowledge of the underlying network architecture of hippocampus, and of its physiological properties, has facilitated the design of such experiments and the interpretation of their results. The main drawback of these “acute” models is that little structural pathological change is apparent in these normal circuits. In contrast, most investigators believe that in temporal lobe epilepsy, the network architecture has been considerably modified (cell loss, sprouting, protein modifications etc.). The “rules” and insights derived from acute studies must therefore be reviewed with caution in trying to understand the mechanisms of chronic epilepsies. In the following sections, I describe a number of the acute models generated in hippocampal slices and briefly comment on the use of slices to study chronic models. Before doing so, however, it is important to consider the issue of the age of the animal from which the slice is made. Rats 5 to 6 weeks old are considered adults. Before this age, the rat’s neuronal networks in the hippocampus are still developing and undergo considerable modifications. During the first postnatal week in the rat, GABA acts as an excitatory neurotransmitter (Ben Ari, 2002), GABAergic interneurons are the source and targets of the first synapses (Gozlan and Ben Ari, 2003), and neuronal network activity results from the synergistic excitatory actions of GABA and glutamate (Ben Ari et al., 1989; Leinekugel et al., 2002). At these young ages, many connections are lacking, and neurons continue to proliferate and migrate to their proper target regions. The state of such a developing network and its functioning mode are quite different from the adult brain. This early period of rodent development corresponds roughly to the last trimester of gestation in infrahuman primates (Khazipov et al., 2001) and (perhaps) in humans (Clancy et al., 2001). Because GABA switches from a depolarizing to a hyperpolarizing mode well before birth in infrahuman primates (Khazipov et al., 2001), studies performed in rodents during the first 2 postnatal weeks may be relevant primarily to in utero epilepsy in human. The following weeks are characterized by a refinement of connections, myelination, changes in receptor subunit composition, channel expression, etc. (Fritschy et al., 1994; Katz & Shatz, 1996; Meier et al., 2004; Ritter et al., 2002; Stellwagen and Shatz, 2002; Tansey et al., 2002). How the time points of these changes correspond to human developmental stages is unknown.
This issue of age relevance is important for many slice studies because recordings are typically carried out on slices from animals between postnatal days 14 and 21. This timing choice is dictated largely by technical considerations, an empirical consensus that it is easiest to prepare slices from these young animals and that slice viability is superior (e.g., compared with slices from more mature animals). As suggested, it is possible to argue that experiments performed on 6-week-old rodents are relevant to human (adult?) epilepsy because the hippocampal neuronal networks have been stabilized in their mature forms. However, in slice studies on rats (or mice) between the second and the sixth postnatal week, it is particularly difficult to extrapolate the data to human epileptic phenomena. Not only is “matching” ages difficult, but the situation is also complicated by the fact that maturation of different cells, different systems, and different molecular controls occur at different speeds (and span different periods) in rodents and in humans. While there is no “solution” to this problem, it is important to keep these issues in mind while designing experiments and interpreting in vitro results.
Acute Models of Epilepsy Several in vivo models of ictal-like and inter-ictal-like activity have been developed in hippocampal slices, including slices challenged with high-frequency electrical stimulation (Somjen et al., 1985), GABAA-receptor antagonists (Schwartzkroin and Prince, 1978; Swann and Brady, 1984), kainic acid (KA) (Fisher and Alger, 1984; Westbrook and Lothman, 1983), K+ channel blockers (Galvan et al., 1982), low [Ca2+]o (Jefferys and Haas, 1982; Taylor and Dudek, 1982), no [Mg2+]o (Anderson et al., 1986) or high [K+]o (Traynelis and Dingledine, 1988). As detailed later, some models reproduce some features of interictal activity and others produce tonic-clonic discharges. These models have several advantages. They are easy to implement, and pathological discharges emerge with high reproducibility. The slice preparation allows easy access to numerous parameters, including activity patterns of neuronal populations, propagation of signals from one subregion to the next, synaptic inputs in individually recorded cells, and pharmacologic responsiveness of different patterns of paroxysmal discharges. Electrical Stimulation-Induced Afterdischarge Ictal-like afterdischarges displaying tonic- and cloniclike phases can be evoked following tetanic stimulation (40–50 pulses at 100 Hz) applied in CA1 stratum radiatum (Figure 2A). The tonic-clonic phase appears after several stimulations, suggesting a long-term potentiation-like effect (Rafiq et al., 1993). The afterdischarge is composed of several epochs, with a sequence of slow and fast oscillations
Studying Epilepsy Using the Slice Preparation
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A
B
C
D
FIGURE 2 A: Progression of afterdischarge waveform and duration, recorded in the CA1 region in response to stimulation of the Schaffer collaterals. (Adapted from Rafiq et al., 1993.) The graph at the right shows the gradual increase in afterdischarge duration with repeated stimuli. B: Interictal-like and ictal-like activities recorded in the disinhibited CA1 minislice. Left panel: Simultaneous intracellular recordings of a pyramidal cell soma (top) and dendrite (bottom), showing an interictal discharge (shown at a faster time scale below) followed by an ictal-like discharge (duration, 7 seconds). Upper right panel: Experimental design showing the cuts performed to isolate the CA1 region and the positions of the recording electrodes. Lower right panel: Bar graph showing the duration of ictal-like activity as a function of the g-aminobutyric acid (GABA) antagonist used. Bic, bicuculline; PTX, picrotoxin; GBZ, gabazine 100 mm. (Adapted from Karnup and Stelzer, 2001.) C: Simultaneous field and intracellular recordings in the CA1 region showing interictal-like activity induced by 1 mm kainic acid. (Adapted from Fisher and Alger, 1984.) D: Simultaneous field and whole-cell patch-clamp recordings in the CA3 region, showing ictal-like activity induced by 100 mm 4-AP in toto. (Adapted from Luhmann et al., 2000.)
followed by a silent postictal depression and a secondary discharge. This pattern is similar to that found in afterdischarges evoked in vivo (Bragin et al., 1997). The mechanisms underlying physiologic oscillations often involve interneurons (Freund and Buzsáki, 1996), and so afterdischarges constitute a good model for studying synchronization via local circuits (including the role of N-methyl-d-aspartate [NMDA] receptors) (Stasheff et al., 1985, 1993), interneurons and depolarizing GABA (Bracci
et al., 1999; Fujiwara-Tsukamoto et al., 2003, 2004; Kaila et al., 1997; Staley et al., 1995; Velazquez and Carlen, 1999; Whittington et al., 1997) as well as ephaptic interactions (Bracci et al., 1999). Combining electrophysiology (intracellular recording) and morphology (intracellular labeling) allows the investigator to dissect out key features of specific hippocampal sub-networks (Fujiwara-Tsukamoto et al., 2004). This model of electrically induced afterdischarge has been discussed as potentially relevant to ictal discharges in
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Chapter 6/Hippocampal Slices: Designing and Interpreting Studies in Epilepsy Research
human temporal lobe epilepsy (Stasheff et al., 1985). As mentioned previously, these experiments are performed on hippocampal slices from normal animals, and translating the results to chronic epilepsy is somewhat dangerous. For example, CA1 stratum oriens interneurons are directly involved in the synchronization process during the afterdischarge (Fujiwara-Tsukamoto et al., 2004); yet a large number of these interneurons are lost in chronic epilepsy (Cossart et al., 2001; Dinocourt et al., 2003). Nevertheless, it is intriguing to note that NMDA receptors and depolarizing GABA play an important role in evoked afterdischarges (Staley et al., 1995; Stasheff et al., 1985). In chronic epilepsy, the NMDA receptor-dependent component of synaptic transmission is considerably increased (Turner and Wheal, 1991) and fast GABAergic neurotransmission becomes, in part, excitatory (Cohen et al., 2002).
as CA3 pyramidal cells (Westbrook and Lothman, 1983) and interneurons (Cossart et al., 1998; Frerking et al., 1998), leading them to generate action potentials. KA also has a multiplicity of presynaptic effects on glutamatergic and GABAergic terminals (Huettner, 2003) as well as on ionic channels (Melyan et al., 2002). Despite its epileptogenicity in vivo (Ben Ari and Cossart, 2000) and its ability to generate g-oscillations (40 Hz) at nanomolar concentration in vitro (Buhl et al., 1998; Fisahn et al., 1998), KA has not been extensively used to study the initiation and propagation of interictal-like discharges in the hippocampal slice preparation. A study performed in the intact (in toto) hippocampus (in vitro) described the development of a “mirror focus” in contralateral (naïve) hippocampus when the ipsilateral hippocampus was challenged with KA (Khalilov et al., 2003). Blocking K+ Channels
GABAergic Disinhibition Pharmacologic blockade of GABAA receptors has been used extensively to study epileptiform activity in vitro. In the absence of fast GABAergic neurotransmission, synchronized inter-ictal-like bursts can occur (Miles and Wong, 1986, 1987; Schwartzkroin and Prince, 1978; Wong et al., 1986). Not surprisingly, these bursts depend on fast glutamatergic neurotransmission to activate (alpha) amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA and NMDA receptors (Simpson et al., 1991; Williamson and Wheal, 1992). In general, the generation of ictal-like events in vitro requires additional pharmacologic manipulations (at least in mature tissue), such as elevating [K+]o (Traub et al., 1996). However, ictal-like events can be recorded in slices from the immature brain (Khalilov et al., 1997; Swann and Brady, 1984). Further, ictal-like activity is present in the disinhibited CA1 minislice of adult guinea pigs (Figure 2B) (Karnup and Stelzer, 2001). The usefulness of the disinhibited hippocampal slice model, at least for studying ictogenesis, is questionable because GABAergic neurotransmission appears to be quite robust (even increased) in chronic temporal lobe epilepsy (Bernard et al., 2000; Cossart et al., 2001; Esclapez et al., 1997). However, blocking fast GABAergic neurotransmission may be a useful approach to unravel changes in excitatory circuits in chronic epilepsy (Esclapez et al., 1999; Meier and Dudek, 1996; Patrylo and Dudek, 1998). Kainic Acid-Induced Epileptiform Activity Bath application of kainic acid KA induces spontaneous interictal-like activity in hippocampal slices (Figure 2C). KA has multiple presynaptic and postsynaptic effects, and so the underlying bases for this KA effect remains unclear (Ben Ari and Cossart, 2000; Huettner, 2003). KA can directly depolarize neurons that express KA receptors, such
Blocking K+ channels with 10 to 30 mm 4-aminopyridine (4-AP) produces ictal-like discharges in the olfactory cortex (Galvan et al., 1982). In the hippocampus, 50 mm 4-AP depolarizes neurons (Perreault and Avoli, 1989) and induces interictal-like (Voskuyl and Albus, 1985) or ictal-like (Chesnut and Swann, 1988) discharges (Figure 2D). This model can be used to study the propagation of paroxysmal discharges between different regions (Luhmann et al., 2000), the role of GABAergic neurotransmission in such discharges (Perreault and Avoli, 1992), the transition between interictal- and ictal-like discharges (Dzhala and Staley, 2003b), and synchronization mechanisms (Netoff and Schiff, 2002). However, it appears particularly useful in combined hippocampus—EC slices (see chapter 4). Low Extracellular Ca2+ Lowering [Ca2+]o (nominally to 0 mM) abolishes neurotransmission and results in spontaneous paroxysmal discharges (Figure 3A) (Jefferys and Haas, 1982; Taylor and Dudek, 1982). These events arise focally and spread through the CA1 region (Konnerth et al., 1984). This model may be particularly valuable for the study of synchronization mechanisms (Bikson et al., 2003) and for investigating interventions to abort/stop these discharges (Ghai et al., 2000). Epileptiform Discharges in the Absence of [Mg2+]o Removing Mg2+ from aCSF results in the appearance of spontaneous interictal-like discharges in slices from juvenile/adult animals (Anderson et al., 1986). This model could be clinically relevant because low levels of Mg2+ have been associated with human epilepsy (Durlach, 1967). Removing [Mg2+]o allows NMDA receptors to respond directly and robustly to glutamate excitation (Figure 3B) (Mody et al., 1987). This model allows for the study of propagation of
Studying Epilepsy Using the Slice Preparation
A
67
B
D C
FIGURE 3 A: Ictal-like discharges recorded in low [Ca2+]o in the CA1 region. Traces are shown at different time scales (expanded time frame in middle trace, slower time frame below). (Adapted from Konnerth et al., 1986.) B: Spontaneous epileptiform discharges recorded in Mg2+-free artificial cerebrospinal fluid (aCSF). These discharges were modulated by application of the N-methyl-d-aspartate (NMDA) receptor antagonist APV. (Adapted from Mody et al., 1987.) C: Spontaneous ictal-like discharge recorded in the CA1 region in toto, in Mg2+-free aCSF. (Adapted from Quilichini et al., 2002.) D: Spontaneous ictal-like discharges recorded in the CA1 region in slices exposed to elevated extracellular [K+]. Different portions of the discharge are displayed at different time scales. (Adapted from Traynelis and Dingledine, 1988.)
epileptiform activity and the role of interneurons in those discharges (Perez Velazquez, 2003). Interestingly, although not present in acute hippocampal slices, ictal-like discharges can be recorded in Mg2+-free aCSF in the in toto immature hippocampal preparation (Figure 3C) (Quilichini et al., 2002). This preparation is useful for testing antiepileptic drugs (Quilichini et al., 2003). The electrographical signature of the discharge is very similar to that recorded in patients. Elevated K+ Model of Epilepsy
complex network questions such as the mechanisms of fast ripples at seizure onset (Dzhala and Staley, 2004) and the transition from interictal to ictal activity (Dzhala & Staley, 2003b). This model is particularly useful because it causes an elevation of the general excitability of all neural networks; it is noteworthy that elevations of [K+]o comparable in magnitude to the K+ changes imposed on hippocampal slices occur during seizures in vivo (Fisher and Alger, 1984; Lothman, 1976; Moody, 1974).
Chronic Models of Epilepsy
Bathing slices with 8.5 mM K results in the occurrence of spontaneous tonic-clonic discharges (Figure 3D) (Traynelis and Dingledine, 1988). Under these conditions, it is possible to investigate the role of GABAergic neurotransmission (Dzhala and Staley, 2003a), the behavior of the different hippocampal cell types (McBain, 1994), and +
Hippocampal slices can be obtained from chronic animal models of epilepsy. The technical advantages of the slice to investigate detailed cellular and synaptic phenomena has been particularly important in studying models of TLE and epileptogenesis, such as the kindling, kainate, and pilocarpine models. Two epochs following an epileptogenic
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Chapter 6/Hippocampal Slices: Designing and Interpreting Studies in Epilepsy Research
event or stimulus have been investigated: the chronic phase of epilepsy, during which the animals display spontaneous recurrent seizures (epilepsy), and the latent period, the interval between the initial insult and the first spontaneous seizure (epileptogenesis). The hippocampus undergoes considerable modifications during epileptogenesis as well as during the chronic phase. In designing experiments focused on these issues, the investigator must consider this “reactive plasticity” to be a dynamic process and assume that results may vary, depending on what parameters are measured and when. An obvious advantage of studying hippocampal slices from chronic animal models (as opposed to studying epileptiform phenomena in “normal” tissue) is the fact that these animals are epileptic. This fact allows the investigator to analyze parameters that are correlated with the epileptic state or may be causally related to ictogenesis. Unfortunately, as suggested already, interictal-like or ictal-like discharges have not been reported to occur—under physiologic conditions—in slices obtained from epileptic animals. However, slices from the ventral hippocampus, in which spontaneous waves can be recorded, remain to be tested (Colgin et al., 2004; Kubota et al., 2003). Further, the epileptogenic challenges described in the previous section (e.g., GABAA receptor blockade, high K+, low Mg2+, etc.) can be used to unveil the epileptic propensity of slices from chronic models. Comparing the pattern and properties of interictallike or ictal-like activity between control and epileptic slices may reveal important information about functional reorganizations (e.g., sprouting of excitatory axons) that take place during epileptogenesis or during the chronic phase of epilepsy (Cronin et al., 1992; Hardison et al., 2000; Lynch and Sutula, 2000; Patrylo and Dudek, 1998; Patrylo et al., 1999; Wuarin and Dudek, 1996). Another use of these slices is to investigate the details of modifications of hippocampal circuitry associated with the epileptic state. Questions are usually related to the fate of glutamatergic and GABAergic pathways as well as of ionic channels. Many studies are based on a multidisciplinary approach that combines electrophysiology, functional morphology, or molecular biology (Bernard et al., 2004; Brooks-Kayal et al., 1998; Buhl et al., 1996; Chen et al., 2001, 2003; Cossart et al., 2001; Esclapez et al., 1997, 1999; Nusser et al., 1998; Ratzliff et al., 2004; Scharfman et al., 2000; Su et al., 2002). As indicated already, the main difficulty in applying slice approaches to studying chronic animal models lies in interpretation of the data. Seizures in temporal lobe epilepsy usually involve several limibic regions. The exact anatomic location(s) of seizure initiation and of the propagation patterns remain unknown, and there is high variability from one patient to another. Thus it is still unclear where the investigator should look in the hippocampal slice for TLE-related abnormalities. It is perhaps more useful to focus on epilepsyinduced plasticity and to use the slice preparation to eluci-
date the details of these changes. Even here, however, much of the connectivity is lost in the slice preparation—even from normal animals—and so correlating slice abnormalities with epilepsy-related pathology may be hazardous. Nevertheless using the hippocampal slice preparation to identify parameters in which alterations could be potentially “proepileptic” may provide new therapeutic targets (Bernard et al., 2004; Su et al., 2002).
FUTURE DIRECTIONS Seizures recruit several regions and thousands of neurons. Thus, ideally one should use a model system that maintains those regions and their connections, that is, an in vivo approach. Historically many investigators lost interest in such complex preparations because in vitro slices were developed to offer more powerful technical approaches. However, advances have suggested that cellular electrophysiologic techniques, for example, patch-clamp recordings (Khazipov and Holmes, 2003), can now be applied to intact model systems (although here the use of anaesthetics is an important issue to consider). The in toto hippocampal in vitro preparation represents an interesting “compromise” between in vitro slice and intact approaches. Most experiments performed in the slice can be done in toto, where network properties can be more effectively investigated (Khalilov et al., 1997, 2003). This preparation is limited, however, to the first postnatal week in rats (Khalilov et al., 1997) and (with some adaptation) to adult mice (Wu et al., 2002). The slice preparation allows high throughput in terms of data generation, but it has been limited to the fine analysis of single cells (with intracellular recordings) or the gross behavior of small populations of neurons (with extracellular recordings). Recent technical advances, particularly with imaging methods, now allow for the study of network properties in the slice preparation. For example, it is possible to record the activity of thousands of neurons simultaneously with high-speed two-photon calcium imaging systems (Figure 4A) and then to record from individual neurons to obtain access to more microscopic properties (Cossart et al., 2003). Another technical approach is to apply voltagesensitive dyes to the preparation. Their use is still limited because of their deleterious effects and low signal-to-noise ratio, but they can provide useful information about the mechanisms underlying the initiation and propagation of ictal and interictal discharges (Figure 4B) (Otsu et al., 2000).
CONCLUSION In summary, the main limitation of the slice preparation is the massive loss of intrinsic and extrinsic connectivity.
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References
FIGURE 4 A: Two-photon imaging of the CA1 region of a hippocampal slice from a mouse. Pyramidal cell somata are clearly visible in the pyramidal cell layer (P); interneurons are visible in stratum oriens (O) and stratum radiatum (R). Neurons are filled with a Ca2+ indicator, and variations in [Ca2+]i are measured. Electrophysiologic properties of any individual neuron can also be recorded. For example, the inset shows the spontaneous activity recorded in a stratum oriens interneuron (Rosa Cossart, personal communication). B: Spatiotemporal patterns of neuronal activity evoked in the dentate gyrus in control and kainic acid (KA)-treated rat hippocampal slices. Level of neuronal activity is displayed according to the pseudocolor code (from background gray to highly activated red). (Adapted from Otsu et al., 2000.) (See color insert.)
Hence the main pitfall lies in the interpretation of the results and their applicability to epileptogenic conditions in intact systems. Slice preparations, however, can provide some insight into the connectivity and the transfer of information from one hippocampal subregion to another. Despite the issues of interpretation and applicability, comparing control and “epileptic” hippocampal slices has proven very useful for unraveling seizure-related modifications in network properties. Arguably the best use of the hippocampal slice preparation is for analysis of the reorganizations that take place within the various hippocampal networks during the latent period and the chronic phase of epilepsy as established within intact animal models. Such modifications give profound insights into epilepsy-associated brain plasticity. The future of this research focus lies, at least in part, in the simultaneous analysis of group and individual cell properties (e.g., with fluorescent dyes and patch-clamp recordings) in relevant models (e.g., a transgenic animal carrying the same mutation found in patients and displaying the same type of epilepsy). In this way the hippocampal slice constitutes an adjunctive approach to studying many of the models that are presented in other chapters in this volume.
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Taylor, C.P., and Dudek, F.E. 1982. Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses. Science 218: 810–812. Traub, R.D., Borck, C., Colling, S.B., and Jefferys, J.G. 1996. On the structure of ictal events in vitro. Epilepsia 37: 879–891. Traynelis, S.F., and Dingledine, R. 1988. Potassium-induced spontaneous electrographic seizures in the rat hippocampal slice. J Neurophysiol 59: 259–276. Turner, D., and Wheal, H. 1991. Excitatory synaptic potentials in kainic acid-denervated rat CA1 pyramidal neurons. J Neurosci 11: 2786–2794. Velazquez, J.L., and Carlen, P.L. 1999. Synchronization of GABAergic interneuronal networks during seizure-like activity in the rat horizontal hippocampal slice. Eur J Neurosci 11: 4110–4118. Voskuyl, R.A. and Albus, H. 1985. Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminopyridine. Brain Res 342: 54–66. Westbrook, G.L., and Lothman, E.W. 1983. Cellular and synaptic basis of kainic acid-induced hippocampal epileptiform activity. Brain Res 273: 97–109. Whittington, M.A., Stanford, I.M., Colling, S.B., Jefferys, J.G., and Traub, R.D. 1997. Spatiotemporal patterns of gamma frequency oscillations tetanically induced in the rat hippocampal slice. J Physiol (Lond) 502: 591–607. Whittington, M.A., and Traub, R.D. 2003. Interneuron diversity series: inhibitory interneurons and network oscillations in vitro. Trends Neurosci 26: 676–682. Wieser, H. G. (1983). Depth recorded limbic seizures and psychopathology. Neu Neurosci. Biobehav. Rev. 7: 427–440. Williamson, R., and Wheal, H.V. 1992. The contribution of AMPA and NMDA receptors to graded bursting activity in the hippocampal CA1 region in an acute in vitro model of epilepsy. Epilepsy Res 12: 179–188. Witter, M.P., and Groenewegen, H.J. 1984. Laminar origin and septotemporal distribution of entorhinal and perirhinal projections to the hippocampus in the cat. J Comp Neurol 224: 371–385. Wong, R.K., Traub, R.D., and Miles, R. 1986. Cellular basis of neuronal synchrony in epilepsy. Adv Neurol 44: 583–592. Wu, C., Shen, H., Luk, W.P., and Zhang, L. 2002. A fundamental oscillatory state of isolated rodent hippocampus. J Physiol 540: 509–527. Wuarin, J.P., and Dudek, F.E. 1996. Electrographic seizures and new recurrent excitatory circuits in the dentate gyrus of hippocampal slices from kainate-treated epileptic rats. J Neurosci 16: 4438–4448. Yamamoto, C., and McIlwain, H. 1966. Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro. J Neurochem 13: 1333–1343.
A
]~ Controlrat 1
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FIGURE 6--4 A: Two-photon imaging of the CAI region of a hippocampal slice from a mouse. Pyramidal cell somata are clearly visible in the pyramidal cell layer (P); interneurons are visible in stratum oriens (0) and stratum radiatum (R). Neurons are filled with a Ca 2+ indicator, and variations in [Ca2+]~ are measured. Electrophysiologic properties of any individual neuron can also be recorded. For example, the inset shows the spontaneous activity recorded in a stratum oriens interneuron (Rosa Cossart, personal communication). B: Spatiotemporal patterns of neuronal activity evoked in the dentate gyms in control and kainic acid (KA)-treated rat hippocampal slices. Level of neuronal activity is displayed according to the pseudocolor code (from background gray to highly activated red). (Adapted from Otsu et al., 2000.)
rO.06
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7 Thalamic, Thalamocortical, and Corticocortical Models of Epilepsy with an Emphasis on Absence Seizures THOMAS BUDDE, HANS-CHRISTIAN PAPE, SANJAY S. KUMAR, AND JOHN R. HUGUENARD
dominant inheritance of the “spike-wave discharge” (SWD) trait (Engel, 2001). Seizures start at 3 to 8 years of age, may occur several hundred times per day, and remit near the onset of adolescence in roughly 70% of the patients. Major neuropathologic deficits have not been found. Linkage studies in absence epilepsy patients have led to the identification of two gene mutations localized to the voltage-gated Ca2+ channel a1A-subunit gene (CACNA1A, chromosome 19p) (Jouvenceau et al., 2001) and the GABAA receptor g subunit gene (GABRG2, chromosome 5q) (Baulac et al., 2001; Wallace et al., 2001). A number of susceptibility loci and polymorphisms in various types of ion channels, transmitter receptors, and other proteins have also been described, although the linkage to CAE is not always clear (see Crunelli and Leresche, 2002).
Absence epilepsy is an idiopathic, generalized, and nonconvulsive form of epilepsy with an as yet unknown polygenic background (for review, see Crunelli and Leresche, 2002). A typical absence episode consists of a sudden epileptic seizure with severe impairment of consciousness. Although the interictal electroencephalogram (EEG) appears normal, the ictal EEG is characterized by phases of bilateral, synchronous 2.5- to 4.0-Hz spike and slow-wave discharges (SWDs). As defined by the current International League against Epilepsy (ILAE) classification (Engel, 2001), typical absence seizures are an integral part of several types of idiopathic generalized epilepsies, including childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and generalized tonic-clonic seizures (GTCS), all of which display complex partially overlapping phenotypes. A single-model pharmacologic manipulation or genetic alteration can hardly account for the integral nature of various types of absence epilepsies, their polygenic background, and the pathophysiologies of the underlying interacting synaptic networks. In fact, different experimental approaches have been used to investigate various aspects of absence epilepsy. Following a brief outline of genetic models and the pathophysiology of absence epilepsy, we review here thalamic, thalamocortical, and corticocortical models (with particular reference to CAE) and discuss their usefulness and limitations. For details on the epidemiology, the genetic etiology, and the pathophysiologic mechanisms of CAE, the reader is referred to a number of excellent recent reviews (Crunelli and Leresche, 2002; Destexhe and Sejnowski, 2003; McCormick and Contreras, 2001; Snead, 1995; Steriade et al., 1997). In brief, CAE is genetically determined, with incomplete penetrance and complex autosomal-
Models of Seizures and Epilepsy
MODELS OF ABSENCE EPILEPSY IN INTACT RATS Genetic Models Spontaneous point mutations have resulted in mutant mice that develop SWDs on the EEG accompanied with behavioral arrest. These mutant mice (see Chapter 17) are considered suitable models of CAE. The best studied of these mutant mice, their genotype, and their phenotype are listed in Table 1. Single-locus abnormalities have been identified in these mutants; the mutant genes and the pathways in which the aberrant gene products are involved provide important insights into the underlying bases for human absence epilepsy. A number of different mutations involving the genes encoding four separate subunits of the
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Copyright © 2006, Elsevier Inc. All rights of reproduction in any form reserved.
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Chapter 7/Thalamic, Thalamocortical, and Corticocortical Models of Epilepsy
TABLE 1 Comparison of Features of Pharmacologically Induced and Genetic Animal Models of Absence Epilepsy with Human Childhood Absence Epilepsy Pharmacologic models
Model
Species
Seizure characteristics
Penicillin
Cat
GHB
Rat, cat, monkey
Onset of seizures 1 h after injection of antibiotic (300,000 IU/kg, IM); duration of seizures 6–8 h SWDs appear within 2–5 min of administration of the prodrug GBL to rats; maximum sensitivity for rats at P28
SWD frequency (Hz)
Attenuation by ETX
3
Yes
7–9
Yes
Genetic mouse models (single gene mutation)
Model Leaner Rocker Tottering Ducky Lethargic
Chromosome gene product
Phenotype
Chromosome 8/Ca2+ channel a1A-subunit Chromosome 8/Ca2+ channel a1A-subunit Chromosome 8/Ca2+ channel a1A-subunit Chromosome 9/Ca2+ channel a2 b2-subunit Chromosome 2/Ca2+ channel b4-subunit
Stargazer
Chromosome 15/Ca2+ channel g2-subunit
Coloboma Mocha
Chromosome 2/SNAP25 Chromosome 10/adaptorlike protein complex (AP-3) b-subunit Chromosome 4/Na+/H+ exchanger (Nhe1)
Slow-wave epilepsy mouse
SWD frequency (Hz)
Attenuation by ETX
Severe ataxia; SWDs
5–7
Yes
Ataxia; SWDs
6–7
Not determined
Ataxia and motor seizures at 3 wk; SWDs Ataxia and dyskinesia; SWDs
6–7
Yes
6
Yes
Lethargy, ataxia and loss of motor coordination at P15; focal motor seizures and SWDs Ataxia and impaired vestibular function; frequent and prolonged SWDs Hyperactivity; SWDs Hyperactivity; SWDs
5–6
Yes
6
Yes
5–6 6
Yes Yes
Ataxia; tonic-clonic seizures; SWDs
3–4
Yes
Inbred rat models (polygenetic)
Model GAERS WAG/Rij
Onset of seizures
Remission with age
At P30–40; all animals reveal seizures at 13 wk Seizures in all animals at 4 mo
SWD frequency (Hz)
Attenuation by ETX
No
7–10
Yes
No
7–11
Yes
Remission with age
Ictal EEG
Attenuation by ETX
CAE
Clinical symptoms Severe impairment of consciousness; no response to commands or recollection of ictal events; eyes are open; automatisms
Onset of seizures 4–10 yr
Seizures will remit in the majority of patients (~70 %) by adolescence; some might go on to develop myoclonic jerks and generalized tonic-clonic seizures
Spike (mostly 1, max. 3) and slow-wave discharge is generalized, bilateral and synchrnous; frequency is 3 Hz (range 2.5–4 Hz); duration is ~10 s (range 4–20 s); abrupt onset and cessation
Yes
CAE, childhood absence epilepsy; EEG, electroencephalogram; ETX, ethosuximide; GHB, g-hydroxybutyrate; IM, intrasmuscularly; SWD, spike-wave discharge.
Pathophysiologic Mechanisms of Childhood Absence Epilepsy
multimeric neuronal Ca2+ channel complex (tottering, leaner, rocker, lethargic, ducky, stargazer), the Na+/H+ exchanger (slow-wave epilepsy mouse), adapter-like proteins (Mocha), and SNAP25 (coloboma) have been described (for reviews see Pietrobon, 2002; Felix, 2002). In most mouse mutant strains, SWDs occur at slightly higher frequencies (5–7 Hz) compared with SWD in humans and are abolished by treatment with the antiabsence drug ethosuximide. It is important to note that these mouse mutants display neurologic deficits in addition to the SWD trait, the most obvious being a dysfunction of the motor system (e.g., ataxia) (Table 1). Given that the identified genes have potentially multiple functions, it is important to consider the broad implications of alterations in protein functions when interpreting the causative role of these gene mutations with respect to the absence phenotype. Two genetic rat models, termed Genetic Absence Epilepsy Rats from Strasbourg (GAERS) (for review, see Danober et al., 1998; Marescaux et al., 1992) and Wistar Albino Glaxo Rats from Rijswik, WAG/Rij (for review, see Coenen et al., 1992; Renier and Coenen, 2000; van Luijtelaar et al., 2002), were independently derived from inbreeding of the Wistar strain (see also Chapter 18). Their genotypes are not known, although there is some evidence of an autosomal-dominant inheritance of the SWD trait and a polygenic background that may modify seizure characteristics (Danober et al., 1998; Renier and Coenen, 2000). The absence phenotype is similar in both strains (although subtle differences have been reported for the developmental and electrophysiologic profile of the seizures) and largely resembles that in human CAE. The SWDs occur spontaneously on a normal EEG (mostly during quiet wakefulness) and are associated with a severe reduction in sensory responsiveness and a mild facial myoclonus; there are no major neuropathological abnormalities (Danober et al., 1998; Renier and Coenen, 2000). Furthermore, classic absence antiepileptic drugs (ethosuximide, valproate, benzodiazepines) suppress SWDs, whereas drugs specific for convulsive or focal seizures (carbamazepine, phenytoin) are ineffective or aggravate SWDs in these rats (Danober et al., 1998; Renier and Coenen, 2000). Differences from human CAE relate to the higher frequency of the SWDs (7–11 Hz), the relatively late appearance of absence seizure activity during development, and the persistence of SWDs through rat “adolescence” (Danober et al., 1998; Renier and Coenen, 2000). The g-Hydroxybutyrate Model One established thalamic model of absence epilepsy is the administration of g-hydroxybutyrate (GHB) or the prodrug g-butyrolactone (GBA) to thalamic nuclei, like the ventrobasal (VB) nucleus (Snead, 1991) (see also Chapter 10). GHB is a short-chain fatty acid that is synthesized from
75
GABA and occurs naturally in the mammalian brain. This compound has the ability to induce absence-like seizures in a number of species (for review, see Snead, 1995). GHBtreated animals show an arrest of activity with staring associated with bilaterally synchronous SWDs, ranging in frequency from 2.5 Hz in monkeys to 6–7 Hz in rats. The cellular mechanisms underlying the actions of GHB are not fully understood but are currently emerging from experimental data; the activity of GHB is thought to be mediated through a GHB receptor, which may be distinct from the GABAB receptor (Gervasi et al., 2003; Wong et al., 2004). The experimental procedures for the injection of GHB and GBA in the VB of young (200–300 g) male Sprague-Dawley rats have been detailed by Snead and colleagues (Snead, 1991) (see also Chapter 10). Intrathalamic injections are made in awake animals; recording of the electrocorticogram (ECoG) are carried out in freely moving animals. About 25 seconds after intrathalamic administration of GHB, the ECoG reveals brief bursts of SWDs (duration ~60 s). The drug threshold for this effect is about 25 mg per side. GHB-treated animals represent a reproducible, consistent, and pharmacologically specific model for the study of generalized absence seizures and allow the investigator to assess the effect of pharmacologic and transgenic manipulations of neuronal channels or receptors on the expression of this epileptic activity (Kim et al., 2001; Snead et al., 2000). GHB administration to horizontal thalamic slices (see later) induces self-sustained intrathalamic oscillations at a frequency of ~3 Hz, resembling SWDs (Gervasi et al., 2003).
PATHOPHYSIOLOGIC MECHANISMS OF CHILDHOOD ABSENCE EPILEPSY Clinical evidence indicates that absence seizures are associated with states of decreased vigilance, such as drowsiness (Crunelli and Leresche, 2002). In an early model of generalized epilepsy that used systemic or focal cortical application of penicillin (penicillin-generalized epilepsy model; for review, see Gloor and Fariello, 1988), investigators observed that SWDs on the EEG (e.g., Figure 1A) developed gradually from sleep spindles during early stages of slow-wave sleep (Kostopoulos et al., 1981). From those and subsequent studies in the mutant mice and the genetic rat models mentioned previously, an overall scenario evolved that can be summarized along two lines: (1) The neurons and synaptic interconnections of the thalamocortical system (Figure 1B) that normally sustain rhythmic activities during slow-wave sleep are also critically involved in the generation of SWDs. (2) Seizure activity arises from a concerted interaction within this network, with initial sites most likely residing in the cortex; the corticothalamocortical loop plays a pivotal role in the generation and synchronization of SWD at an extended spatiotemporal scale.
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Chapter 7/Thalamic, Thalamocortical, and Corticocortical Models of Epilepsy
A
EEG
B
cortex +
+
thalamocortical network
+
+
-
TC
-
dorsal thalamus 20 mV
RT nucleus
C 0.5 s
20 mV
-60 mV
D 1s
20 mV
10 mV
-60 mV
100 ms LTS
E
20 ms
EPSPs -71 mV
20 mV
-66 mV
EPSP
0.5 s
-50 mV
10 mV
-60 mV IPSPs 20 ms
FIGURE 1 Intracellular counterparts of spike-wave discharges (SWDs) in GAERS model of absence epilepsy. A: Electroencephalographic (EEG) recording of spontaneous SWDs. B: Schematic diagram of the thalamocortical network. C: Layer V cortical neurons reveal rhythmic depolarizations, which elicit one to three action potentials, superimposed on a long-lasting hyperpolarization. D: In reticular thalamic (RT) neurons, SWD-associated activity starts with a hyperpolarization (see expanded trace bottom left), followed by rhythmic generation of low-threshold Ca2+ spikes (LTSs) associated with bursts of action potentials (see expanded trace bottom right). Note that excitatory postsynaptic potentials (EPSPs) lead to the generation of a LTS. E: Thalamocortical (TC) neurons of the ventrobasal thalamus show rhythmic sequences of EPSP and inhibitory postsynaptic potentials (IPSPs), with occasional firing of action potentials. An EPSP/IPSP sequence is expanded in the bottom panel. (C–E adapted, with permission, from Crunelli and Leresche, 2002.) (See color insert.)
It should be stressed that neither cortical nor thalamic networks alone can generate or sustain SWDs (Steriade and Contreras, 1995, 1998) and that influences exerted by brain structures other than the thalamocortical system can modulate SWD generation (Danober et al., 1998). Of additional interest is the notion that the hippocampal formation is exempt from SWDs in models of pure absence (Snead, 1995). Studies in various models, including GAERS and WAG/Rij, have indicated that spontaneous SWDs occur first in the somatosensory cortex and then invade other cortical and thalamic areas (Inoue et al., 1993; Manning et al., 2004; Meeren et al., 2002; Richards et al., 2003; Seidenbecher et al., 1998; Steriade and Contreras, 1995, 1998). Pathophysiologic mechanisms in the cortex may relate to an increase in excitatory synaptic transmission mediated via N-methyl-d-aspartate (NMDA) receptors (Pumain et al., 1992), a decrease in GABAergic inhibition (Luhmann et al., 1995) and an altered expression of the hyperpolarizationactivated cyclic-nucelotide-gated cation channels (HCN) (Strauss et al., 2004). Cortical SWDs (Figure 1C) reach the reticular thalamic (RT) nucleus via corticofugal fibers (Figure 1B). The response of RT neurons consists of bursts of excitatory postsynaptic potentials (EPSPs) and a regenerative low-threshold Ca2+ spike (LTS; Figure 1D) crowned by a burst of fast spikes (Slaght et al., 2002; Steriade, 1997; Steriade et al., 1993a; Timofeev et al., 1998). The Ca2+ potential is produced by activation of a Ca2+ current with low threshold of activation, termed IT, which requires membrane hyperpolarization for de-inactivation (Huguenard, 1996). The overall response is synchronized burst activity in RT neurons coinciding with the SWDs on the EEG. Pathophysiologic alterations that have been reported with respect to SWD generation include an increase in strength of corticofugal inputs (Blumenfeld and McCormick, 2000), an increase in T-current amplitude associated with an increased expression of the Cav3.2 subunit in RT neurons (Tsakiridou et al., 1995; Talley et al., 2000), and an imbalance of GABAA receptor- and gap junction-mediated synaptic interactions involved in synchronization of local synaptic networks (Bal et al., 1995a, 1995b; Huntsman et al., 1999). Circuitry within the thalamus promotes recurrent network activity. RT neurons are GABAergic in nature and are reciprocally connected with excitatory thalamocortical (TC) neurons of the corresponding sensory relay nuclei (Figure 1B), for instance, the VB thalamic complex of the somatosensory system (for review, see Steriade et al., 1993b, 1997). In response to the incoming RT volleys, TC neurons produce inhibitory postsynaptic potentials (IPSPs; Figure 1E) mediated via GABAA and GABAB receptors, which result in de-inactivation of the T-type Ca2+ current (Crunelli and Leresche, 2002; Steriade et al., 1993b, 1997). The
In Vitro Models of Absence Epilepsy
current is activated on repolarization of the membrane potential during IPSP decay, resulting in a regenerative lowthreshold calcium spike triggering a burst of fast spikes, which is then relayed to the RT nucleus and cortex. As a corollary of this activity, the cortico-thalamo-cortical network produces synchronized burst discharges that are temporally locked to the spike component of the SWDs on the EEG (Crunelli and Leresche, 2002; Steriade et al., 1993b, 1997). The pivotal role of the T-type Ca2+ conductance in thalamic neurons is supported by the findings that knockout of the Cav3.1 subunit results in resistance to gbutyrolactone-induced SWDs (Kim et al., 2001). Further, an imbalance of GABAB over GABAA receptor-mediated inhibition has been reported to shift activity in TC neurons toward SWD-like discharges (Bal et al., 1995a, b; Crunelli and Leresche, 1991; Lui et al., 1992; Snead, 1992; Snead et al., 1992), although GABAA receptor-mediated influences dominate during spontaneous SWDs in rat genetic models (Staak and Pape, 2001), and the balance between GABAA and GABAB seems to relate to the prevailing frequency of the SWDs (3–5 Hz in humans versus 7–11 Hz in most animal models) (Destexhe, 1998). It also should be noted that slightly different conclusions have been reached in various models with respect to the relative contribution of lowthreshold burst firing and of GABA potentials to the SWDs in TC neurons (Pinault et al., 1998). Furthermore, there is evidence for an involvement of neurons in intralaminar thalamic nuclei to the SWDs (Figure 2), although their exact role remains to be delineated (Seidenbecher and Pape, 2001). Because the relative roles played by the different components of the thalamocortical system in the generation of SWDs are still a matter of debate, experimental models with different network connectivity (i.e., thalamic, cortical, and thalamocortical models) have been developed, thereby helping to assess their specific contributions.
IN VITRO MODELS OF ABSENCE EPILEPSY The Ferret Dorsal Lateral Geniculate Nucleus Slice: A Model for the Transformation of Spindle Waves into Spike-Wave Discharges A number of thalamic in vitro models have been developed to investigate the mechanisms underlying the generation and spread of spike wave (SW)-like discharges in this brain structure. The ferret primary visual thalamic relay nucleus (dorsal part of the lateral geniculate nucleus, LGNd) and the associated section in the RT nucleus (termed the perigeniculate nucleus, PGN) have proven particularly useful. A sufficient part of the synaptic network is preserved in a slice preparation in vitro to generate spontaneous recur-
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rent, spindle-like activity (Figure 3A), and this pattern of activity can be acutely transformed into SW-like discharges (Figure 3B) on pharmacologic manipulation (Bal et al., 1995a, b; von Krosigk et al., 1993). Ferret thalamic slices can be obtained and studied using standard brain slicing and electrophysiologic recording techniques. Preparation includes decapitation of deeply anaesthetized (pentobarbital, 30–40 mg/kg intraperitoneally) male or female animals 3 months to 3 years old. Sagittal slices (400-mm thickness) from the forebrain of one hemisphere are prepared using a vibratome or vibroslicer. During preparation, tissue should be placed in a chilled solution in which NaCl is replaced with sucrose while maintaining an osmolarity of ~305 mOsm. After preparation, slices are placed in an interface style recording chamber and allowed at least 2 hours to recover. The bathing solution contains (in mM): NaCl, 126; KCl, 2.5; MgSO4, 1.2; NaH2PO4, 1.25; CaCl2, 2; NaHCO3, 26; dextrose, 10. A pH of 7.4 is achieved by gassing the solution with 95% O2 and 5% CO2. Bathing of the slices in an equal mixture of normal NaCl and the sucrose-substituted solutions for the first 20 minutes in the recording chamber may be beneficial. Intraspindle and particularly interspindle frequencies as well as the duration of each spindle wave are temperature sensitive; a bath temperature of 34 to 35° C is optimal for studying the cellular basis of spindle waves and SWDs in vitro. Typically each hemisphere of the ferret LGNd yields two or three slices that exhibit robust spontaneous spindling activity in more than 95% of experiments. Extracellular multiunit recordings from ferret LGNd slices, using standard electrophysiologic techniques, revealed the occurrence of spontaneous spindle waves that were remarkably similar to those recorded in vivo in anaesthetized ferrets (interspindle period of 3–30 s, intraspindle frequency 6–10 Hz, spindle duration 2–5 s). Intracellular recordings obtained with bevelled micropipettes pulled from medium-walled borosilicate glass and filled with 4 M potassium acetate allowed the identification of the cellular mechanisms underlying spindling in TC neurons of the LGNd. It was shown that spindle waves are associated with barrages of IPSPs occurring at a frequency of 6 to 10 Hz, occasionally resulting in the generation of low-threshold Ca2+ spikes (Figure 3A). Most important, the bath application of 20 mM (—)-bicuculline methiodide resulted in a prolongation and an increase in amplitude of these IPSPs, leading to a highly synchronized 2- to 4-Hz oscillation in which each TC cell generated action potential bursts on every cycle, thus forming a paroxysmal event resembling SWDs (Figure 3B). Bath application of the GABAB antagonist 2-OH-saclofen (250 mM) abolished synchronized 2- to 4-Hz oscillations in ferret LGNd slices (Bal et al., 1995a); the effect of the classic antiabsence drug ethosuximide (ETX) has not been reported.
A
C
B
D
E
FIGURE 2 Correlated cellular activities in thalamus and neocortex during spike-wave discharges (SWDs). A: Simultaneous recording of multiunit activities in ventrobasal nucleus (VPM), rostral reticular thalamic nucleus (rRT), and somatosensory cortical sites. Epidural recordings of the bilateral electroencephalogram (EEG) (r, right; l, left hemisphere) from frontoparietal cortical areas. Calibration bars indicate 500 mV for EEG recording, 100 mV for unit activity. B: Inset demonstrates the temporal relationship of SWDs and burst-like activity at a faster time scale. C–E: Multisite unit recordings were simultaneously obtained from deep layers of the somatosensory cortex, rRT and caudal reticular thalamic (cRT) nucleus (C), cortex, rRT, ventroposterolateral thalamic (VPL) nucleus (D), and cortex, ventroposteromedial (VPM) and ventrolateral (VL) thalamic nucleus (E). Peristimulus-time histograms of unit activity (1-ms bins) triggered by the spike component on the EEG (upper traces in C—E show averaged SWDs) demonstrate phase locked unit activity. Stimulus-time histograms were averaged from 40 trials; n indicates number of animals. (Reprinted, with permission, from Seidenbecher et al., 1998.)
In Vitro Models of Absence Epilepsy ferret LGNd slice
A control -71 mV
B 20 µM bicuculline -70 mV
20 mV 1s
FIGURE 3 Block of g-amino-butyric acid (GABA)A receptors leads to spike-wave discharge (SWD)-like synchronized oscillations in ferret dorsal part of the lateral geniculate nucleus (LGNd) slices. Intracellular recording from a thalamocortical (TC) neuron during the generation of a normal spindle wave. B: Bath application of (-)- bicuculline methiodide (20 mM) results in a slowing of the frequency of oscillations from 5 to 2.4 Hz and to a marked enhancement of low-threshold spike (LTS)-associated burst firing. (Adapted from Bal et al., 1995.)
The Horizontal Thalamic Slice: A Model for Intrathalamic Rhythmicity A rodent in vitro thalamic slice preparation has been developed that contains interconnected VB and RT neurons and that is capable of generating sustained low-frequency (2–4 Hz) oscillations (Huguenard and Prince, 1994b). The horizontal thalamic slice provides a preparation that can be obtained easily and that allows straightforward analyses of genetic mutations or pharmacologic interventions on intrathalamic rhythmicity. Horizontal thalamic slices are taken through the middle portion of the RT nucleus (stereotaxic levels 2.1–4.1 mm posterior from bregma), which contains the adjacent ventral posterior lateral nucleus (see Paxinos and Watson, 1997). Rat pups P8–P26 are anesthetized (50 mg/kg pentobarbital) and decapitated. Brains are rapidly removed and placed in chilled (4° C) low-Ca2+/low-Na+ slicing solution consisting of (in mM): sucrose, 234; glucose, 11; NaHCO3, 24; KCl, 2.5; NaH2PO4, 1.25; MgSO4; CaCl2, 0.5; equilibrated with a mixture of 95% O2, 5% CO2. Thereafter a block of brain containing the thalamus is transferred to a vibratome, and 400-mm slices are obtained in the horizontal plane, hemisected and submerged in preheated (33° C), oxygenated physiological saline containing (in mM): NaCl, 126; glucose, 11; NaHCO3, 26; KCl, 2.5; NaH2PO4, 1.25; MgSO4, 0.63; CaCl2, 2; equilibrated with a mixture of 95% O2, 5% CO2. After 1 hour the heat is turned off and slices are allowed to cool to room temperature. For electrophysiologic recordings, slices are placed in an interface-type recording chamber with continuous perfusion (2 ml/min) of physiologic saline at 33° C. Multiunit extracellular recordings can be obtained from thalamic relay neurons in VB and from GABAergic neurons from the RT
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nucleus using sharpened tungsten electrodes (0.1 MW resistance). Recordings are amplified and filtered above 100 Hz. Intrathalamic network oscillations driven by synaptic interactions between RT and VB neurons can be evoked by stimulation of the internal capsule. Bicuculline methiodide (1–10 mM) added to the bathing medium results in a prolongation and increase in synchronization of oscillatory activity. On the other hand, application of the classic antiabsence drug ETX has a robust anti-oscillatory effect. Typical responses evoked by stimulating the internal capsule (40 ms shocks of 40-V intensity) consist of one to five repetitive bursts, at a frequency of 2 to 4 Hz, that last for 2 to 8 seconds. Recordings are typically stable for more than an hour. The horizontal thalamic slice preparation represents a simple experimental setup that is ideal for screening of antiepileptic drugs and the effects of gene knockout (Huguenard and Prince, 1994a, b; Huntsman et al., 1999; Yue and Huguenard, 2001). Besides the extracellular recordings described previously, the horizontal thalamic slice can be used in conjunction with conventional sharp intracellular (Sohal et al., 2003) or patch-clamp recording techniques (use of a submerged rather than an interface-type chamber is highly recommended for the latter). Whereas blind patchrecordings were described originally (Huguenard and Prince, 1994b), the use of a standard upright microscope equipped with infrared differential interference contrast optics (Dodt and Zieglgänsberger, 1990) allows for the visual identification of single neurons.
The Thalamocortical Slice: A Model for Synchronous Thalamocortical Activity To account for the joint contribution of both thalamus and cortex to the generation of SWDs, in vitro preparations including both regions of the brain have been developed. Originally the mouse thalamocortical slice was introduced as a suitable system for studying the physiology and pharmacology of the thalamocortical synapse and for exploring the synaptic circuitry of the somatosensory cortex (Agmon and Connors, 1991). Later a similar preparation was introduced for rats (Tancredi et al., 2000; Zhang et al., 1996). This thalamocortical slice preparation is a reproducible model that enables investigators to analyze thalamocortical synchronization and to understand the pathogenesis of absence epilepsy (D’Arcangelo et al., 2002; Tancredi et al. 2000). To achieve thalamocortical slices Wistar rats (P15–P28) are decapitated under halothane anesthesia, and their brains are quickly removed and placed in cold, oxygenated artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 124; KCl, 2; KH2PO4, 1.25; MgSO4, 0.5; CaCl2, 2; NaHCO3, 26; glucose, 10; a pH of 7.4 is achieved by bubbling
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with 95% O2/5% CO2. Combined thalamocortical slices (550–650 mm), involving a hybrid coronal plane forming a 45-degree angle with the sagittal plane, can be obtained on a vibratome (Figure 4). Typically two slices are obtained from each hemisphere at the level of the VB. After cutting, slices are transferred to an interface tissue chamber perfused with ACSF (32–35° C). Extracellular field potentials are recorded with ACSF-filled electrodes (2–8 MW) positioned under visual control in cortex and thalamus (VB or RT nucleus). Signals are fed to high-impedance amplifiers and processed through second-stage amplifiers with filtering capability. Bipolar stainless steel electrodes can be used to deliver extracellular stimuli (50–150 ms; <200 mA) to selected areas of the thalamocortical slice. Single-shock stimuli delivered in VB, the internal capsule, or the white matter are able to elicit a complex series of field potential waves in layers IV to V of the cortex, resulting from antidromic activation of corticothalamic axions and synaptic activation of thalamocortical afferents. An additional short latency (12–18 ms) response, resulting from reactivation of thalamocortical neurons in VB, can be observed. Spontaneous synchronous field potentials do not occur in thalamocortical slices bathed in normal ACSF. However, the use of low concentrations of the K+ channel blocker 4-aminopyridine (4-AP, ~1 mM) increases transmitter release at both excitatory and inhibitory synapses and
B
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45-55° Th
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D Cx
thus enhances the background activity of cortical and thalamic neurons. Indeed, with 4-AP, rhythmic oscillatory field potential activity occurs either spontaneously or following electrical stimulation of either thalamus or cortex. These oscillations can be recorded at both cortical (500–1200 mm from the pia) locations and thalamic sites (VB or RT nucleus) and are characterized by bursts of field potential waves of negative-positive polarity at frequencies ranging from 9 to 16 Hz (0.4–3.5 s duration; recurrence of the bursts every 6–24 s). Furthermore, other experimental procedures known to increase neuronal excitability (nominal Mg2+-free ACSF; elevation of the total extracellular K+ concentration to 8.25 mM) are able to induce synchronous oscillatory activity in thalamocortical slices of epileptic WAG/Rij and nonepileptic control rats (with different characteristic features) (D’Arcangelo et al., 2002). Although the effects of different glutamate receptor antagonists on 4-AP-induced oscillatory activity in thalamocortical slices has been described, no data are available for the classic anti-absence drug ETX in this preparation. Epileptiform discharge induced by nominal Mg2+ free ACSF was reduced by ETX (Zhang et al., 1996).
IN VITRO MODELS OF CORTICOCORTICAL CIRCUITRY IN EPILEPSY Whereas thalamocortical involvement is implicated in most forms of typical absence seizures, in some pharmacologic feline epilepsy models, SWDs have been observed in isolated cortical circuits (Marcus and Watson, 1966; Steriade and Contreras, 1998), suggesting that the cortical circuitry itself is capable of generating bilaterally synchronous 3- to 5-Hz electrographic activity. As a preliminary step in testing the involvement of long-range intracortical connections in widespread neocortical synchronization, Kumar and Huguenard (2001) refined a callosal slice preparation based on earlier work of Vogt and Gorman (1982).
3.0 mm 600 µm VB Hip
FIGURE 4 Preparation of rat thalamocortical slices. A: The brain is placed on a ramp with the caudal end elevated above the rostral end at an angle of 10 degrees. Th, approximate location of the thalamus. B: Dorsal view showing angle of initial vertical cut made with a razor blade. C: The cut surface of the brain is then glued to the vibratome stage. Horizontal lines indicate the approximate position of collected slices. Typically two or three slices (550–650 mm in thickness) retain full thalamocortical connectivity. D: Typical positions of recording (left, in cortical barrel field) and stimulating (right, in the external part of the ventrobasal (VB) near the border with the reticular thalamic nucleus (RTN) electrodes in a schematic thalamocortical slice. Cx, cortex; Hip, hippocampus; RTN, reticular thalamic nucleus; VB, ventrobasal thalamus. (After Agmon and Connors, 1991; and Tancredi et al., 2000.)
What Does the Corticocortical Preparation Model? The mammalian neocortex is characterized by extensive recurrent excitatory circuitry. Roughly 85% of synapses are excitatory, and an almost equal percentage of synapses made by excitatory neurons are onto other excitatory neurons (Braitenberg and Schüz, 1991). Such neural networks with prominent positive feedback are prone to become hyperexcitable and seizure generating following disruption of inhibition. The study of epilepsy necessitates model systems in which excitatory corticocortical synapses can be studied in isolation so that response properties of individual neurons and their contribution to the overall excitability of the
In Vitro Models of Corticocortical Circuitry in Epilepsy
network can be characterized. Not only are such model systems useful in assessing pathophysiology, but they are also essential in characterizing normal physiologic properties of excitatory-to-excitatory synapses and in elucidating their receptor compositions. This background is essential for understanding the changes in synaptic connectivity during epileptogenesis, changes that are difficult to detect given the complexity of the underlying circuitry. Attempts to isolate and study synaptic excitatory responses in brain tissue have been frustrating in many models of experimental epilepsy because attempts to block inhibition invariably lead to conditions of “runaway” excitation or hyperexcitability. By contrast, studies of inhibition are more readily accomplished because excitatory receptors can be readily blocked (using glutamatergic receptor antagonists), yielding “residual” synaptic responses that are (for the most part) inhibitory responses mediated by GABA receptors. In the following sections we provide an overview of the development of a callosal slice model that lends itself quite well to the study of long-range cortical excitatory connectivity.
Importance of the Corpus Callosum in a Model for the Study of Intracortical Excitability The corpus callosum is the principal commissural pathway in the forebrain linking the two cerebral hemispheres. The cells of origin of neocortical callosal projections are almost entirely pyramidal neurons, located mainly in layers III and V; the projection terminates exclusively with excitatory asymmetric synapses on spines of pyramidal neurons in homotopic and heterotopic regions of the contralateral cortex (Akers and Killackey, 1978; Jacobson, 1965; Jacobson and Trojanowski, 1974; Seggie and Berry, 1972; Wise and Jones, 1976). Callosal projections can be reliably stimulated (Vogt and Gorman, 1982); because they are purely excitatory, activation of this pathways aids greatly in the study of excitatory-to-excitatory cortical synapses (Aram and Lodge, 1988) and intracortical excitation. Furthermore, the callosum is itself considered a primary substrate for intrahemispheric spread of discharges in generalized epileptic seizures (Gazzaniga et al., 1975; Reeves and O’Leary, 1985; Wilson et al., 1975). Clinical studies have shown that cutting of this pathway (callosotomy) can eliminate seizure activity or decrease its severity and frequency, suggesting that transcortical excitation can initiate or perpetuate epileptiform activity. Excitatory corticocortical projections may play a crucial role in determining the strength and extent of seizure generalization, especially during critical periods in early maturation when neocortical tissue is vulnerable to epileptiform activity (Luhmann and Prince, 1990; Moshe et al., 1983; Swann et al., 1993). However, despite the long history of callosal
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studies in epilepsy, and a wealth of anatomic information on callosal organization, a neocortical preparation involving hemispheric connection via the corpus callosum has only recently been recognized as a viable model for studying intracortical excitation. This delay is due in part to limitations in our knowledge of the functional properties and receptor composition of the synapses made by callosal fibers and the mechanisms that control their efficacy. To overcome this deficiency, Kumar and colleagues (2001) undertook a study to assay the usefulness of callosal activation in a model system for studying intracortical excitability by (1) investigating the receptor composition of the callosal synapse; (2) characterizing the voltage dependence of the pharmacologically isolated components of excitatory postsynaptic currents (EPSCs) evoked by minimal stimulation of the callosum; (3) determining whether kinetic properties of spontaneous and evoked EPSCs recorded in these neurons can reveal differences in function of the underlying receptor populations at callosal and noncallosal synapses; and (4) identifying changes in the physiologic properties of these receptors as a function of early postnatal age (Kumar and Huguenard, 2001). Kumar and colleagues (2001) found that midline stimulation of the corpus callosum in callosally connected acute brain sections of the rat neocortex (Figure 5) could evoke inward EPSCs in whole-cell voltage-clamped (-70 mV) pyramidal neurons in layer V of the agranular frontal cortex. In the presence of the GABAA receptor antagonist picrotoxin (to isolate the EPSCs) and a low concentration of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo [f] quinoxaline7-sulfonamid (NBQX) (0.1 mM) (to block partially a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and thereby prevent epileptiform activity), purely glutamatergic EPSCs were obtained. These isolated EPSCs had fixed latencies from stimulus onset, could follow stimulus trains (1–20 Hz) without changes in kinetic properties, and were completely antagonized with high concentrations of NBQX (10 mM), indicating that they were mediated by AMPA receptors. Depolarization of the recorded neurons revealed a late, slowly decaying component that reversed at ~0 mV and that could be blocked by D-2 amino-5-phosphonovaeric acid (D-APV). These studies showed clearly that callosal synapses involve both AMPA and NMDA receptors. Analysis of the callosal EPSCs revealed age-dependent changes in the composition of the underlying AMPA receptors. In particular, AMPA receptor-mediated responses in immature cortex (tissue from younger than 15 day old rats) were characterized by inward rectification—the hallmark of GluR2-deficient AMPA receptors. Direct measurement of Ca2+ permeability, another characteristic of GluR2 deficiency, confirmed this finding (Kumar et al., 2002). An interesting finding was that the functional properties of AMPA receptors were similar at callosal and noncallosal excitatory connections. By contrast, NMDA receptor-mediated
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response properties were pathway-specific, suggesting differences in subunit composition of the underlying receptors (Kumar and Huguenard, 2003).
Corticocortical Slice Preparation General Considerations
FIGURE 5 The callosal slice preparation and schematic. A1–A5: lowpowered images of a coronal cortical section, obtained with a 10¥ objective, depicting placement of a bipolar stimulating electrode (S in A1) at different locations (•) along the dorsal—ventral extent of the callosum. The corresponding whole-cell responses of a layer V pyramidal neuron (R in A1), evoked by stimulation of the callosum at these locations, are shown as insets. Traces are averaged responses evoked at a holding potential of -70 mV; stimulus intensity was constant throughout. Patch pipette in A1 is visible during whole-cell recording from pyramidal neuron on the right (R, scale bar is 40 mm). Note that responses were especially robust for one nearmidline stimulus location (* in A2). Postsynaptic currents (PSCs) with longer latency and similar kinetic properties could also be evoked from the contralateral hemisphere (A5). Typically the recording region was closer to midline, in the frontal-cortex areas 1 and 2 (Fr1 and Fr2, A1). B: schematic of the slice preparation, showing typical placement of the stimulating (S) and recording (R) electrodes (S1 and S2 for callosal and local intracortical stimulation, respectively) on the slice. Anatomic structures that can be easily recognized are labeled: CC, corpus callosum; CA1 and CA3 regions of the hippocampus; neocortical laminae indicated by Roman numerals. (Reprinted with permission from the American Physiological Society, 2001.)
The primary consideration in making acute callosal slices for studying corticocorticol synapses is to retain and optimize intrahemispheric connectivity. All experiments were carried out in coronal sections of rat (Sprague-Dawley) brains, retaining both hemispheres and the callosal tract. Preserving callosal connectivity in brain slices is complicated owing to the curvature of the corpus callosum. We recommend using straightforward coronal sections (300– 350 mm thick) obtained on a vibratome. We have found that during slicing the presentation angle of the sectioning blade is a critical factor for optimal slice health and connectivity; an angle of 18 degrees relative to the plane of the section is optimal. Using coronal slices prepared in this manner, we have been able to evoke responses in neurons located as lateral as the somatosensory cortex via midline stimulation of the callosum. In general, however, we prefer recording closer to the midline—from the agranular frontal cortex— to maximize callosal connectivity (Figure 6). The thickness of the slice is also a critical factor in determining connectivity; the range of slice thickness we recommend is chosen as a compromise so that we can maintain connectivity but still record from visually identified neurons deep in the tissue. Another important parameter that affects connectivity is the age of the experimental animals. We have used animals in three different developmental age groups for studying the ontogeny of callosally evoked responses: neonates (P6–7), in which callosal fibers just approach their final cortical target lamina; juveniles (P12–16), in which neocortical synaptogenesis approaches adult levels of maturity; and young adults (P20–28), in which synaptogenesis is almost complete and myelination has begun to develop. It is worth noting that these demarcations of age groups overlap a period of early postnatal maturation between P11–20, during which there is transient but well-defined temporal manifestation of strong excitatory polysynaptic activity (NMDA receptor-mediated) leading to an enhanced sensitivity for epileptogenesis. Although the callosal model represents long-range intrahemispheric connections, layer V pyramidal neurons also receive a diverse set of short-range intracortical excitatory afferents that arise locally from neurons in close vicinity to the cells of interest (Burkhalter and Charles, 1990). These latter fibers can also be activated concomitantly with the callosal afferents by using stimulating electrodes placed intracortically in close proximity to the recorded neurons (either off- or on-column), to study any pathway-specific differences
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FIGURE 6 Orthograde transport of biocytin reveals callosal projection in coronal slices. A: A biocytin crystal (~0.5 mm diameter) was placed in layer V of the contralateral cingulate cortex (right side, crystal placement not shown in this figure). The slice was incubated for 3 hours in an interface chamber and then processed to obtain a final horseradish peroxidase reaction product. The arrows in A indicate labeling of the callosal fibers. The square area marked in A is expanded in B. B: A dark-field image showing the callosal fibers coursing upward through the ipsilateral layer V. (See color insert.)
in synaptic properties. Previous work suggests that although functional attributes of AMPA receptors are uniform among synaptic connections onto layer V pyramidal neurons (i.e., callosal and noncallosal excitatory inputs onto these cells are indistinguishable), properties of NMDA receptors at these inputs can be functionally distinct (Kumar and Huguenard, 2003). How Sections Are Prepared Standard techniques for preparing and maintaining neocortical slices are employed (reviewed in Kumar and Huguenard, 2001). Briefly, Sprague-Dawley rats of the desired age are anesthetized (50 mg/kg pentobarbital), decapitated, and the brain rapidly removed and transferred to a chilled (4° C) low Ca2+, low Na+ slicing solution equilibrated with a 95% : 5% mixture of O2 and CO2. The brain is subsequently blocked and coronal slices, 350mm thick, are prepared on a vibratome with the blade set at the appropriate cutting angle. All slices are incubated at 32° C in oxygenated artificial cerebrospinal fluid (ACSF) for 1 hour before being transferred to the recording chamber. Generally, four to six slices are obtained per animal; slices remain viable for up to 6 hours. Electrophysiologic Techniques, Data Collection, and Analysis Recordings are obtained at 32 ± 1° C from layer V pyramidal neurons in the agranular frontal cortex (Paxinos and
Watson, 1997). Patch recording electrodes (1.2–2 mm tip diameters; 3–6 MW) typically contained the following electrolyte (in mM): for voltage-clamp experiments, 120 cesium gluconate, 1 MgCl2, 1 CaCl2, 11 KCl, 10 HEPES, 2 NaATP, 0.3 NaGTP, 1 QX-314, and 11 EGTA (pH 7.3 was corrected with Cs-OH, 290 mOsm); for current-clamp experiments, 105 potassium gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 MgATP, and 0.3 GTP (adjusted to pH 7.3 with KOH). Slices are maintained in oxygenated (95% O2-5% CO2) ACSF, and drugs and chemicals are applied via the perfusate (2 ml/min) or through a local perfusion system that allows fast exchange of media at the level of the synapse (Kumar et al., 2002). Concentric bipolar electrodes (e.g., CB-XRC75 from Frederick Haer Company, Bowdoinham, ME), with 75-mm-tip diameters, are positioned on the callosal tract or intracortically in close proximity to the recorded neuron; constant current pulses (50–300 ms in duration and 100–500 mA in amplitude) are applied at low frequencies (0.1–0.3 Hz). Callosal stimulation activates fibers potentially in both orthodromic and antidromic directions, each of which in turn activates monosynaptic excitatory connections onto the recorded pyramidal neuron (Kumar and Huguenard, 2001). Thus this model consists of activating a well-defined, relatively homogeneous population of intracortical excitatory connections. Stimulation parameters are determined by increasing current strength until postsynaptic responses can be evoked (minimal stimulation); stimulus intensity is held constant at about 1.2 times the threshold for obtaining a detectable response throughout the duration of the experiment (thresholds are characterized by
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a large proportion of failures). Characterization of synaptic currents mediated by AMPA and NMDA receptors requires the isolation of purely excitatory monosynaptic responses with minimal contribution from polysynaptic events, such as those arising from feed-forward excitation. Toward this end, the minimal stimulation paradigm outlined previously can be used in conjunction with a pharmacologically controlled blockade of inhibition. Callosally evoked responses can be compared with EPSCs evoked during stimulation of local excitatory circuitry in the vicinity of the recorded neuron to determine differences in the biophysical properties of the underlying receptor subtypes and the input specificity of their responses. Evoked responses are recorded with a patch amplifier filtered at 2 to 5 kHz and digitized at 10 kHz. Solutions The low-Ca2+, low Na+ slicing solution consists of (in mM): 234 sucrose, 11 glucose, 24 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2. The composition of the ACSF used in the incubation and perfusion (bathing medium) of the slices during recording is as follows: 126 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, and 10 glucose, pH 7.4. The following are bath-applied as required for specific protocols: APV, 40 mM; NBQX, 0.05–0.1 mM; picrotoxin (PTX), 50 mM. Biocytin (0.1%) can be included in the patch solution in experiments in which histologic processing is required.
Using the Corticocortical Model: An Example Intrinsic excitatory connectivity is the primary mediator for the generation and spread of epileptiform activity within the neocortex. During maturation, there is a critical period during which normal cortical tissue appears to be most epileptogenic (Luhmann and Prince, 1990; Moshe et al., 1983; Swann et al., 1993). This maturational dynamic presents an opportunity to study changes in the functional properties of intrinsic excitatory connections and the cellular mechanisms that lead to epileptiform activity. To this end, we have used the callosal model to determine how the ontogeny of excitatory connections compares with the development of intrinsic neuronal excitability and epileptogenicity within the frontal cortex. Given that the incidence of seizure generalization is critically dependent on the maturation of corticocortical connections, we used animals from developmentally distinct age groups to assay the ontogeny of synaptic responses in visually identified layer II/III and V pyramidal neurons. We then determined whether changes in the postsynaptic properties of these excitatory connec-
tions lead to alterations in synaptic efficacy, thereby making the tissue more likely to engage in epileptiform activity. The goals of these studies included the hope that our findings would aid in the identification of candidate cellular mechanisms that produce changes in synaptic efficacy, thereby providing useful information about the role of excitation in the developmental regulation of epileptiform events. We also anticipated that these studies would help address two critical basic neurobiological questions: (1) Do maturational changes in corticocortical synaptic efficacy parallel the transient manifestation of the period of hyperexcitability in the neocortex? (2) Do the functional properties and relative contribution of the different excitatory receptors (AMPA vs. NMDA) change during this critical period? Using the callosal-connected cortical model, we discovered that synaptic AMPA receptors of excitatory layer 5 pyramidal neurons in the rat neocortex were deficient in GluR2 in early development (before P16), as evidenced by their inwardly rectifying current-voltage relationship, blockade of AMPA receptor-mediated EPSCs by external and internal polyamines, permeability to Ca2+, and GluR2 immunoreactivity. Our results indicated that neocortical pyramidal neurons underwent a developmental switch in the Ca2+ permeability of their AMPA receptors through an alteration of the AMPA subunit composition (Kumar et al., 2002). This finding led us to explore the general question of whether all cortical glutamatergic receptors change their subunit composition during maturation? If that were so, we could begin to understand how these alterations influence the development of intrinsic neuronal excitability and epileptogenicity. Early low expression of GluR2 subunits or the failure to incorporate the GluR2 subunit into AMPA receptors during the developmental critical period might underlie increased seizure susceptibility of the immature brain (Moshe et al., 1983; Schwartzkroin and Prince, 1980; Sensi et al., 1999). Indeed, one study (Sanchez et al., 2001) demonstrated that hypoxia-induced seizures in neonatal rats (P10–12) are linked with maturational and seizure-induced changes in AMPA receptor composition and function, particularly involving the GluR2 subunit (see Chapter 25). These results indicated that seizures induce an increased expression of Ca2+-permeable AMPA receptors and an increased capacity for AMPA receptor-mediated epileptogenesis. The AMPA receptor switch described in our study occurs approximately midway through the period of maximum synaptogenesis in rats (P11–20) (Sutor and Luhmann, 1995) and results in changes in the functional properties of AMPA receptors that depend on the presence or absence of GluR2. Failure to switch the subunit composition of these receptors to incorporate GluR2 at this juncture might indeed have deleterious consequences for neocortical excitability (Feldmeyer et al., 1999; PellegriniGiampietro et al., 1997).
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Conclusions
FURTHER DEVELOPMENT OF THE MODEL Insights into Human Conditions Callosal projections (i.e., excitatory synapses onto excitatory cells) have long been known to provide a primary pathway for intrahemispheric generalization of seizure activity. Callosotomy has emerged as one of the only surgical treatment for patients with medically intractable seizures who are not eligible for resective surgery. A variety of seizure types have been considered as appropriate for callosotomy surgery, including complex partial seizures and frontal lobe epilepsies (Reeves and O’Leary, 1985). The reported efficacy of such intervention for epilepsy (such as a reduced frequency of generalized seizures) varies from one study to another. Currently there are no good models available to address precisely how seizures propagate and generalize. What are the underlying cellular events? In vitro models such as the ones considered in this chapter constitute an invaluable resource for studying such questions that are directly related to the human condition. For instance, it is possible, using the corticocortical model, to generate an epileptic focus in one hemisphere of the brain section (e.g., by disrupting inhibition focally) to explore the precise features of its spread to the other hemisphere; this model allows the investigator to elucidate synaptic changes at the single cell level (intracellular recording), to detail the mechanisms underlying seizure propagation, and to describe “global” changes at the level of the network (field recordings). The callosal model is also amenable to exploration of therapeutic approaches, pharmacologic or otherwise, for preventing intrahemispheric spread of epileptic discharges. In addition to pathophysiology, structural changes following callosotomy can also be investigated using a combined in vivo and in vitro approach in which the effects of callosotomy in vivo can be followed both anatomically (for neuropathology) and physiologically in vitro using brain sections obtained from such animals.
LIMITATIONS OF THE THALAMIC, THALAMOCORTICAL, AND CORTICOCORTICAL MODEL Several technical and methodological difficulties can be expected in the course of using the thalamic, thalamocortical, and corticocortical models: (1) Perhaps most critically, it is sometimes difficult to obtain intrathalamic, thalamocortical, or callosal connectivity, despite the intact appearance of VB-RT, thalamocortical, or callosal axons. In such instances it may not be possible to evoke any responses in the recorded neurons, despite the presence of normal spontaneously occurring postsynaptic currents. (2) Neurons that contribute axons to the corpus callosum (or internernal
capsule) may not be uniformly distributed throughout the neocortex, and different cortical areas may have different distribution profiles that might be subject to change during development (Ivy and Killackey, 1981; Olavarria and van Sluyters, 1986). This limitation therefore restricts the number of anatomically distinct regions that can be studied using coronal sections. (3) As is the case for many preparations, it may not be possible to adequately voltage-clamp— from a single point source—neurons with extensive dendritic arborizations. Recordings of PSCs may be contaminated by errors resulting from the extended electrotonic structure of the recorded neurons. (4) As is commonly found for slice studies, whole-cell recording from neurons in tissue from older animals can be technically more challenging than those in younger animals; obtaining long-term, high-quality recordings is simply more difficult in mature tissue. (5) Finally, one must acknowledge the in vitro nature of the preparation and exercise all necessary precautions that go into interpreting results and extending them to situations in vivo. One potentially effective means of improving connectivity and extending the number of areas that can be studied using these models is to increase the thickness of the brain sections. However, there is a tradeoff between connectivity and visualization. Clear visualization of neurons, especially those located deep within the sections (usually the ones with the best intrahemispheric connectivity in callosal slices), is generally difficult in thicker sections (despite the use of infrared video microscopy). This limitation is particularly problematic in thalamic areas, such as RT and lateral VB, which contain densely packed myelinated axons. Another potentially powerful approach to improving the extent of callosal connectivity in brain sections is to take into account the global curvature of the corpus callosum and design sectioning techniques that follow curved trajectories. For instance, the callosal projections to areas OC1 and OC2 of the rat visual cortex are generally inaccessible to studies using brain slices on account of their curved pathway. However, Berry et al. (1990) developed a slice preparation in which curved cutting blades were used to circumvent this problem; they have obtained slices in which callosal fibers projecting to OC1 or OC2 are preserved.
CONCLUSIONS We have described various in vitro model systems that allow for careful dissection of cellular, synaptic, and network interactions that occur during seizure genesis. Each of these has advantages and disadvantages; as pointed out by in a review of absence epilepsy by Crunelli and Leresche (2002): “There is no established in vitro model that is capable of fully reproducing human SWDs.” Some models may accurately reproduce the types of neural networks that
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become activated during seizures, but insights regarding the mechanisms of initiation of seizures have remained quite elusive. In the intact brain, absence seizures arise spontaneously, often from a normal EEG background. This type of sudden transition is rarely seen in vitro, likely because of the disconnected (deafferented) state of brain slices. It is hoped that future models will incorporate more complete circuitry that will approximate the intact brain and yet still allow the quantitative intracellular recordings that are required for elucidation of epileptic mechanisms. For example, a preparation retaining the entire cerebral cortex has recently been developed by Kilb and Luhmann (2003).
References Agmon, A., and Connors, B.W. 1991 Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro. Neuroscience 41: 365–379. Akers, R.M., and Killackey, H.P. 1978. Organization of corticocortical connections in the parietal cortex of the rat. J Comp Neurol 181: 513–537. Aram, J.A., and Lodge, D. 1988. Validation of a neocortical slice preparation for the study of epileptiform activity. J Neurosci Methods 23: 211–224. Bal, T., von Krosigk, M., and McCormick, D.A. 1995a. Role of the ferret perigeniculatate nucleus in the generation of synchronized oscillations in vitro. J Physiol (Lond) 483(3): 665–685. Bal, T., von Krosigk, M., and McCormick, D.A. 1995b. Synaptic and membrane mechanisms underlying synchronized oscillations in the ferret lateral geniculate nucleus in vitro. J Physiol (Lond) 483(3): 641–663. Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud’homme J.F., Baulac, M. et al. 2001. First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2subunit gene. Nat Genet 28: 46–48. Berry, R.L., Nowicky, A., and Teyler, T.J. 1990. A slice preparation preserving the callosal projection to contralateral visual cortex. J Neurosci Methods 33: 171–178. Blumenfeld, H., and McCormick, D.A. 2000. Corticothalamic inputs control the pattern of activity generated in thalamocortical networks. J Neurosci 20: 5153–5162. Braitenberg, V., and Schüz, A. 1991. Anatomy of the Cortex. Berlin: Springer-Verlag. Burkhalter, A., and Charles, V. 1990. Organization of local axon collaterals of efferent projection neurons in rat visual cortex. J Comp Neurol 302: 920–934. Coenen, A.M., Drinkenburg, W.H., Inoue, M., and van Luijtelaar, E.L. 1992. Genetic models of absence epilepsy, with emphasis on the WAG/Rij strain of rats. Epilepsy Res 12: 75–86. Crunelli, V., and Leresche, N. 1991. A role for GABAB receptors in excitation and inhibition of thalamocortical cells. TINS 14: 16–21. Crunelli, V., and Leresche, N. 2002. Childhood absence epilepsy: genes, channels, neurons and networks. Nat Rev Neurosci 3: 371–382. D’Arcangelo, G., D’Antuono, M., Biagini, G., Warren, R., Tancredi, V., and Avoli, M. 2002. Thalamocortical oscillations in a genetic model of absence seizures. Eur J Neurosci 16: 2383–2393. Danober, L., Deransart, C., Depaulis, A., Vergnes, M., and Marescaux, C. 1998. Pathophysiological mechanisms of genetic absence epilepsy in the rat. Prog Neurobiol 55: 27–57. Destexhe, A. 1998. Spike-and-wave oscillations based on the properties of GABAB receptors. J Neurosci 18: 9099–9111. Destexhe, A., and Sejnowski, T.J. 2003. Interactions between membrane conductances underlying thalamocortical slow-wave oscillations. Physiol Rev 83: 1401–1453.
Dodt, H.U., and Zieglgänsberger, W. 1990. Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res 537: 333–336. Engel, J. 2001. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classification and Terminology. Epilepsia 42: 796–803. Feldmeyer, D., Kask, K., Brusa, R., Kornau, H.C., Kolhekar, R., Rozov, A., Burnashev, N., et al. 1999. Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B. Nat Neurosci 2: 57–64. Felix, R. 2002. Insights from mouse models of absence epilepsy into Ca2+ channel physiology and disease etiology. Cell Mol Neurobiol 22: 103–120. Gazzaniga, M.S., Risse, G.L., Springer, S.P., Clark, D.E., and Wilson, D.H. 1975. Psychologic and neurologic consequences of partial and complete cerebral commissurotomy. Neurology 25: 10–15. Gervasi, N., Monnier, Z., Vincent, P., Paupardin-Tritsch, D., Hughes, S.W., Crunelli, V., and Leresche, N. 2003. Pathway-specific action of gammahydroxybutyric acid in sensory thalamus and its relevance to absence seizures. J Neurosci 23: 11469–11478. Gloor, P., and Fariello, R.G. 1988. Generalized epilepsy: some of its cellular mechanisms differ from those of focal epilepsy. TINS 11: 63–68. Huguenard, J.R. 1996. Low-threshold calcium currents in central nervous system neurons. Annu Rev Physiol 58: 329–3248. Huguenard, J.R., and Prince, D.A. 1994a. Clonazepam suppresses GABAB-mediated inhibition in thalamic relay neurons through effects in nucleus reticularis. J Neurophysiol 71: 2576–2581. Huguenard, J.R., and Prince, D.A. 1994b. Intrathalamic rhythmicity studied in vitro: nominal T-current modulation causes robust antioscillatory effects. J Neurosci 14: 5485–5502. Huntsman, M.M., Porcello, D.M., Homanics, G.E., DeLorey, T.M., and Huguenard, J.R. 1999. Reciprocal inhibitory connections and network synchrony in the mammalian thalamus. Science 283: 541–543. Inoue, M., Duysens, J., Vossen, J.M., and Coenen, A.M. 1993. Thalamic multiple-unit activity underlying spike-wave discharges in anesthetized rats. Brain Res 612: 35–40. Ivy, G.O., and Killackey, H.P. 1981. The ontogeny of the distribution of callosal projection neurons in the rat parietal cortex. J Comp Neurol 195: 367–389. Jacobson, S. 1965. Intralaminar, interlaminar, Callosal and thalamocortical Connections in frontal and parietal areas of the albino rat cerebral cortex. J Comp Neurol 124: 131–145. Jacobson, S., and Trojanowski, J.Q. 1974. The cells of origin of the corpus callosum in rat, cat and rhesus monkey. Brain Res 74: 149–155. Jouvenceau, A., Eunson, L.H., Spauschus, A., Ramesh, V., Zuberi, S.M., Kullmann, D.M., and Hanna, M.G. 2001. Human epilepsy associated with dysfunction of the brain P/Q-type calcium channel. Lancet 358: 801–807. Kilb, W., and Luhmann, H.J. 2003. Carbachol-induced network oscillations in the intact cerebral cortex of the newborn rat. Cereb Cortex 13: 409–421. Kim, D., Song, I., Keum, S., Lee, T., Jeong, M.J., Kim, S.S., McEnery, M.W. et al. 2001. Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels. Neuron 31: 35–45. Kostopoulos, G., Gloor, P., Pellegrini, A., and Siatitsas, I. 1981. A study of the transition from spindles to spike and wave discharge in feline generalized penicillin epilepsy: EEG features. Exp Neurol 73: 43–54. Kumar, S.S., Bacci, A., Kharazia, V., and Huguenard, J.R. 2002. A developmental switch of AMPA receptor subunits in neocortical pyramidal neurons. J Neurosci 22: 3005–3015. Kumar, S.S., and Huguenard, J.R. 2001. Properties of excitatory synaptic connections mediated by the corpus callosum in the developing rat neocortex. J Neurophysiol 86: 2973–2985.
References Kumar, S.S., and Huguenard, J.R. 2003. Pathway-specific differences in subunit composition of synaptic NMDA receptors on pyramidal neurons in neocortex. J Neurosci 23: 10074–10083. Luhmann, H.J., Mittmann, T., van Luijtelaar, G., and Heinemann, U. 1995. Impairment of intracortical GABAergic inhibition in a rat model of absence epilepsy. Epilepsy Res 22: 43–51. Luhmann, H.J., and Prince, D.A. 1990. Transient expression of polysynaptic NMDA receptor-mediated activity during neocortical development. Neurosci Lett 111: 109–115. Lui, Z., Vergnes, M., Depaulis, A., and Marescaux, C. 1992. Involvment of intrathalmic GABAB neurotransmission in the control of absence seizures in the rat. Neuroscience 48: 87–93. Manning, J.P., Richards, D.A., Leresche, N., Crunelli, V., and Bowery, N.G. 2004. Cortical-area specific block of genetically determined absence seizures by ethosuximide. Neuroscience 123: 5–9. Marcus, E.M., and Watson, C.W. 1966. Studies of the bilateral cortical callosal preparation. Trans Am Neurol Assoc 91: 291–293. Marescaux, C., Vergnes, M., and Depaulis, A. 1992. Genetic absence epilepsy in rats from Strasbourg–A review. J Neural Transm 35: 37–69. McCormick, D.A., and Contreras, D. 2001. On the cellular and network bases of epileptic seizures. Annu Rev Physiol 63: 815–46. Meeren, H.K., Pijn, J.P., van Luijtelaar E.L., Coenen, A.M., and Lopes da Silva, F.H. 2002. Cortical focus drives widespread corticothalamic networks during spontaneous absence seizures in rats. J Neurosci 22: 1480–1495. Moshe, S.L., Albala B.J., Ackermann, R.F., and Engel, J. Jr. 1983. Increased seizure susceptibility of the immature brain. Brain Res 283: 81–85. Olavarria, J., and van Sluyters, R.C. 1986. Axons from restricted regions of the cortex pass through restricted portions of the corpus callosum in adult and neonatal rats. Brain Res 390: 309–313. Paxinos, G., and Watson, C. 1997. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press. Pellegrini-Giampietro, D.E., Gorter, J.A., Bennett, M.V., and Zukin, R.S. 1997. The GluR2 (GluR-B) hypothesis: Ca(2+)-permeable AMPA receptors in neurological disorders. Trends Neurosci 20: 464– 470. Pietrobon, D. 2002. Calcium channels and channelopathies of the central nervous system. Mol Neurobiol 25: 31–50. Pinault, D., Leresche, N., Charpier, S., Deniau, J.M., Marescaux, C., Vergnes, M., and Crunelli, V. 1998. Intracellular recordings in thalamic neurones during spontaneous spike and wave discharges in rats with absence epilepsy. J Physiol (Lond) 509: 449–456. Pumain, R., Louvel, J., Gastard, M., Kurcewicz, I., and Vergnes, M. 1992. Responses to N-methyl-D-aspartate are enhanced in rats with petit mallike seizures. J Neural Transm Suppl 35: 97–108. Reeves, A., and O’Leary, P. 1985. Total corpus callosotomy for control of medically intractable epilepsy. In Epilepsy and the Corpus Callosum. Ed. A. Reeves. pp. 269–280. New York: Plenum Press. Renier, W.O., and Coenen, A.M.L. 2000. Human absence epilepsy: The WAG/Rij rat as a model. Neurosci Res Commun 26: 181–191. Richards, D.A., Manning, J.P., Barnes, D., Rombola, L., Bowery, N.G., Caccia, S., Leresche, N. et al. 2003. Targeting thalamic nuclei is not sufficient for the full anti-absence action of ethosuximide in a rat model of absence epilepsy. Epilepsy Res 54: 97–107. Sanchez, R.M., Koh, S., Rio, C., Wang, C., Lamperti, E.D., Sharma, D., Corfas, G. et al. 2001. Decreased glutamate receptor 2 expression and enhanced epileptogenesis in immature rat hippocampus after perinatal hypoxia-induced seizures. J Neurosci 21: 8154–8163. Schwartzkroin, P.A., and Prince, D.A. 1980. Changes in excitatory and inhibitory synaptic potentials leading to epileptogenic activity. Brain Res 183: 61–76. Seggie, J., and Berry, M. 1972. Ontogeny of interhemispheric evoked potentials in the rat: significance of myelination of the corpus callosum. Exp Neurol 35: 215–232.
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Seidenbecher, T., and Pape, H.C. 2001. Contribution of intralaminar thalamic nuclei to spike-and-wave-discharges during spontaneous seizures in a genetic rat model of absence epilepsy. Eur J Neurosci 13: 1537–1546. Seidenbecher, T., Staak, R., and Pape, H.-C. 1998. Relations between cortical and thalamic cellular activities during absence seizures in rats. Eur J Neurosci 10: 1103–1112. Sensi, S.L., Yin, H.Z., Carriedo, S.G., Rao, S.S., and Weiss, J.H. 1999. Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production. Proc Natl Acad Sci USA 96: 2414–2419. Slaght, S.J., Leresche, N., Deniau, J.M., Crunelli, V., and Charpier, S. 2002. Activity of thalamic reticular neurons during spontaneous genetically determined spike and wave discharges. J Neurosci 22: 2323–2234. Snead, O.C.3. 1991 The gamma-hydroxybutyrate model of absence seizures: correlation of regional brain levels of gamma-hydroxybutyric acid and gamma-butyrolactone with spike wave discharges. Neuropharmacology 30: 161–167. Snead, O.C. III 1992. Evidence for GABAB-mediated mechanisms in experimental generalized absence seizures. Eur J Pharmacol 213: 343–349. Snead, O.C. III 1995. Basic mechanisms of generalized absence seizures. Ann Neurol 37: 146–157. Snead, O.C. III, Banerjee, P.K., Burnham, M., and Hampson, D. 2000. Modulation of absence seizures by the GABA(A) receptor: a critical role for metabotropic glutamate receptor 4 (mGluR4). J Neurosci 20: 6218–6224. Snead, O.C. III, Depaulis, A., Banerjee, P.K., Hechler, V., and Vergnes, M. 1992. The GABAA receptor complex in experimental absence seizures in rat: an autoradiographic study. Neurosci Lett 140: 9–12. Sohal, V.S., Keist, R., Rudolph, U., and Huguenard, J.R. 2003. Dynamic GABA(A) receptor subtype-specific modulation of the synchrony and duration of thalamic oscillations. J Neurosci 23: 3649–3657. Staak, R., and Pape, H.C. 2001. Contribution of GABA(A) and GABA(B) receptors to thalamic neuronal activity during spontaneous absence seizures in rats. J Neurosci 21: 1378–1384. Steriade, M. 1997. Synchronized activities of coupled oscillators in the cerebral cortex and thalamus at different levels of vigilance. Cereb Cortex 7: 583–604. Steriade, M., and Contreras, D. 1995. Relations between cortical and thalamic cellular events during transition from sleep patterns to paroxysmal activity. J Neurosci 15: 623–642. Steriade, M., and Contreras, D. 1998. Spike-wave complexes and fast components of cortically generated seizures. I. Role of neocortex and thalamus. J Neurophysiol 80: 1439–1455. Steriade, M., Contreras, D., Curro Dossi, R.C., and Nunez, A. 1993a. The slow (<1 Hz) oscillation in reticular thalamic and thalamocotical neurons: Scenario of sleep rhythm generation in interacting thalamic and neocortical networks. J Neurosci 13: 3284–3299. Steriade, M., Jones, E.G., and McCormick, D.A. 1997. Thalamus. Amsterdam: Elsevier. Steriade, M., McCormick, D.A., and Sejnowski, T.J. 1993b. Thalamocortical oscillations in the sleeping and aroused brain. Science 262: 679–685. Strauss, U., Kole, M.H., Brauer, A.U., Pahnke, J., Bajorat, R., Rolfs, A., Nitsch, R. et al. 2004. An impaired neocortical Ih is associated with enhanced excitability and absence epilepsy. Eur J Neurosci 19: 3048–3058. Sutor, B., and Luhmann, H.J. 1995. Development of excitatory and inhibitory postsynaptic potentials in the rat neocortex. Perspect Dev Neurobiol 2: 409–419. Swann, J., Smith, K., Brady, R., and Pierson, M. 1993. Epilepsy: Models, Mechanisms, and Concepts. Cambridge: Cambridge University Press.
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Talley, E.M., Solorzano, G., Depaulis, A., Perez-Reyes, E., and Bayliss, D.A. 2000. Low-voltage-activated calcium channel subunit expression in a genetic model of absence epilepsy in the rat. Brain Res Mol Brain Res 75: 159–165. Tancredi, V., Biagini, G., D’Antuono, M., Louvel, J., Pumain, R., and Avoli, M. 2000. Spindle-like thalamocortical synchronization in a rat brain slice preparation. J Neurophysiol 84: 1093–1097. Timofeev, I., Grenier, F., and Steriade, M. 1998. Spike-wave complexes and fast components of cortically generated seizures. IV. Paroxysmal fast runs in cortical and thalamic neurons. J Neurophysiol 80: 1495–513. Tsakiridou, E., Bertollini, L., De Curtis, M., Avanzini, G., and Pape, H.C. 1995. Selective increase in T-Type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy. J Neurosci 15: 3110–3117.
van Luijtelaar, E.L., Drinkenburg W.H., van Rijn, C.M., and Coenen, A.M. 2002. Rat models of genetic absence epilepsy: what do EEG spike-wave discharges tell us about drug effects? Methods Find Exp Clin Pharmacol 24(Suppl D): 65–70. Vogt, B.A., and Gorman, A.L. 1982. Responses of cortical neurons to stimulation of corpus callosum in vitro. J Neurophysiol 48: 1257– 1273. von Krosigk, M., Bal, T., and McCormick, D.A. 1993. Cellular mechanisms of a synchronized oscillation in the thalamus. Science 261: 361– 364. Wallace, R.H., Marini, C., Petrou, S., Harkin, L.A., Bowser, D.N., Panchal, R.G., Williams, D.A. et al. 2001. Mutant GABAA receptor g2-subunit in childhood absence epilepsy and febrile seizures. Nat Genet 28: 49–52.
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FIGURE 7 - 1 Intracellular counterparts of spike-wave discharges (SWDs) in GAERS model of absence epilepsy. A: Electroencephalographic (EEG) recording of spontaneous SWDs. B: Schematic diagram of the thalamocortical network. The different colors of neuronal cell types also apply to the intracellular in vivo traces recorded during SWDs in C-E: Green, neurons of reticular thalamic (RT) nucleus; blue, thalamocortical (TC) neuron of the ventrobasal thalamus; red, neuron of the cortical layer V. C: Layer V cortical neurons reveal rhythmic depolarizations, which elicit one to three action potentials, superimposed on a long-lasting hyperpolarization. D: In reticular thalamic (RT) neurons, SWDassociated activity starts with a hyperpolarization (see expanded trace bottom left), followed by rhythmic generation of low-threshold Ca 2+ spikes (LTSs) associated with bursts of action potentials (see expanded trace bottom right). Note that excitatory postsynaptic potentials (EPSPs) lead to the generation of a LTS. E: Thalamocortical (TC) neurons of the ventrobasal thalamus show rhythmic sequences of EPSP and inhibitory postsynaptic potentials (IPSPs), with occasional firing of action potentials. An EPSP/IPSP sequence is expanded in the bottom panel. (C-E adapted, with permission, from Crunelli and Leresche, 2002.)
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FIGURE 7 - 6 Orthograde transport of biocytin reveals callosal projection in coronal slices. A: A biocytin crystal (-0.5 mm diameter) was placed in layer V of the contralateral cingulate cortex (right side, crystal placement not shown in this figure). The slice was incubated for 3 hours in an interface chamber and then processed to obtain a final horseradish peroxidase reaction product. The arrows in A indicate labeling of the callosal fibers. The square area marked in A is expanded in B. B: A dark-field image showing the callosal fibers coursing upward through the ipsilateral layer V.
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8 Studying Epilepsy in the Human Brain In Vitro RÜDIGER KÖHLING, PHILIP A. SCHWARTZKROIN, AND MASSIMO AVOLI
ments on human epileptic brain can be carried out only in vitro (or to a rather limited extent, in the operating room in situ on tissue due to be resected), and so the phenomena available for study reflect the activity of a “reduced” system that does not reveal the overall properties and capabilities of the intact epileptic brain. These limitations are addressed in more detail below. In this chapter we concentrate mainly on electrophysiologic methods for studying resected human epileptic tissue and review much of the work carried out over the past 30 years. Although most of these studies have focused on the question of functional alterations associated with epileptic activity, these studies also illustrate how general principles of neuronal function can be derived from such studies. In this respect, investigations in human epileptic tissue, despite their limitations, offer an exciting possibility to explore many aspects of both normal and pathological human brain function.
OVERVIEW The goal of this chapter is to provide an overview of the methods employed, useful experimental goals, and limitations of investigating human chronically epileptic brain tissue in vitro. Such tissue is often available in the course of epilepsy surgery, particularly for temporal lobe resection associated with intractable mesial temporal lobe epilepsy (MTLE) but also for lesionectomies and other types of epileptogenic “foci.” Human specimens can be maintained in vitro in a slice preparation, and thus they can be studied with many of the technical advantages applied to tissue from animal models. Because human brain slices do not really model epileptic phenomena in the human epileptic brain (i.e., this tissue cannot serve as a model of itself), the format of this chapter departs somewhat from that of the other chapters in this book. In particular we spend little time drawing comparisons to clinical seizure types, elaborating on the challenge of establishing an epilepsy model, or discussing peculiarities of a model in different species. However, we focus not only on the technical aspects of human slice experiments but also on the conceptual issues involved in evaluating the advantages of this particular approach to elucidating basic mechanisms of epilepsy or for advancing drug discovery. Research on human tissue faces specific problems that are not evident in animal experiments. First, for obvious reasons, experiments can be carried out exclusively on tissue that is therapeutically resected and thus per se abnormal. This means that human brain samples are chronically affected by seizures, exposed to wide variations in pharmacologic treatment, and (in almost all cases) resistant to pharmacotherapy. Second, studies on human tissue slices lack proper controls. Third, for obvious ethical reasons, experi-
Models of Seizures and Epilepsy
RELATIONSHIP OF IN VITRO ACTIVITY TO CLINICAL SEIZURE PATTERNS Discussion of seizure types and their relationship to the International League Against Epilepsy (ILAE) classification is not particularly useful within the context of in vitro human slice preparations. It is, to start, important to note that no ictal-like events are observed spontaneously in such preparations (a general property of slices from animals as well as from humans). In contrast, brief interictal-like discharges have been reported (see section on Spontaneous Epileptiform Activity) by several laboratories. However, questions remain regarding the significance of these discharges, especially because we have no “normal” human tissue for
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comparison (see section on Limitations). Although such events could reflect tissue epileptogenicity, they may also reflect (1) normal discharge properties of the tissue of origin, (2) aberrant discharge resulting from presurgical treatment (e.g., drug washout), or even (3) tissue pathology resulting from slice preparation and maintenance. These spontaneous events appear to be quite variable from patient to patient and even across slices within the tissue sample from a given patient. It is notable that there has been no careful attempt to relate such discharges to the activity of that tissue region before resection (i.e., relationship to regional interictal discharge or seizure initiation in the intact brain). A variety of interictal-like and ictal-like electrographic phenomena can be elicited from human tissue samples with experimental manipulations (see section on Evoked Epileptiform Discharges as Models of Epileptiform Synchronization). Similar patterns can, however, also be elicited from “normal” animal tissue. Thus these patterns may not be useful in characterizing the tissue with respect to a particular epilepsy type or manifestation. What may be more significant, in human slices as in slices from animal models, are the sensitivity and threshold of such tissue to epileptogenic manipulations. Unfortunately, here too the absence of appropriate normal controls makes interpretation of threshold data very difficult.
METHODOLOGIC APPROACHES In this section we discuss general aspects of obtaining, slicing, and maintaining the human tissue for both neocortical and hippocampal specimens. In addition, we will consider some of the ethical and health-hazard issues involved in handling human tissue.
resections, this is most easily accomplished with a diagram or photograph showing the resection site. When tissue is removed from deeper structures (e.g., hippocampus), it is useful to get a verbal description from the surgeon regarding the location and extent of the resection. If the tissue is to be divided (e.g., with some going to the clinical pathologist), it is important to maintain a record of which part of the tissue is retained for the slice experiment. In all cases a key piece of information is how the resected tissue is related to the epileptogenic zone. When electrocorticography has been carried out as part of the surgery, electrode positions should be documented relative to the cortical surface so that spiking and nonspiking areas can be identified (Avoli and Olivier, 1989; Colder et al., 1996; Williamson et al., 1995; Schwartzkroin et al., 1983). If electroencephalographic (EEG) monitoring has been carried out before the surgery (e.g., with implanted strips or grids), an attempt should be made to relate the resected tissue to the site(s) of seizure initiation. Clinical, Imaging, and Pathology Background Data A record of presurgical clinical workup is also helpful. The investigator must know patient age and sex, the onset of seizure activity (at what age and for how many years), the clinical seizure diagnosis (seizure type, frequency), and current and past medications. Also useful is information from magnetic resonance imaging (MRI) or other imaging procedures (especially if that helps to localize the resection with respect to abnormal structure and/or function). The pathology report based on the resected tissue is also important and may complement the investigator’s own histology efforts. Relating Tissue Features to Surgical Outcome
Obtaining Human Brain Tissue from Epilepsy Surgery Resections Human tissue samples in most cases result from surgical procedures aimed at treating focal, pharmacoresistant epileptic disorders. These samples derive from partial temporal or extratemporal lobectomy, excision of lesions, or selective hippocampectomy. Rarely, complete lobectomies or functional or anatomic hemispherectomies are undertaken, during which tissue for experimental purposes can be obtained. In some studies, specimens from tumor resections have been investigated (Lücke et al., 1995; Patt et al., 1996; Straub et al., 1992; Williamson et al., 2003). Location and Source of the Tissue and Its Relationship to the Epileptogenic Zone It is critical to obtain a detailed record of precisely where the resected tissue comes from. In the case of neocortical
Although surgical outcome is not necessarily a determinant of whether the resected tissue is actually “epileptogenic,” it is helpful to learn whether the surgical procedure led to cessation of seizures. Presumably, if the answer to this question is yes, the investigator can conclude that the resected tissue contributed to the patient’s epileptic state. Tissue Procurement and Handling The general approach to obtain the tissue is similar across laboratories. As the patient is undergoing surgery, the investigator sets up a tissue-processing station in or adjacent to the operating room. It is useful for the experimenter and the surgeon to have conferred about the nature of the resection beforehand so that the surgeon can deliver the requested samples without the need for discussion at the time of surgery. Tissue samples (often more than one) are excised and immediately placed in a beaker filled with ice-cold
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Methodologic Approaches
(4° C) artificial cerebrospinal fluid (ACSF) saturated with a 95% O2/5% CO2 gas mixture (pH 7.4). The composition of the ACSF, as with animal slice experiments (Dingledine, 1984), varies slightly from group to group. A basic ACSF solution consists of (in mM): NaCl, 124–129; KCl, 2–4; CaCl2, 1.6–2.4; MgSO4 1.3–2, NaH2PO4 1.24–1.25, NaHCO3 21–26 and glucose, 10 (e.g., Schwartzkroin et al., 1983). Instead of NaH2PO4, KH2PO4 can be used; in this instance, the KCl concentration should be 2 mM. Some groups (e.g., Kivi et al., 2000; Köhling et al., 1998b) have used one or more of the following variations as protective solution for transport and slicing procedures of the tissue: (1) 0.1 mM atocopherol as radical scavenger; (2) high-Mg2+ concentration (addition of 2 mM MgCl2) to reduce excitability; (3) low Na+ concentrations (omitting NaCl completely and replacing it with 200 mM sucrose) to reduce neuronal toxicity; and (4) low (1 mM) CaCl2 levels to protect against hypoxia-induced depolarization and Ca2+-mediated cytotoxicity. Tissue handling, from its removal from the patient through all experimental manipulations, must be carried out in accordance with the institutional review board policies for experiments with humans (or human materials). In addition, appropriate precautions should be taken to protect the experimenters from human pathogen exposure. Although rare, the danger of exposure to tissue containing pathogens (e.g., prions associated with Jacob-Creutzfeldt disease) warrants special precautions. Thus it is important not only to adhere to common-sense laboratory practices (e.g., use of gloves, eye shields, etc) but also to clean and disinfect all surfaces exposed to the tissue. It is also highly recommended to have a separate set of instruments (and a separate slice chamber) devoted to human studies.
Slicing Procedures and Tissue Maintenance Tissue samples may come from the neurosurgeon in small blocks (of approximately 1 cm3) or as larger resections (e.g., en bloc hippocampus). If the tissue is to be used for functional (e.g., electrophysiologic or imaging) studies, it should be rapidly sliced into thinner sections (maximally 600 mm in thickness) to ensure proper oxygen supply by diffusion from the surrounding ACSF. In all cases, special care should be taken to slice the block of resected tissue as quickly as possible, maximally within 5 minutes after excision. Preferably this tissue preparation is carried out at a site adjacent to the operating room. Coronal slices are cut with a conventional chopper (e.g., McIllwain type) or with a vibratome (e.g., Campden, Leica, TSE, WPI, etc.), using the same procedures as used for making slices from animal tissue. If network activity is to be investigated, thicker (450–600 mm) slices should be obtained to maintain a maximum degree of connectivity within the slices; for isolated neurones (see later), slices can
be made thinner. In some cases, intense gliotic reaction will render the tissue hard and rubbery and make it difficult to cut through the tissue to make thin, even slices. In such cases, the experimenter is left to his or her own devices. It is usually the case, however, that such tissue will yield little useful electrophysiologic data because it will be difficult to penetrate with microelectrodes. After slicing, the tissue can be transferred directly to the recording chamber, or it can be maintained viable at room temperature in carbogenated ACSF (placing slices on a nylon mesh immersed in a fluid-filled beaker). Good viability, particularly for longer transport of the tissue slices from the operating room to the laboratory, can be achieved by using a portable chamber with ACSF at 28° C (Köhling et al., 1996a). Such a chamber, illustrated in Figure 1, consists of multiple wells (with nylon-mesh bottoms) and a central funnel through which the carbogen-bubbled ACSF can rise. The fluid circulation stabilizes the slices on the nylon mesh. Recording chambers are identical to those used for animal tissue slices and may be of either a submerged type, with slices resting on the bottom of an ACSF; a perfused flow chamber; or a standard interface-type chamber with the tissue resting on a nylon mesh at the interface of humidified carbogen and ACSF. Recognition of slice orientation within the tissue chamber is essential for carrying out interpretable experiments. If hippocampal slices are used, identification of the different hippocampal subregions is often visually possible, although the convoluted nature of the very anterior tip of hippocampus may make such identification difficult. In many cases, the hippocampus may show severe atrophy of areas CA3 and CA1 (Ammon’s horn sclerosis, or AHS), it may be damaged from surgical procedures, or it may be intensely gliotic. These factors can determine which areas are accessible and appropriate for further investigation. In studying neocortical samples, it is useful to not only identify pial and white matter edges but also to estimate cortical layers.
Electrophysiologic Monitoring Field Potential Recordings Extracellular recording of network activity can be performed in human tissue very much the same way as it is done in animal preparations. To obtain field potential recordings, low impedance (1–2 MW) glass microelectrodes filled with ACSF usually are used. For special purposes (e.g., current-source-density analyses, which necessitate multiple electrode recordings at short equidistant positions), insulated, etched tungsten or platinum-nickel wire (diameter, 30–40 mm) or carbon fibers can be used (Cohen et al., 2002; Köhling et al., 1999; Louvel et al., 2001). The disadvantage of such electrodes is that that high-frequency signal components (>50 Hz) are not detected because of the impedance
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FIGURE 1 Schematic diagram of slice maintenance chamber. (Modified from Köhling et al., 1996a, with permission.)
properties, and direct coupled (DC) recordings are not possible because of the polarizing properties of metal or carbon. Field potential recordings have been used to address several experimental issues in human tissue slices (see section on Characteristics of the Activity Generated by Human Epileptic Neurons). Intracellular Recordings The first intracellular studies in human tissue were performed with sharp-electrode recordings. For impalements of both neocortical and hippocampal cells, sharp microelectrodes with a resistance of 30 to 150 MW and filled with 2 to 4 M of K-acetate or K-methylsulphate were used. Several experimental issues can be addressed with this technique: 1. Resting and passive properties of neurons. Most investigators have concluded that resting membrane potential, input resistance, and time constants of human neurons are not different from corresponding cells in animal preparations. Most laboratories failed to observe any conspicuous increase in intrinsic bursting; firing properties on current injection generally did not differ from region—specific
“controls” from rodents (or monkeys) (Avoli and Olivier, 1989; Foehring et al., 1991; McCormick and Williamson, 1989; Schwartzkroin, 1987; Schwartzkroin and Prince, 1976; Tasker et al., 1996). One exception to this generality is that Dietrich et al. (1999a) found that some dentate granule cells in AHS tissue appeared to display properties of hilar interneurones. 2. Spontaneous synaptic activity. Spontaneous activity was demonstrated to occur regularly in human slices, apparently without sufficient network synchronization to generate field potential discharges. Such activity, particularly in mesial structures, was found to be dependent on glutamatergic and g-aminobutyric acid (GABA)ergic transmission (Knowles et al., 1992; Schwartzkroin and Haglund, 1986; Schwartzkroin and Knowles, 1984). An interesting finding was that in mesial temporal tissue where such network synchronization was seen, this spontaneous activity reflected bursting-discharging neurones in which there was a positively-shifted GABA reversal potential (and hence a depolarizing GABA response) (Cohen et al., 2002). 3. Synaptic activation by focal stimulation. For such studies, a stimulating electrode (monopolar or bipolar) is placed into pathways afferent to the recorded cell. In the
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hippocampus, these investigations have focused mainly on perforant path activation of granule cells (Dietrich et al., 1999a; Isokawa et al., 1991). In neocortical tissue, stimulation of underlying white matter or focal activation of tangential association fibers have been used (Avoli and Olivier, 1989; Avoli et al., 1994a; Schwartzkroin et al., 1983; Strowbridge et al., 1992). These studies revealed graded evoked bursts and, in rare cases, paroxysmal depolarization shifts or very prolonged excitatory postsynaptic potentials with relatively weak inhibitory activity. For further analysis of both synaptic responses and modulation of voltage-gated channels, voltage-clamp experiments have been performed in human tissue slices, either using switch-clamp amplifiers and sharp electrodes (McCormick and Williamson, 1989, Wuarin et al., 1992), or patch-clamp electrodes (2–4 MW, with Lucifer yellow filling for cell identification (Isokawa et al., 1997, Isokawa, 1998). These studies revealed the existence of pharmacologically isolated NMDA-receptor mediated currents and the existence of Mg2+-block, as known from animal experiments (Wuarin et al., 1992) and modulation of different K+ currents via acetylcholine, adenosine, and other modulators (McCormick and Williamson, 1989). Perhaps more importantly, NMDA-receptor mediated responses were particularly variable in amplitude, whereas GABA-mediated inhibitory potentials were reduced after high-frequency activation (Isokawa, 1998; Isokawa et al., 1997). These results underscore an increased excitability of the human epileptic hippocampus. Also, human astrocytes have altered properties (i.e., a larger proportion of AMPA-receptor flip variant, enhanced inward-rectifying K+ currents similar to juvenile rodent astrocytes, and even generation of slow action potentials) in sclerotic hippocampus (Bordey and Sontheimer, 2004; Hinterkeuser et al., 2000; Schröder et al., 2000). Measuring Extracellular Ionic Concentrations As an extension of field potential recordings, the concentrations of ions in the extracellular space can be monitored using modified, double-barrelled micropipettes. The methods used to manufacture these electrodes have been described in detail in studies on animal tissue, where this technique has been in use for many years (Heinemann et al., 1977). Briefly, double-barrelled theta-glass capillaries are pulled (tip diameter, 2–6 mm), and one channel is backfilled with of NaCl solution equimolar to the ACSF to be used as field potential and reference channel. The tip of the other barrel channel’s tip is silanized, filled with ion exchanger (typically Fluka 60398, 60031, or Corning 477317 for K+ and Fluka 21196, 21048, or 21191 for Ca2+) and backfilled with corresponding KCl or CaCl2 solutions (100 mM). Recordings with these electrodes have shown that although basal levels of K+ are relatively normal in human neocorti-
cal or hippocampal tissue (Kivi et al., 2000; Köhling et al., 1998b), K+ spatial buffering can be disturbed, particularly in sclerotic hippocampus (Kivi et al., 2000a). High levels of K+ are also reached in human neocortical tissue when the tissue exhibits pathological function, such as spreading depression (Avoli et al., 1991; Gorji et al., 2001); however, these levels do not substantially differ from those seen in animal preparations undergoing similar treatments. Isolated Neurons Another technique, again adapted from animal experiments, is the use of acutely isolated neurons from neocortex or hippocampal tissue. This approach has been used to investigate voltage-gated currents, which are difficult to be analyze in situ because of space-clamp problems. The methods of cell isolation are essentially the same as in animal tissue (see Chapter 2). First, slices are cut from the resected block and then microdissected to yield the areas of interest. These slabs are then incubated with proteases, for example, trypsin (type XI, 0.5 mg/ml for 1–2 hours in ACSF at 29° C) or pronase (2–3 mg/ml, 25 minutes, 28° C, in piperazine diethanesulfonic acid (PIPES)-buffered ACSF) and then washed with PIPES- or 4-(2-hydroxyethyl)-1piperazine-ethanesulfonic acid (HEPES)-buffered solution. To isolate neurons, the predigested tissue slabs are triturated through fire-polished pipettes and then incubated in appropriate solutions (Beck et al., 1996, 1997a, b, 1998; Cummins et al., 1994; Remy et al., 2003; Rüschenschmidt et al., 2004; Vreugdenhil et al., 1998, 2004). These studies have characterized current properties in human cells and studied the mechanism of action of antiepileptic drugs. Although K+, Na+ and Ca2+ currents are essentially similar to those described in animal tissue, some peculiarities have been identified. For instance, human subicular cells possess a large, persistent Na+ current that may predispose them to bursting (Vreugdenhil et al., 2004); moreover, effects of carbamazepine on Na+ currents are reduced in cells from sclerotic, but not from nonsclerotic tissue (Remy et al., 2003, Vreugdenhil et al., 1998).
Other Methodologic Approaches Optical Imaging Optical imaging techniques can be employed in human tissue in vitro very much the same way as in animal preparations. Both voltage-sensitive dyes and intrinsic optical imaging can be used to monitor spatiotemporal patterns of network activity. For voltage-sensitive imaging, the styryl dye RH795 has already been employed (1 hour incubation in 12.5 mg/ml, 450-mm- thick slices; (Köhling et al., 2000, 2002)); other fast dyes of the ANEP-type should also be informative. This method has the advantage of yielding high
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temporal resolution, but it allows for limited recording times because of phototoxicity effects. Studies using this technique have shown that spontaneous network activity goes along with heterogeneously distributed excitation within the neuronal network which it can be initiated in minimal foci up to 50 ms before activation of the rest of the network (Köhling et al., 2000, 2002). Furthermore, about 30% of the slices tested displayed epileptic responses after focal stimulation (Straub et al., 2003). Intrinsic optical imaging makes use of alterations in reflectance and absorbance properties of brain tissue as a consequence of neuronal activity. These changes, as recorded in in vitro slices, presumably reflect modifications in the extracellular space, for example, as a function of K+ accumulation and cell swelling (Andrew et al., 1996, Holthoff and Witte, 1996; MacVicar and Hochman, 1991). To date the temporal resolution of this method appears to be somewhat limited; however, it does not necessitate any tissue manipulation other than illumination. Hence, it can be used to monitor spread of activity not only in vitro but also in the patient’s brain in the operating room (as a diagnostic tool for guidance of tissue resection) (Haglund et al., 1992, Haglund and Hochman, 2004). Histologic Analysis A large number of histochemical and immunochemical techniques have been used to characterize resected human tissue. It is beyond the scope of this chapter to describe such analyses, but they are a critical part of the study of human tissue and provide information that can help to interpret the electrophysiologic data (see chapter 50). The importance of identifying individual neurons from which intracellular data have been obtained is noteworthy. A number of dye injection techniques for cellular labeling have been used, including fluorescent dyes (e.g., Lucifer yellow) and tracers to be detected histologically (e.g., biocytin, neurobiotin). These dyes are added to the intracellular recording solution both in sharp and patch pipettes.
iments with antibodies directed against specific GABA and metabotropic glutamate receptor (mGluR) subtypes have shown up-regulation of specific GABA and mGluR subunits (Lie et al., 2000; Loup et al., 2000). Finally, by employing a similar approach, Lie et al. (1999) discovered that Ca2+ channel b1 and b2 subunits are increased in sclerotic hippocampus. Molecular methods can also provide information on the mRNA expression of receptors and channels in the human tissue. These studies involve the use of reverse transcription reaction followed by polymerase chain reaction (PCR) and by restriction enzyme assays. Such experiments have shown that the relative amount of edited RNA of the AMPA receptor GluR2 was significantly increased in the hippocampal tissue, whereas no changes were found in neocortical tissues from epilepsy patients (Musshoff et al., 2000) and that NMDA-receptor mRNA splicing was unchanged compared with autopsy control material (Vollmar et al., 2004). Similar changes have been found in neurones and in hippocampal astocytes, where real-time PCR has revealed an increase in flip-to-flop ratio of the GluR1 AMPA-receptor mRNA matching functional results (Seifert et al., 2004). An alternative approach involves expresson monitoring by microarrays (Becker et al., 2002, 2003). Using this method, investigators have found some genes are up-regulated in human epileptic hippocampus (ataxin-3 and glial acidic fibrillary protein), whereas some are down-regulated (e.g., calmodulin) (Becker et al., 2002). In these studies, the findings were pinpointed to cell populations by using single-cell real-time PCR. Another application of molecular techniques to the analysis of ligand-gated currents in human tissue consists of injecting mRNA or cell membranes extracted from epileptic patients into frog oocytes (Palma et al., 2002, 2004). This procedure led to the expression of ionotropic receptors for GABAA, kainate, and AMPA. These investigators have reported that GABAA receptor-mediated currents in oocytes injected with “epileptic” mRNA or cell membranes are characterized by a strong run-down after repetitive ligand applications; this phenomenon can be abolished by phosphatase inhibitors (Palma et al., 2004).
Analysis of Channel and Receptors A number of nonelectrophysiologic techniques can be used to address changes of intrinsic excitability or synaptic physiology in human tissue. Although these methods do not directly show functional effects, they are nevertheless valuable for identifying the density of receptors or the abundance and localization of channel proteins. In many cases, and in contrast to functional studies, control tissue is available (e.g., from autopsy), so that direct comparisons between epileptic and nonepileptic tissue can be drawn. Receptor autoradiography using [3H]-tagged ligands have revealed up-regulation of AMPA-receptors in human epileptogenic neocortex (Zilles et al., 1999). Immunohistochemical exper-
CHARACTERISTIC OF THE ACTIVITY GENERATED BY HUMAN EPILEPTIC NEURONS Spontaneous Epileptiform Activity One important topic in studying human epileptic tissue is whether spontaneous epileptiform network activity is maintained in vitro after tissue excision. Field potential recordings have demonstrated network synchronization as reflected in population spikes (Figure 2). These spontaneous discharges, which resemble epileptiform spikes seen with
Characteristic of the Activity Generated by Human Epileptic Neurons
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FIGURE 2 Spontaneous field potential discharges and associated intracellular responses in human neocortical (A) and subicular (B) tissue in vitro are sensitive to GABAA receptor blockade by bicuculline (bic). A: Field potential discharges (FP) at condensed (bottom) and expanded time scale (top inset) before (control), during (Bic), and after (washout) bicuculline application. Inset demonstrates that, generally, hyperpolarizing membrane potential (MP) fluctuations of single neurons accompany field discharges. B: FP discharges at condensed time scale (bottom) before (control), during (Bic), and after (washout) bicuculline application. Top insets show two different responses of single pyramidal cells (top recording of each inset) related to field discharges (bottom recording of each inset): Most responses (as in A) are hyperpolarizing (inhibited pyramidal), but a fraction of neurons shows depolarizing (excited pyramidal) potentials, at times leading to bursts. (Modified from Köhling et al., 1998b (A), and Cohen et al., 2002 (B), with permission.)
intracranial EEG recordings, have been found in neocortical (Köhling et al., 1998b, 1999, 2000) and hippocampal preparations (Cohen et al., 2002). Because in the latter study spontaneous events appeared to be triggered by pacemaker neurons, they indeed might reflect intrinsic epileptogenicity of the tissue.
Evoked Epileptiform Discharges as Models of Epileptiform Synchronization As in animal studies, electrical stimulation, pharmacologic manipulations, or changes in the ionic microenviron-
ment disclose epileptiform activity in human tissue maintained in vitro. For example, Masukawa et al. (1989, 1996) reported that 1-Hz repetitive stimulation of the perforant path induces epileptiform afterdischarges in the dentate gyrus. Pharmacologic manipulations such as bath application of the GABAA receptor antagonist bicuculline (10 mM) also lead to short-lasting epileptiform discharges that resemble interictal events, correspond to intracellular bursts of action potentials, and are accompanied by afterdischarges (Avoli and Olivier, 1989; Franck et al., 1995; Hwa et al., 1991; McCormick, 1989; Tasker et al., 1992). Apart from constituting a model of epileptiform activity, these findings
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FIGURE 3 Spontaneous synchronous activity induced by 4-aminopyridine (4-AP) in neocortical slices obtained from patients with mesial temporal lobe epilepsy (MTLE) (A) and focal cortical dysplasia (FCD) (B and C). A: Isolated field potentials occur spontaneously in a MTLE slice analyzed with field potential and [K+]o recordings at 1000 mm from the pia. Note that each field potential event is associated with a transient increase in [K+]o. B: Spontaneous field potential discharges recorded in two slices (a and c panels) obtained from two FCD patients; in both cases the activity is characterized by isolated interictal field potentials (asterisks) and sustained epileptiform events resembling ictal discharges (continuous lines). Note also that the onset of the ictal event is associated with the occurrence of a negative field potential (arrow) followed by a slow negative event (arrowhead) leading to ictal discharge oscillations. The different characteristics of the isolated negative field events (1) and of the ictal discharge onset (2) are shown in (b) for three or four graphically superimposed samples obtained from the experiment in (a); note that the isolated interictal events are of lower amplitude compared with those leading to ictogenesis. C: Temporal relation between slow interictal events and ictal discharge onset during 4-AP application to slices from FCD cortex. In (a), histogram of the probability of occurrence of the interictal activity over a period of 50 seconds before ictal onset normalized to epoch 5 seconds before ictal event; data were obtained from 64 epochs recorded in 11 FCD slices. One of these epochs is shown in (b). Arrow points to time zero (i.e., ictal on set); asterisks identify slow interictal events.
Characteristic of the Activity Generated by Human Epileptic Neurons
suggest the presence of GABAA receptor-mediated inhibition within the human neocortical network, along with its ability to control epileptiform synchronization. However, one study using this technique suggested an impaired GABAergic inhibitory system in some hippocampi with dentate abnormalities; in these experiments, bicucullineinduced bursting occurred at a lower drug concentration in epileptic hippocampal tissue with mossy fiber sprouting than
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in tissue with a relatively “normal” dentate (Franck et al., 1995). Another important pharmacologic manipulation has involved the application of the K+ channel blocker 4aminopyridine (4-AP). As in normal animal tissue, 4-AP produces recurrent interictal events in human neocortical slices removed from temporal lobe epilepsy patients (Figure 3A) (Avoli et al., 1994); this tissue is characterized by no
FIGURE 4 Field potential and intracellular characteristics of the synchronous epileptiform activity and of the spreading depression (SD)-like episodes generated by human neocortical slices superfused with Mg2+-free medium. A: Field potential of spontaneous and stimulus-induced (arrow) epileptiform discharges. B: Typical intracellular and field potential activity associated with an ictal-like event. Note that a long-lasting hyperpolarization follows the end of the epileptiform discharge. C: intracellular (top trace) and [K+]o (bottom trace) recordings during an epileptiform discharge (a, left portion of the trace) and two SD-like episodes (a, right portion of the trace and b). The SD in (a) was induced by a train of low-frequency (5 Hz) stimuli (continuous line).
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obvious structural aberration, including abnormal lamination. In contrast, a similar dose of 4-AP elicits NMDA receptor-mediated ictal discharges in neocortical tissue obtained from patients with Taylor’s type, focal cortical dysplasia (FCD) (Figure 3B). FCD corresponds to a localized disruption of the normal cortical lamination with an excess of large, dysmorphic neurons (Spreafico et al., 1998; Taylor et al., 1971). These studies (Avoli et al., 1999; D’Antuono et al., 2004) have shown that epileptiform synchronization leading to in vitro ictal activity in the human FCD tissue is initiated by a synchronizing mechanism that paradoxically relies on GABAA receptor activation, causing sizeable increases in [K+]o. Moreover, this mechanism may be facilitated by the decreased ability of GABAB receptors to control GABA release from interneuron terminals. In addition to the bicuculline and 4-AP, epileptiform discharges are also recorded with field potential and intracellular techniques in human neocortical (Avoli et al., 1991, 1995; Köhling et al., 1998b, 1999) and hippocampal slices (Remy et al., 2003) during the application of Mg2+-free bathing medium. These events are often characterized by similarities in duration and waveform to the electrographic seizures recorded in vivo (Figure 4A and B). Moreover, human neocortical slices superfused with Mg2+-free medium generate spreading depression (SD)-like episodes (Figure 4C). SDs have also been recorded during hypoxia (Köhling et al., 1996b, 1998a) and following local local pressure ejection of 3 M KCl (Gorji et al., 2001). As reported to occur in animal experiments, Mg2+-free epileptiform events recorded in human tissue are readily blocked by NMDA receptor antagonists. Because Mg2+-freeinduced epileptiform activity has been considered as a model of therapy-resistant discharges in vitro (Heinemann et al., 1994; Zhang et al., 1995), Mg2+-free-induced discharges have been used to test the efficacy of standard and new antiepileptic drugs in human tissue. These experiments demonstrated that carbamazepine exerts only a moderate antiepileptic action on Mg2+-free-induced epileptiform activity, whereas vigabatrin is highly effective (Musshoff et al., 2000b); retigabine and melatonin also displayed a suppressive action in this model (Fauteck et al., 1995; Straub et al., 2001).
Synaptic Plasticity Using field potential recordings, several groups have revealed that in epileptic hippocampus, repetitive stimulation leads to disproportionately large responses and afterdischarges. Moreover, synaptic depression mediated via group III (but not group II) metabotropic glutamate receptor (mGluR) is impeded in sclerotic but not in nonsclerotic human epileptic hippocampus (Dietrich et al., 1999b, 2002; Masukawa et al., 1989, 1996). Further, studies using paired stimuli in the dentate gyrus to test the strength of recurrent
inhibition or presynaptic modulation confirmed that inhibition in epileptic tissue is presumably intact and dependent on presynaptic group II metabotropic glutamate receptors (as in animal tissue; Dietrich et al., 2002; Masukawa et al., 1996; Swanson et al., 1998; Uruno et al., 1995). In these studies, as in other investigations, a distinction between sclerotic and nonsclerotic hippocampus may help to circumvent the problem of lacking appropriate control tissue. As proposed by Dietrich et al. (1999, 2002), sclerotically altered hippocampus can differ from nonsclerotic tissue; for the purpose of comparison, the latter specimens may be viewed as “control” tissue. Lastly, perforant path long-term potentiation elicited by high-frequency stimulus trains was also lost in sclerotic but not in nonsclerotic hippocampus (Beck et al., 2000).
LIMITATIONS A general limitation of all slice preparation models of epileptiform activity is their isolation from the distributed system that is presumably involved in seizure generation. This is an especially important consideration in dealing with human tissue because we ignore which brain region is actually responsible for seizure generation or even if there is a localized epileptogenic zone. At the clinical level, surgical procedures have been justified by their effective ends. However, it is not the case that if seizure activity is interrupted by removal of, for example, hippocampus, that result “proves” that the hippocampus is responsible for the seizure activity. That may be the case, or the hippocampus simply may have been a part (perhaps a normal part) of a larger seizure circuit. Thus when we remove a piece of tissue from the human brain, we do not know that tissue is “epileptic.” A major problem in interpreting data from human slice experiments is the absence of “normal” controls. Obviously, for ethical reasons, we are not able to remove apparently normal tissue. In some cases, it may be possible to obtain “nonepileptic” cortical samples, for example, when the surgeon must remove a deep-lying brain tumor. However, even in such cases, it seems unlikely that this tissue is “normal.” Investigators have adopted a variety of strategies to deal with the control issue, but none is entirely adequate. For instance, a relatively easy approach is to compare the human epileptic tissue to “comparable” animal tissue. Thus, resected tissue from MTLE cases may be compared with chronically epileptic animal models such as kainate- or pilocarpine-treated rodents. However, this approach ignores what are likely to be significant species differences as well as the difficulty of determining what rat brain region corresponds to a given region of the human brain. Another approach is to compare human tissue that is electrographically very active (“hot”) on presurgical electrocorticography with human tissue that is relatively inactive (e.g., spiking vs.
References
nonspiking cortex). It is unclear what is being compared in this analysis because it is highly likely that even “nonspiking” tissue is abnormal. Further, this comparison ignores the likelihood that different regions of the same brain—even normal brain—may have quite different functional characteristics. Ideally, regional differences should be recognized and characterized. A further strategy is to make comparisons on the same area of resection across patients and to use as a basis for comparison some identifiable epilepsy marker, for example, sclerotic versus nonsclerotic hippocampus (or lesional vs. nonlesional cortex). Unfortunately, it is often unclear if and how tissue epileptogenicity is connected to such markers (e.g., both sclerotic and nonsclerotic hippocampus may be “epileptic”; both lesional and nonlesional neocortex may be “epileptic”). A third significant area of concern revolves around the variability of phenomenology from patient to patient. Whereas variables in animal models can be controlled, the clinical variability is often overwhelming, and it prevents most investigators from collecting tissue samples from a significant number of patients with similar clinical disorders. Age, sex, age of seizure onset, duration of the seizure disorder, history of medication, location and clinical characteristics of the epilepsy, etc., all contribute to making each clinical case “unique.” Finally, given our inability to “see” spontaneous seizure events in resected tissue, it may be unclear to what extent the human tissue slice provides advantages over slices from well-characterized animal models. To say that we can produce similar patterns of epileptiform activity in human tissue (“epileptic” human tissue) and in tissue from animal models does not constitute, in itself, a rationale for the human slice studies.
INSIGHTS INTO CLINICAL DISORDERS Given the limitations discussed herein, it is extremely important that investigators who choose to carry out studies on human epileptic tissue identify and justify their experiments carefully. Certainly most experimental manipulations used for animal preparations in vitro can also be adapted to investigations on human tissue. Thus few technical problems arise when electrophysiologic or other techniques are employed. Technology is not the obstacle. The real challenge is to design experiments so that the data we obtain are useful and interpretable.
References Andrew, R.D., Adams, J.R., and Polischuk, T.M. 1996. Imaging kainate and NMDA-induced intrinsic optical signals from the hippocampal slice. J Neurophysiol 76: 2707–2717. Avoli, M., and Olivier, A. 1989. Electrophysiological properties and synaptic responses in the deep layers of the human epileptogenic neocortex in vitro. J Neurophysiol 61: 589–606.
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Fauteck, J.-D., Bockmann, J., Böckers, T.M., Wittkowski, W., Köhling, R., Lücke, A., Straub, H. et al. 1995. Melatonin reduces low-Mg2+ epileptiform activity in human temporal slices. Exp Brain Res 107: 321–325. Foehring, R.C., Lorenzon, N.M., Herron, P., and Wilson, C.J. 1991. Correlation of physiologically and morphologically identified neuronal types in human association cortex in vitro. J Neurophysiol 66: 1825–1837. Franck, J.E., Pokorny, J., Kunkel, D.D., and Schwartzkroin, P.A. 1995. Physiologic and morphologic characteristics of granule cell circuitry in human epileptic hippocampus. Epilepsia 36: 543–558. Gorji, A., Scheller, D., Straub, H., Tegtmeier, F., Köhling, R., Höhling, J.M., Tuxhorn, I. et al. 2001. Spreading depression in human neocortical slices. Brain Res 906: 74–83. Haglund, M.M., and Hochman, D.W. 2004. Optical imaging of epileptiform activity in human neocortex. Epilepsia 45(Suppl 4): 43–47. Haglund, M.M., Ojemann, G.A., and Hochman, D.W. 1992. Optical imaging of epileptiform and functional activity in human cerebral cortex. Nature 358: 668–671. Heinemann, U., Lux, H.D., and Gutnick, M.J. 1977. Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat. Exp Brain Res 27: 237–243. Heinemann, U., Draguhn, A., Ficker, E., Stabel, J., and Zhang, C.L. 1994. Strategies for the development of drugs for pharmacoresistant epilepsies. Epilepsia 35(suppl 5): S10–21. Hinterkeuser, S., Schröder, W., Hager, G., Seifert, G., Blümcke, I., Elger, C.E., Schramm, J. et al. 2000. Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances. Eur J Neurosci 12: 2087–2096. Holthoff, K., and Witte, O.W. 1996. Intrinsic optical signal in rat neocortical slices measured with near-infrared dark-field microscopy reveal changes in extracellular space. J Neurosci 16: 2740–2749. Hwa, G.G.C., Avoli, M., Olivier, A., and Villemure, J.G. 1991. Bicucullineinduced epileptogenesis in the human neocortex maintained in vitro. Exp Brain Res 83: 329–339. Isokawa, M. (1998) Modulation of GABAA receptor-mediated inhibition by postsynaptic calcium in epileptic hippocampal neurons. Brain Res 810: 241–250. Isokawa, M., Avanzini, G., Finch, D.M., Babb, T.L., and Levesque, M.F. 1991. Physiologic properties of human dentate granule cells in slices prepared from epileptic patients. Epilepsy Res 9: 242–250. Isokawa, M., Levesque, M.F., Babb, T.L., and Engel, J. Jr. 1993. Single mossy fiber axonal systems of human dentate granule cells studied in hippocampal slices from patients with temporal lobe epilepsy. J Neurosci. 13: 1511–1522. Isokawa, M., Levesque, M., Fried, I., and Engel, J. 1997. Glutamate currents in morphologically identified human dentate granule cells in temporal lobe epilepsy. J Neurophysiol 77: 3355–3369. Kivi, A., Lehmann, T.N., Kovács, R., Eilers, A., Jauch, R., Meencke, H.J., Von Deimling, A., et al. 2000. Effects of barium on stimulus-induced rises of [K+]o in human epileptic non-sclerotic and sclerotic hippocampal area CA1. Eur J Neurosci 12: 2039–2048. Knowles, W.D., Awad, I.A., and Nayel, M.H. 1992. Differences of in vitro electrophysiology of hippocampal neurons from epileptic patients with mesiotemporal sclerosis versus structural lesions. Epilepsia 33: 601–609. Köhling, R., Lücke, A., Straub, H., and Speckmann, E.J. 1996a. A portable chamber for long-distance transport of surviving human brain slice preparations. J Neurosci Methods 67: 233–236. Köhling, R., Schmidinger, A., Hülsmann, S., Vanhatalo, S., Lücke, A., Straub, H., Speckmann, E.-J. et al. 1996b. Anoxic terminal negative DC-shift in human neocortical slices in vitro. Brain Res 741: 174–179. Köhling, R., Greiner, C., Wölfer, J., and Speckmann, E.-J. 1998a. Optical monotoring of PO2 changes and simultaneous recording of bioelectric activity in human and animal brain slices. J Neurosci Methods 85: 181–186.
Köhling, R., Lücke, A., Straub, H., Speckmann, E.-J., Tuxhorn, I., Wolf, P., Pannek, H. et al. 1998b. Spontaneous sharp waves in human neocortical slices excised from epileptic patients. Brain 121: 1073–1087. Köhling, R., Qu, M., Zilles, K., and Speckmann, E.J. 1999. Current-sourcedensity profiles associated with sharp waves in human epileptic neocortical tissue. Neuroscience 94: 1039–1050. Köhling, R., Höhling, H.-J., Straub, H., Kuhlmann, D., Kuhnt, U., Tuxhorn, I., Ebner, A. et al. 2000. Optical monitoring of neuronal activity during spontaneous sharp waves in chronically epileptic human neocortical tissue. J Neurophysiol 84: 2161–2165. Köhling, R., Reinel, J., Vahrenhold, J., Hinrichs, K., and Speckmann, E.J. 2002. Spatio-temporal patterns of neuronal activity: analysis of optical imaging data using geometric shape matching. J Neurosci Methods 15: 114: 17–23. Lie, A.A., Blümcke, I., Volsen, S.G., Wiestler, O.D., Elger, C.E., and Beck, H. 1999. Distribution of voltage-dependent calcium channel beta subunits in the hippocampus of patients with temporal lobe epilepsy. Neuroscience 93: 449–456. Lie, A.A., Becker, A., Behle, K., Beck, H., Malitschek, B., Conn, P.J., Kuhn, R. et al. 2000. Up-regulation of the metabotropic glutamate receptor mGluR4 in hippocampal neurons with reduced seizure vulnerability. Ann Neurol 47: 26–35. Loup, F., Wieser, H.G., Yonekawa, Y., Aguzzi, A., and Fritschy, J.-M. 2000. Selective alterations in GABAA receptor subtypes in human temporal lobe epilepsy. J Neurosci 20: 5401–5419. Louvel, J., Papatheodoropoulos, C., Siniscalchi, A., Kurciewicz, I., Pumain, R., Devaux, B., Turak, B. et al. 2001. GABA-Mediated synchronization in the human cortex: Elevations in extracellular potassium and presynaptic mechanisms. Neuroscience 105: 803–813. Lücke, A., Köhling, R., Straub, H., Moskopp, D., Wassmann, H., and Speckmann, E.J. 1995. Changes of extracellular calcium concentration induced by application of excitatory amino acids in the human neocortex in vitro. Brain Res 671: 222–226. MacVicar, B., and Hochman, D. 1991. Imaging of synaptically evoked intrinsic optical signals in hippocampal slices. J Neurosci 11: 1458–1469. Masukawa, L.M., Wang, H.W., O’Connor, M.J., and Uruno, K. 1996. Prolonged field potentials evoked by 1 Hz stimulation in the dentate gyrus of temporal lobe epileptic human brain slices. Brain Res 721: 132–139. Masukawa, L.M., Higashima, M., Kim, J.H., and Spencer, D.D. 1989. Epileptiform discharges evoked in hippocampal brain slices from epileptic patients. Brain Res 493: 168–174. McCormick, D.A. 1989. GABA as an Inhibitory neurotransmitter in human cerebral Cortex. J Neurophysiol 62: 1018–1027. McCormick, D.A., and Williamson, A. 1989. Convergence and divergence of neurotransmitter action in human cerebral cortex. Proc Natl Acad Sci USA 86: 8098–8102. Musshoff, U., Schünke, U., Köhling, R., and Speckmann, E.-J. 2000a. Alternative splicing of the NMDAR1 glutamate receptor subunit in human temporal lobe epilepsy. Mol Brain Res 76: 377–384. Musshoff, U., Lücke, A., Köhling, R., Speckmann, E.-J., Tuxhorn, I., Wolf, P., Pannek, H. et al. 2000b. Vigabatrin reduces epileptiform activity in brain slices from pharmacoresistant epilepsy patients. Eur J Pharmacol 401: 167–172. Palma, E., Esposito, V., Mileo, A.M., Di Gennaro, G., Quarato, P., Giangaspero, F., Scoppetta, C. et al. 2002. Expression of human epileptic temporal lobe neurotransmitter receptors in Xenopus oocytes: An innovative approach to study epilepsy. Proc Natl Acad Sci U S A 99: 15078–15083. Palma, E., Ragozzino, D.A., Di Angelantonio, S., Spinelli, G., Trettel, F., Martinez-Torres A., Torchia, G. et al. 2004. Phosphatase inhibitors remove the run-down of gamma-aminobutyric acid type A receptors in the human epileptic brain. Proc Natl Acad Sci U S A 101: 10183–10188. Patt, S., Labrakakis, C., Bernstein, M., Weydt, P., Cervòs-Navarro, J., Nisch, G., and Kettenmann, H. 1996. Neuron-like physiological prop-
References erties of cells from human oligodendroglial tumors. J Neurosci 71: 601–611. Remy, S., Gabriel, S., Urban, B.W., Dietrich, D., Lehmann, T.N., Elger, C.E., Heinemann, U. et al. 2003. A novel mechanism underlying drug resistance in chronic epilepsy. Ann Neurol 53: 469–479. Rüschenschmidt, C., Köhling, R., Schwarz, M., Straub, H., Gorji, A., Siep, E., Ebner, A. et al. 2004. Characterization of a fast transient outward current in neocortical neurons from epilepsy patients. J Neurosci Res 75: 807–816. Schröder, W., Hinterkeuser, S., Seifert, G., Schramm, J., Jabs, R., Wilkin, G.P., and Steinhäuser, C. 2000. Functional and molecular properties of human astrocytes in acute hippocampal slices obtained from patients with temporal lobe epilepsy. Epilepsia 41: S181–S184. Schwartzkroin, P.A. 1987. The electrophysiology of human brain slices resected from “epileptic” brain tissue. In: Fundamental mechanisms of human brain function. Ed. J. Engel. pp. 145–154. New York: Raven Press. Schwartzkroin, P.A., and Haglund, M.M. 1986. Spontaneous rhythmic synchronous acivity in epileptic human and normal monkey temporal lobe. Epilepsia 27: 523–533. Schwartzkroin, P.A., and Knowles, W.D. 1984. Intracellular study of human epiieptic cortex: in vitro maintenance of epileptiform activity? Science 223: 709–712. Schwartzkroin, P.A., and Prince, D.A. 1976. Microphysiology of human cerebral cortex studied in vitro. Brain Res 115: 497–500. Schwartzkroin, P.A., Turner, D.A., Knowles, W.D., and Wyler, A.R. 1983. Studies of human and monkey “epileptic” neocortex in the in vitro slice preparation. Ann Neurol 13: 249–257. Seifert, G., Hüttmann, K., Schramm, J., and Steinhäuser, C. 2004. Enhanced relative expression of glutamate receptor 1 flip AMPA receptor subunits in hippocampal astrocytes of epilepsy patients with Ammon’s horn sclerosis. J Neurosci 24: 1996–2003. Spreafico, R., Battaglia, G., Arcelli, P., Andermann, F., Dubeau, F., Palmini, A., Olivier, A. et al. 1998. Cortical dysplasia: an immunocytochemical study of three patients. Neurology 50: 27–36. Straub, H., Lücke, A., Köhling, R., Moskopp, D., Pohl, M., Wassmann, H., and Speckmann E.J. 1992. Low-magnesium-induced epileptiform activity in the human neocortex maintained in vitro: Suppression by the organic calcium antagonist verapamil. J Epilepsy 2: 166–170. Straub, H., Köhling, R., Höhling, J.M., Rundfeldt, C., Tuxhorn, I., Ebner, A., Wolf, P. et al. 2001. Effects of retigabine on rhythmic synchronous activity of human neocortical slices. Epilepsy Res 44: 155–165. Straub, H., Kuhnt, U., Höhling, J.-M., Köhling, R., Gorji, A., Kuhlmann, D., Tuxhorn, I. et al. 2003. Stimulus-induced patterns of bioelectric activity in human neocortical tissue recorded by a voltage sensitive dye. Neuroscience 121: 587–604.
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Strowbridge, B.W., Masukawa, L.M., Spencer, D.D., and Shepherd, G.M. 1992. Hyperexcitability associated with localizable lesions in epileptic patients. Brain Res 587: 158–163. Swanson, T.H., Sperling, M.R., and O’Connor, M.J. 1998. Strong paired pulse depression of dentate granule cells in slices from patients with temporal lobe epilepsy. J Neural Transm 105: 613–625. Taylor, D.C., Falconer, M.A., Bruton, C.J., and Corsellis, J.A.N. 1971. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiatry 34: 369–387. Tasker, J.G., Peacock, W.J., and Dudek, F.E. 1992. Local synaptic circuits and epileptiform activity in slices of neocortex from children with intractable epilepsy. J Neurophysiol 67: 496–507. Tasker, J.G., Hofman, N.W., Kim, Y.I., Fisher, R.S., Peacock, W.J., and Dudek, F.E. 1996. Electrical properties of neocortical neurons in slices from children with intractable epilepsy. J Neurophysiol 75: 931–939. Uruno, K., O’Connor, M.J., and Masukawa, L.M. 1995. Effects of bicuculline and baclofen on paired-pulse depression in the dentate gyrus of epileptic patients. Brain Res 695: 163–172. Vollmar, W., Gloger, J., Berger, E., Kortenbruck, G., Köhling, R., Speckmann. E.-J., and Musshoff, U. 2004. RNA editing (R/G site) and flipflop splicing of the AMPA receptor subunit GluR2 in nervous tissue of epilepsy patients. Neurobiol Dis 15: 371–379. Vreugdenhil, M., van Veelen, C.W.M., van Rijen, P.C., Da Silva, F.H.L., and Wadman, W.J. 1998. Effect of valproic acid on sodium currents in cortical neurons from patients with pharmaco-resistant temporal lobe epilepsy. Epilepsy Res 32: 309–320. Vreugdenhil, M., Hoogland, G., van Veelen, C.W., and Wadman, W.J. 2004. Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy. Eur J Neurosci 19: 2769–2778. Williamson, A., Patrylo, P.R., Lee, S., and Spencer, D.D. (2003) Physiology of human neurons adjacent to cavernous malformations and tumors. Epilepsia 44: 1413–1419. Williamson, A., Spencer, S.S., and Spencer, D.D. 1995. Depth electrode studies and intracellular dentate granule cell recordings in temporallobe epilepsy. Ann Neurol 38: 778–787. Wuarin, J.-P., Peacock, W.J., and Dudek, F.E. 1992. Single-electrode voltage-clamp analysis of the N-methyl-d-aspartate component of synaptic responses in neocortical slices from children with intractable epilepsy. J Neurophysiol 67: 84–92. Zhang, C.L., Dreier, J.P., and Heinemann, U. 1995. Paroxysmal epileptiform discharges in temporal lobe slices after prolonged exposure to low magnesium are resistant to clinically used anticonvulsants. Epilepsy Res 20: 105–111. Zilles, K., Qü, M.S., Köhling, R., and Speckmann, E.-J. 1999. Ionotropic glutamate and GABA receptors in human epileptic neocortical tissue: quantitative in vitro receptor autoradiography. Neuroscience 94: 1051–1061.
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9 In Vitro Isolated Guinea Pig Brain MARCO DE CURTIS, LAURA LIBRIZZI, AND LAURA UVA
The isolated guinea pig brain preparation maintained in vitro by arterial perfusion has been utilized to develop a model of acute epileptogenesis in the temporal lobe. The technique is ideal for performing high-resolution electrophysiologic and optical imaging studies of the generation and the propagation patterns of interictal and ictal epileptiform discharges induced by different acute pharmacologic treatments in vitro. The preparation potentially could be used to study long-range network interactions, also in brains isolated from guinea pigs, in which a chronic model of epilepsy has been developed. Preservation of the neuronal and vascular compartments in this preparation allows investigation of the role of neurovascular interactions in the control of epileptiform discharges.
mental conditions: the isolated guinea pig brain preparation maintained in vitro by arterial perfusion. The preparation was introduced and originally developed by Rodolfo Llinas in collaboration with Yosef Yarom, Matsuyuki Sugimori, Kerry Walton, and Michael Muhlethaler at the New York University (Llinás et al., 1981, 1989; Muhlethaler et al., 1993). In 1991 the preparation was set up at the Istituto Nazionale Neurologico in Milano, Italy, where it was further developed and characterized (de Curtis et al., 1994, 1996, 1998a, 1998b; Forti et al., 1997; Librizzi and de Curtis, 2003). Multisite eletrophysiologic recordings combined with intracellular recordings (Biella et al., 2001; Forti et al., 1997; Pare et al., 1992), optical imaging of both intrinsic signals (Federico and MacVicar, 1996; Federico et al., 1994), and voltage-generated fluorescent signals (Biella et al., 2003; de Curtis et al., 1999b) have been performed since in the isolated guinea pig brain in conditions of normal excitability and after diverse epileptogenic challenges. In addition, given that the whole brain is perfused in vitro via the arterial system integrally preserved in situ during the isolation procedure, it is feasible to utilize this preparation to evaluate the reciprocal interactions between the vascular and neuronal compartments during the generation of epileptiform discharges (Librizzi and de Curtis, 1999). With this purpose in mind, the functional and structural preservation of the cerebral vascular system, in particular of the blood-brain barrier, has been extensively characterized in the isolated guinea pig brain during the last few years (Librizzi and de Curtis, 1999; Librizzi et al., 2000, 2001; Mazzetti et al., 2004).
GENERAL DESCRIPTION OF THE MODEL The identification of neuronal circuits involved in the generation and propagation of interictal and ictal epileptiform discharges is central to understanding the dynamic process of epileptogenesis. The analysis of network interactions in epileptogenesis has been approached in various in vivo and in vitro experimental conditions that allow exploration of brain activity with different degrees of detail and complexity. For instance, in vivo experimental conditions allow performance of electrophysiologic studies of population activities sampled from a restricted number of recording sites over a wide cortical area. On the other hand, in vitro studies performed on sliced cerebral tissue allow a finer cellular analysis of epileptiform activity, but these studies are spatially restricted to the network interactions spared by the slicing procedure. This chapter describes a preparation that has been used in the last 15 years to study epileptogenesis in different experi-
Models of Seizures and Epilepsy
WHAT DOES IT MODEL? The in vitro isolated guinea pig brain has been used to analyze propagation of epileptiform discharges induced in
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the olfactory-limbic cortices either by tetanic cortical stimulation (Pare et al., 1992) or by acute pharmacologic application of epileptogenic agents, such as the g-aminobutyric acid (GABA) receptor antagonists bicuculline, penicilline, and picrotoxin (de Curtis et al., 1994, 1999b, 1998; Forti et al., 1997) and the muscarinic agonists carbachol and pilocarpine. These drugs were applied either locally in the brain parenchyma or by arterial perfusion. In both conditions epileptiform discharges with a focal onset were observed. Therefore the preparation has been largely used as a model of acute, focal epileptogenesis. As for studies conducted on in vitro slices, whole brains could be acutely isolated ex vivo from animals in which a chronic epileptic condition has been established. These experiments would allow study of complex interactions that occur between remote areas in a chronically epileptic brain. Preliminary studies in this direction are in progress in our laboratory (Librizzi et al., 2005).
METHODS OF PREPARATION AND GENERATION OF EPILEPTIFORM DISCHARGES The methods used to isolate and maintain in vitro a guinea pig brain have been extensively described (de Curtis et al., 1991, 1998; Llinás et al., 1981, 1989; Muhlethaler et al., 1993). Young adult guinea pigs weighing 150 to 250 g are anesthetized with 70 mg/kg tiopenthal, administered intraperioneally. A study of the time course of barbiturate washout in this preparation demonstrated that within 1 hour from the in vitro isolation, the brain concentration of the anesthetic measured by high-performance liquid chromatography (HPLC) is reduced to values close to zero (Librizzi et al., unpublished observations), which exclude possible pharmacologic interference with the recorded activity. After exposing the heart, intracardiac perfusion with a cold saline solution (see later discussion) at 14° C is performed to reduce brain metabolism and to preserve the brain tissue during the dissection. The animal is decapitated after 3 minutes, and the brain is carefully isolated and transferred to the incubation chamber. The surgical maneuver to isolate the brain does not differ substantially from the technique used to prepare brain slices, but it must be performed rapidly (i.e., in 6 to 8 minutes). The details are reported in Muhlethaler et al. (1993). After dissection the isolated brain is positioned in the incubation chamber with its ventral surface upward to visualize the base of the brain and the vascular system formed by the basilar artery and the circle of Willis that is removed en bloc with the brain. The isolated brain is held down by two silk threads secured to the bottom of the incubation chamber to improve mechanical stability. The dura that enfolds the basilar artery is removed, a cannula (PE 60 tubing tapered to about 300-mm-tip diameter) is
inserted into the basilar artery, and the cannula is secured by tying a thin silk thread around the artery. Arterial perfusion at a rate of 5.5 ml per minute is restored with the following solution: NaCl 126 mM, KCl 2.3 mM, NaHCO3 26 mM, MgSO4 1.3 mM, CaCl2 2.4 mM, KH2PO4 1.2 mM, glucose 15 mM, 4(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) 5 mM, and 3% dextran 70.000 (pH 7.3) saturated with a 95 to 5% O2-CO2 gas mixture. The same solution is used for the intracardiac perfusion with a slightly acidic pH (7.1) to enhance protection of the tissue during dissection. In the perfusion chamber, the hypophyseal and carotid arteries are ligated with silk knots. The temperature of the incubation chamber is slowly increased to 32° C (0.2°C per minute). The arterial pressure of the isolated brain, as measured by a pressure transducer interposed along the perfusion line, ranges between 45 and 65 mm Hg. Brains are commonly isolated from young adult guinea pigs, 15 days to 4 months of age. No attempts have been performed to date to isolate the brains of younger and older animals. Viable isolated guinea pig brains can be reliably reproduced by expert experimenters. Since setting up the method at the Department of Experimental Neurophysiology of the Istituto Nazionale Neurologico, more than 10 young scientists have been trained to isolate guinea pig brains; all have mastered the isolation technique within 2 to 3 months. Brains can be isolated in vitro from species such as guinea pigs that show a peculiar arrangement of the communication between the posterior (vertebrobasilar) and the anterior (carotid) arterial systems that form the circle of Willis. The basilar artery in the guinea pig divides into two large-diameter posterior communicating arteries (Figure 1), from which the posterior cerebral arteries originate. The guinea pig is the only animal analyzed so far in which the presence of these large-capacity posterior communicating arteries and the arrangement of the circle of Willis are compatible with perfusion of the entire brain via the basilar artery. In other animal species, such as the rat and the mouse (see Figure 1A), the small diameter of the posterior communicating arteries does not allow good brain perfusion in vitro when the basilar artery is cannulated. No attempts have been made to evaluate the feasibility of the brain isolation technique in animal species other than the rat, the mouse, and the guinea pig. An acute epileptogenic condition can be easily and reliably established in the isolated guinea pig brain preparation. Interictal and ictal discharges can be consistently reproduced by brief arterial applications of proconvulsive compounds that are permeable to the blood-brain barrier. Three-minute applications of bicuculline (50 mM), penicillin (1000 units/ml), or picrotoxin (1 mM) diluted in the perfusion solution induce interictal spikes in the piriform-entorhinal cortex; these applications are followed, within 5 to 10 minutes, by ictal discharges that typically originate in the
Methods of Preparation and Generation of Epileptiform Discharges
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FIGURE 1 A: Schematic representation of the circle of Willis of the rat (left) and the guinea pig (right). 1, Anterior communicating artery; 2, anterior cerebral artery; 3, middle cerebral artery; 4, carotid artery; 5, hypophyseal artery; 6, posterior communicating artery; 7, posterior cerebral artery; 8, Superior cerebellar artery; 9, basilar artery. B: Arteriographic image obtained by perfusing an isolated guinea pig brain with 0.2 ml iodine contrast medium in the basilar artery. Large arteries of the circle of Willis are shown during an early perfusion time of the contrast medium.
hippocampus-entorhinal cortex and secondarily invade the perirhinal cortex (Figure 2). Unlike results reported for cats in vivo (Avoli and Gloor, 1982; Gloor et al., 1977), GABAergic antagonists in the isolated guinea pig brain preparation induce epileptiform discharges that demonstrate a clear focal onset in the mesial temporal lobe. Typical ictal events are characterized by fast activity (around 20 to 25 Hz) that originates from the hippocampus-entorhinal cortex and builds up in 2 or 3 seconds (Figure 2b) superimposed on a slow extracellular voltage shift. The fast activity is followed by phasic runs of high-amplitude afterdischarges (0.5 to 1 second in duration) widely distributed in the hippocampus and parahippocampal regions (Figure 2c) that progressively become more regular and increase in amplitude. Afterdischarge can propagate to regions in which the early part of the seizure was never observed, such as the piriform cortex or the neocortex. After 2 to 4 minutes, afterdischarges decrease in duration and periodicity and ultimately disappear. A postictal depression of several tenths of minutes follows, during which epileptiform discharges could not be induced, but afferent stimulation can evoke responses in the limbic region. Ictal epileptiform discharges repeat for several minutes or hours after a single arterial perfusion with GABA-receptor antagonists. A normal excitability condition resumes within 2 to 3 hours after bicuculline application. Ictal activity generated in the temporolimbic region does not propagate diffusely into the brain; for instance, ictal epileptiform discharges were never observed in the neocortex and seldom occurred in the piriform cortex (Librizzi and de Curtis, 2003; Uva et al., unpublished observations). For this reason the experimental procedure can be proposed as
a model of acute focal epileptogenesis of the temporal lobe. The ictal pattern generated in the isolated brain preparation is similar to that commonly observed during stereo electroencephalographic (EEG) recordings from human epileptogenic areas (Francione et al., 1994, 2003; Lieb et al., 1976; Pacia and Ebersole, 1999; Spencer and Spencer 1994; Tassi et al., 2002; Towsend and Engel, 1991; Wendling et al., 2002; Young Jung et al., 1999) characterized by the appearance of fast activity at the onset, followed by afterdischarges. Such an ictal onset differs from the pattern usually induced by the application of epileptogenic drugs in slices. In most acute pharmacologic models developed in slices of hippocampus or cortex, ictal-like events are formed by large-amplitude afterdischarges characterized by repeated, fast-onset paroxysmal depolarizing shifts (PDSs) that gradually decline in frequency to about 2 to 10 Hz, interpreted as a preictal event or subclinical “embryo seizure” (Ralston, 1958). The differences between the pattern observed in the isolated guinea pig brain and that observed in slices probably depends on the restricted connectivity preservation in brain tissue slices. Muscarinic receptor agonists (carbachol, pilocarpine) do not induce epileptiform activity unless they are co-perfused with a GABA-receptor antagonist. Unlike the case for cortical slices (Nagao et al., 1996), arterial perfusion with pilocarpine alone at concentrations between 10 and 100 mM, for periods up to 2 hours, never resulted in epileptiform discharges, even though intraperitoneal application of pilocarpine in the guinea pig in vivo induced a prolonged status epilepticus.
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FIGURE 2 Representative ictal discharge recorded in the olfactory and limbic cortices of the isolated guinea pig brain preparation after arterial perfusion of 50 mM bicuculline. Recordings were performed in the piriform cortex (PC), in the entorhinal cortex (EC), in the CA1 region of the hippocampus (hip), and in the perirhinal cortex (PRC), as illustrated on the ventral view of the guinea pig brain on top. Expanded traces in a, b, c, and d are sampled from the upper traces. The ictal onset of the discharge features fast activity that originates in the hippocampus. For details see text.
Focal hypersynchronous potentials can be reliably induced by local application of proconvulsive compounds. Intraparenchymal injection of bicuculline (1 to 2 mM) or picrotoxin (10 mM) in the piriform cortex induces largeamplitude interictal spikes that repeat with a 5- to 10-second periodicity and propagate to synaptically connected regions, such as the amygdala, the periamygdaloid cortex, and the lateral entorhinal cortex (Biella et al., 1996, 2003). Unlike systemic applications, local drug treatment in the piriform cortex did not induce ictal discharges but could promote the generation of brief afterdischarges. To date no attempts have been performed to trigger epileptiform activity by local applications of drugs in structures other than the piriform region. An alternative procedure that has been used to generate epileptiform discharges in the hippocampal-parahippocampal area is represented by direct application of tetanic stimulation to the cerebral tissue at 100 Hz for 1 second, as
reported by Paré and colleagues (1992). When such prolonged tetanic stimuli are repeated for three to five cycles at 0.5 Hz, self-sustained epileptiform afterdischarges of brief duration are generated that only occasionally develop into seizure-like activity with features similar to those described above for the pharmacologic model.
ADVANTAGES, LIMITATIONS, AND FUTURE DEVELOPMENTS The advantages of the isolated guinea pig brain with respect to other in vitro and the in vivo conditions and preparations have been discussed extensively in previous articles (de Curtis et al., 1991; Llinás et al., 1981; Muhlethaler et al., 1993). The most obvious advantage is preservation of the tridimensional connections between close and remote brain areas, which allows evaluation of the unrestricted expres-
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Advantages, Limitations, and Future Developments
sion of network interactions during epileptiform discharges. Moreover, use of the in vitro brain preparation allows (1) approaching brain regions that are otherwise difficult to access in vivo; (2) evaluating the tangential propagation of activity across the surface of the brain with a more direct and facilitated approach than in vivo by means of electrophysiologic recordings and optical imaging; (3) performing multisite recordings at extracellular and intracellular levels; (4) performing pharmacologic tests by perfusing drugs via the arterial system in a close-to-in vivo situation in which the blood-brain barrier is functionally active; (5) applying drugs that cannot be used in vivo because of their severe, if not lethal, systemic effects. Finally, because of the in situ preservation of the vascular system in the isolated guinea pig brain, the mechanisms of interactions between neuronal activity and the vascular compartment can be analyzed. It is well known that bidirectional neurovascular-neuronal interactions regulate brain excitability. Neuronal and glial activities are coupled to localized changes in cerebral blood flow that rely on several mechanisms, such as changes in local concentration of ions and the release of classic neurotransmitters or neuromodulators (e.g., nitric oxide or adenosine). Further changes in blood flow and in blood-brain barrier permeability influence metabolism and activity of the brain, which result in dramatic excitability changes (Akgoren et al., 1996; Gaillard et al., 1995; Logothetis et al., 2001; Magistretti et al., 1999; Iadecola et al., 1997; Malonek and Grinvald, 1996; Villringer and Dirnagl, 1995; Zonta et al., 2003). The neuronal and vascular compartments during epileptiform activation have been studied simultaneously in the past during pioneer studies in vivo (Caspers and Speckmann, 1972; Jasper and Erickson, 1941; Paulson and Sharbrough, 1974; Penfield et al., 1940) that have been largely overlooked for several decades in epilepsy research field in favor of the study of intrinsic excitability properties and synaptic interactions between neurons. With the spread of diagnostic imaging technology, such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and functional magnetic resonance imaging (MRI), the interactions between blood flow, brain metabolism, and neuronal activity have been reconsidered (Arthurs and Boniface, 2002; Duncan, 1992; Logothetis et al., 2001). Very preliminary attempts to correlate brain activity with changes in blood flow during an ictal epileptiform discharge have been carried out on the isolated brain preparation (de Curtis et al., 1998). As illustrated in Figure 3, the time course of seizure-like events induced by arterial application of bicuculline can be correlated with the simultaneous changes in extracellular ion concentrations (potassium and protons/pH) and changes in resistance to blood flow, measured with a pressure transducer positioned along the perfusion line. These studies could be further detailed by measuring changes in the size of pial vessels with a video microscopy
FP
1 mV
[K ]o
+
2 mM
pH
0.05 pH
VT
5 mmHg 1 min
FIGURE 3 Simultaneous recording of ictal discharges elicited by arterial application of penicillin (1000 units/ml). Two-barrel electrodes recorded field potentials (FP) simultaneously with extracellular potassium ([K+]o) and proton (pH) concentration in the extracellular space by means of ion-selective electrodes. The changes in brain blood flow associated with seizure activity were simultaneously measured as changes in the resistance to flow by a pressure transducer inserted along the perfusion line just upstream of the insertion of the polyethylene cannula in the basilar artery. VT, vascular tone. Downward deflections represent vasodilation associated with an increase in blood flow.
system to evaluate changes in local cerebral blood flow during brain activity. Even though a detailed neuropathologic study of isolated brains after the induction of repetitive seizures has never been performed, no obvious damage was observed in the ictal onset region (hippocampus and entorhinal cortex) after thionine staining of the tissue (performed to identify the position of the recording and stimulating electrodes at the end of the electrophysiologic experiment). Histochemical studies of the isolated brains after induction of epileptiform discharges could be performed, in principle, to analyze acute changes in brain tissue, such as induction of immediate early genes, inflammatory molecules, edema, etc. In addition, MRI scans of in vitro isolated or postfixed guinea pig brains (Figure 4) can be performed with a spatial resolution that allows identification of an altered signal in the gray and white matter, such as postepileptic brain edema or alterations associated with sclerosis or gliosis (not shown). The main limitation of the currently available technique is that it models an acute epileptogenic condition. Future developments include the possibility of isolating brains from animals in which a chronic epileptic condition has been established. Preliminary experiments on the pilocarpine model have been performed (Turski et al., 1989). Intraperitoneal injection of 380 mM of pilocarpine induces in guinea pigs subcontinuous partial and secondary generalized seizures lasting several hours this activity can be effectivey terminated by intraperitoneal injection of benzodiazepines. Brains isolated just after the epileptic status produce
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FIGURE 4 High-definition magnetic resonance T2-weighted images of the isolated brain of the guinea pig performed with a 1.5 Tesla Philips instrument (courtesy of Dr. Maria Grisoli and Dr. Maria Grazia Bruzzone).
spontaneous epileptiform activity without further pharmacologic or stimulation procedures. Ideally brain isolation could be performed at different times during the “latent period” as well as before and after establishment of a chronic epileptic condition. The use of an in vitro isolated brain for such a study would allow precise reconstruction of the patterns of generation and propagation of epileptiform discharges using microphysiologic and imaging techniques, under conditions that preserve the tridimensional connectivity among brain structures.
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References Iadecola, C., Yang, G., and Ebner, T. 1997. Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex. J Neurophysiol 78: 651–659. Jasper, H., and Erickson, T.E. 1941. Cerebral blood flow and pH in excessive cortical discharge induced by metrazol and electrical stimulation. J Neurophysiol 4: 333–347. Librizzi, L., and de Curtis, M. 1999. Simultaneous recording of changes in pH, K+, and vasular tone during epileptiform activity in the olfactorylimbic cortex. Soc Neurosci Abst 25: 842. Librizzi, L., and de Curtis, M. 2003. Epileptiform ictal discharges are prevented by periodic interictal spiking in the olfactory cortex. Ann Neurol 53: 382–389. Librizzi, L., Folco, G., and and de Curtis, M. 2000. NO-synthase inhibitors block acetylcholine-mediated dilation of cerebral arteries in the in vitro isolated guinea pig brain. Neuroscience 101: 283–287. Librizzi, L., Janigro, D., De Biasi, S., and de Curtis, M. 2001. Blood brain barrier preservation in the in vitro isolated guinea-pig brain preparation. J Neurosc Res 66: 289–297. Lieb, J.P., Walsh, G.O., and Babb, T.L. 1976. A comparison of EEG seizure patterns recorded with surface and depth electrodes in patients with temporal lobe epilepsy. Epilepsia 17: 137–160. Llinás, R., Muhlethaler, M., and Walton, K. 1989. Electrophysiology of the isolated adult guinea pig in vitro. J Physiol (Lond) 414: 16P. Llinás, R., Yarom, Y., and Sugimori, M. 1981. Isolated mammalian brain in vitro: new technique for analysis of electrical activity of neuronal circuit function. Fed Proc 40: 2240–2245. Logothetis, N.K., Pauls, J., Augath, M., Trinath, T., and Oeltermann, A. 2001. Neurophysiological investigation of the basis of the fMRI signal. Nature 412: 150–157. Magistretti, P.J., Pellerin, L., and Rothman, D. 1999. Energy on demand. Science 283: 496–497. Malonek, D., and Grinvald, A. 1996. Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping. Science 272: 551–444. Mazzetti, S., Librizzi, L., Frigerio, S., de Curtis, M., and Vitellaro-Zuccarello, L. 2004. Molecular anatomy of the cerebral microvessels in the isolated guinea-pig brain. Brain Res 999: 81–90. Muhlethaler, M., de Curtis, M., Walton, K., and Llinas, R. 1993. The isolated and perfused brain of the guinea-pig in vitro. Eur J Neurosci 5: 915–926. Nagao, T., Alonso, A., and Avoli, M. 1996. Epileptiform activity induced by pilocarpine in the rat hippocampal-entorhinal slice preparation. Neuroscience 72: 399–408.
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Pacia, S.V., and Ebersole, J.S. 1999. Intracranial EEG in temporal lobe epilepsy. J Clin Neurophysiol 16: 399–407. Pare, D., de Curtis, M., and Llinas, R. 1992. Role of the hippocampalentorhinal loop in temporal lobe epilepsy: extra- and intracellular study in the isolated guinea pig brain in vitro. J Neurosci 12: 1867–1881. Paulson, O.B., and Sharbrough, F.W. 1974. Physiologic and pathophysiologic relationship between the electroencephalogram and the regional cerebral blood flow. Acta Neurol Scand 50: 194–220. Penfield, W., von Santha, K., and Cipriani, A. 1940. Cerebral blood flow during induced epileptiform seizures in animals and man. J Neurophysiol 3: 257–267. Ralston, B.L. 1958. The mechanism of transition of interictal spiking foci into ictal seizure discharges. Electroencephalogr Clin Neurophysiol Suppl 10: 217–232. Spencer, S.S., and Spencer, D.D. Entorhinal-hippocampal interactions in medial temporal lobe epilepsy. Epilepsia 35: 721–727, 1994. Tassi, L., Colombo, N., Garbelli, R., Francione, S., Lo Russo, G., Mai, R., Cardinale, F. et al. 2002. Focal cortical dysplasia: neuropathological subtypes, EEG, neuroimaging and surgical outcome. Brain 125: 1719–1732. Towsend, J.B., and Engel, J. 1991. Clinico-pathological correlations of low voltage fast and high amplitude spike and wave medial temporal stereoencephalographic ictal onset [abstract]. Epilepsia 32: 21. Turski, L., Ikonomidou, C., Turski, W.A., Bortolotto, Z.A., and Cavalheiro, E.A. 1989. Review: cholinergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy. Synapse 3: 154–171. Villringer, A., and Dirnagl, U. 1995.Coupling of brain activity and cerebral blood flow: basis of functional neuroimaging. Cerebrovascular Brain Metab 7: 240–276. Wendling, F., Bartolomei, F., Bellanger, J.J., and Chauvel, P. 2002. Epileptic fast activity can be explained by a model of impaired GABAergic dendritic inhibition. Eur J Neurosci 15: 1499–508. Young Jung, W., Pacia, S.V., and Devinsky, O. 1999. Neocortical temporal lobe epilepsy:intracranial EEG features and surgical outcome. J Clin Neurophysiol 16: 419–425. Zonta, M., Angulo, M.C., Gobbo, S., Rosengarten, B., Hossmann, K.A., Pozzan, T., and Carmignoto, G. 2003. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 6: 43–50.
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10 Pharmacologic Models of Generalized Absence Seizures in Rodents MIGUEL A. CORTEZ AND O. CARTER SNEAD III
in Table 2, criteria designed to mirror accurately human absence seizures (see Table 1). Pharmacologic animal models of absence seizures are defined by the electrobehavioral characteristics produced by acute administration of a specific compound to an animal, usually a rat or a mouse. Data from these pharmacologic models typically have a limited time frame for collection that is dependent on the half-life of the compound administered. Most of the pharmacologic models of absence, such as the 4,5,6,7 tetrahydroxyisoxazolo (4,5,c) pyridine 3-ol (THIP) model (Fariello and Golden, 1987), the penicillin model (Fisher and Prince, 1977; Gloor, 1984), the low-dose pentylenetetrazole (PTZ) model (Marescaux et al., 1984; Snead, 1988), and the GHB model (which utilizes the biologically inactive prodrug of GHB, g-butyrolactone [GBL]) (Snead, 1988, 2002, 1996; Hu et al., 2000, 2001a) are selflimited and resolve within a defined period of administration of the respective drugs. The exceptions to this rule are the AY-9944 (Cortez et al., 2001) and the methylazoxymethanol acetate (MAM)-AY-9944 models (Serbanescu et al., 2004), in which the atypical absence seizures induced by AY 9944 persist long after administration of the compound. Acute pharmacologic models of absence seizures have expanded our understanding of thalamocortical mechanisms (Banerjee et al., 1993; Cortez et al., 2001; Gloor, 1984; Gloor and Fariello, 1988; McCormick and Bal, 1997; Steriade and Llinas, 1988), absence-seizure ontogeny (Schickerova et al., 1984; Snead, 1984 b, 1994, 2002), GABAergic mechanisms (Smith and Biercamper, 1990; Snead, 1984a, 1990), and the molecular changes that may participate in the generation and maintenance of absence seizures (Banerjee et al., 1998a, b; Hu et al., 2001a, b; Kim et al., 2001). The pharmacologic animal models of general-
GENERAL DESCRIPTION OF MODEL Generalized absence seizures are defined as a paroxysmal loss of consciousness of abrupt and sudden onset and offset that is associated with bursts of bilaterally synchronous three cycles per second or 3 Hz spike-wave discharge (SWD) recorded on the electroencephalogram (EEG). There is no aura or postictal state. This particular type of seizure usually occurs in children between the ages of 4 years and adolescence, although they can occur at either ends of that age spectrum (Snead, 1995; Snead et al., 1999). Generalized absence seizures are pharmacologically unique, responding only to ethosuximide, trimethadione, valproic acid, or benzodiazepines and being resistant to or worsened by phenytoin, barbiturates, or carbamazepine (Snead et al., 1999) (Table 1). The unpredictable occurrence of absence seizures and the limitations of the clinical investigation on mechanisms of seizure generation constitute two fundamental challenges that led to the development of animal models of absence seizures. Since the development of the pentylenetetrazole animal model of acutely induced seizure and the evolution of this model as a standard tool for the screening and development of antiepileptic drugs (AEDs) with antiabsence properties, a number of additional pharmacologic models of absence seizures have been developed, along with considerable debate over their relevance to human absence epilepsy. As with all other epilepsy syndromes, there is no perfect animal model of epilepsy (for reviews see Mody and Schwartzkroin, 1997; Snead et al., 1999). Rather, the investigation of the determinants of both the subtlety and the complexity of human absence seizure phenomenology can be approached only by the rational use of multiple animal models that have in common the basic requirements outlined
Models of Seizures and Epilepsy
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TABLE 1 Characteristics of Generalize Absence Seizures in Humans Occur in children with onset between 4 and 15 yr of age EEG findings of bilaterally synchronous 3 Hz SWDs Clinical findings of staring, behavioral arrest, occasional myoclonus, eye movements, automatisms Brief, no aura, no postictal state Treated with ethosuximide, trimethadione, or valproic acid Aggravated by carbamazepine and phenytoin Aggravated by GABA agonists (progabide, vigabatrin, tiagabine, baclofen) c/s, cycles per second; EEG, electroencephalographic; GABA, gaminobutyric acid; SWD, spike-and-wave discharges.
TABLE 2 Criteria for Experimental Generalized Absence Seizures EEG and behavior similar to the human condition
International League Against Epilepsy (ILAE). Both clinicians and basic scientists have contributed to the dynamic evolution of the classifications of epileptic seizures (ILAE, 1981) and epileptic syndromes (ILAE, 1989). For a more detailed account of the rationale of these changes and for the terminology we use in this chapter, we refer you to the work of the Task Force on Classification and Terminology (ILAE, 2001). This chapter focuses on the pharmacologic models of absence seizures. All the pharmacologic models of typical absence seizures are acute and self-limited. The models of atypical absence seizures are chronic models. The epilepsy syndromes discussed in the context of the animal models reviewed are childhood and juvenile absence epilepsy and Lennox Gastaut syndrome. The latter is included because atypical absence seizures are an integral part of the symptom complex of this syndrome.
METHODS OF GENERATION
Reproducibility and predictability Quantifiable Appropriate pharmacology Unique developmental profile Exacerbated by GABAergic drugs Involvement of thalamocortical mechanisms Blocked by GABAB receptor antagonists EEG, electroencephalographic; GABA, g-aminobutyric acid.
ized bilaterally synchronous SWDs in rats are reproducible and have been standardized across models for the quantification of SWDs (Depaulis et al., 1989). Chronic animal models of absence epilepsy represent the semiology EEG correlates and pharmacologic profile of human typical (Snead 1995; 1996) and atypical absence seizures (Nolan et al., 2004). The former group includes the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) (Marescaux et al., 1984) and Waj/Rij (Wistar Albino Glaxo strain bred in Rijswijk, the Netherlands) (Coenen and Van Luijtelaar, 1987) rat models of typical absence epilepsy; the latter consists of the AY-9944 and AY9944/MAM (Cortez et al., 2004) rat models of atypical absence seizures (Cortez et al., 2001; Serbanescu et al., 2004; Wu et al., 2004).
WHAT DOES IT MODEL? To facilitate communication among clinicians and basic scientists and to enable the development of animal models relevant to human epilepsy, an agreement on the terminology to designate clinical seizures was required. This standardization was perhaps the major contribution of the
Acute pharmacologic models of typical absence seizures are derived from systemic administration of a single pharmacologic compound (THIP, GHB, PTZ, or penicillin) that results in bilaterally synchronous SWD associated with behavioral arrest, facial myoclonus, and vibrissal twitching. In the animal models of typical absence, there is a precise correlation between the onset and offset of the behavioral manifestations of the experimental absence seizure and that of the SWD. The seizure ends abruptly, and the animal resumes its preictal activity with no impairment in consciousness (Snead et al., 1999). In the models under review, the onset of absence seizures occurs reliably within 5 minutes of administration of drug. The acquired chronic models of atypical absence seizures are derived from a timely prenatal administration (MAM) and postnatal systemic administration of an inhibitor of cholesterol, AY-9944. As with all animal models of absence seizures, both acute and chronic pharmacologic models of absence seizures require EEG, and preferably EEG-video, recordings to measure accurately seizure duration, frequency, and severity (Depaulis et al., 1989). The methods of generation for the pharmacologic models, acute or chronic, are illustrated in Table 3, where the pharmacologic models of absence seizures are compared with standard genetic models of absence epilepsy, such as the GAERS and the WAG/Rij rats.
The THIP Model THIP is a GABA agonist that induces bilaterally synchronous SWDs in rats (Fariello and Golden, 1987). THIP is administered intraperitoneally (IP) in a dose of 5 to 10 mg per kilogram of weight and leads to bilaterally synchronous SWDs lasting 7 to 9 seconds that occur in bursts lasting 1
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Methods of Generation
TABLE 3 Acute Seizure Models and Chronic Epilepsy Models Dose (mg/kg)
Species
Strain
Acute PCL THIP LD-PTZ GHB
600Ta 10 20 100 (GBL)
Cat Rat Rat Rat
?
Genetic GAERS WAG/Rij
-
Chronic AY-9944 MAM-AY
7.5 100/7.5
Model
Age Specificity
Procedure
Monitoring
Effort (%)
Reliability
Seizures
Remission
SD, W SD, W
Adult Adult Adult Adult
IM IP IP IP
EEG EEG EEG EEG/VEEG
++ + + +
100 100 100 100
TAS ?TAS TAS TAS
Yes Yes Yes Yes
Rat Rat
W W
Adult Adult
Genetic Genetic
EEG/VEEG EEG/VEEG
+++ +++
100 100
TAS TAS
No No
Rat Rat
LEH LEH
Prepuberty Prepuberty
SC IP/SC
EEG/VEEG EEG/VEEG
++ +++
100 100
CAAS CRAAS
No No
AY-9944, AY; BCC, bicuculline; CAAS, chronic atypical absence seizures; CRAAS, chronic refractory atypical absence seizures; EEG, electroencephalography; GAERS, Genetic Absence Epilepsy in Rats from Strasbourg; GBL, gamma butyrolactone; GHB, gamma-hydroxybutyrate; HD, high dose; LD, low dose; MAM, methylazoxymethanol acetate; PCL, penicillin; PTZ, pentylenetetrazole; SD, Sprague-Dawley; TAS, typical absence seizures; VEEG, video electroencephalography; W, Wistar. a 600.000 units per kilogram.
to 7 seconds. The dose of THIP to elicit SWDs is 7.5 mg/kg of THIP given IP in a volume of normal saline of 1 ml/kg. The model is quantitated in the same manner as described later herein for the GHB model (Depaulis et al., 1989) (see Table 3).
The GHB Model GHB is a GABA metabolite that occurs naturally in the mammalian brain (Roth and Giarman, 1969). After IP administration of GHB, a predictable sequence of electrographic and behavioral events occurs, mirroring generalized absence seizures. This phenomenon has been well described in cats, rats, and monkeys (Bearden et al., 1980; Godschalk et al., 1977; Snead et al., 1976, 1978a–c, 1980, 1988). Use of the prodrug of GHB (i.e., GBL) enhances the reproducibility and predictability of the GHB model of absence seizures. GBL has been shown to be biologically inactive (Roth et al., 1966; Snead, 1991). An active lactonase that rapidly converts GBL to GHB is present in serum and liver but not brain or cerebrospinal fluid (Roth and Giarman, 1966; Roth et al., 1967). GBL is used because of the consistency and rapidity of onset of its effect (Bearden et al., 1980) and has been shown to produce exactly the same EEG and behavioral effect as that of GHB (Snead, 1991; Snead et al., 1980). The regional brain concentration of both GHB and GBL has been determined in time-course and dose-response studies after IP administration of GBL as well as at the onset of EEG changes induced by both GHB and GBL. Also, EEG and behavior were assessed following bilateral intrathalamic microinjection of either GHB or GBL in the rat. IP administration of GBL resulted in rapid onset of bilaterally synchronous SWDs in rat that correlated with an almost immediate appearance of GHB in the brain. In
animals that received IP GHB, the EEG changes did not occur until 20 minutes after GHB administration, when GHB levels in brain were peaking. The threshold brain concentration of GHB for EEG changes in both GHB- and GBL-treated animals was 240mM. GBL concentration in brain peaked 1 minute after GBL administration and fell rapidly to undetectable levels within 5 minutes. Bilateral microinjection of GHB into thalamus resulted in brief bursts of SWD, whereas GBL administered into the thalamus had no effect. These data confirm the hypothesis that GBL is biologically inactive in brain and support the validity of the use of GBL as a prodrug for GHB in this model of absence seizures (Snead, 1991). The GHB model of generalized absence seizures meets all criteria outlined in Table 2 (Snead, 2002; Snead et al., 1999). The model is quantitated similar to other electrographic models of generalized absence seizures (Depaulis et al., 1989). GBL-induced SWDs can be quantitated in terms of cumulative duration (in seconds) per 20-minute epoch of time or as a percent of control SWD duration. In this way the GHB model of absence can be compared with any other rodent model of generalized absence seizures using the same pharmacologic paradigm (Depaulis et al., 1989). The GHB rat model of generalized absence seizures is a useful experimental model for the study of the mechanisms of bilaterally synchronous SWD production and can be used to screen for antiabsence activity of potential antiepileptic drugs (Tables 1–3).
The PTZ Model PTZ is the most commonly used GABAA receptor (GABAAR) antagonist used to induce absence-like seizures with low doses. However, all GABAAR antagonists have this
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property (Snead et al., 2000). Low doses (20–30 mg/kg) of PTZ induce absence-like seizures that meet all the criteria for experimental absence set forth in Table 2, and the PTZ model is indistinguishable from the GHB model in this regard. The dose-response curves of GABAAR antagonists indicate that the EEG and behavior changes induced by GABAAR antagonists in mice represent a highly dose-specific continuum. Low doses induce absence-like seizures that meet the criteria for absence seizure models (see Table 2) (Marescaux et al., 1984; Snead, 1988; Snead et al., 2000). The dose-response curves for GABAAR antagonist-induced absence seizures indicate that the CD100 is 30 mg/kg for PTZ, 3 mg/kg for bicuculline, and 1.5 mg/kg for picrotoxin. As the dosage of GABAAR antagonists is increased, clonic seizures appear. In fact, even though the seizures do not meet the criteria outlined in Table 2 for experimental absence seizures, these clonic seizures are used to screen for antiabsence activity of putative anticonvulsant drugs in the PTZ screening model. With a further increase in dosage, tonic seizures emerge. The CD95 for the induction of clonic seizures with PTZ, bicuculline, and picrotoxin in the mGluR4+/+ mice is 45, 4.5, and 1.8 mg/kg, respectively (Snead et al., 2000). The clonic component observed with intermediate doses of PTZ (40–60 mg/kg) may be correlated with the work of Browning and Nelson (1986), who showed that clonus restricted to the face and forelimbs depends on seizure discharge emanating from structures within the forebrain for expression. Facial clonus mirrors the forebrain involvement and is typical of all rat models of generalized absence seizures (Table 3). The tonic seizures manifested in the face of high doses of GABAAR antagonists represent brainstem seizures (Browning and Nelson, 1986).
The Penicillin Model Intramuscular (IM) administration of penicillin (300,000–600,000 units/kg) to the cat consistently produces generalized, bilaterally synchronous SWD discharges associated with blinking, myoclonus, and staring (Fisher and Prince, 1977; Gloor, 1984; Guberman et al., 1975; TaylorCourval and Gloor, 1984). This model shows pharmacologic specificity for antiabsence drugs (Guberman et al., 1975) and is exacerbated by PTZ (Gloor and Testa, 1974), photic stimulation (Quesney, 1984), and GABAergic agonists (Fariello, 1979) (Table 3). When given IM to rodents, penicillin does not consistently produce bilaterally synchronous SWDs similar to that seen in cats. Rather, this drug produces multifocal spikes with only occasional bursts of bilaterally synchronous SWDs associated with a decrease in vigilance (Avoli, 1980). The penicillin model in rodents has not been as well characterized as the GHB and PTZ models and is of limited usefulness because of inconstant penetration of penicillin into the brain through the blood-brain barrier. This model,
however, has been shown to be exacerbated by GHB. Conversely, pretreatment with penicillin prolongs GHB-induced SWDs (Snead, l988). When using this model of absence, the same general experimental design as described previously is used. The dose of penicillin is from 300,000 to 600,000 units/kg given IM. There are no antiabsence, antiepileptic drugs or ontogeny data for the penicillin model in rat, but there is some evidence of involvement of thalamocortical mechanisms in penicillin-induced SWDs in rats (Avoli, 1980). Because of the limited usefulness of the penicillin model in rat and the fact that this chapter focuses on pharmacologic models in rodents, the penicillin model in rat will not be considered further in this review.
The AY-9944 Model Subcutaneous administration AY-9944 (7.5 mg/kg), a compound that inhibits the reduction of 7-dehydrocholesterol to cholesterol (Cenedella, 1980; Dvornik and Hill, 1968), to suckling rats every 6 days at postnatal days 2, 6, 8, 14, and 20 leads to absence-like seizures during the adult period (Cortez et al., 2001) (see Table 3). Seizures in this model represent human atypical absence seizures and are associated with an abnormal cognitive outcome (Chan et al., 2004; Nolan et al., 2004), with a prepubescent seizure onset (Persad et al., 2002) and maximum peak in the adult period (Cortez et al., 2002). These seizures are reduced by antiabsence drugs and exacerbated by phenytoin and GABA agonists (Cortez et al., 2001; Smith and Bierkamper, 1990). Developmentally the seizures in this model emerge at postnatal day (P) 21 (Persad et al., 2002; Snead and Cortez, 1999). The role of cholesterol inhibition in epileptogenesis is still under investigation (Serbanescu et al., 2004) (see Table 3). The AY-treated rat represents a predictable, reproducible, and clinically relevant animal model of atypical absence seizures that can be used to investigate the pathogenesis and treatment of this malignant disorder.
The MAM-AY Model (Double-Hit Model) Rats exposed to the antimitotic agent MAM on gestational day (G) 15 develop a neuronal migration disorder similar to the cortical dysplasias seen in the human brain. MAM-exposed suckling rats then undergo the AY model protocol as described previously. The result is that the animal presents with spontaneous, recurrent, atypical absence seizures that are characterized by bilaterally synchronous slow spike-wave discharges (SSWDs) with a frequency of 4 to 6 Hz (see Table 3). The MAM-AY-induced atypical absence seizures are refractory to ethosuximide and sodium valproate. Histologic examination of brains from MAM-treated rats showed hippocampal heterotopias in addition to atrophy and abnormalities of cortical lamination. The MAM-AY-treated rat represents a reproducible model
Characteristics and Defining Features
of refractory atypical absence seizures in children with brain dysgenesis (Serbanescu et al., 2004) (see Table 3).
CHARACTERISTICS AND DEFINING FEATURES Behavioral and Clinical Features 1. The THIP model. Animals demonstrate immobility and some vibrissal twitching. This model appears to be a generalized absence model based on electroclinical correlation; however, to date no pharmacologic data support this hypothesis, nor are there ontogeny data or data concerning possible thalamocortical mechanisms in the generation of these discharges. This model differs from others described in this review because THIP-induced absence-like seizures in rats are exacerbated by valproate (Vergnes et al., 1985). 2. The GHB model. The behavioral correlate of SWD is complete behavioral arrest with facial myoclonus and vibrissal twitching. GHB-induced SWDs most similar to those seen in humans are produced by intravenous (IV) administration of GHB to prepubescent monkeys. In this animal, an IV dose of 200 mg/kg of GHB results in 2.5 Hz SWDs associated with behavioral immobility, head drops, staring, pupillary dilation, eyelid fluttering, rhythmic eye movements, and stereotypical automatisms (Snead, 1978a). In the
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rat, a standard dose of 0.09 ml (100 mg) of GBL per kilogram given IP reliably produces onset of bilaterally synchronous SWDs within 2 to 5 minutes of GBL administration (Figure 1). The frequency of the SWD is 7 to 9 Hz. Associated with these hypersynchronous electrographic changes are behavioral arrest, facial myoclonus, and vibrissal twitching. Therefore this model meets the criteria outlined in Table 2 in that it is predictable, reproducible, and produces electrographic and behavioral events similar to the human condition. The GHB meets all the pharmacologic criteria (Snead, 1988) and involves thalamocortical circuitry (Banerjee et al., 1993). An additional advantage of the GHB model is that it affords control of pharmacokinetic variables in any pharmacologic study in that the concentration of GBL and GHB can be determined in the brain and the kinetics is known (Snead, 1991). 3. The low-dose PTZ model. This model would seem to meet all the criteria set forth in Table 2. A dose of 20 mg/kg of PTZ results in bursts of bilaterally synchronous SWDs with a frequency of about 7 to 9 Hz. The behavior seen in the PTZ animal is exactly the same as that described for GHB-treated animals, and it too meets pharmacologic criteria (Snead, 1988). 4. The AY-9944 model. This is a model of spontaneous, recurrent, atypical absence seizures. Clinically, atypical absence seizures are more complex than typical absence seizures. They present with a clinical behavioral change that
FIGURE 1 A: Baseline electrocorticogram (ECoG) recordings in controls are characterized by 35 to 50 uV, 7 to 11 Hz intermingled 6 to 9 Hz oscillations in awake resting conditions. B: The ECoG recording 5 minutes following 100 mg/kg GBL illustrates two consecutive high-amplitude 7 to 9 Hz bilaterally synchronous SWDs. The ictal behavior during SWD consisted of frozen stare, vibrissal twitching, and facial myoclonus with complete behavioral arrest. GBL, g-butyrolactone; LF-P, left frontal-parietal; RF-P, right frontal-parietal; SWDs, spike-and-wave discharges.
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is gradual in both onset and offset. During atypical absence seizures children retain some ability for purposeful movement and speech but with fogging of consciousness. The ictal EEG discharge in atypical absence seizures is slower than the 3 Hz that characterizes typical absence seizures, and it is not time-locked with the ictal behavior (Figure 2). Similarly, the AY9944 model shows a gradual onset and offset of ictal behavior and the ability to move purposefully during the seizures. Also reminiscent of the human condition, the ictal EEG discharge in the AY 9944 model is not time-locked with the ictal behavior, and it is slower in frequency than the epileptiform activity that characterizes typical absence seizures (Cortez et al., 2001). The phenotypic expression of atypical absence seizures in the AY 9944 model is highly significant because there is a major difference in outcome in children with typical compared with atypical absence seizures. Children with typical absence seizures have a good outcome and are spared any cognitive deficit, perhaps because of the limitation of the SWDs to the thalamocortical circuitry. In distinct contrast, atypical absence seizures are associated with a severely abnormal cognitive and neurodevelopmental outcome in children (Nolan et al., 2004). Therefore, whether absence seizures are typical or atypical is a critical predictor of outcome in children with absence epilepsy. The AY 9944 model also shows evidence of cognitive impairment (Chan et al., 2004) and thus represents a well-characterized model of atypical absence seizures that can be used to investigate mechanistic reasons for why atypical absence seizures confer such a poor long-term outcome on children afflicted by this disorder. 5. The MAM-AY model. The behavioral and EEG features of the MAM-AY model are exactly the same as the AY
model. The difference is that the MAM-AY treated rat is a model of medically refractory atypical absence seizures, whereas the AY model responds to ethosuximide and valproic acid (Serbanescu et al., 2004). Seizure Severity (Racine or Modified Racine Scale) The original rating scale of convulsive seizures presented by Racine in (1972a, b) was based on amygdala kindling and may not be applicable to kindling from other sites (McIntyre et al., 2002). Status Epilepticus (SE): Defining the Type of SE There are few data concerning animal models of generalized nonconvulsive status epilepticus (NCSE) (Hosford, 1999). Experimentally, PTZ-induced generalized NCSE leads to a subtle deficit-in-place learning in rats, with no demonstrable long-term behavioral effects on spatial learning or sensorimotor function. However, at 1 week followup, these animals showed an increase in absence seizures in response to a repeat dose of PTZ compared with controls. There was no detectable brain damage, but rats continued to show neuronal functional changes characterized by alteration of electrical excitability of neural circuits after generalized NCSE (Erdogan et al., 2004; Wong et al., 2003). This finding is consistent with our previous report on repeated induction of GHB absence seizures (Hu et al., 2001a) and is distinct from the pilocarpine-induced NCSE, which is associated with attendant seizure-related brain damage (Krsek et al., 2001).
FIGURE 2 Baseline electrocorticogram (ECoG) recordings at postnatal day 60 AY-9944-treated rat illustrates the spontaneous bilaterally synchronous and high-amplitude 5- to 6-Hz SSWDs from cortex, thalamus, and hippocampal monopolar electrodes. The ictal behavior during SSWDs consisted of frozen stare, vibrissal twitching, and facial myoclonus with the ability to move during seizures. L, left; R, right; Ctx, cortex; Th, thalamus; Hi, hippocampus. SSWD, slow spike-and-wave discharges.
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Characteristics and Defining Features
Forebrain Versus Hindbrain Seizures Experimental absence seizures are constrained within thalamocortical circuitry and therefore are exclusively forebrain seizures (Banerjee et al., 1993; Crunelli and Leresche, 2002; McCormick and Bal, 1997; Snead et al., 1999, 2000; Vergnes et al., 1990). As mentioned, increasing doses of the GABAAR antagonists bicuculline, picrotoxin, or pentylenetetrazole results in a dose-dependent phenomenon in which the lower doses first involve thalamocortical circuitry and produce absence-like seizures. Intermediate doses involve more widely distributed forebrain structures and produce forelimb clonus. Higher doses result in recruitment of brainstem circuitry with resultant tonic seizures. One could postulate that the same progression is present in Lennox-Gastaut syndrome, which is characterized by atypical absence (thalamocortical-hippocampal circuitry), clonic and myoclonic seizures (widespread neocortical circuitry), and tonic seizures (brainstem circuitry).
Electrographic and Electroencephalogric Features McQueen and Woodbury (1975) attempted to produce bilaterally synchronous SWDs in the electrocorticogram of rats by using several experimental paradigms, including administration of pentylenetetrazole, picrotoxin, conjugated estrogens, and bilateral intracerebral cobalt implants. In their hands, no pharmacologic modality produced consistent bilaterally synchronous SWDs. The authors concluded, therefore, that the rodent was not suitable for any detailed study of the pathophysiology of SWD. However, that same year, spontaneous SWDs were first reported in rodent (Vanderwolf, 1975) and have been described in a number of strains of rats since that time (Buzsaki et al., 1988; Kaplan, 1985, 1990; Kleinlogel, 1985). With a few notable excep-
tions (Cox et al., 1997), rats do not usually generate 3 Hz SWDs; rather, the usual frequency of SWDs in the THIP, PTZ, and GHB models range from 7 to 9 Hz, whereas the SSWD frequency in the AY-9944 and MAM-AY models is 4 to 6 Hz (Table 4).
Neuropathology Cell Loss There are no reports on cell loss in pharmacologic models of generalized absence seizures comparable to those reported in the other models of status that are associated with severe hippocampal damage. The reason for this is probably related to the fact that absence seizures are constrained within thalamocortical circuitry. Reactive Gliosis To date, there are no reports on reactive gliosis following experimental absence seizures of any kind except for the recently described two-hit MAM-AY9944 model of refractory atypical absence epilepsy (Serbanescu et al., 2004). However, further pathological and molecular investigation of the morphologic changes observed in animal models of limbic epilepsy may provide further understanding of the chronicity and refractoriness observed in MAM-atypical absence seizure model because atypical absence seizures appear to involve limbic as well as thalamocortical circuitry (Chan et al., 2004; Cortez et al., 2001). Plasticity Cognitive abnormalities are reported to occur in patients with atypical absence seizures (Nolan et al., 2004) and in the AY model (Chan et al., 2004). In typical absence seizures
TABLE 4 Electrographic features pharmacological models Models
Latency (minutes)
Frequency (Hz)
Seizure Duration (Sec/Hour)
8–9 2–4 3–5 4–5
7–9 7–9 7–9 7–9
300 400 400 300
TAS TAS TAS AAS
Prince, 1978 De Deyn, 1992 Snead, 2002 Depaulis et al; 1989
Genetic GAERS WAG/Rij
Spontaneous Spontaneous
9–11 7–9
450 560
CTAS CTAS
Marescaux, 1992 van Luijtelaar, 2001
Acquired AY-9944 MAM-AY
Spontaneous Spontaneous
4–6 4–6
600 560
CAAS CRAAS
Cortez et al; 2001 Serbanescu et al; 2004
Acute PCL LDPTZ GHB THIP
Human Seizure Type
References
Chronic
AS, Absence Seizures; AY-9944, AY; CAAS, Chronic Atypical Absence Seizures; CRAAS, Chronic Refractory Atypical Absence Seizures, CTAS, Chronic Typical Absence Seizures; GAERS, Genetic Absence Epilepsy Rat from Strasbourg; LD, Low Dose; PCL, Penicillin; PTZ, Pentylenetetrazole; TAS, Typical Absence Seizures; WAG/Rij, rat strain.
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and the experimental models of typical absence, there is no cognitive deficit. One of the great conundrums in clinical and basic epilepsy research, particularly in regard to children, in whom neurodevelopment is critical, is whether there is a cause and effect relationship between seizure frequency and severity and cognitive deficits. In the AY model the hypothesis was tested that ongoing AY-induced atypical absence seizures are wholly or partially responsible for the observed learning and memory by determining the effect of treating the seizures with ethosuximide on cognitive outcome. These data indicate that the cognitive deficits in this model of atypical absence seizures are largely seizure dependent, but seizure-independent mechanisms also may be at play in the genesis of the perturbation of cognition in this model. The most definitive way to test this hypothesis would be to accomplish complete, long-term seizure control in the AY model to tease out the effect of seizure versus intrinsic brain dysfunction on cognition (Chan et al., 2004).
Imaging and Metabolic Changes Electrophysiologic studies in WAG/Rij rats, have shown SWDs over the perioral somatosensory cortex but not over the visual cortex. Functional magnetic resonance imaging (fMRI) studies showed localized increases in fMRI signals in the perioral somatosensory cortex and thalamus during SWD. Furthermore, there was a parallel increase in neuronal activity and cerebral blood flow (CBF) during SWD in the whisker somatosensory (barrel) cortex, whereas the visual cortex showed no significant changes. These measurements were repeated during somatosensory (whisker) stimulation and bicuculline-induced GTCS in the same animals. These findings suggest that even in regions with intense neuronal activity during epileptic seizures, oxygen delivery exceeds metabolic needs, enabling fMRI to be used for investigation of dynamic cortical and subcortical network involvement in this disorder (Abo et al., 2004; Mueggler et al., 2001; Nersesyan et al., 2004). EEG-triggered blood oxygen level dependant (BOLD) fMRI has confirmed an anatomic correlation between areas in which an increased BOLD signal is found and those in which SWDs have been recorded and no negative BOLD signal was associated with these spontaneous SWDs (Tenney et al., 2004). In GAERS, the early studies using [C]2-deoxyglucose (2DG) autoradiographic method demonstrated a lack of correlation between the occurrence of SWDs and local cerebral metabolic rates for glucose (LCMRglcs) and favor normal or decreased ictal metabolism and increased interictal glucose utilization by the brain in rats with absence epilepsy (Nehlig et al., 1993). The diffuse increase in cerebral energy metabolism was not directly related the occurrence of SWDs (Nehlig et al., 1991). The generalized increase in cerebral glucose metabolism occurs in both at the glycolytic and at
the oxidative step. It is unclear how the ubiquitous mutation(s) generates SWDs only in the thalamocortical circuit (Dufour et al., 2003). During development and prior seizure onset by P21, the basal local cerebral metabolic rates for glucose (LCMRglcs) indicate that the genetic mutation(s) underlying the cellular and molecular events responsible for the expression of SWDs in adult GAERS is(are) able to increase metabolic activity in limbic structures and in the nigral inhibitory system before the occurrence of absence seizures. Conversely, the full electrocortical maturation seems necessary for the expression of SWDs with the concurrent increase in CMRglcs in adult GAERS (Nehlig et al., 1998 a, b). A recent study suggested the mediodorsal nucleus of the thalamus to be involved in absence seizures modulation and that this nucleus could participate in the control of the basal ganglia over generalized epileptic seizures (Riban et al., 2004). Rates of glucose utilization, measured by the quantitative autoradiographic 2-DG in rats at 10, 14, 17, and 21 days of postnatal life, indicate that maturation of connections, of metabolic activity, and of neurotransmitter interaction within the brain, occurs mainly during the third week of postnatal life (el Hamdi et al., 1992). 2-DG studies in the PTZ model (systemic administration of PTZ, first 40 mg/kg, followed 10 minutes later by 20 mg/kg, and later every 10 minutes, additional injections of PTZ 10 mg/kg until the onset of SE) have shown changes over the cerebral cortex, hippocampus, sensory regions, as well as scattered thalamic and hypothalamic nuclei; and they occur in the absence of visible neuronal death, most likely related to changes in the final arborization and synaptic organization of the developing brain (Nehlig et al., 1996). Brain extraction of (18)Flabeled 2-fluoro-2-deoxy-d-glucose (FDG) was significantly higher in (PTZ)-treated rats, and the transporter V(max) and blood brain barrier (BBB) glucose permeability increased by 30 to 40% (Cornford et al., 2000). In the THIP model, the magnitudes and distribution of in vivo cerebral metabolic responses were not correlated simply with markers for GABAergic synapses, suggesting that additional factors, such as neural circuitry, regulate the local cerebral glucose utilization (LCGU) responses to GABAergic drugs (Kelly et al., 1982; Palacios et al., 1982). Genetic and Molecular Changes Reciprocal thalamocortical projections play a critical role in the generation of SWDs in GAERS. The epileptic phenotype apparent in adult GAERS may result in part from elevations in T-type calcium channel mRNA levels (Rogawski and Loscher, 2004; Talley et al., 2000; Tsakiaridou et al., 1995). Similarly, quantification of channel expression indicates that the development of SWDs in WAG/Rij rats is concomitant with an increase of Ca(v)2.1 channels in the rhomboid thalamic nucleus (RhTN). These channels are
Characteristics and Defining Features
mainly presynaptic, as revealed by double immunofluorescence involving the presynaptic marker syntaxin (van de Bovenkamp-Janssen et al., 2004). Conversely, animals made deficient in T-type calcium channels are resistant to GHBinduced absence seizures (Kim et al., 2001). Some early immediate gene studies were done with the PTZ model. At 15 minutes following PTZ injection, only transcription for c-fos was increased. By 6 hours following PTZ treatment, transcription for all immediate early genes and for dynorphin and neuropeptide Y was increased; however, this increase was transient in that transcription of all genes returned to control values by 48 hours after PTZ treatment, which suggests that additional posttranscriptional regulation of gene expression occurs in hippocampal neurons (Yount et al., 1994). When the expression profiles of N-methyl-daspartate (NMDA) receptor subunits in rats were examined, the expression of NMDA receptors was found to undergo subunit- and region-related changes in the developmental and kindled seizure of rats induced by PTZ (Zhu et al., 2004). The ability of the rodent brain to support plasticityrelated phenomena declines with increasing age. Old rats retain the capacity to initiate transcription for immediate early genes, particularly as it relates to brain plasticity, in response to a strong stimulus such as PTZ. Although the aging brain retains the capacity to respond to chemically induced seizures, the induction of tissue plasminogen activator (TPA) mRNA is temporarily delayed and the levels are diminished with increasing age. Because TPA has been implicated in neuronal plasticity, this finding suggests that immediate early genes are important factors in the limited plasticity of the aging brain (Popa-Wagner et al., 2000). Transient changes in transcription of the GABA(A)receptor delta-subunit gene occur after acute PTZ-induced seizures, but not after kindling. (Penschuck et al., 1997). Zhang et al. (1991) examined fos oncoprotein expression in the rat thalamus with fos antibody immunohistochemistry after GHB-induced absence-like seizure activity. There was a progressive involvement of the bilateral thalamic paraventricular nuclei (PV), the lateral habenular nucleus (LHb), the PV, the rhomboid thalamic nucleus, and the intralaminar nuclei of the thalamus, which suggest that the LHb and the midline and intralaminar thalamic nuclei are very likely involved in the pathophysiology of absence seizures in the GHB model. GHB-induced absence seizures interact in a number of ways with GABAAR-mediated activity in brain, although GHB itself has no affinity for the GABAAR (Snead and Liu, 1993). The two synthetic neuroactive steroids, alphaxalone (5a-pregnane 3a-ol-11,20-dione) and tetrahydrodeoxycorticosterone both exacerbate GHB-induced absence seizures when given intrathalamically (Banerjee and Snead, 1998). These same neuroactive steroids inhibit [3H]GHB binding in a dose-dependent fashion. This inhibition is limited to the thalamus and increases after the onset of GHB-induced
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absence seizures, suggesting that the enhancement of inhibition of GHB binding was absence seizure induced (Banerjee et al., 1998a). GHB-induced absence seizures also regulate GABAA receptor a1 and a4 gene expression in thalamic relay nuclei (Banerjee et al., 1998b) and decrease steroid modulation of the binding of ligands to the GABAAR in thalamus (Banerjee et al., 1998c). Also, there is decreased release of GABA in thalamus in the GHB model of absence seizures (Banerjee and Snead, 1995). There also are perturbations of glutamate-mediated excitation in the GHB model of absence. GluR2 protein expression significantly decreases after onset of absence seizures in the GHB model, and this alteration of GluR2 was accompanied by a redistribution of GluR2 expression from laminae V to IV in the cerebral cortex (Hu et al., 2001b). The duration of SWDs was also significantly decreased in GluR2 knockout mice compared with wild-type controls (Hu et al., 2001a). Systemic administration of GBL has been associated with a decrease in K+-evoked glutamate release in thalamus, the onset and duration of which correlates with that of GHB-induced absence seizures. However, basal release of glutamate was unaltered in these experiments (Banerjee and Snead, 1995). Using the same experimental design, no alteration in basal and in K+-evoked glutamate release was observed in superficial laminae of cerebral cortex, that region of cortex from which SWDs emanate in the GHB model of absence seizures (Hu et al., 2000). There is reduction of sonic hedgehog (Shh) signaling in AY9944-treated embryos, resulting in the definition of a narrower ventral domain. Later in development, this reduction of Shh signaling led to a complete interruption of the pathway in the rostral hindbrain and caudal midbrain. Other regions, such as the forebrain and the spinal cord, appeared less sensitive to the reduction of Shh signaling, and interruption of the pathway was observed only in a subset of embryos (Gofflot et al., 2001). The steroidal analogue GW707, the oxidosqualene cyclase inhibitor U18666A, the 3-b-hydroxysterol delta(7)-reductase inhibitor AY-9944, and the vacuolar-type adenosine triphosphatase (ATPase) inhibitor bafilomycin A1 induced sequestration of free cholesterol in the endosomal-lysosomal compartment, leading to a positive filipin staining pattern and a complete inhibition of cholesterol ester synthesis (Issandou et al., 2004). It is unclear whether these mechanisms are involved in the AY model. MAM-induced apoptosis in the external granule cell layer of the rat is associated with strong c-Jun expression, which is restricted to apoptotic cells, and with the formation of high-molecular-weight c-Jun. c-Jun may participate in the genetic cascade of events leading to apoptotic cell death in the developing cerebellum (Ferrer et al., 1997). Molecular analysis revealed that MAM-induced heterotopic cells do not express mRNA markers normally found in hippocampal pyramidal cells or dentate granule cells (SCIP, Math-2, Prox-1, neuropilin-2). In contrast, Id-2 mRNA, normally
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abundant in layer II and III supragranular neocortical neurons but not in CA1 pyramidal neurons, was prominently expressed in hippocampal heterotopia (Castro et al., 2002).
Response to Antiepileptic Drugs and Usefulness in Screening Drugs Historically, the classic screening tests, such as maximal electroshock (MES) and PTZ, have used nonepileptic rodents to identify AEDs and their mechanisms of action and side effects. Clearly no single model can be used to identify potential compounds adequately for development, provided the full pharmacologic profile is demonstrated (Kupferberg, 2001). Optimal screening of novel AEDs, both for efficacy and side effects, requires models with receptor and ion channel changes similar to those in the target human syndrome (Meldrum, 2002); however, all the pharmacologic models, with the exception of the THIP model, have a predicted pharmacologic profile for absence seizures; that is, the seizures are blocked or attenuated by ethosuximide, trimethadione, benzodiazepines, and valproic acid and worsened by phenytoin or carbamazepine. Therefore the pharmacologic models are useful in screening putative antiepileptic drugs for antiabsence seizure activity.
LIMITATIONS How Easy to Develop Are They? The acute pharmacologic models of absence seizures are relatively easy to reproduce. The major obstacle for an investigator inexperienced in these models is the EEG monitoring required for the identification and quantitation of the experimental absence seizure. The chronic pharmacologic models that represent the human (AY and MAM-AY models) entail more demanding and labor intensive laboratory work. In both cases, acute or chronic systemic administration of the proabsence compounds leads to an epileptic phenomenon amenable to EEG or vigilance-controlled electroencephalogram (VEEG) monitoring and further circuitry and molecular investigations. The genetic models of absence continue to be the standard reference by which comparisons are made, but the AY and MAM-AY models compare quite favorably to the genetic models in terms of reliability, spontaneity, recurrence of seizures, and chronicity of the epilepsy induced. The AY and AY-MAM models have the additional advantage of representing a unique seizure type for which there is no genetic model, atypical absence seizures.
Mortality There is virtually no mortality involved in the administration of proabsence compounds at the recommended
doses. Mortality becomes an issue rather in the postoperative condition of rodents with chronically implanted electrodes. To minimize mortality in developing rodents, halothane anesthesia is recommended. Pentobarbital or ketamine anesthesias at the recommended doses also minimize mortality in adult rodents.
Reproducibility Acute pharmacologic models of generalized absence seizures are user friendly and quite reproducible. These acute models are particularly useful for illustrating the EEG correlates of absence to young scientists newly interested in epilepsy. Also, these models can be used to formulate hypotheses of mechanisms of epileptogenesis in absence seizures. The chronic models are also highly reproducible, although they require careful and timely administrations of either MAM at G 15 or AY at P2, P8, P14, and P20 to obtain 100% reproducibility of an accurate experimental representation of refractory atypical absence seizures with neuronal heterotopias (MAM-AY) or atypical absence seizures alone AY). The spontaneous occurrence of chronic SSWDs in these models is remarkably constant and age dependent, as described already. SSWD onset as early as P21, with maximum peak of atypical absence seizures during adulthood, is followed by severe exacerbation of SSWDs associated with similar seizure type and head clonus at the frequency of SSWDs, which is at 4 to 6 Hz. The reproducibility of the acquired chronic models is comparable only to that of the genetic models of absence epilepsy (Table 5).
Age-Related Effects The AEDs must be tested during development because it may not be possible to extrapolate age-specific anticonvulsant effects from studies in adult animals (Veliskova et al., 1996). Chronic postnatal administration of 1-phenylcyclohexylpiperidine (phencyclidine, or PCP), a NMDA channel blocker, alters PTZ-induced seizure susceptibility in an agedependent manner, leading to long-term changes that persist into adulthood (Sircar et al., 1994). GHB in threshold doses of 100 mg/kg was observed to produce bilaterally synchronous SWDs in rats initially at P18. Earlier than that, GHB produces varying degrees of slowing and, in very young animals, a profound burst suppression seen only at doses greater than 200 mg/kg in adult animals (Snead, 1984b, 1994). This ontogeny is similar to the developmental profile of the low-dose PTZ model of typical absence seizures (Schickerova et al., 1984) and the AY 9944 model of atypical absence epilepsy (Cortez et al., 2001). Seizure onset in the AY model occurs at postnatal day 21 (Cortez et al., 2001; Persad et al., 2002).
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Limitations
TABLE 5 Experimental Rat Models of Absence Seizures Compared with Those of Clinical Absence Seizures Typical
Atypical
Experimental Features General Reproducibility Standardized, quantitative Appropriate ontogeny Strain/Ethnic differences Gender differences Other seizure typesa Associated cognitive deficitsa Neurophysiologic Bilaterally synchronous SWD SWD frequencya, Hz SWD from thalamus and cortex SWD from hippocampusa Spontaneous, recurrent SWD Recurrent spontaneous absences
Experimental
GAERS
GHB
Clinical CTAE
AY-9944
MAM-AY
Clinical CAAS
+ + + -
+ + + ND ND -
NA NA NA NA -
+ + + + + + +
+ + + ND ND + +
NA NA NA + + +
+ 7–11 + -
+ 7–9 + -
+ 3 + ND
+ 4–6 + +
+ 4–6 + +
+ 1.5 ND +
+
Behavioral Immobility, staring and myoclonus Precise EEG/behavioral correlationa Movement during SWDa SWD and myoclonus during sleep
+ + -
+ + -
+ + + -
+ + + +
+ + + +
+ + +
Pharmacologic Blocked by ETX, VPA, TMD Exacerbated by GABAA, B R agonists Blocked by GABABR antagonists
+ + +
+ + +
+ + +
+ + +
+ +
+ + ND
a Characteristics that separate atypical absence seizures from typical absence seizures. These are also the features that define the AY-9944 and the MAM-AY treated rats as models of atypical absence epilepsy. AY-9944, AY; CAAS, Chronic Atypical Absence Seizures; CTAE, Clinical typical absence epilepsy; ETX, ethosuximide; GABAA, B R, Receptors; GAERS, Genetic Absence Epilepsy in Rats from Strasbourg; GHB, gamma-hydroxybutyrate model; MAM, methylazoxymethanol acetate; NA, non applicable; ND, no clinical data; SWD, spike-and-wave discharge; TMD, trimethadione; VPA, valproic acid. Modified from Cortez et al; 2001.
Need for Future Development to Improve the Model or Characterize its Features Common strategies used currently for both the development of antiepileptic drugs and the investigation of the basic pathogenesis of epilepsy entail the use of animal models of seizures that are known to respond to currently marketed antiepileptic drugs (White, 2002). Numerous problems have emerged with this approach (Serbanescu et al., 2004). First, the question arises about the utility of existing models in discovering novel and efficacious antiepileptic drugs. The use of standard animal models of seizures in adult rodents (e.g., electroshock, pentylenetetrazole, kindling, strychnine) to screen for anticonvulsant efficacy and a spectrum of seizures against which the potential AED is effective has become a self fulfilling prophecy. Putative AEDs shown to be efficacious against seizures in these animal-model screens have proven to be possessed of a clinical efficacy similar to other drugs that have been predicted to be effective by the animal
models. The data to support this supposition are that the rates of remission in patients who receive an established AED are similar to those who are treated with a new AED (Kwan and Brodie, 2000). Therefore the likelihood is remote that new drugs developed with existing strategies utilizing standard animal models will be beneficial for the 30% of patients with medically refractory epilepsy. The second problem with current strategies of AED development is the unlikelihood that the use of the standard models will give rise to new insights into clinically relevant pathogenic processes involved in epileptogenesis. There are at least three reasons for this concern. First, acute animal models of seizures lend themselves to hypothesis testing that addresses the underlying seizure events and not epileptogenesis, the latter requiring an animal to have spontaneous, recurrent seizures over a long period. Second, the fundamental question of what makes epilepsy refractory cannot be addressed with an animal model that responds to known AEDs. Finally, existing models that utilize adult animals do
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not address the unique problems of medically refractory epilepsy in children, in whom it is highly likely that the refractoriness of the epilepsy is related to a perturbation in brain development. Selection criteria for laboratory models of intractable epilepsy have been proposed (Löscher, 1997), including similarity to the clinical condition, paroxysmal EEG abnormalities associated with the behavioral ictal event in the animal, resistance of the seizures to standard AEDs, and the ability to use the putative model for long-term studies on anticonvulsant drug efficacy. The MAM-AY model meets all these criteria (Serbanescu et al., 2004). An additional advantage is that the MAM-AY model is uniquely applicable to the investigation of refractory epilepsy in children. Specifically, the MAM-AY model of medically refractory epilepsy is particularly relevant to the Lennox-Gastaut syndrome in children. The ictal event in the MAM-AY model consists of spontaneous recurrent, medically refractory atypical absence seizures that occur in the presence of a congenitally dysplastic brain, a finding observed in 75% of patients with the Lennox-Gastaut syndrome (Sillinpää, 1995; Zifkin, 1990).
INSIGHTS INTO HUMAN DISORDERS Underlying Mechanisms Primary Generalized Epilepsies In both rodent and feline models of absence seizures, the evidence suggests that the mechanisms that underlie the SWD bursts that characterize this seizure type may be related to the thalamocortical mechanism that mediates spindles and recruiting responses (Crunelli and Leresche, 2002). Seizures with SWD complexes preferentially evolve from sleep oscillations. They are initiated in the neocortex and spread to the thalamus after a few seconds (Sitnikova and Van Luijtelaaar, 2004; Steriade and Amzica, 2003; Timofeev et al., 2004). Because human (Williams, 1953) and animal data both strongly suggest that generalized absence seizures arise from aberrant thalamocortical rhythms, it may prove helpful to consider the functional aspects of thalamocortical circuitry. The EEG is state dependent because the electrical activity of mammalian forebrain as recorded on the EEG varies with the state of consciousness. When the animal is alert, the EEG is characterized by desynchronization or a replacement of synchronized rhythms by lower amplitude and faster wave forms. Alternatively, certain altered states of consciousness (e.g., slow-wave sleep) are associated with synchronous EEG activity, such as high-amplitude oscillations with relatively slow frequencies (Avoli et al., 1993; Steriade et al., 1990). These state related alterations in EEG are a reflection of fundamental and dynamic underlying changes
in the activity of forebrain neurons in response to interplay between the intrinsic activity of thalamocortical circuitry with ascending neurotransmitter systems that project on thalamocortical structures (Steriade et al., 1990). The regional distribution of GHB-induced SWD has been determined by the use of EEG mapping and lesional studies. To carry out the EEG mapping studies, bipolar depth electrodes were implanted in discrete regions of thalamus, cortex, and hippocampus in rat. With the advent of GHBinduced absence seizures, the ventroposterolateral (VPL), ventroposteromedial (VPM), medial, and reticular nuclei (RT) of the thalamus discharged synchronously with layers I through IV of cerebral cortex. No SWDs were recorded from deeper layers (V–VI) of cerebral cortex. Hippocampal structures were completely silent during the GHB-induced SWDs (Banerjee et al., 1993). The effect of bilateral electrolytic lesions of various thalamic nuclei on the GHB-induced absence seizures also has been determined. Bilateral lesions in mediodorsal (MD) and intralaminar thalamic nuclei abolished GHB-induced SWDs from both the cortex and the thalamus. Bilateral lesions of the VPL and RT suppressed but did not eliminate GHBinduced SWDs. The emanation of SWD from superficial layers of cortex during GHB-induced absence suggests that the projections from mediodorsal thalamic nuclei to those superficial cortical laminae rich in [3H]GHB binding sites form an integral part of thalamocortical circuitry involved in GHB-induced absence seizures (Banerjee and Snead, 1994; Banerjee et al., 1993). Other subcortical structures such as the mamillary bodies and their projections (Mirski and Ferrendelli, 1986; Mirski et al., 1986), the superior colliculus (Depaulis et al., 1990), and the substania nigra (Depaulis et al., 1988a, b, 1989) may also have an important role in generalized absence seizures experimentally, but their involvement in human absence has yet to be established. Symptomatic Generalized Epilepsies Recent experimental observations involving the chronic model of atypical absence seizures led us to revisit the clinical spectrum of atypical absence seizures. We concluded that differentiation between children with only atypical absence seizures and children with multiple seizure types may be useful with respect to potential academic ability (Nolan et al., 2004). Human atypical absence seizures produce a 1 to 2-Hz SSWD that is concomitant with other abnormal background activity. Although patients with atypical absence do not have an aura or postictal state, the behavioral arrest is not sudden. The gradual entrance into ictal state prevents the patient from losing complete consciousness; thus some mobility and interaction with others can occur. During the seizure, the patient may become atonic or have other seizure types, such as tonic or clonic activity.
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Atypical absence seizures are not as easily controlled by AEDs and frequently become intractable to drug treatment. The prognosis is usually poor, and patients are usually developmentally delayed, often displaying neurologic and cognitive deficits. Both clinical and experimental data indicate that the hippocampus may play a substantial role in epileptogenesis and cognitive outcome (Chan et al., 2004; Nolan et al., 2004). A great dead of work on the chronic models will be required to determine the molecular perturbations that lead to the SSWD and cognitive impairment associated with atypical absence seizures. Loss of selective types of interneurons, alteration of GABA receptor configuration, or a decrease in dendritic inhibition could contribute to the development of spontaneous seizures (Morimoto et al., 2004).
Usefulness for Treatment Assessment, Development, and Screening Animal and human data suggest that generalized seizures involve selective thalamocortical networks. We are confident that a greater understanding of these molecular and network mechanisms will ultimately lead to improved targeted therapies for generalized epilepsy (Blumenfeld, 2003). For example, the findings that mGluR4 knockout mice are resistant to absence seizures induced by low doses of GABAAR antagonists and that this phenotype is reproduced by the intra-nRT administration of an mGluR4 antagonist suggest that mGluR4 antagonists may potentially be useful in the treatment of absence epilepsy.
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Roth, R.H., and Giarman, N.J. 1969. Conversion in vivo of gammaaminobutyric acid to gamma-hydroxybutyrate in the rat. Biochem Pharmacol 18: 247–250. Roth, R.H., Delgado, J.M.R., and Giarman, N.J. 1966. g-Hydroxybutyric acid and g-butyrolactone. The metabolically active form. Int J Neuropharmacol 5: 421–428. Roth, R.H., Levy, R., and Giarman, N.J. 1967. Dependence of rat serum lactonase upon calcium. Biochem Pharmacol 16: 596–598. Schickerova, R., Mares, P., and Trojan, S. 1984. Correlation between electrocorticographic and motor phenomena induced by pentylenetetrazol during ontogenesis in rats. Exp Neurol 84: 153–164. Serbanescu, I., Cortez, M.A., McKerlie, C., and Snead, O.C. III. 2004. Refractory atypical absence seizures in rat: a two hit model. Epilepsia Res. 62(1): 53–63. Sillinpää, M. 1995. Epidemiology of intractable epilepsy in children. In Intractable Epilepsy. Eds. S.I. Johannessen, L. Gram, M. Sillinpää, and T. Tomson. pp.13–25. Petersfield, UK: Wrightson Biomedical Publishing. Sircar, R., Veliskova, J., and Moshe, S.L. 1994. Chronic neonatal phencyclidine treatment produces age-related changes in pentylenetetrazolinduced seizures. Brain Res Dev Brain Res 81: 185–191. Sitnikova, E., and van Luijtelaar, G. 2004. Cortical control of generalized absence seizures: effect of lidocaine applied to the somatosensory cortex in WAG/Rij rats. Brain Res 1012: 127–137. Smith, K.A., and Bierkamper, G.G. 1990. Paradoxical role of GABA in a chronic model of petit mal (absence)-like epilepsy in the rat. Eur J Pharmacol 176: 45–55. Snead, O.C. III. 1978a. Gammahydroxybutyrate in the monkey. I. Electroencephalographic, behavioral, and pharmacokinetic studies. Neurology 28: 636–642. Snead, O.C. III. 1978b. Gammahydroxybutyrate in the monkey. II. Effect of chronic oral anticonvulsant drugs. Neurology 28: 643–648. Snead, O.C. III. 1978c. Gammahydroxybutyrate in the monkey. III. Effect of intravenous anticonvulsant drugs. Neurology 28: 1173–1178. Snead, O.C. III. 1984a. Gamma-hydroxybutyric acid, gamma-aminobutyric acid and petit mal epilepsy. In Neurotransmitters, Seizures, and Epilepsy, vol II. Eds. R.G. Fariello, P.L. Morselli, K.G. Lloyd, L.F. Quesney, and J. Engel. pp. 37–38. New York: Raven Press. Snead, O.C. III. 1984b. Ontogeny of gamma-hydroxybutyric acid. II. Electroencephalographic effects. Brain Res 317: 89–96. Snead, O.C. III. 1988. The g-hydroxybutyrate model of generalized absence seizures: further characterization and comparison to other absence models. Epilepsia 29: 361–368. Snead, O.C. III. 1990. The ontogeny of GABAergic enhancement of the gamma-hydroxybutyrate model of generalized absence seizures. Epilepsia 31: 363–368. Snead, O.C. III. 1991. The g-hydroxybutyrate model of absence seizures: correlation of regional brain levels of g-hydroxybutyric acid and gbutyrolactone with spike-wave discharges. Neuropharmacology 30: 161–167. Snead, O.C. III. 1994. The ontogeny of [3H]gamma-hydroxybutyrate and [3H]GABAB binding sites: relation to the development of experimental absence seizures. Brain Res 659: 147–156. Snead, O.C. III. 1995. Basic mechanisms of generalized absence seizures. Ann Neurol 37: 146–147. Snead, O.C. III. 1996. Antiabsence seizure activity of specific GABAB and g-hydroxybutyric acid receptor antagonists. Pharmacol Biochem Behav 53: 73–79. Snead, O.C. III. 2002. g-Hydroxybutyric acid and absence seizure activity. In Gamma Hydroxybutyrate. Eds. G. Tunnicliff, C.D. Cash. pp. 132–141. New York: Taylor and Francis. Snead, O.C., III, and Liu, C.C. 1993. GABAA receptor function in the ghydroxybutyrate model of generalized absence seizures. Neuropharmacology 32: 401–409.
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Snead, O.C. III, and Cortez, M.A. 1999. Ontogeny of spike and wave discharge (SWD) in the chronic model of atypical absence seizures. Neurology 52: A49. Snead, O.C. III, Yu, R.K., and Huttenlocher, P.R. 1976. Gamma-hydroxybutyrate. Correlation of serum and cerebrospinal fluid levels with electroencephalographic and behavioral effects. Neurology 26: 51–56. Snead, O.C. III, Bearden, L.J., and Pegram, V. 1980. Effect of acute and chronic anticonvulsant administration on endogenous gamma-hydroxybutyrate in rat brain. Neuropharmacology 19: 47–52. Snead, O.C. III, Depaulis, A., Vergnes, M., and Marescaux, C. 1999. Absence epilepsy: advances in experimental animal models. Adv Neurol 79: 253–278. Snead, O.C. III, Banerjee, P.K., Burnham, M., and Hampson, D. 2000. Modulation of absence seizures by the GABA(A) receptor: a critical role for metabotropic glutamate receptor 4 (mGluR4). J Neurosci 20: 6218–6224. Steriade, M., Amzica, F. 2003. Sleep oscillations developing into seizures in corticothalamic systems. Epilepsia 44(Suppl 12): 9–20. Steriade, M., and Llinas, R.R. 1988. The functional states of the thalamus and the associated neuronal interplay. Physiol Rev 68: 649–742. Steriade, M., Gloor, P., Llinas, R.R., Lopes da Silva, F.H., and Mesulam, M.M. 1990. Basic mechanisms of cerebral rhythmic activities. Electroencephalogr Clin Neurophysiol 76: 481–508. Talley, E.M., Solorzano, G., Depaulis, A., Perez-Reyes, E., and Bayliss, D.A. 2000. Low-voltage-activated calcium channel subunit expression in a genetic model of absence epilepsy in the rat. Brain Res Mol Brain Res 175: 159–165. Taylor-Courval, D., and Gloor, P. 1984. Behavioral alterations associated with generalized spike and wave discharges in the EEG of the cat. Exp Neurol 83: 167–186. Tenney, J.R., Duong, T.Q., King, J.A., and Ferris, C.F. 2004. FMRI of brain activation in a genetic rat model of absence seizures. Epilepsia 45: 576–582. Timofeev, I., Grenier, F., and Steriade, M. 2004. Contribution of intrinsic neuronal factors in the generation of cortically driven electrographic seizures. J Neurophysiol 92: 1133–1143. Tsakiridou, E., Bertolini, L., DeCurtis, M., Avanzini, G., and Pape, H.C. 1995. Selective increasein T-type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy. J Neurosci 153: 3110–3117. van de Bovenkamp-Janssen, M.C., Scheenen, W.J., Kuijpers-Kwant, F.J., Kozicz, T., Veening, J.G., van Luijtelaar, E.L., McEnery, M.W. et al.
2004. Differential expression of high voltage-activated Ca2+ channel types in the rostral reticular thalamic nucleus of the absence epileptic WAG/Rij rat. J Neurobiol 58: 467–478. Vanderwolf, C.H. 1975. Neocortical and hippocampal activation in relation to behavior effects of atropine, eserine, phenothiazines, and amphetamine. J Comp Physiol Psychol 88: 300–323. Veliskova, J., Velisek, L., and Moshe, S.L. 1996. Age-specific effects of baclofen on pentylenetetrazol-induced seizures in developing rats. Epilepsia 37: 718–722. Vergnes, M., Marescaux, C., Micheletti, G., Rumbach, L., and Warter, J.M. 1985. Blockade of “antiabsence” activity of sodium valproate by THIP in rat model of genetic petit mal-like seizures. Comparison with ethosuximide. J Neural Transm 63: 133–141. Vergnes, M., Marescaux, C., Depaulis, A., Micheletti, G., and Warter, J.M. 1990. The spontaneous spike and wave discharges in Wistar Rats: a model of genetic generalized nonconvulsive epilepsy. In Generalized Epilepsy: Neurobiological Approaches. Eds. M. Avoli, P. Gloor, G. Kostopoulos, and R. Naquet. pp. 238–253. Boston: Birkhauser. White, H.S. 2002. Animal models of epileptogenesis. Neurology 59(Suppl 5): S7–S14. Williams, D. 1953. A study of thalamic and cortical rhythms in petit mal. Brain 76: 50–69. Wong, M., Wozniak, D.F., and Yamada, K.A. 2003. An animal model of generalized nonconvulsive status epilepticus: immediate characteristics and long-term effects. Exp Neurol 183: 87–99. Wu, J., Ellsworth, K., Ellsworth, M., Schroeder, K.M., Smith, K., and Fisher, R.S. 2004. Abnormal benzodiazepine and zinc modulation of GABAA receptors in an acquired absence epilepsy model. Brain Res 1013: 230–240. Yount, G.L., Ponsalle, P., and White, J.D. 1994. Pentylenetetrazole-induced seizures stimulate transcription of early and late response genes. Brain Res Mol Brain Res 21: 219–224. Zhang, X., Ju, G., and Le Gal La Salle, G. 1991. Fos expression in GHBinduced generalized absence epilepsy in the thalamus of the rat. Neuroreport 2: 469–472. Zhu, L.J., Chen, Z., Zhang, L.S., Xu, S.J, Xu, A.J., and Luo, J.H. 2004. Spatiotemporal changes of the N-methyl-d-aspartate receptor subunit levels in rats with pentylenetetrazole-induced seizures. Neurosci Lett 356: 53–56. Zifkin, B.G. 1990. The Lennox-Gastaut syndrome. In Comprehensive Epileptology. Eds. M. Dam and L. Gram. pp. 123–131. New York: Raven Press.
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11 Models of Chemically-Induced Acute Seizures ˇEK LIBOR VELÍS
This chapter reviews models of generalized seizures induced by systemic administration or focal application of chemical agents. For some substances, there is an unavoidable overlap with the preceding chapter on absence models or with subsequent chapters on exploring status epilepticus (SE) or the effects of repeated seizures. This chapter intends to help the investigator develop and use a particular model. Chemical agents are organized by the route of administration and then by the prevailing mechanism of action or a major feature. This chapter is not an all-exhaustive review of available convulsive chemicals, however, so references to appropriate reviews are provided (Fisher, 1989; Sarkisian, 2001). Whenever possible, a reference to a general neurotoxicology text that summarizes the neurotoxic effects in the experimental models and in humans is linked (Spencer and Schaumburg, 2000). Because seizures in humans most frequently occur during childhood and I have extensively studied developmental issues of seizure models, special emphasis is placed on developing animals, if such information is available. Significant material for this chapter was retrieved from the lifetime studies of Pavel Maresˇ and his co-workers, whose contributions to the field of developmental seizure models should be acknowledged irrespective of the fact that I worked on my Ph.D. thesis in Pavel Maresˇ’s laboratory.
is the most widely used route to create models of seizures. The procedure is convenient, straightforward, and simple. Sub-Q injection is commonly applied in the skin fold on the back of the neck. A syringe with a small, 25- to 26-gauge, needle is usually sufficient. The onset of action after sub-Q administration of the bolus of the drug is usually slow compared with the same bolus administered IP or IV. The doses producing seizures in 50% of subjects (CD50) usually follow the same order, from highest to the lowest: sub-Q, IP, and IV administration (Petersen, 1983). For IP injection, a larger needle (22- to 23-gauge) is more convenient for its capability to penetrate all involved layers: skin, muscle wall, and peritoneum. After inserting the needle, gentle aspiration will verify whether the inferior vena cava or abdominal aorta has not been hit. If this is the case and the drug is administered, seizures develop within seconds. Some drugs (such as bicuculline) are heavily metabolized in liver and, if the liver enzymatic system is developed, IP doses for the same effect to occur may be substantially higher than sub-Q doses (firstpass effect). For IV administration of the drug, the tail vein is usually used. Administration requires complete restraint of the rat. Effects of the bolus dose will occur faster than after a similar dose administered IP. Sometimes a catheter in the tail is implanted for continuous convulsant drug delivery. This arrangement allows for determining the seizure threshold based on the amount of delivered drug (Orlof et al., 1949). For basic experiments, only experimental animals, a syringe with a needle, a convulsant substance in a solution, and a stopwatch are required. However, stress resulting from the systemic drug administration (e.g., handling, potentially painful injection, etc.) may interfere with the model expression.
SYSTEMIC INJECTION OF CONVULSANT SUBSTANCES Systemic administration (subcutaneous [sub-Q], intraperitoneal [IP], or intravenous [IV]) of convulsant agents
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GABA-Related Substances The g-aminobutyric acid (GABA) drugs are probably the most commonly used. The group of chemicals includes GABAA receptor antagonists, chloride channel blockers, inhibitors of GABA synthesis, convulsant benzodiazepines and drugs with prevailing or suspected effects on the GABAA receptor, including flurothyl, which is commonly administered by inhalation (although IP and IV administrations are also possible) and has certain GABA-related effects. Figure 1 provides a simplified view of a GABAA receptor and the convulsant drugs described here. For a brief overview of the models, see Table 1.
GABA site Bicuculline
Barbiturate site
Cl-
Benzodiazepine site b-Carbolines
Steroid site
Picrotoxin site Picrotoxin Ro 5-3663 TBPS site PTZ
FIGURE 1 Simplified scheme of the g-aminobutyric acid (GABA)A receptor with its binding sites and identified sites of action for some convulsant drugs. PTZ, pentylenetetrazol; TBPS, t-butyl-bicyclo-phosphorothionate.
Behaviorally all these drugs induce different phenomena, depending on the dose, delay after administration, and developmental stage of the experimental animal (for detailed analysis, see Chapter 48). Behavioral phenomena usually occur in the following sequence: (1) freezing behavior, (2) myoclonic twitches, (3) clonic seizures, and (4) tonic-clonic seizures. After high doses of convulsant drugs, the rats usually die. In the electroencephalogram (EEG), four different patterns appear: 1. Isolated spikes. These spikes may be initially focal and eventually may generalize. Sometimes they are associated with mycolonic twitches, but the association is loose. Developing animals are unable to generate spikes. During the first 2 to 3 postnatal weeks, slow and sharp waves are seen instead. 2. Spindles of spike and wave activity. In the rat, this crescendo-decrescendo EEG pattern usually runs with a frequency around 5 to 6 Hz, and it is associated with freezing (motionless stare) behavior. Occurrence of these spindles is developmentally regulated and also depends on the drug used. 3. Decrescendo spike and wave. This pattern is commonly associated with clonic seizures and thus may also be regulated developmentally and depend on the drug. Dissociation of this EEG pattern from behavioral seizures is frequent. 4. Polyspike, polyspike, and wave. This EEG pattern frequently occurs at the beginning of tonic-clonic seizures and is followed by regular spike and wave discharges (SWDs). Early in development, only sharp waves may be present.
TABLE 1 Acute Seizure Models Induced by GABA-Related Drugs in Adult and Immature Rats Administration route
Bolus doses adult rats (mg/kg)a
Bolus doses PN12–18 rats (mg/kg)a
Saline
Sub-Q IP
40–120 40–100
40–100 30–100
0.1 N HCl
IP IV
6–8 1–2
2–4 1–2
Picrotoxin
Saline
IP, IV, sub-Q
4–6
3–4
Isonicotinehydrazide
Saline
IP
300–400
200–400
Drug Pentylenetetrazol (PTZ) Bicuculline
3-Mercaptopropionic acid (3-MPA)
Solubility
Already liquid
IP
30–60
20–60
Allylglycine
Saline
IP
100–250
100–250
b-carbolines
Acetic acid
IP Sub-Q
1–2 1–5
N/A N/A
Ro 5-3663
0.1 N HCl
IP
5–15
5–15
GABA, g-aminobutyric acid; IP, intraperitoneally; IV, intravenously; N/A, information not available; PN, postnatal day; sub-Q, subcutaneously. a See the text for additional information about the correlation of convulsant drug doses and elicited seizure types. Doses for younger ages are available in the text.
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Neuropathology is usually negligible. These drugs are commonly used to produce acute seizures without long-term survival. Although high doses may lead to prolonged tonicclonic seizures, adult animals usually die during these seizures. Minor changes in the cerebellum have been described after convulsions induced by pentylenetetrazol and bicuculline (Ben-Ari et al., 1981b). In adult rats, induction of SE as a prerequisite for occurrence of neuropathologic changes is difficult when using these drugs. However, in immature rats it is possible to sustain SE using repeated administrations of subconvulsive doses (Nehlig and de Vasconselos, 1996; Pereira de Vasconcelos et al., 1995). Resulting neuronal injury is only transient and does not lead to neuronal death (Pineau et al., 1999). Relationship to Human Seizure Disorders Differential features of seizures induced by GABArelated substances can be related to different seizures in humans (Engel, 2001). Thus motionless stare accompanied by rhythmic EEG spindles, which involves thalamocortical circuits, is considered a model of generalized seizures— typical absence seizures (Depaulis et al., 1989). Clonic seizures are considered a model of generalized seizures— myoclonic seizures (Loscher and Schmidt, 1988). Finally, tonic-clonic seizures are believed to represent generalized seizures—tonic-clonic seizures (Sarkisian, 2001).
FIGURE 2 CD50 for clonic and tonic-clonic seizures induced by the subcutaneous administration of pentylenetetrazol (PTZ) during postnatal development of Wistar rats (according to Velísˇek et al., 1992). Mean (M) ± standard error of the mean (SEM) (in mg/kg) are displayed. Clonic seizures were regularly induced by PTZ from the third postnatal week; tonic-clonic seizures occurred throughout development.
Pentylenetetrazol Methods of Generation Originally a cardiostimulant, pentylenetetrazol (Cardiazol, Leptazol, Metrazol, pentamethylenetetrazol, pentetrazol, pentazol), or PTZ, has significant convulsant potency in mice, rats, monkeys, and humans (Reinhard and Reinhard, 1977; Swinyard et al., 1989; Vernadakis and Woodbury, 1969a, b). PTZ-induced clonic seizures represent a routine test for screening anticonvulsants (Swinyard et al., 1989). PTZ freely dissolves in saline or water and can be administered sub-Q, IP (most commonly), or IV (less commonly, usually via the tail vein). Repeated low doses of PTZ administered IP may be used to induce SE in immature rats (Nehlig and de Vasconselos, 1996; Pereira de Vasconcelos et al., 1995). Developmental CD50 levels for clonic and tonicclonic seizures in male Wistar rats are shown in Figure 2 (according to (Velísˇek et al., 1992). CD50 levels for IP and IV administration are lower compared with those of sub-Q doses (Fisher, 1989). For practical purposes, doses around 100 mg/kg IP or sub-Q are usually used. With these doses, seizures develop within 20 minutes after application. Defining Features PTZ induces all four behavioral phenomena: freezing, myclonic twitches, clonic seizures, and tonic-clonic sei-
FIGURE 3 Rhythmic, spindle-shaped discharges induced by a low systemic dose of pentylenetetrazol (PTZ) in a Wistar rat. These discharges with a crescendo-decrescendo pattern were associated with freezing behavior (motionless stare). Electrocorticograms from RF, right frontal (sensorimotor) cortex; RO, right occipital (visual) cortex; LF, left frontal cortex; LO, left occipital cortex.
zures. Twitches and tonic-clonic seizures are recorded throughout development. However, there is limited occurrence of freezing behavior and clonic seizures during the first 2 postnatal weeks of the rat. PTZ-induced seizures can be easily scored according to the appropriate scoring table (see Chapter 48). Graded doses of PTZ in adult rats will induce specific seizures. Thus low doses can be titrated to induce only freezing with underlying EEG spindles (Figure 3), somewhat higher doses for kindling, even higher doses for clonic seizures, and finally doses over 100 mg/kg for tonic-clonic seizures. Using a constant PTZ dose, latency to onset of seizures is also age specific (de Casrilevitz et al., 1971; Velísˇek et al., 1992; Vernadakis and Woodbury, 1969a; Weller and Mostofsky, 1995). The whole spectrum of EEG changes can be observed after PTZ
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administration (Schickerová et al., 1984; Zouhar et al., 1980); however these changes are developmentally regulated: EEG spindles occur from the third postnatal week and beyond (Marescaux et al., 1984; Ono et al., 1990; Schickerová et al., 1984). Metabolic [14C]2-deoxyglucose (2DG) studies demonstrate that there is an increased uptake (ergo metabolic activation) in the motor and limbic cortex after PTZ-induced status epilepticus during the first postnatal week and in the brainstem areas at postnatal day (PN)10. In PN17, PN21, and adult rats, there is a redistribution of glucose uptake from the cortex and hippocampus to the midbrain, brainstem, hypothalamus, and septum (Ben-Ari et al., 1981a; Nehlig et al., 1992; Pereira de Vasconcelos et al., 1992). In young rats (
as a solvent, followed by careful titration using a weak base (0.1 N NaOH) up to the resulting pH of approximately 5.6 (de Feo et al., 1985; Velísˇek et al., 1995). Because of the low final pH, it is recommended that bicuculline solution be administered IP because the sub-Q injection is very painful and stressful. However, IP injection of bicuculline in mature rats may require significantly higher doses than young rats require because of the “first-pass” effect. This disadvantage can be avoided by using IV administration of bicuculline (Zouhar et al., 1989). For IP administration, 2- to 4-mg/kg doses are used for developing rats; 6- to 8-mg/kg doses are used for prepubertal and adult rats (Velísˇková et al., 1990). However, if administered IV, a dose of 2 mg/kg is sufficient for the adult rats (Zouhar et al., 1989). After these doses, seizures occur within 20 minutes. An additional bicuculline model is worth mentioning. A derivative, bicuculline methiodide, is almost freely soluble in saline. However, this compound does not cross the blood-brain barrier. Nevertheless, in developing rats, in which the blood-brain barrier is still imperfect, systemic administration of bicuculline methiodide induces seizures. Required doses are between 2 and 20 mg/kg IP for rats up to PN18 (Maresˇ et al., 2000). Defining Features Bicuculline produces all behavioral phenomena described previously. The EEG pattern also fits the description (Figure 4). The only difference compared with other drugs is that clonic seizures induced by bicuculline begin to occur during the second postnatal week of the rat. Metabolic studies with 2DG identified changes after bicuculline seizures similar to those seen after PTZ seizures (Ben-Ari et al., 1981a). A study using 2DG in paralyzed, ventilated rats, however, did not show any increases in cerebral glucose metabolic rate (Evans and Meldrum, 1984).
FIGURE 4 Example of several episodes of rhythmic, spindle-shaped discharges induced by intraperitoneal administration of bicuculline in a Wistar rat. Discharges were generalized over all cortical recording. Arrows mark these discharges in RF cortical area recording. Electrocorticograms from the RF, right frontal (sensorimotor) cortex; RO, right occipital (visual) cortex; LF, left frontal cortex; LO, left occipital cortex.
Systemic Injection of Convulsant Substances
Limitations The difficulties in dissolving and the acidic pH of the solution, along with the significant first-pass effect somewhat limit the usefulness of systemically administered bicuculline for seizure models. EEG spindles can vary not only as a function of age but also as a function of sex (Mateˇ jovská et al., 1998). Insights into Human Disorders The mechanisms of action of bicuculine are well understood (competitive antagonism at GABA binding site of the GABAA receptor), although additional effects of bicuculline, such as prolongation of Ca2+ action potential and blockade of K+ channels, have been reported (Seutin and Johnson, 1999). Classic anticonvulsant drugs have good effects against bicuculline-induced seizures with superb activity of GABAA receptor-acting drugs, such as benzodiazepines (De Deyn et al., 1992). Except for the well-known mechanism of action, there is no other advantage to using bicuculline over PTZ for screening for anticonvulsant drugs. Picrotoxin Methods of Generation In contrast to bicuculline, picrotoxin (Ludolph and Spencer, 2000) can be dissolved in saline (Velísˇková et al., 1990, 1993), although some groups prefer dissolving in 10% dimethylsulfoxide (Hiscock et al., 1996). Route of administration can be sub-Q, IP, or IV. Doses for IP bolus administration range between 3 and 6 mg/kg through all developmental stages of the rat. Development of seizures is slower than in PTZ and bicuculline. Seizures usually occur within 30 to 40 minutes. Continuous infusion of picrotoxin solution in the jugular vein has been used to characterize regional EEG patterns and the EEG power spectra (Mackenzie et al., 2002). The addition of a small dose of picrotoxin to pilocarpine has been used to intensify development of pilocarpine-induced SE, thus helping to decrease the pilocarpine dose (Hamani and Mello, 1997, 2002). Defining Features Behavioral and EEG patterns after picrotoxin resemble to those found after PTZ and bicuculline administration (Mackenzie et al., 2002). After a single picrotoxin seizure, early gene c-fos expression was found in the frontal and parietal cortex as well as in the piriform and entorhinal cortex. Neurons with c-fos expression were mostly calbindin D28K-positive cells and many of the calcium-binding, protein-unlabeled neurons, which were spiny and presumed excitatory, (Hiscock et al., 1996). Limitations Picrotoxin seizures develop at a slower pace than PTZ and bicuculline seizures, and they also occur less reliably
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(Velísˇek et al., 1995). However, the very specific mechanism of action (even compared with bicuculline) and the very good solubility provide a small advantage over bicucullineinduced seizures. Insights into Human Disorders Picrotoxin seizures are well defined mechanistically. They arise from GABAA receptor chloride channel blockade. Available data on anticonvulsant drugs suggest a similar efficacy as against bicuculline-induced seizures (Velísˇková et al., 1990; De Deyn et al., 1992; Velísˇková et al., 1993). The usefulness for screening of putative antiepileptic drugs is similar as in bicuculline-induced seizures. Glutamic Acid Decarboxylase (GAD) Inhibitors Methods of Generation The GAD inhibitors (isonicotinehydrazide, 3-mercaptopropionic acid, and allylglycine), which inhibit GABA synthesis, can be potent convulsants. Isonicotinehydrazide can be dissolved in saline in the concentration of 100 mg/ml. Doses used for IP induction of seizures range between 150 and 400 mg/kg throughout development in the rat (Maresˇ and Trojan, 1991). Seizures usually occur within 40 to 60 minutes. 3-Mercaptopropionic acid is a liquid and doses between 30 and 60 mg/kg can be used for IP seizure induction throughout development (Maresˇ et al., 1993). In this model, seizures occur within 10 to 15 minutes at all developmental stages except for the first postnatal week (within 25 to 40 minutes). Finally, allyglycine can be dissolved in saline; and 100 to 250 mg/kg can produce seizures. The solution should be used within 2 hours, though (Horton and Meldrum, 1973; Ashton and Wauquier, 1979, 1981; de Feo et al., 1985; Thomas and Yang, 1991). After this dose, seizures develop between 60 and 80 minutes after administration. Defining Features All drugs invariably induce a sequence of myoclonic twitches as well as clonic and tonic-clonic seizures. In the isonicotinehydrazide model, in younger rats, tonic-clonic seizures continuously follow after clonic seizures (Maresˇ and Trojan, 1991). In the EEG, all drugs induce spikes (sharp waves in immature animals), polyspikes, and spike and wave patterns. In the 3-mercaptopropionic acid model, the correlation between behavioral seizures and EEG (recorded in the neocortex) is poor even in adult rats (Maresˇ et al., 1993). Tonic extension may be pronounced in seizures induced with allylglycine (Thomas and Yang, 1991). During l-allylglycine seizures, there is significantly increased glucose cerebral metabolic rate determined by the 2DG method in the cortex, caudate/putamen, amygdala, hippocampus, thalamus, and inferior colliculus (Evans and
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Meldrum, 1984); however, this study was carried out on paralyzed and ventilated rats.
Beta-carbolines and convulsant benzodiazepine Ro 5-3663 Methods of Generation
Limitations The slow development of isonicotinehydrazide and allylglycine-induced seizures may become inconvenient. Reliability of seizure production is very good for all three drugs. Insights into Human Disorders Figure 5 shows a simplified scheme of GABA synthesis and the effects of main GAD inhibitors. Isonicotinehydrazide (Schröder, 2000) acts most likely via inhibition of the GAD coenzyme pyridoxine (Williams and Bain, 1961; Woodbury, 1980b). 3-Mercaptopropionic acid (Spencer, 2000b) and allylglycine (Ludolph, 2000a) are direct inhibitors of GAD (Lamar, 1970; Horton and Meldrum, 1973; Loscher, 1979; Meldrum et al., 1987). As a result, GABA production is decreased and both GABAA and GABAB receptors are less activated. Use of these models for screening purposes does not provide advantages other than the known mechanisms of action at a particular stage of GABA synthesis.
Isonicotinehydrazide
pyridoxine glutamate
GAD
GABA
3-Mercaptopropionic acid Allylglycine FIGURE 5 Simplified diagram of biosynthesis pathway for g-aminobutyric acid (GABA) with sites of action of some convulsant drugs. X marks a blockade of the enzyme. GAD, glutamic acid decarboxylase.
Among beta-carbolines (Collins and Neafsey, 2000), methyl-b-carboline-3-carboxylate (b-CCM) and methyl6,7-dimethoxyl-4-ethyl-b-carboline (DMCM) were shown to induce convulsions (Braestrup and Nielsen, 1982; Braestrup et al., 1980, 1982; Jensen et al., 1983; Prado de Carvalho et al., 1984). Effective doses of b-CCM are 1 to 10 mg/kg administered IP in mice. CD50 for b-CCM for clonic seizures in mice was calculated at about 1 mg/kg IP (Chapman et al., 1985) and 5 mg/kg sub-Q (Prado de Carvalho et al., 1984). DMCM can be dissolved in minimal amounts of acetic acid and diluted with saline (Chapman et al., 1985). Effective doses of DMCM to elicit clonic seizures in rats are about 1 mg/kg IV (Ableitner and Herz, 1987) and up to 15 mg/kg IP to elicit tonic-clonic seizures in mice (Chapman et al., 1985).The CD50 for clonic seizures in mice is 1.5 mg/kg IP (Chapman et al., 1985). Petersen’s study (Petersen, 1983) demonstrates the differences of CD50 levels for clonic and tonic-clonic seizures as well as LD50 after IP, SC, IV, and oral (PO) administration in NMR1 mice strain. Ro 5-3663 can be dissolved in 0.1 N HCl. Effective doses range between 5 and 15 mg/kg IP with seizures occurring within 30 minutes (Maresˇ et al., 1987). b-CCM can be also used as an add-on convulsant with picrotoxin and strychnine (for details, see Zhao et al., 1996). Defining Features Both beta-carbolines and Ro 5-3663 induce clonic and tonic-clonic seizures. EEG analysis of Ro 5-3663 effects determined that this drug also produces EEG spindles associated with behavioral arrest. A functional (2DG) study demonstrated an increase in local glucose utilization in the hippocampus, dentate gyrus, and basal ganglia, including the substantia nigra reticulate, and in the ventral and lateral thalamic nuclei (Ableitner and Herz, 1987). Limitations These drugs represent less commonly used GABArelated agents for production of seizures. Additional information on the effects of these drugs can be found in the following studies (Chapman et al., 1987; Croucher et al., 1984; Meldrum, 1984; Meldrum and Chapman, 1986; Petersen 1983). Insights into Human Disorders Beta-carbolines have a high affinity to benzodiazepine receptors and act as inverse agonists (Braestrup et al., 1982, 1983). The result is seizures resulting from attenuated GABAA inhibition. A benzodiazepine derivative, Ro 5-3663 is also a potent convulsant; however, it acts via a picrotoxin-
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binding site in the chloride channel of the GABAA receptor (Harrison and Simmonds, 1983; Maresˇ et al., 1987).
Excitatory Amino Acid–Related Substances Excitatory features of some of the excitatory amino acid (EEA) drugs were described more than 50 years ago (Hayashi, 1952). EAAs are very popular for the induction of SE and studies of neuropathologic changes. However, these features are described in detail elsewhere in this book (see Chapter 34). In addition to the systemic administration (Collins et al., 1980), microamounts of these drugs are frequently administered (microinfused) in the lateral ventricles for central, intracerebroventricular (ICV) administration (Ben-Ari et al., 1980). The advantage is that only a very small amount of the drug is required for a convulsant response and the lethal effects associated with systemic administration are diminished. Early studies showed feasibility of seizure induction by systemically administered EAA in the adult and infant rats (Albala et al., 1984; BenAri et al., 1981b; Fagg et al., 1986; Nitecka et al., 1984; Schoepp et al., 1990; Zaczek and Coyle, 1982; Zaczek et al., 1981). For an overview of these models, see Table 2. Behaviorally, these drugs produce various automatisms and clonic and tonic-clonic seizures. The occurrence of all these behavioral features is drug and age specific. Additionally, special seizure patterns may be recorded. The EEG patterns are also very similar to EEGs already described, with spikes, spike and waves, polyspikes, and wave complexes. However, an extra feature can be observed after the administration of all principal EAA agonists: largeamplitude slow waves with superimposed spikes (“serrated waves”) frequently recorded from the hippocampus. Sometimes periods of EEG suppression occur. There is no special timing for the occurrence of the discharges and EEG suppression; frequently they are observed during motor convulsions but they can be also recorded when the rat is motionlessness.
The neuropathology is very complicated and still is subjected to extensive studies. In general, all drugs can produce neuronal damage resulting from neurotoxic features (BenAri, 1985). In addition, all these drugs can produce SE (prolonged continuous seizures longer than 30 minutes), which has an additional capability to produce seizure-induced damage (Albala et al., 1984; Pitkanen and Sutula, 2002; Sutula et al., 1988). Current data indicate that the neuronal damage resulting from EAA-related drug-induced convulsions has age- and brain-region specific features (Haut et al., 2004). Relationship to human seizure disorders (Engel, 2001): Undoubtedly, automatisms, which may reflect very focal activation of the limbic system (entorhinal cortex, dentate gyrus, hippocampus proper) may serve as a model of focal motor seizure with typical automatisms. The occurrence of clonic or tonic-clonic seizures may be considered the secondary generalization from a focus. Special seizure types may become a useful model for intractable epilepsy syndromes, as described later.
Kainic Acid Methods of Generation Kainic acid (KA) is the most common neurotoxin (McGeer et al., 1978) used to produce SE and related neuro-pathologic changes (Baran, 1985; Ben-Ari et al., 1981b; Fariello et al., 1980; Fuller and Olney, 1979; Heggli and Malthe-Sorenssen, 1982; Lothman and Collins, 1981; Sperk, 1985; Sperk et al., 1983; Worms et al., 1981; Zaczek et al., 1981). KA can be dissolved in normal saline; however, the resulting pH is on the acidic side. Therefore phosphatebuffered saline, pH 7.4, and the IP route of administration are preferred. Doses depend on the maturational state of the animal because there is somewhat limited penetration of KA to the brain. Usually for adult rats doses of 10 to 14 mg/kg are sufficient to produce seizures and SE. In young rats,
TABLE 2 Acute Seizure Models Induced by Excitatory Amino Acid (EAA)-Related Drugs in Immature and Adult Rats Drug
Solubility
Kainic acid (KA)
PBS buffer
Quisqualic acid
TRIS buffer, water
N-methyl-D-aspartic acid (NMDA)
Saline up to 50 mg/ml
Homocystein Homocysteic acid
Administration route
Bolus doses: adult rats (mg/kg)a
Bolus doses: PN12–18 rats (mg/kg)a
IP
10–14
2–8
ICV, IP
10 mg/min ICV infusion
2–12 mg/kg IP
IP
150–250
10–60
Saline
IP
6–16 mmol/kg
6–16 mmol/kg
Saline, adjust pH
IP
>13 mmol/kg
3–13 mmol/kg
ICV, intracerebroventricularly; IP, intraperitoneally; IV, intravenously; N/A, information not available; PBS, phosphate-buffered saline; PN, postnatal day. a See text for additional information about the correlation of convulsant drug doses and elicited seizure types. Doses for younger ages are available in the text.
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doses of 1 to 4 mg/kg may be sufficient. For doses in developing rats, see Table 3. It should be noted that mice generally require higher doses of KA; doses of 20 to 60 mg/kg are usual. For additional information on KA, see Chapter 34. Defining Features Systemically administered KA produces hypoactivity for about 20 to 30 minutes (Ben-Ari et al., 1981b). Then agespecific automatisms occur. In rats, during the first 2 weeks, profuse scratching prevails (Albala et al., 1984; Cherubini et al., 1983; Tremblay et al., 1984; Velísˇková et al., 1988).
TABLE 3 Developmental Doses of Kainic Acid (KA): Rat Strain Differences
Age (postnatal days; PN)
KA dose mg/kg IP 90–100% status epilepticus Sprague-Dawley ratsa
KA dose mg/kg IP (age in parentheses) 50–100% status epilepticus Wistar ratsb
PN5 (7)
1–2
4–8
PN10 (12)
2–3
4–8
PN20 (18)
8
8–10
PN30 (25)
10–11
8–14
PN60 (90)
10–12
10–16
“adult” a b
From Stafstrom et al., 1992. From Velísˇková et al., 1988.
During the third postnatal week, “wet-dog shakes” (WDS) begin to emerge; their frequency increases with aging. Clonic seizures can be induced only exceptionally during first 2 postnatal weeks, regularly appear during the third and fourth postnatal week, and are the exclusive pattern in the adult rats. Profound salivation frequently occurs. Tonicclonic seizures decrease in incidence with increasing age. They represent the only seizure type in 2-week-old and younger rats and become extremely rare even after high doses of KA (60 mg/kg; unpublished observation) in adult rats. Seizures usually occur within the first 60 minutes after KA administration. EEG patterns consist of spikes, spike and waves, and polyspikes and waves (Figure 6). Hippocampal recordings may show serrated waves. There is very poor correlation between KA-induced motor and EEG activity. EEG activity significantly prevails (Figure 7). Electrographic seizures may last many hours after the behavioral seizures have stopped (Giorgi et al., 2005). Metabolic studies in the adult rats revealed an increase in 2DG uptake in the hippocampal formation and lateral septum during early stages (<1 hour) of KA seizures (Ben-Ari et al., 1981a, 1984). Substantia nigra part reticulata also displayed increased 2DG uptake (Albala et al., 1984). Later putamen, anterolateral thalamic nuclei, nucleus accumbens, rostral limbic complex, perirhinal and piriform cortex, and amygdala also display 2DG uptake increases (Ben-Ari et al., 1981a, 1984). Additionally, there is a decrease in 2DG uptake in the neocortex, forebrain, mesencephalon, and brainstem. In immature, PN15 to 18 rats, KA seizures increase 2DG uptake in the hippocampus and lateral septum, deep cortical layers, and
FIGURE 6 Discharges induced by i.p. administration of kainic acid (KA; 6–14 mg/kg) in a Wistar rat. Arrow indicates the onset of clonic motor seizures. There were many more discharges than motor seizure activity. For quantification, see Figure 7. Electrocorticogram and stereo electroencephalogram (EEG) from RO, right occipital (visual) cortex; LF, left frontal (sensorimotor) cortex; LO, left occipital cortex; RHi, right hippocampus; LHi, left hippocampus.
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been described in adult rats of different strains: Thus hippocampal CA1 damage prevails in Wistar rats and either CA1 or CA3 damage prevails in Sprague-Dawley rats (Albala et al., 1984; Sperber, 1996b; Brandt et al., 2003). Additionally, the damage may be sex specific (Brandt et al., 2003). Immature 2-week-old rats are very resistant to a classic KA seizure-induced damage seen in adult rats (hilus of the dentate gyrus, CA3 pyramidal cells (Albala et al., 1984; Nitecka et al., 1984; Sperber, 1996a; Sperber et al., 1999b; Haas et al., 2001; Sperber and Moshé, 2001). Insights into Human Disorders
FIGURE 7 Quantification of the occurrence of discharges in the hippocampus and neocortex along with behavioral activity and motor seizures after intraperitoneal administration of 14 mg/kg of kainic acid (KA). Duration of phenomena in seconds was recorded within each 5-minute interval and plotted as a histogram (means from eight rats are shown, standard error of the means were omitted for clarity). Occurrence of discharges prevailed over the occurrence of behavioral activity, especially motor seizures. This indicates significant dissociation of electroencephalographic and motor seizures after systemic KA.
KA is an agonist for the KA subtype of ionotropic glutamate receptor with highest density in the hippocampus, especially in the CA3 region, also in the amygdala, perirhinal and entorhinal cortex (Miller et al., 1990). These areas are preferentially affected after systemic administration of KA. Therefore KA serves as a valuable model of partial (focal) seizures with complex symptomatology and secondary generalization from the limbic focus (Ben-Ari, 1985; Engel, 2001) as well as a model of epileptogenesis after status epilepticus (Cavalheiro et al., 1982; Stafstrom et al., 1993). For these features, KA-induced seizures may be a valuable tool in assessment of the antiepileptic drug efficacy in complex partial seizures. Another glutamate analogue structurally related to KA, domoic acid, produced temporal lobe seizures in humans after ingestion of infested mussels (Cendes et al., 1995), providing an additional support for use of KA in modeling limbic seizures. Quisqualic Acid/a-Amino-3-Hydroxy-5-Methyl-4Isoxasole Propionic Acid Methods of Generation
ventromedial thalamic nuclei. There are no changes in the 2DG uptake in the substantia nigra pars reticulata (Albala et al., 1984). A study in paralyzed, ventilated rats showed significant increases in glucose metabolism only throughout the hippocampus (Evans and Meldrum, 1984). Neuropathology is age specific. After PN18 to 21, there is a significant cell loss after KA in the hippocampus associated with synaptic remodeling (Albala et al., 1984; Sperber et al., 1999b). Other areas of neuronal damage include pyriform and entorhinal cortex, medial, cortical, and lateral posterior nuclei of amygdala and thalamic nuclei (mediodorsal, paraventricular and parafascicular) (Ben-Ari et al., 1981b). In younger animals, only minimal nonspecific changes in limbic structures can be observed (Albala et al., 1984; Nitecka et al., 1984). For additional details, see Chapter 34. Limitations Doses of KA may be quite specific for the rat strain (see Table 3). Similarly, significant differences in the prevailing location and extent of KA seizure–induced damage have
Quisqualic acid (QA) and amino-3-hydroxy-5-methyl-4isoxasole propionic acid (AMPA) are almost exclusively used for ICV administrations in minute amounts. QA can be dissolved easily in tris(hydroxyymethyl)-aminomethane (TRIS) buffer and pH balanced to 7.4 (Thurber et al., 1994). The ICV dose for QA is 10 mg per minute of 2.94 mg/ml solution. Schoepp and colleagues (1990) described convulsant effects of QA after systemic administration in PN7 and PN11 rats. QA seizures can also be induced in mice (Jurson and Freed, 1990; Schwarz and Freed, 1986). The ICV dose for AMPA is 1 to 5 mg (Turski et al., 1981). Defining Features After a period of either hypoactivity (PN23) or agitation (older rats), QA administered unilaterally ICV produces contraversive circling throughout development (PN23 adult). Later the rats display facial clonus, salivation, and tail tonus. In the EEG, spikes, spikes and waves, and serrated waves can be recorded (Thurber et al., 1994). Histologic evaluation after 2 weeks revealed that QA caused
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massive neuronal loss in hippocampal CA2 and CA3 regions. There was also a cell loss in the hilus and CA1 region of both the injected and contralateral side, which indicates seizure-induced damage. Limitations Blood-brain barrier permeability is an obstacle for systemic use of QA/AMPA in older animals. Insights into Human Disorders QA and AMPA act as agonists at the AMPA subtype of ionotropic glutamate receptors expressed especially in the hippocampal CA3 region and in the amygdala (Insel et al., 1990). Thus seizures after systemic administration may arise from these structures. However, the usefulness of these two drugs administered systemically for screening purposes is limited by their cost and very poor penetration to the brain. N-methyl-d-aspartic acid (NMDA) Methods of Generation NMDA dissolves well in normal saline up to 50 mg/ml. Seizures are usually induced intracerebrally administered, 2 to 10 nmol (Ishimoto et al., 2000; Lees, 1995) administered NMDA because of poor blood-brain barrier permeability. However, appropriate doses of NMDA (>100 mg/kg) administered systemically will induce seizures even in mature animals with a fully developed blood-brain barrier. In mice, CD50 is around 110 mg/kg administered IP (Budziszewska et al., 1998), whereas in rats, it is estimated between 150 to 200 mg/kg IP (Maresˇ and Velísˇek, 1992). In immature rats, doses required for seizure induction are much lower than in adults (Schoepp et al., 1990; Maresˇ and Velísˇek, 1992) (for developmental CD50, see Figure 8). Seizures occur within 15 to 45 minutes, depending on the dose. Defining Features The first symptom is increased locomotor activity, especially in prepubertal and adult rats. Wild running is the most prominent feature. If rats are allowed enough space, they run the “8”-shaped trajectory. After the period of hyperactivity, automatisms occur at all developmental stages. These usually start with tail flicking beginning at the tip and continuing sigmoidally to the trunk. Sometimes biting of forelimbs or hindlimbs occurs. In rats younger than 3 weeks of age, NMDA induces a special seizure pattern consisting of hyperflexion of the head, body, and tail while the rat is lying on its side. These seizures are termed emprosthotonic. Additionally, NMDA induces tonic-clonic but not clonic seizures throughout development. The tonic phase of tonic-clonic seizures may not be developed. However, in this model, clonus regularly precedes tonus, which is indicative of imminent death (Maresˇ and Velísˇek, 1992). (For further
FIGURE 8 CD50 values for tonic-clonic seizures induced by i.p. administration of N-methyl-d-aspartic acid (NMDA) during postnatal development in Wistar rats (according to Maresˇ and Velísˇek, 1992). Tonic-clonic seizures were the only seizure occurring throughout development after systemic administration of NMDA. CD50 for postnal day 60 (PN60: young adult) rats was estimated due to few experimental groups used for this age.
details, see Chapter 48.) The EEG pattern is nonspecific. Long periods of EEG suppression occur in cortical and hippocampal recordings (Figure 9). During these almost isoelectric recordings, various behaviors can be observed, including hyperactivity and tonic-clonic seizures. Later, serrated EEG waves may occur (Kábová et al., 1999). Frequently, chaotic activity in the EEG appears between motor seizures. Although intracerebrally administered NMDA induces severe neuronal damage (Lees, 1995; McDonald et al., 1988) in adult rats, no overt neuronal damage is found after seizures induced by systemic NMDA in young rats (Kábová et al., 1999; Stafstrom and Sasaki-Adams, 2003). Systemic administration of NMDA in rats induces c-fos expression, especially in the piriform cortex and dentate gyrus of the hippocampus, irrespective of seizure occurrence (Morgan and Linnoila, 1991). Limitations Follow-up studies after NMDA-induced emprosthotonus are quite restricted by the very low survival rates. Administration of the NMDA receptor antagonist ketamine (50 mg/kg IP) after a defined (15–30 minutes) period of seizure duration may help to increase survival. Insights into Human Disorders NMDA is a prototype agonist at the NMDA subtype of the ionotropic glutamate receptor. These receptors are prominently expressed in the hippocampal CA1, dentate gyrus, and striatum (Insel et al., 1990). Based on the specific motor pattern of hyperflexion, significant age specificity (Maresˇ and Velísˇek, 1992), resistance to pharmacotherapy (Kábová et al., 1999; Velísˇek and Maresˇ, 1995), and long-term learning impairments (Stafstrom and
Systemic Injection of Convulsant Substances
137
normal saline and effective dose ranges between 6 and 16 mmol/kg in rats (Kubová et al., 1995). D,l-homocysteic acid can be also dissolved in saline; however, the pH needs to be adjusted by alkalinization. CD50 for PN7–25 rats are between 1.5 and 12.5 mmol/kg (Folbergrová et al., 2000; Maresˇ et al., 1997). Defining Features
FIGURE 9 Electrocorticogram and stereo electroencephalographic (EEG) recording in a postnatal day 18 (PN18) Wistar rat before and after intraperitoneal administration of N-methyl-d-aspartate (NMDA). Top traces: baseline recordings before NMDA administration. Bottom traces: EEG suppression, which developed after systemic NMDA administration. During these periods of EEG suppression, different associated motor behaviors or seizures were recorded from motionlessness to emprosthotonus and tonic-clonic seizures. RF, right frontal (sensorimotor) cortex; RO, right occipital (visual) cortex; LF, left frontal cortex; LO, left occipital cortex; RHi, right hippocampus.
Sasaki-Adams, 2003), we believe that NMDA-induced emprosthotonic seizures in the immature rats are one of the closest models currently available for the West syndrome, although with some reservations (Lado and Moshé, 2002; Stafstrom and Holmes, 2002). NMDA seizures have been previously proposed as a model of refractory seizures (Loscher, 1997). NMDA induces only a tonic-clonic seizure pattern, and this seizure type in various chemically induced models is significantly suppressed by NMDA receptor antagonists; therefore we further propose that the tonicclonic seizure pattern uses NMDA receptor neurotransmission for its expression (Velísˇek and Maresˇ, 1990, 1992; Velísˇek et al., 1989, 1990 1991, 1997). Other Drugs (Homocysteine, Homocysteic Acid) Methods of Generation Homocysteine seizures can be induced in rats and in mice (Blennow et al., 1979). Homocysteine can be dissolved in
D,l-homocysteic acid induces seizures similar to NMDA during development. A special feature of D,l-homocysteic acid and both its stereoisomers is the occurrence of barrel rotations in PN12 rats (Maresˇ et al., 1997). Homocysteine induces different seizures. The first sign is decreased locomotion; then clonic seizures may occur. Younger rats may display a status of clonic seizures. Flexion (emprostohotonic) seizures develop later but regularly in rats younger than PN15 and only rarely in older age groups. Tonic-clonic seizures can be observed in all age groups. The efficacy of homocysteine increases with age (Kubová et al., 1995). In the EEG, slow waves (young rats), spikes, and spike and wave patterns occur (in prepubertal and older rats). All these phenomena have very poor electroclinical correlation, which slightly improves with age. Metabolic studies show early (1 hour after ICV infusion) decreases in glucose and glycogen and increases in lactate in the cerebral cortex and late (24 hours after administration) increases in glucose and glycogen (Folbergrová et al., 2000). Limitations The profile of action is practically the same as with NMDA seizures, including EEG patterns (Maresˇ et al., 2004). All the mechanisms of action of homocysteine remain to be elucidated. Because of high mortality after systemic homocysteic acid administration in developing rats (similar to NMDA), investigators may use ICV administration, which induces long-lasting but not lethal tonic-clonic seizures (Folbergrová et al., 2000). Insights into Human Disorders Both homocysteine and homocysteic acid are related to the EAA system. Although l-homocysteic acid is an agonist at the NMDA subtype of the ionotropic glutamate receptor, homocysteine has broader nonspecific agonistic features on the EAA receptors. Homocysteic acid can be used instead of NMDA for flexion (emprosthotonic) seizures.
Acetylcholine (ACh)-Related Substances The cholinergic system has been practically explored for seizure induction far before the experimental approach was used. Organophosphorus-based nerve gasses tabun, sarin, soman, cyclosarine, VX, and VR produce seizures on their way to lethal effects and mechanistically block acetylcholine (ACh) degradation in the synaptic cleft by inhibiting
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acetycholinesterase (Shih and McDonough, 1999). Experimentally, in addition to the use of nerve gasses, muscarinic receptor agonists are widely employed for induction of clonic seizures, especially of the long-lasting SE with neuropathologic consequences (Turski et al., 1989b). Issues of pilocarpine-induced seizures and SE are analyzed in detail in Chapter 35. Behaviorally all these drugs induce akinesia, tremor, olfactory automatisms, wet-dog shakes, and clonic seizures, culminating in SE (Turski et al., 1983). These symptoms can be clearly observed only with pilocarpine because nerve gasses have extremely fast onset of seizures, consistent with their intended effects: Only in animals that do not develop seizures and thus do not die is it possible to record automatisms (Shih et al., 2003). In the EEG, the hippocampus is activated before the amygdala and cortex are, with prevailing high-voltage spiking. Neuropathologically, pilocarpineinduced SE produces an extensive and age-specific injury.
cortex, septum, and neocortex (Cavalheiro et al., 1987). However, in immature rats at the lower limit of this developmental interval (PN12), neuronal damage is found in thalamic nuclei (Kubová et al., 2001). Limitations Pilocarpine seizures are very persistent and long-lasting, and they result in severe neuropathologic damage that far exceeds the damage seen in human mesial temporal lobe sclerosis. The pilocarpine or Li-pilocaprine model has become very popular during the period of very limited (practically nonexistent) availability of KA, which until then had been a drug of choice for models of temporal lobe seizures with SE, resulting in spontaneous seizures and brain damage. The administration of a peripheral cholinergic antagonist further complicates the use of high doses of pilocarpine for acute seizure induction. On the other hand, the low cost of pilocarpine compared with KA represents a significant advantage.
Pilocarpine
Insights into Human Disorders
Methods of Generation
Pilocarpine is an agonist of muscarinic ACh receptors expressed especially in the hippocampus, striatum, and cortex (Kuhar and Yamamura, 1976). Therefore its seizureproducing effect arises from the increased activation of these receptors. The pilocarpine model may be useful for testing of the drugs potentially effective against complex partial seizures.
Systemic (IP) injection of pilocarpine, 300 to 400 mg/kg, produces seizures in rats and mice (Turski et al., 1983). However, this very high dose has significant peripheral effects. Therefore concomitant treatment with a peripheral muscarinic antagonist (not crossing the blood-brain barrier), such as scopolamine methylbromide (1 mg/kg sub-Q), is necessary. To decrease pilocarpine doses (and thus peripheral effects), pretreatment with lithium chloride (LiCl, 3 mEq) may be used 24 hours before the pilocarpine dose of 30 to 60 mg/kg. This paradigm significantly limits peripheral side effects (Jope et al., 1986). For detailed information on all aspects of pilocarpine-induced seizures, see Chapter 35. Defining Features Pilocarpine induces automatisms, WDS, and clonic seizures developing into SE as described previously. In the EEG, fast spikes occur in the hippocampus and spread to the cortex at all studied ages PN3 through adulthood (Cavalheiro et al., 1987). Metabolic studies determined the involvement of the hippocampus, dentate gyrus, globus pallidus, substantia nigra, ventrobasal and mediodorsal thalamus, pyriform, and visual and frontal cortex (Clifford et al., 1987). Features of the Li-pilocarpine model are very similar to those of pilocarpine (Turski et al., 1989a). Neuropathologic changes are age specific. In adult rats, the hippocampus is predominantly damaged (Turski et al., 1989a), but other areas (e.g., amygdala, pyridorm cortex, thalamic nuclei, and the substantia nigra pars reticulate) are also affected. In immature animals at PN11–21, some damage can be found in the hippocampus, thalamus, olfactory
Weapon-Grade Organophosphorus Compounds Methods of Generation All nerve agents (organophosphates) (Lotti, 2000; Spencer et al., 2000) can be dissolved in saline. Seizures were described after administration in rats and guinea pigs. All the drugs are extremely toxic (Lotti, 2000; Spencer et al., 2000); the median lethal dose (LD)50 ranges from 8 mg /kg to 300 mg/kg, depending on the potency (from the lowest to the highest: tabun, cyclosarin, sarin, soman, VR, and VX; see Table 4) and on the experimental animals used. Administration is usually sub-Q (Shih et al., 1999). No data for immature rats are available. Features Convulsions begin as a tremor, twitching, and shivering, continuing to strong convulsions (Tuovinen, 2004) associated with loss of righting reflex (Shih et al., 1999). Cortical EEG displays fast spiking (Shih et al., 2003). Limitations These drugs are extremely toxic and dangerous for humans even in minimal doses. The popularity of this model is limited. There is no available detailed description of seizures and associated EEG.
Systemic Injection of Convulsant Substances
TABLE 4 LD50 of Weapon-Grade Organophosphates Nerve agent
LD50 rats (mg/kg sub-Q)a
LD50 guinea pigs (mg/kg sub-Q)b
300 110 125 210 16 N/A N/A
120 42 28 72 8 52 11
Tabun Soman Sarin GF VX Cyclosarin VR a
From Shih et al., 1999; Shih and McDonough, 1999. From Shih and McDonough, 1999; Shih et al., 2003. N/A, information not available; sub-Q, subcutaneous.
b
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be seen from PN7 onward. Tonic-clonic seizure pattern either fully developed or without the tonic phase can be recorded in all age groups, including adults. Sharp waves and spikes are the predominant features in the EEG of PN12–25 rats. In younger rats, slow waves with very poor electroclinical correlation occur (Kubová and Maresˇ, 1994; Pylkkö and Woodbury, 1961). Limitations Seizures induced by strychnine are considered by some investigators as a model of therapy-resistant seizures (Löscher, 1997). However, it is difficult to fit these seizures into the classification of human seizures and epilepsy (Engel, 2001).
Insights into Human Disorders
Insights into Human Disorders
Organophosphates are potent and irreversible inhibitors of acetylcholine esterase (AChE), the ACh degrading enzyme. Therefore their administration increases the availability of ACh for general activation of all subtypes of the ACh receptors.
The mechanism of the convulsant action of strychnine has been defined as a blockade of chloride channel associated with glycine receptors (Curtis et al., 1971). These inhibitory receptors can be found mostly in the spinal cord and brainstem (Young and Snyder, 1973; Zarbin et al., 1981). Therefore strychnine can serve as a model of therapyresistant seizures arising from the lower brainstem and spinal cord.
Other Drugs This section combines several other drugs, the administration of which is associated with seizures. Although these drugs are unrelated to one another, they are worth mentioning either for their mechanisms of convulsive action or because of relevant occurrence in humans. Some of these drugs induce conditions with epileptic seizures that do not require a diagnosis of epilepsy: drug or other chemically induced seizures (Engel, 2001). However, hypoglycemia is not included in the current classification (Engel, 2001), although one might argue that this condition associated with seizures is commonly caused by excessive insulin (i.e., a drug) of exogenous or endogenous origin.
Aminophylline Methods of Generation Both aminophylline (theophylline and ethylenediamine) and caffeine can induce convulsions (Chu, 1981; Stone and Javid, 1980; Walker, 1981a, b). Aminophylline can be dissolved in normal saline. Doses from 150 to 350 mg/kg IP induce seizures in rats from PN7 to adulthood with CD50 values ranging between 180 and 280 mg/kg IP throughout development (Maresˇ et al., 1994). Defining Features
Strychnine Methods of Generation Strychnine (Allen, 2000) has been used for practical human toxicology for centuries. Victims of strychnine poisoning die in convulsions. Strychnine sulfate can be dissolved in normal saline (1 mg/ml). Doses between 1 and 4 mg/kg IP are used for 3- to 25-day-old rats. CD50 values for the same ages are between 1.8 and 0.5 mg/kg and decrease with age (Kubová and Maresˇ, 1995). Doses for adult rats are around 2 to 3 mg/kg administered sub-Q (Kubová et al., 1990). Defining Features In very young rats at PN3–5, circling and barrel rotations are observed. A higher incidence of myoclonic twitches can
Aminophylline induces dose-dependent clonic seizures, tonic-clonic seizures, and lethality throughout development. Limitations This is an unusual model of symptomatic seizures. Theophylline-induced seizures have been described in humans (Jensen et al., 1984). Insights into Human Disorders Theophylline acts as an antagonist of adenosine receptors. According to the classification published in 2001 (Engel, 2001), aminophylline-induced seizures are a model of conditions with epileptic seizures that do not require a diagnosis of epilepsy (drug or other chemically induced seizures).
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Insulin-induced Hypoglycemia Methods of Generation In experimental animals, seizures can be induced by administration of insulin, leading to hypoglycemia (Urion et al., 1979). Insulin can be dissolved in distilled water at a concentration of 15 international units (IU) per milliliter. The usual dose for seizure production is 5 to 30 IU/kg IP (Urion et al., 1979; Vannucci and Vannucci, 1978). After this dose, rats develop seizures within 4 hours. Our unpublished data show that the prior fasting (for 24 hours) improves the incidence and decreases the latency to onset of insulininduced seizures. Thus, in overnight fasted rats, hypoglycemic seizures usually fully develop within 2 to 3 hours, and their incidence is close to 100%. In our experience, it is very difficult to produce hypoglycemic seizures in the immature rats. We were able to induce seizures in PN21 and older rats (post weaning) but only extremely rarely in suckling rats (Vannucci and Vannucci, 1978), even during observation periods as long as 10 hours after insulin administration. Defining Features During severe hypoglycemia, a sequence of events occurs. Onset of seizures usually correlates with a peripheral glucose drop down to 20 mg/100 ml (1.1 mM). First, with decreasing systemic glucose, rats are hypoactive and flaccid. Individual twitches then occur. Jumps, usually restricted to the hindlimbs, are also observed. Barrel rotations are a very typical expression of hypoglycemic seizures
(see Chapter 48). Sometimes clonic and tonic seizures occur, including an opisthotonic position. All these seizures are associated with a loss of posture. EEG shows generalized spike and spike and wave activity (Figure 10). Metabolic studies demonstrate significant changes in the 2DG uptake in hypothalamic nuclei (belonging to food-intake control group) and also in the midbrain and brainstem structures, such as subthalamic nucleus, substantia nigra pars reticulata, pedunculopontine tegmental nucleus, and vestibular nuclei. The c-fos immunopositivity is especially prominent in the substantia nigra reticulata and subthalamic nucleus in rats experiencing hypoglycemic seizures. Neuronal injury has been studied in rats with 10 to 30 minutes of isoelectric EEG during severe hypoglycemia. The injury involves the neocortex, hippocampal CA1, and amygdala regions as well as cerebellar Purkinje cells (Auer and Siesjo, 1993; Auer et al., 1984). Limitations This model is not important for the studies of the mechanisms of anticonvulsant drug action. However, the mechanisms underlying hypoglycemic seizures and the brain structures responsible for excessive and synchronized firing during conditions of general metabolic shutdown of neuronal activity are still worth further exploration (Lewis et al., 1974). Insights into Human Disorders Hypoglycemia is frequently associated with neurologic side effects (Davis et al., 1997). From these side effects,
FIGURE 10 Discharges recorded during severe insulin-induced hypoglycemia (blood glucose <20 mg/100 ml) in a Sprague-Dawley rat. Arrow marks time of onset of barrel rotations. This pattern illustrates dissociation of electroencephalographic (EEG) discharges and motor seizures induced by insulin-induced hypoglycemia. Electrocorticograms and stereo EEG from sensorimotor cortex, hippocampus, and deep midbrain/brainstem structures: SNR, substantia nigra pars reticulate; PPTG, pedunculopontine tegmental nucleus.
Inhalation of Convulsant Substances
141
seizures occur most frequently (Kaufman, 1998; Pocecco and Ronfani, 1998). Hypoglycemic seizures represent a model of symptomatic seizures frequently occurring in humans, especially with type 1 diabetes mellitus or metabolic conditions affecting carbohydrate metabolism (Cornblath and Schwartz, 1991). Treatment for these seizures is correction of blood glucose; therefore no antiepileptic drugs are required (Auer and Siesjo, 1988).
INHALATION OF CONVULSANT SUBSTANCES Inhalation is a less usual route of delivery for a convulsant drug. For inhalation, the drug must have some special features, such as easy evaporation at room temperature. This is true for all ether-like substances. Indeed, ethers have excitatory features, reflected in the original classification of ether-induced anesthesia as stage II. However, in most of these drugs, stage III of surgical anesthesia develops quickly and covers the excitatory stage II. Although delivery of inhalatory convulsants requires some special equipment, it also has significant advantages over administration by injection. Minimum requirements for the equipment are an airtight glass cylinder, which serves as the inhalation chamber, and a fume hood for draining away excessive fumes of the convulsant. More sophisticated systems (and less hazardous for the laboratory worker) consist of an airtight chamber with sufficient inside volume (Figure 11). This chamber is attached to the vacuum exhaust system through the system of valves, which allows the chamber to be flushed after each experiment. Therefore the convulsant fumes do not escape to the surrounding atmosphere. The chamber is placed in the fume hood for additional protection. The convulsant drug is delivered using a constant infusion rate pump, and it evaporates at the filter paper with a standard size placed at the top of the chamber. Latency to onset of seizures from the beginning of the convulsant drug delivery is recorded. The major advantage of inhalation delivery is that no potentially painful injection and stressful restraint of the animal is involved, which results in minimal interference of stressors with the seizure model. Flurothyl (Indoklon) Methods of Generation Flurothyl (bis-2,2,2-trifluoroethyl ether) seizures have been described in mice, rats, gerbils, and humans. The procedure consists of placing the animal (rat or mouse) in an airtight cylinder of a sufficient volume and injecting a specific volume of flurothyl, which then evaporates and forms convulsant fumes (Prichard et al., 1969; Truitt et al., 1960). Our laboratory uses 9.34 l airtight chamber for the rat exper-
FIGURE 11 Scheme of the chamber for the induction of flurothyl seizures. Flurothyl is delivered by precision microinfusion pump onto a filter paper, where it evaporates. After the experiment, the chamber is flushed with vacuum. The entire chamber, including the microinfusion pump, is placed in the fume hood for increased protection of the crew because flurothyl vapors have the capability of inducing seizures in humans.
iments. Flurothyl is delivered using a microinfusion pump at a constant rate; for this size chamber, the usual rate is 20 ml/min. Latency to onset of seizures from the application of the convulsant is measured. In our paradigm, the exact amount of flurothyl required to produce a certain seizure type can be calculated (flurothyl seizure threshold). It is imperative to run the tests in control and experimental conditions back to back because atmospheric factors may affect evaporation of flurothyl. Mortality after acute flurothyl exposure is low. However, if the adult rats are subjected to long-term flurothyl exposure to induce SE, they regularly die, in contrast to immature PN14 rats (Huang et al., 1999; Sogawa et al., 2001; Sperber et al., 1999a). Defining Features Seizures induced by flurothyl are age specific (Sperber and Moshé, 1988). Flurothyl induces initial agitation and increased exploratory activity in rats throughout development (Gatt et al., 1993). In rats during the first postnatal week, swimming movement occurs, followed by a tonus. Clonic seizures develop after PN10. After this age, rats usually display several myoclonic twitches followed by a clonic seizure, which at between PN10 and 20 usually evolves into a tonic-clonic seizure with loss of posture. After
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PN20, several separated episodes of clonic seizures appear before the development of tonic-clonic seizures. It should be noted that the sequence and seizure phenomena after flurothyl exposure are very similar to the seizures occurring after systemic administration of GABA-related drugs (especially PTZ and bicuculline). However, in contrast to these drugs, flurothyl induces seizures that are more violent: Clonic seizures often involve tonic components of axial muscles, which may cause a temporary loss of posture. Clonic seizure may involve all four limbs simultaneously, which also interferes with the upright posture. However, in all these cases, the rat struggles to get up. This is in contrast to the loss of posture in tonic-clonic seizure, in which no active effort is made to regain posture. During the tonic phase, cyanosis and acidosis may occur, with fast correction within 5 minutes after the end of the seizure (McCabe et al., 2001). EEG shows rhythmic discharges (Figure 12) associated with a motionless stare from the third postnatal week onward. Additionally, individual spikes (or sharp waves in immature animals) are recorded. In young rats, these discharges very loosely correlate with myoclonic twitches; the correlation improves with age. Bilateral forelimb clonus is associated with long periods of spikes or sharp waves (Figure 13). In adult rats, discharges are found in the cortex, whereas in immature rats the discharges occur in both the cortex and the hippocampus (Sperber et al., 1999a). Metabolic studies using 2DG in adult rats demonstrate complex involvement of basal ganglia structures, including the
substantia nigra reticulata. The pattern of metabolic involvement depends on the seizure type and whether in ictal or postictal state (Velísˇková et al., 2005, in press). In immature rats, mild seizures induce decreases in 2DG uptake while severe seizures produce increases in the brainstem and decreases in the cortex (Szot et al., 2001). In adult rats, c-fos immunoreactivity after flurothyl seizures was found throughout the cortex; PN10 rats had immunopositivity only in the deep neocortical layers (Jensen et al., 1993). Limitations A more sophisticated airtight chamber is required to produce flurothyl seizures by inhalation of flurothyl vapors. Atmospheric factors such as barometric pressure, air humidity, and temperature may affect the evaporation rate of flurothyl. Therefore the experiments should be carefully planned. Sometimes it is difficult to compare corresponding data collected over different periods. Insights into Human Disorders The mechanisms of action of flurothyl are uncertain. Increased opening of sodium channels has been suggested (Woodbury, 1980a). Antagonism at the GABAA receptor may also contribute to the convulsant potency of flurothyl (Araki et al., 2002). Additionally, activation of cholinergic system has been proposed (Eger et al., 2002). Because flurothyl induces a motionless stare with accompanying
FIGURE 12 Rhythmic, spindle-shaped discharge induced by inhalation of flurothyl in sensorimotor cortex of an adult Sprague-Dawley rat. This discharge was associated with freezing behavior. Bipolar recording from the sensorimotor cortex is shown.
FIGURE 13 Spike-and-wave and polyspike discharges induced by inhalation of flurothyl in sensorimotor cortex of an adult Sprague-Dawley rat. Arrow indicates onset of clonic seizure and rearing. The onset of this discharge significantly preceded the onset of motor seizures. However, the occurrence of high-frequency polyspike discharges was associated with the onset of clonic seizure.
Topical (Focal) Application of Substances
EEG spindles, this can be considered a model of generalized seizures—typical absence seizures (Engel, 2001). Clonic seizures can be a model of generalized myoclonic seizures. Finally, tonic-clonic flurothyl seizures are a model of generalized tonic-clonic seizures (Engel, 2001).
TOPICAL (FOCAL) APPLICATION OF SUBSTANCES This approach is frequently used to create an acute or chronic seizure focus in a particular brain area, usually in the cortex. Additional structures may be injected with a convulsant substance, such as the hippocampus, amygdala, substantia nigra, area tempestas, etc. Many of the convulsant drugs can be injected in a solution into the brain ventricular system. Some of these ICV administrations have already been mentioned herein. The advantage of this approach is that it produces seizures very reliably and only microamounts of the drug are required. However, there is no exact control of the focus of the seizure onset. All structures surrounding the injected ventricle are affected by the drug; thus seizure onset can be multifocal. For precise localization of the focus, intraparenchymal administration of a convulsant substance is preferred. Many different convulsant substances are used for production of seizure focus, and its major representatives are briefly mentioned as follow. Metals (Cobalt, Zinc, Antimony, Alumina Cream, Iron) Methods of Generation Cobalt can be implanted as a powder or rod, or it can be injected as a solution of CoCl2 (Cooper and Legare, 1997). ICV injection of cobalt chloride has the CD50 of 10 ml of 0.45 mM solution. Seizures develop very slowly in powdered or rod cobalt implants and vanish in about a month. After ICV injection of a soluble cobalt salt, seizures develop within an hour and last for about a week (Zhao et al., 1985). Other metals such as nickel and antimony have high epileptogenic potency; iron has a somewhat lower potency than cobalt (Colasanti and Craig, 1992; Craig and Colasanti, 1992; Dambinova et al., 1998; DeDeyn et al. 1992; Doi et al., 2000; Kabuto et al., 1998; Pei and Koyama, 1986; Ueda and Willmore, 2000a, b, c; see also Fisher, 1989). Additional technique of application includes positioning of a filter paper soaked with a soluble salt (e.g., saturated solution of NiCl2·6H2O) on the cortex (Cooper et al., 2001). Alumina cream (Kopeloff et al., 1942, 1954; Ward, 1969; Wyler et al., 1978), 4% AlOH3 (Spencer, 2000a), is a very potent and frequently used topical convulsant; however, its effects are limited to monkeys and rabbits (see Chapter 14). The latent period is several months and seizures may last for up to several years (Louis et al., 1987).
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Defining Features In the rat, unilateral cobalt implant produces seizures within 5 days. Simple partial seizures develop first and consist of contralateral clonus associated with EEG spikes above the implant. Although very frequent (more than 20 per day), these seizures disappear within 9 days. Generalization is characterized by a bilateral clonus, occurring about 10 times a day, most frequently 1 week after cobalt implantation. Behavioral arrest may also occur and is associated with gradual buildup of EEG activity (Chang et al., 2004). ICV administration of soluble cobalt salt produces seizures resulting from irritation of the hippocampus. Therefore these seizures are similar to KA-induced seizures (Zhao et al., 1985). Nickel salt applied on the filter paper on the cortex produces contralateral myoclonic twitches within an hour; these twitches last about 2 hours (Cooper et al., 2001). After cobalt implantation in the posterior cortex, dark patches of increased 2DG uptake appear around the implant site and in the lateral geniculate nucleus of the thalamus. This is, however, not true for anterior cortex cobalt implants and for implants of other convulsogenic metals such as nickel and antimony (Cooper and Legare, 1997). Metal implants cause focal necrosis. Limitations These models are labor intensive and expensive to prepare, especially for the monkey alumina cream model. The difference of clinical seizure foci may be the direct irritation of the brain tissue by the metal deposit. Cobaltinduced seizures are more sensitive to antiepileptic drugs used against absence seizures in humans and thus cannot be a predictive model of refractory focal epilepsy (Loscher, 1997). Except for the alumina cream model in monkeys, foci spontaneously deactivate after several weeks. Insights into Human Disorders Mechanisms of action of these metal deposits are largely unclear. Metal deposition models depend on the localization of the deposit; these are good models of focal motor seizures with elementary clonic motor signs or focal motor seizures with typical (temporal lobe) automatisms (Engel, 2001). Except for cobalt, metal deposit models may have good predictive value for screening anticonvulsant drugs effective against focal onset seizures. Convulsant Drugs Methods of Generation All convulsant drugs mentioned previously in this section, plus cholinergics, anticholinergics, estrogens, and strychnine can act focally if applied on the cortex or in a particular seizure-prone structure, such as amygdala, hippocampus, substantia nigra, or area tempestas (Campbell
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and Holmes, 1984). Older studies usually used focal administration of the drug on the surface of the neocortex of paralyzed and ventilated or anesthetized animals (Velísˇková et al., 1991). In that case, a filter paper of an exact geometry is used. Its area is either soaked with a water solution of a convulsant drug or a defined volume is delivered using a microsyringe to the filter paper positioned on the cortex. More recent studies use chronic cannulation with microinfusions of the drugs in freely moving animals (Gale, 1995; Halonen et al., 1994; Soukupová et al., 1993; Velísˇek et al., 1993).
initial automatisms followed by generalization of seizure activity and clonic seizures (Sierra-Paredes and SierraMarcuno, 1996b). Application of the drugs on the motor cortex will produce myoclonic twitches and, later, clonic seizures (Soukupová et al., 1993). Finally, drugs with high affinity for limbic structures will produce automatisms and clonic seizures even when the injection site is remote to the limbic system (such as the substantia nigra pars reticulata (Sawamura et al., 2001). EEG usually displays individual discharges (Figure 14), which may cumulate over time and develop into ictal activity.
Defining Features
Limitations
Behavioral features depend on the following: (1) the drug used and (2) the exact placement of the microinfusion. For doses of commonly used convulsant drugs, see Table 5. Microinfusions of any drug in the limbic system will induce
These models are used less frequently because they are relatively labor intensive. Further, the use of NMDA as a focal convulsant drug may be surprising in some brain structures because NMDA has a very high propensity to
TABLE 5 Concentrations of Some Focally Applied Convulsant Drugs Drug
Structure
Dose
Frequency
Reference
Bicuculline methiodide
Neocortex
5 ml of 1–2 mM solution
1–2 times
Soukupová et al., 1993
Pentylenetetrazol
Neocortex
2.5 ml of 200 mg/ml solution
Single
Velísˇek et al., 1993
Bicuculline methiodide
Neocortex
2.5 ml of 4 mM solution
Single
Velísˇ ková et al., 1991
Picrotoxin
Hippocampus
100–500 mM
Continuous
Sierra-Paredes and Sierra-Marcuno, 1996a, b
Kainic acid
Amygdala
0.4–2.0 mg in 0.1–0.4 ml
Single
Ben-Ari et al., 1980
Kainic acid
Substantia nigra
1 mg in 1 ml
Single
Sawamura et al., 2001
Bicuculline
Area tempestas
118 pmol
Single
(Wardas et al., 1990)
Carbachol
273 pmol
Kainic acid
117 pmol
FIGURE 14 Discharges induced by bilateral application of 2.5 ml of the 200 mg/ml pentylenetetrazol (PTZ) solution on the sensorimotor cortex in a freely moving adult Wistar rat. Individual discharges (sharp waves) are well synchronized between both frontal areas; transfer to the occipital areas (especially left one) is incomplete. RF, right frontal (sensorimotor) cortex; RO, right occipital (visual) cortex; LF, left frontal cortex; LO, left occipital cortex.
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induce spreading depression (for review, see Somjen, 2001). This feature may account for some of its paradoxical anticonvulsant effects when it is applied focally (Turski et al., 1987).
neurons in the epileptic focus (Matsumoto and Ajmonemarsan, 1964; Prince, 1968). Neurons around the focus exert substantial inhibitory activity in an attempt to limit the spread of the seizure (Prince and Wilder, 1967).
Insights into Human Disorders
Limitations
Mechanisms of action for these drugs were mentioned during the description of their systemic use (see preceding). All these models represent models of focal seizures with possible secondary generalization. According to the localization of the initial injection site or the preferential activity of the drug, the model can be of focal motor seizures with elementary clonic motor signs or focal seizures with typical automatisms (Engel, 2001). Focal administration of convulsant drugs may serve as a model for screening antiepileptic drugs potentially useful in treatment of focal-onset seizures.
If a high concentration of a potassium salt of the antibiotic drug is directly applied onto the cortex, spreading depression may develop (for review, see Somjen, 2001), masking thus the onset of discharges and interfering with the experiment. Therefore sodium salts are preferred, if they are available.
Antibiotics (Penicillins and Cephalosporins) Methods of Generation Epileptogenic effects of antibiotic drugs were first described in monkeys and later in humans (Johnson et al. 1946; Walker and Johnson, 1945; Walker et al., 1945). Locally administered penicillin (Velísˇek and Moshé, 2000a) produces an epileptic focus in many other species, including rats and cats. The potency of antibiotic drugs to induce epileptiform discharges has been studied repeatedly both in vivo (Gutnick et al., 1976) and in vitro (Grondahl and Langmoen, 1993). After application on the cortex, the convulsant potency is the following: benzyl penicillin (threshold concentration ~25 mmol/L), phenoxymethylpenicillin (~100 mmol/L), oxacilin (~150 mmol/L), methicillin (~150 mmol/L), and ampicillin (~175 mmol/L) (Gutnick et al., 1976). Sodium and potassium salts of penicillins are preferred for application because their solubility is superior to the antibiotic bases. Cephalosporins, especially the first generation, have also substantial epileptogenic potency if applied focally (Kamei et al., 1983); for review see (Velísˇek and Moshé, 2000b). Some epileptogenic antibiotic drugs the cross blood barrier, although in limited amounts. Therefore, it is possible to induce seizures by systemic injections of high doses of some penicillins or cephalosporins. Cats are more sensitive to systemic antibiotics than rats or mice (Chen et al., 1986; Eng et al., 1989; Gloor and Testa, 1974; Nistico et al., 1978; Yu et al., 1984).
Insights into Human Disorders Low doses of penicillin selectively block GABAA mediated inhibitory postsynaptic potentials as a result of their GABAA receptor antagonism (Dingledine and Gjerstad, 1980; Wong and Prince, 1979). Higher doses may have additional nonspecific effects (Ayala et al., 1970). Focally administered epileptogenic antibiotics produce a model of focal seizures with initial symptoms that depend on the exact localization (Engel, 2001). After generalization of the electrical seizure activity from the focus, clonic seizures may occur. Tetanus Toxin Methods of Generation Focal administration of tetanus toxin (Griffin and Oyler, 2000) introduced by Mellanby and colleagues (Mellanby et al., 1977) can induce seizures in both mice and rats. Hippocampal localization of tetanus toxin injection is used to produce recurrent seizures (see Fisher, 1989). After the infusion of 4 to 5 ng in 1 ml of phosphate buffer (Finnerty and Jefferys, 2002), the seizures occur within a day and recur chronically over weeks (see Chapter 33 for further details). Defining Features In rats a seizure usually begins with behavioral arrest, followed by myoclonic twitches of forelimbs and sometimes with tonic-clonic seizures. Seizures may be quite frequent (up to 100 per day, returning for a month). EEG shows fast spikes or spike and waves (3–20 Hz) (Jefferys and Williams, 1987).
Defining Features
Limitations
Seizures depend on the exact localization of the focus. Interictal focal activity is usually accompanied with isolated twitches, while the ictal discharges may be associated with a clonic seizure. The penicillin focus is very localized (Noebels and Pedley, 1977) and the neurons display the paroxysmal depolarizing shift, the hallmark of epileptic
There is limited neuropathology and temporal limitation for recurrent seizures. Insights into Human Disorders After focal administration, tetanus toxin is picked up by nerve endings and interferes with synaptic vesicular release.
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As per localization of the focus in the hippocampus, this is a model of focal seizures with typical automatisms (Engel, 2001) with further generalization. This model is used with advantage in both immature (Anderson et al., 1999; Rashid et al., 1999; Smith et al., 1998) and adult rats (Jefferys and Williams, 1987) because epileptogenesis develops after initial seizures.
CONCLUSION Acute seizures can be induced by administration of chemoconvulsant drugs via different delivery routes (sub-Q, IP, IV, ICV, or inhalation). Main categories of convulsant drugs used for the production of acute seizures affect the inhibitory (GABA or glycine) transmission, affect excitatory amino acid transmission, or act on ACh receptors. The seizures are primarily or secondarily generalized, and some of their behavioral, EEG, metabolic, and neuropathologic features are age specific. An additional possibility for the induction of acute focal seizures is implantation of convulsant substances into the sensitive brain regions (e.g., neocortex, hippocampus, or area tempestas). For this purpose, convulsant metals, convulsant drugs, or antibiotics with convulsant features can be used. Acute behavioral seizures emanating from an altered GABAergic system represent models of myoclonic seizures and generalized tonic-clonic seizures; rhythmic EEG activity associated with behavioral arrest corresponds to absence seizures. Seizures that are due to hyperactivation of the EAA system may help with models of special seizure syndromes, especially catastrophic epilepsies of childhood. Some of the EAA-based models (such as NMDA seizures) are resistant to antiepileptic drug therapy; thus they may be useful for screening the drugs that are potentially effective in refractory epilepsy.
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12 Electrical Stimulation-Induced Models of Seizures PAVEL MARESˇ AND HANA KUBOVÁ
Seizures induced by electrical stimulation exhibit one advantage: The epileptogenic agent is acting only during application of current and epileptic activity is no longer contaminated by this agent. On the other hand, electrical current stimulates all neuronal and nonneuronal elements in the stimulated nervous tissue. Electrically induced seizures may be divided according to the mode of stimulation into two main types: (1) those elicited by stimulation of the whole brain (electroshock seizures) and (2) those induced by local stimulation of a defined brain structure (epileptic afterdischarges).
induced by threshold and slightly suprathreshold stimulation through corneal electrodes. With increasing intensity of stimulation, generalized tonic-clonic seizures are elicited. Maximal electroshock seizures (MESs) are a model of generalized tonic-clonic seizures. They represent hindbrain seizures; using auricular stimulation, even the threshold current evokes this type of seizures (Browning and Nelson, 1985).
Animals Mostly mice or rats are used. Other species are used only exceptionally. There are no data indicating differences among mouse or rat strains. This may be due to extremely high intensity of stimulation current; a difference might be expected at threshold or slightly suprathreshold intensities. Screening of anticonvulsant drugs is usually done in mice; their body weight is approximately 10 times less than that of rats.
ELECTROSHOCK SEIZURES Methods of Generation There are various modifications of this model (Swinyard, 1972), but only two are commonly used: (1) stimulation with an alternating current of 60- or 50-Hz (according to the local current) frequency and (2) low-frequency stimulation (usually 6 Hz). Two types of seizures might be induced in relation to the localization of stimulation electrodes and intensity of stimulation current (Figure 1): (1) minimal clonic seizures involving muscles of head and forelimbs (righting ability is preserved) and (2) maximal (i.e., generalized tonic-clonic) seizures with a loss of righting reflexes. Both stimulation frequencies may be used for estimation of seizure threshold as well as for elicitation of maximal seizures. Minimal seizures probably represent a model of myoclonic seizures (Löscher and Schmidt, 1988). Minimal clonic seizures are generated in the forebrain; they are
Models of Seizures and Epilepsy
Procedures Mainly corneal electrodes are used; eyes must be moistened with a drop of physiologic saline after previous local anesthesia with 0.5% tetracaine. Less common are transcranial electrodes or ear clips. Low impedance, that is, good contact of stimulation electrodes, is of primary importance; therefore formulas used for electroencephalographic (EEG) electrodes in human patients are recommended for transcranial as well as ear stimulation. The classic MES test is performed with 0.2-second series of alternating current with 60-Hz frequency and an intensity of 50 mA for mice and 150 mA for rats applied through
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FIGURE 1 Motor seizures elicited by electroshock stimulation. A–F: Minimal seizures induced by corneal suprathreshold stimulation in an 18-day-old rat. Righting ability is preserved, and the animal exhibits clonic movements of forelimbs. A, F: Classified as stage 2 (only head movements, both forelimbs on the floor). C–E: Stage 3 (forelimb clonus). B: Stage 4 (forelimb clonus with rearing; not yet perfect at this age). G: Tonic extension of forelimbs and hindlimbs in an adult rat. H: Tonic flexion of the forelimbs in 7-day-old rat pup (hindlimbs are not involved).
corneal electrodes. The animal is restrained in hand during stimulation. It is necessary to release the animal immediately after the end of stimulation to see all phases of the seizures. Monitoring these tests is visual; the presence or absence of individual components of seizures and their duration may be measured. In the most common MES test, only the presence of tonic hindlimb extension is taken as a criterion. Threshold estimation may be performed using the staircase method (Finney, 1952). Statistical evaluation, that is, estimation of a current eliciting seizures in 50% of animals (CD50) as well as 50% effective doses (ED50) of anticonvulsants can be performed using the Lichtfield and Wilcoxon graphic method (1953) or the Finney’s probit analysis (1952). These tests are very easy to perform. It is necessary to have a good stimulator with a constant current output allowing application of high current intensities (up to 150 mA). Reliability of data is high. It can be affected by the experience of the observer, and video recording could be used if more than tonic hindlimb extension is measured.
TABLE 1 Stages of seizures 1
Mouth and facial movements
2
Head nodding
3
Forelimb clonus
4
Rearing
5
Rearing and falling From Racine, 1972.
Characteristics Behavioral Features Minimal threshold electroshock seizures consist of clonic seizures of the head and the forelimb muscles (Figure 1A–F). To quantify these seizures, the 5-point scale of Racine (1972) can be used (Table 1). Maximal electroshock seizures consist of tonic flexion followed by tonic extension and clonus accompanied by a loss of posture. The endpoint is tonic extension of hindlimb
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with the limbs extended 180 degrees at the plane of the body (Figure 1G). The duration of hindlimb tonic extension is in the range of seconds (tens of seconds at maximum). It is possible but not common to include a 5-second duration as the limit for a positive result. MESs are followed by a period of postictal depression when the same stimulation cannot induce MES (Velísˇek and Maresˇ, 1992). This depression lasts longer in rats than in mice, and its duration depends on the intensity of stimulation (Swinyard, 1972). There are no consistent data on EEG during MES because of the high intensity of stimulation current and violent movements during the clonic phase. Morphologic studies have not found any significant neuropathological changes after single MES (Meldrum, 1986). To induce neuronal death, repeated electroshocks must be applied, but single electroshock seizures are sufficient to induce immediate early gene expression (French et al., 2001). Inducing and evaluating MESs are easy tasks that are not time consuming. They thus represent an ideal screening tool and are used as a routine testing method for potential antiepileptic drugs (AEDs). The AEDs that block voltagegated sodium channels (phenytoin, carbamazepine, valproate, lamotrigine, topiramate, zonisamide, and others) are effective (Swinyard and Woodhead, 1982). There are data not only for all clinically used and potential AEDs but also for many other drugs. The 6-Hz stimulation inducing minimal seizures was also extensively studied pharmacologically (Barton et al., 2001). There are two limitations of the MES test: (1) animals are to be used only once; and (2) evaluation of hindlimb tonic extension only is a restriction of possibilities of this model. Low-frequency electroshock seizures may be repeated, especially if high-stimulation currents are avoided. Electroshock seizures (usually repeated) may serve as a tool in studies of brain chemistry (e.g., Wasterlain and Plum, 1973) or molecular biology (e.g., expression of genes; see French et al., 2001; Altar et al., 2004). Ontogeny Developmental data were published by Millichap (1958) and Vernadakis and Woodbury (1969). The maturation patterns are not the same using the two basic stimulation frequencies. A mature pattern of MES induced by 60-Hz stimulation appears at the age of 16 days in rats; younger animals exhibited only forelimb flexion (10–12 days or earlier) (Figure 1H) or forelimb flexion, followed by forelimb extension and hindlimb flexion (13–15 days). The lowest threshold for elicitation of seizures with the 60-Hz stimulation is around postnatal day 16 in both rats and mice. Low-frequency electroshock threshold with minimal seizures as an endpoint decreases steeply up to the end of
the third postnatal week, and then a moderate decrease continues up to postnatal day 40 (Vernadakis and Woodbury, 1969).
LOCAL ELECTRICAL STIMULATION Methods of Generation Electroencephalographic afterdischarges (ADs) elicited by local electrical stimulation have different electrographic patterns and behavioral correlates according to the structure stimulated. The experiments can be aimed at two different phenomena: (1) measurement of threshold intensities of electrical current necessary for elicitation of ADs or (2) repeated stimulations with the same intensity but with short intervals not leading to kindling. This second type of arrangement can be used for studies of acute anticonvulsant effects. Epileptic ADs represent a model of complex partial seizures (stimulation of limbic structures) or myoclonic seizures (stimulation of sensorimotor cortex). Animals used in these experiments are mostly rats; mice, rabbits, and cats are nowadays rarely used.
Procedures The experiment starts with surgical preparation. Volatile anesthetics (diethylether, halothane, isoflurane) or barbiturates with a short to middle duration of action (e.g., pentobarbitone) are recommended. Dissociative anesthesia (ketamine 100 mg/kg IP [administered intraperitoneally] and xylazine 20 mg/kg IM [administered intramuscularly]) is also possible. Postsurgical recovery takes at least 1 week in adult animals with chronically implanted electrodes. Electrodes can be placed epidurally (flat or ball electrodes; higher stimulation intensities are then needed). If the electrodes are introduced into the cerebral cortex (the same type of electrodes as for subcortical stimulation), a stereotaxic apparatus must be used. Stimulation of subcortical structures can be performed using concentric electrodes or twisted twin electrodes. Care must be given to isolating the electrodes up to the tips. Different frequencies of stimulation can be used similarly to the electroshock model: high frequency (60 or 50 Hz, less frequently higher frequencies) or low frequency (from 3 to 12 Hz). Rectangular pulses are more common, but sinusoid waves may also be used. Biphasic pulses (i.e., those with the same duration of both phases) are preferable to avoid excessive polarization of electrodes. Duration of stimulation series is in an inverse relation to frequency: High-frequency stimulation is usually applied only for 1 to 2 seconds, whereas low-frequency stimulation has to be applied for longer time (up to tens of seconds). Stimulation sessions might be repeated if very high intensities of stimulation are
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not used. It is necessary to avoid initial kindling because progressive lengthening of ADs (see Chapter 28) might interfere with the action of AEDs. Long-lasting stimulation can lead to status epilepticus (see Chapter 36). In the case of pharmacologic experiments, the half-life of the administered drug has to be taken into account. Monitoring requires EEG apparatus; a sample rate of 200 Hz might not be sufficient, or the sharp EEG discharges can be distorted. Digitization at a rate of 500 Hz is recommended. The evaluation focuses on the presence or absence of epileptic ADs (a duration of 5 seconds is sometimes taken as a criterion), their pattern, and accompanying behavioral phenomena. This method is not difficult to develop, but it is reliable only with experience in the field (anesthesia of animals, stereotaxic implantation of electrodes, and concomitant registration of EEG and behavior).
Characteristics Behavioral Features Behavior depends on the stimulated structure and age (see below). Spread of epileptic activity (especially if high stimulation intensities are used) modifies behavioral pattern of seizures (see Chapters 28 and 30). Electroencephalographic Features Afterdischarges may be of different types in relation to the stimulated structure: cortical or thalamic low-frequency stimulation elicits spike-and-wave rhythm, whereas limbic ADs are characterized by trains of fast spikes, large delta waves (sometimes with superimposed low-amplitude spikes), or sharp theta waves. Again the spread of epileptic activity can modify the EEG pattern of ADs (e.g., highintensity neocortical stimulation; see later). The duration of ADs also depends on the stimulated structure: spike-andwave type lasts usually only a few seconds; limbic ADs are substantially longer: from tens of seconds to few minutes. Even a single AD results in plastic changes. Immediately after the end of the Ads, postictal depression (postictal refractoriness) appears (Dyer et al., 1979); its duration may change with stimulated structure and age (see later discussion). Postictal depression prevents elicitation of the second seizure for a maximum of few minutes, with a progressive return to the prestimulation level (i.e., there is an absolute and relative refractory phase). This refractoriness is probably due to prolonged activation of inhibitory systems responsible for termination of seizures. Depression is followed by potentiation; if stimulation is repeated with long intervals, kindling may be induced (see Chapters 28–30). Possible changes in the range of tens of minutes to a few hours were studied only exceptionally, and repeated poten-
tiation and suppression phases cannot be excluded Neuropathology was not studied after a single or a few seizures. Investigators should be aware that even the insertion of electrodes may lead to focal changes (focal hemorrhage, neuronal damage, activation of glia). Penetration of electrodes into the hippocampus (i.e., a mechanical insult) may induce a short-lasting seizure. Response to AEDs was repeatedly studied. There are many studies, but they are far from being comparable and complete because of differences in stimulated structure, stimulation paradigm, methods of evaluation, and selection of AEDs to be studied. Limitations Electrically induced ADs require good equipment and operator experience to be reproducible. In addition, they are time consuming. On the other hand, they provide much more information than electroshock seizures do. Mortality of animals depends mostly on surgical technique and postsurgical care. Afterdischarges can be elicited if a certain level of maturation is achieved. The exact age depends on the structure stimulated (see later discussion). Standardization is necessary in evaluation of behavioral phenomena accompanying ADs. Most common is a scale published by Racine (1972), originally for limbic stimulation (see Table 1); but with slight modification it can be used for ADs elicited from various structures (e.g., cortical ADs) (Maresˇ et al., 2002). Afterdischarges Elicited by Stimulation of Individual Brain Structures Neocortex Two experimental arrangements using rats have been published ( Kubová et al., 1996; Voskuyl et al., 1989, 1992). Voskuyl’s method uses electrodes over right and left motor cortical areas; that is, stimulation of both hemispheres is performed. The intensity of current is progressively increased during the stimulation series. Originally they estimated only the threshold intensity of current necessary to induce clonic movements during stimulation (threshold for localized seizure activity); later this method was extended to the second parameter: spread of convulsions (threshold for generalized seizure activity) (Hoogerkamp et al., 1994; Krupp and Löscher, 1998). An advantage of this method is the possibility of performing repeated measurements. Using daily stimulation, the threshold values initially decreased, but after 10 days they stabilized and did not change further (Della Paschoa et al., 1998). Thus animals can be used for repeated measurements of drug effects. This has been done (with parallel measurement of pharmacokinetic parameters) with oxazepam, midazolam, phenytoin, valproate, lamotrigine, tiagabine, and loreclezole (see Jonker et al., 2003).
Local Electrical Stimulation
The method developed in our laboratory applies both electrodes over the sensorimotor area of the same hemisphere; that is, only one hemisphere is stimulated, but the cortical area is more extensive than in the Voskuyl’s model. Low-frequency stimulation (8 Hz for 15 seconds) is used; it allows evaluation of motor phenomena during stimulation (i.e., induced by direct excitation of the motor cortex) and epileptic ADs appearing after the end of stimulation. High-
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frequency stimulation (50 Hz for 2 seconds) resulted in a similar pattern of ADs, but it cannot be used for studies of the direct effects of stimuli on the motor system (Maresˇ et al., 2002). Repeated stimulations with a stepwise increase of current intensity make it possible to measure four different phenomena: (1) movements during stimulation, (2) spikeand-wave EEG ADs (Figure 2); (3) clonic seizures as a motor correlate of spike-and-wave ADs; (4) transition into
FIGURE 2 Electroencephalographic (EEG) recording of cortical aferdischarges (ADs) in an adult rat. Upper part: Spike-and-wave type of ADs accompanied by stage 4 with a transition into stage 3. Lower part: mixed type of ADs elicited in the same rat by higher stimulation intensity. The right sensorimotor cortical area was stimulated. Individual leads from top to bottom: LF, left sensorimotor (frontal); LP, left parietal; LO, left visual (occipital); and RO, right visual cortical regions. The last second of the 15-second stimulation series is in the frame at the beginning of the recordings. Full arrows mark the end of the spike-and-wave type of ADs; empty arrow denotes the end of the mixed type. Time mark is 2 seconds; amplitude calibration is 0.5 mV.
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another type of AD (Figure 2) similar to that induced by stimulation of limbic structures accompanied by automatisms, mostly wet-dog shakes. Repeated elicitation of spikeand-wave ADs with unchanged stimulation intensity can be used for testing of antiepileptic drugs (Kubová et al., 1996). Ontogeny Ontogenetic study demonstrated reliable elicitation of cortical ADs in rats from the age of 12 days and older if low-frequency stimulation is used or from postnatal day 9 and older if 50-Hz stimulation is applied. Twelve-dayold rat pups are not able to generate spike-and-wave rhythm; the AD is formed by rhythmic sharp delta waves (Maresˇ et al., 2002). The maximal sensitivity to cortical stimulation is around the end of the third postnatal week of life in rats (Maresˇ et al., 2002). In addition, repetition of stimulation in 12-day-old rats with 10- or 20-minute intervals leads to a progressive increase of duration of ADs; postictal depression, which is observed in adult rats, is absent in this age group. Hippocampus Electrodes can be introduced into the dorsal or ventral hippocampus. The pattern of ADs is variable: low-amplitude spikes, delta waves, and delta waves with superimposed low-amplitude spikes. The first AD is followed by a period of EEG depression, and then the secondary (recurrent) AD appears (Dyer et al., 1979; Leung, 1987). The most common behavioral pattern is wet-dog shakes (Le Gal la Salle et al., 1983; Velísˇek and Maresˇ, 2004). Ontogeny It is possible to evoke hippocampal ADs in rats at the age of 7 days (Velísˇek and Maresˇ, 1991). In rat pups 12 to 15 days old, postictal depression is absent; 7-dayold rat pups exhibit postictal refractoriness, probably because of the fatigability of the system, not to a presence of active inhibition (Velísˇek and Maresˇ, 1991). Amygdala There are differences among individual nuclei studied in detail in the kindling model. Epileptic automatisms form a behavioral pattern of these seizures: arrest at the beginning, followed by chewing movements that continue for several seconds after the end of stimulation (Goddard et al., 1969). Ontogeny Ontogenetic studies demonstrated the possibility of eliciting these ADs at the end of the second postnatal week in rats (Moshe, 1981). In contrast to the results in older animals, amygdala ADs are not followed by a period of postictal depression in 2-week-old rats (Moshe and Albala, 1983). Baram et al. (1993) was able to elicit epileptic ADs by amygdala stimulation in 7-day-old rats. Entorhinal Cortex Electrodes can be introduced either into the entorhinal cortex or into the perforant path. It is possible to perform a
preoperative electrophysiologic control using single stimuli and recording responses in the dentate gyrus. The pattern of ADs may be variable: low-amplitude spikes, large delta waves, and delta waves with superimposed low-amplitude spikes. Behavioral concomitants are represented by an intense orienting reaction in a cage where the animal spent tens of minutes and include locomotion, sniffing, and rearing. Such behavior is appropriate for the first 2 to 3 minutes in a new environment. This automatism appears toward the end of the AD and continues even after the EEG epileptic activity is over. Wet-dog shakes are observed less frequently. Ontogeny Afterdischarges can be induced by stimulation of angular bundle in rat pups 10 to 11 days old, and adult characteristics are attained at the age of 21 days (Stringer and Lothman, 1992). Piriform Cortex Afterdischarges and their behavioral correlates elicited by stimulation of this structure do not differ from those induced by amygdala stimulation (Goddard et al., 1969; Honack et al., 1991). A disadvantage of piriform cortex is its anatomy: a very thin, rather long structure difficult for an exact placement of electrodes.
CONCLUSIONS The aforementioned seizures in rodents model different types of human seizures: MES is generally accepted as a model of generalized tonic-clonic seizures and stimulation of limbic structures as models of human complex partial seizures. Seizures elicited by stimulation of the sensorimotor cortex represent a model of focal motor or myoclonic seizures. Changes with repeated stimulations or increasing intensity can be used for studies of seizure spread as well as of secondary generalization (see Chapters 28–30). The MES screening tool is useful because it can provide data for many doses of the tested drug with minimal investment and experience. The other models discussed in this chapter are more time and money consuming, and both recording and evaluation of the data require experience. It is necessary to take into account that we discuss here models of epileptic seizures, not epilepsies, and that these experimental seizures are usually elicited in a healthy brain.
References Altar, C.A., Laeng, P., Jurata L.W., Brockman, J.A., Lemire, A., Bullard, J., Bukhman et al. 2004. Electroconvulsive seizures regulate gene expression of distinct neurotrophic signaling pathways. J Neurosci 24: 2667–2677. Baram, T.Z., Hirsch, E., and Schultz, L. 1993. Short-interval amygdala kindling in neonatal rats. Dev Brain Res 73: 79–83.
References Barton, M.E., Klein, B.D., Wolf, H.H., and White, H.S. 2001. Pharmacological characterization of the 6 Hz psychomotor seizures model of partial epilepsy. Epilepsy Res 47: 217–227. Browning, R.A., and Nelson, D.K. 1985. Variation in threshold and pattern of electroshock-induced seizures in rats depending on site of stimulation. Life Sci 37: 2205–2211. Della Paschoa, O.E., Kruk, M.R., Voskuyl, R.A., and Danhof, M. 1998. Effects of repeated seizure induction on seizure activity, postictal and interictal behavior. Brain Res 814: 199–208. Dyer, R.S., Swartzwelder, H.S., Eccles, C.U., and Annau, Z. 1979. Hippocampal afterdischarges and their post-ictal sequelae in rats: a potential tool for assessment of CNS toxicity. Neurobehav Toxicol 1: 5–19. Finney, D.J. 1952. Probit Analysis. Cambridge: Cambridge University Press. French, P.J., O’Connor, V., Jones, M.W., Davis, S., Errington, M.L., Voss, K., Truchet, B., et al. 2001. Subfield specific immediate early gene expression associated with hippocampal long-term potentiation in vivo. Eur J Neurosci 13: 968–976. Goddard, G.V., McIntyre, D.C., and Leech, C.K. 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 25: 295–330. Honack, D., Wahnschaffe, U., and Loscher, W. 1991. Kindling from stimulation of a highly sensitive locus in the posterior part of the piriform cortex. Comparison with amygdala kindling and effects of antiepileptic drugs. Brain Res 538: 196–202. Hoogerkamp, A., Vis, P.W., Danhof, M., and Voskuyl, R.A. 1994. Characterization of the pharmacodynamics of several antiepileptic drugs in a direct cortical stimulation model of anticonvulsant effect in the rat. J Pharmacol Exp Ther 269: 521–528. Jonker, D.M., van de Mheen, C., Eilers, P.H., Kruk, M.R., Voskuyl, R.A., and Danhof, M. 2003. Anticonvulsant drugs differentially suppress individual ictal signs: a pharmacokinetic/pharmacodynamic analysis in the cortical stimulation model in the rat. Behav Neurosci 117: 1076–1085. Krupp, E., and Löscher, W. 1998. Anticonvulsant drug effects in the direct cortical ramp-stimulation model in rats: Comparison with conventional seizure models. J Pharmacol Exp Ther 285: 1137–1149. Kubová, H., Lansˇtiaková, M., Mocková, M., Maresˇ, P., and Vorlícˇek, J. 1996. Pharmacology of cortical epileptic afterdischarges in rats. Epilepsia 37: 336–341. Le Gal La Salle, G., Cavalheiro, E.A., Feldblum, S., and Maresova, D. 1983. Studies of wet-dog shake behavior induced by septohippocampal stimulation in the rat. Can J Physiol Pharmacol 61: 1299–1304. Leung, L.W. 1987. Hippocampal electrical activity following local tetanization. I. Afterdischarges. Brain Res 419: 173–187.
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Lichtfield, J.T. Jr., and Wilcoxon, F. 1953. The reliability of graphic estimates of relative potency from dose-per cent effect curves. J Pharmacol Exp Ther 108: 18–25. Löscher, W., and Schmidt, D. 1988. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical considerations. Epilepsy Res 2: 95–134. Maresˇ, P., Haugvicová, R., and Kubová, H. 2002. Unequal development of thresholds for various phenomena induced by cortical stimulation in rats. Epilepsy Res 49: 35–43. Meldrum, B.S. 1986. Neuropathological consequences of chemically and electrically induced seizures. Ann N Y Acad Sci 462: 186–193. Millichap, J.G. 1958. Seizure patterns in young animals. II. Significance of brain carbonic anhydrase. Proc Soc Exp Biol Med 97: 606–611. Moshe, S.L. 1981. The effects of age on the kindling phenomenon. Dev Psychobiol 14: 75–81. Moshe, S.L., and Albala, B.J. 1983. Maturational changes in postictal refractoriness and seizure susceptibility in developing rats. Ann Neurol 13: 552–557. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation: II. Motor seizures. Electroencephalogr Clin Neurophysiol 32: 269–279. Stringer, J.L., and Lothman, E.W. 1992. Ontogeny of hippocampal afterdischarges in the urethane-anesthetized rat. Dev Brain Res 70: 223–229. Swinyard, E.A. 1972. Electrically induced convulsions. In Experimental Models of Epilepsy. Eds. D.P. Purpura, J.K. Penry, D. Tower, D.M. Woodbury, and R. Walter. pp. 433–458. New York: Raven Press. Swinyard, E.A., and Woodhead, J. 1982. Experimental detection, quantification and evaluation of anticonvulsants. In Antiepileptic Drugs, 2nd ed. Eds. D.M. Woodburt, J.K. Penry, C.E. Pippenger. pp. 111–126. New York: Raven Press. Velísˇek, L., and Maresˇ, P. 1991. Increased epileptogenesis in the immature hippocampus. Exp Brain Res 20: 183–185. Velísˇek, L., and Maresˇ, P. 1992. Differential effects of naloxone on postictal depression. Epilepsy Res 12: 37–43. Velísˇek, L., and Maresˇ, P. 2004. Hippocampal afterdischarges in rats. I. Effects of antiepileptics. Physiol Res 53: 453–461. Vernadakis, A., and Woodbury, D.M. 1969. The developing animal as a model. Epilepsia 10: 163–178. Voskuyl, R.A., Dingmanse, J., and Danhof, M. 1989. Determination of the threshold for convulsions by direct cortical stimulation. Epilepsy Res 3: 120–129. Voskuyl, R.A., Hoogerkamp, A., and Danhof, M. 1992. Properties of the convulsive threshold determined by direct cortical stimulation in rats. Epilepsy Res 12: 111–120. Wasterlain, C.G., and Plum, F. 1973. Vulnerability of developing brain to electroconvulsive seizures. Arch Neurol 29: 38–45.
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13 Alcohol Withdrawal Seizures PROSPER N’GOUEMO AND MICHAEL A. ROGAWSKI
to generalized tonic seizures (Freedland and McMicken, 1993a). Alcohol withdrawal seizure has been included in the International League Against Epilepsy Classification of Epilepsies and Epileptic Syndromes as a condition with epileptic seizures that does not require a diagnosis of epilepsy (Engel, 2001). These seizures typically occur 6 to 48 hours after discontinuation of alcohol consumption, but they may also occur up to 7 days after the last drink, particularly if the patient has been abusing other psychoactive agents, such as benzodiazepines or barbiturates. Generalized tonic-clonic seizures are the most severe type of alcohol withdrawal seizures in humans. Convenient rodent models that mimic human alcohol withdrawal-related tonic-clonic seizures have been developed, thus providing a substrate to study the underlying seizure mechanisms and for the development of treatment approaches. In these models, the following tonic-clonic seizure types can be observed during a finite period (typically 1 to 3 days) after the cessation of alcohol intake: (1) spontaneous seizures, (2) auditory-evoked seizures (“audiogenic seizures,” or AGS), and (3) handling-induced convulsions (HIC). This chapter describes procedures for inducing alcohol dependence, precipitating withdrawal, and eliciting and scoring the resultant alcohol withdrawal seizures. In these models rats or mice are exposed to alcohol by intragastric intubation, inhalation, or feeding in a nutritionally complete liquid diet for periods of 2 to 21 days. (The use of a seizuresensitive mouse strain that exhibits an increase in seizure severity after a single dose of alcohol is briefly discussed in the section entitled Selection of a Model.) Each of the models is associated with highly reproducible alterations in seizure susceptibility so that they are suitable for basic neurobiological, pharmacologic, and genetic studies. In
Ethyl alcohol (ethanol) is a central nervous system (CNS) depressant that exerts diverse behavioral actions. At low blood concentrations, alcohol produces euphoria and behavioral excitation, and at concentrations greater than 0.08 g/dl (17 mM), it significantly impairs motor skills. Concentrations of 0.15 to 0.30 g/dl induce acute intoxication, which manifests as drowsiness, ataxia, slurred speech, stupor, and coma. The acute effects of alcohol on brain function are believed to result largely from its actions on ligand-gated and voltage-gated ion channels, resulting in alterations in neuronal signaling (Crews et al., 1996; Deitrich and Erwin, 1996; Nevo and Hamon, 1995). Chronic alcohol consumption leads to the development of tolerance and physical dependence, which may result from compensatory changes in neuronal signaling that balance the acute effects of alcohol. Abrupt cessation of chronic alcohol consumption unmasks the compensatory physiologic change, leading to a cluster of neurologic signs and symptoms known as alcohol withdrawal syndrome. In humans alcohol withdrawal syndrome includes blackouts, tremors, muscular rigidity, delirium tremens, and seizures (Hillbom et al., 2003; Kosten and O’Connor, 2003). These various manifestations of alcohol withdrawal may reflect an involvement of different neuronal networks and cellular mechanisms (Schmidt and Sander, 2000). The most prominent and dramatic aspects of alcohol withdrawal syndrome in humans are alcohol withdrawal seizures, which can be life threatening (Hillbom et al., 2003; Mattson, 1983; Peininkeroinen et al., 1992; Ripley, 1990). Alcohol withdrawal seizures are usually generalized tonic-clonic (“grand mal”) seizures, although 60% of subjects experience multiple seizure types (Victor and Brausch, 1967). These additional seizure types include partial seizures and partial seizures progressing
Models of Seizures and Epilepsy
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addition, the models can be of value in evaluating potential therapies for alcohol withdrawal seizures. This chapter also reviews the present understanding of the brain mechanisms underlying withdrawal seizures and considers briefly seizures related to withdrawal of other CNS depressants.
METHODS FOR INDUCTION OF ALCOHOL DEPENDENCE AND FOR MONITORING WITHDRAWAL SEIZURES Species, Strain, Gender, and Age Considerations As in humans, an alcohol withdrawal syndrome that includes generalized tonic-clonic seizures has been observed in the mouse, rat, cat, dog, monkey, and chimpanzee (Ellis and Pick, 1970; Essig et al., 1969; Freund, 1969; Guerrero-Figueroa et al., 1970; Majchrowicz, 1975; Pieper et al., 1972). In all species, the signs of alcohol withdrawal last for 1 to 3 days, after which behavior returns to normal and there is no enhanced seizure susceptibility. Rodents are the most common species used in laboratory studies of alcohol withdrawal seizures. Here we describe methods for the induction of alcohol dependence and for inducing and scoring withdrawal seizures in the rodent species that are most frequently used in laboratory experiments. The intragastric intubation method was originally developed in studies with rats and is mainly applied in this species. In contrast, inhalation methods are most commonly used in mice. The liquid diet procedure is applied in both mice and rats. The experimental methods can be adapted for use with other species. Among rodents, there are substantial differences among strains in the severity of withdrawal seizures (Crabbe, 2002; Metten and Crabbe, 1994). For example, DBA/2 inbred mice exhibit more severe acute alcohol withdrawal seizures than do C57BL/6 mice (Roberts et al., 1992). As discussed in the section entitled Metabolic Changes Following Alcohol Withdrawal, there has been an extensive effort to identify the specific genes that influence alcohol withdrawal seizure severity and other behavioral components of alcohol dependence and withdrawal. Consideration of strain differences is critical when selecting animals for experimental studies, and in the future, as specific susceptibility loci are identified, the specific genotype will also likely be a factor of relevance. There are significant sex differences in the incidence of ethanol abuse and alcoholism, with nearly 20% of adult men affected, in contrast to only about 5 to 6% of adult women (Devaud et al., 1999). Alcohol withdrawal seizures seem to affect predominantly men (Essardas Daryanani et al., 1994; Pilke et al., 1984). These differences have been attributed mainly to societal influences, although neurobiological
factors could also play a role. Both male and female rodents exhibit withdrawal seizures in alcohol-dependence models. However, there may be small sex differences in susceptibility. For example, male mice may experience more severe seizures than females do (Buck et al., 2002). Adult animals are typically used in studies of alcohol withdrawal seizures. Interestingly, however, mice that have been bred for susceptibility to ethanol withdrawal seizures may also show enhanced susceptibility to AGS, but only at young ages (Feller et al., 1994) corresponding with the developmental sensitivity of mouse strains such as DBA/2, which are only susceptible to audiogenic seizures from postnatal days 20 to 80.
Procedures Methods for Alcohol Administration Intragastric Intubation Induction of alcohol dependence in rats by intragastric intubation was first described by Majchrowicz (1975). A key advantage of this approach over other methods of alcohol administration is that dosing is highly reliable, so when the treatment is discontinued, all animals exhibit the major signs of withdrawal. In addition, this method allows physical dependence to be produced in a relatively short time. In the model, 50 mM ethanol (95%) is administered in ISOMIL Soy Infant Formula Concentrate diluted 1 : 1 with water (Riaz and Faingold, 1994). A priming dose of 5 g/kg of ethanol is administered to each animal, and subsequent doses are determined by the degree of intoxication exhibited. Dosing occurs at 8-hour intervals for 4 days, with total daily doses in the range of 9 to 15 g/kg/day. The dosages are adjusted so that the animals exhibit mild to moderate ataxia (Table 1). Ethanol is withdrawn after the second dose on the
TABLE 1 Behavioral Rating Scale for Alcohol Intoxication Stage
Signs
Neutral
No signs of alcohol intoxication or withdrawal
Sedation
Reduced muscle tone; slow locomotor activity
Ataxia 1
Slight impairment of gait and motor coordination
Ataxia 2
Impaired gait and motor coordination; considerable elevation of abdomen and pelvis
Ataxia 3
Sedation; lethargy; impaired motor coordination
Loss of righting reflex
Inability to right after 5 s when placed on back
Coma
No movements; no response to pain stimuli; no eye blink; palpebral closure
Adapted from Majchrowicz (1975).
Methods for Induction of Alcohol Dependence and for Monitoring Withdrawal Seizures
fourth day. Control animals are maintained under similar conditions but are fed the ISOMIL diet without ethanol. This method can be associated with significant mortality (9–23%; N’Gouemo et al., 1996). Inhalation Alcohol intoxication is initiated by administration of a loading dose of ethanol (1.6 g/kg; 8%, wt/vol). Some investigators administer the alcohol dehydrogenase inhibitor pyrazole to enhance and stabilize the blood ethanol concentrations (Goldstein and Pal, 1971). Animals are then placed in a closed inhalation chamber as illustrated in Figure 1, and alcohol dependence is induced by continuous exposure to alcohol vapors for 2 to 7 days. Air is continuously delivered to the chamber at a rate of 10 liters per minute to provide for the respiratory need of the animals. Ethanol (95%) is evaporated into the air so that the experimental chamber holding the animal receives air with an ethanol concentra-
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tion of 7 to 35 mg/liter. Control chambers are similarly configured, but with the absence of ethanol vapor. Liquid Diet Alcohol can also be chronically administered in a liquid diet. Rats have an aversion to alcohol. However, high alcohol intake can be induced with a nutritionally complete liquid diet used as the only food source, such as the LieberDeCarli diet (Dyets, Inc., Bethlehem, PA) (Lieber and DeCarli, 1982). Control animals receive a similar diet in which an isocaloric amount of maltose is substituted for ethanol. The ethanol concentration is typically 6 to 7% (vol/vol) and is administered for 4 to 21 days. At the end of the alcohol exposure period, animals are switched to a regular diet. Mice can be treated with a similar diet, but the ethanol concentration may be lower (4.5 to 6.5%) and the exposure period is typically 2 to 6 days. Mice can also be “kindled” by repeated withdrawal (periods of 1 to 2 days of
FIGURE 1 Schematic representation of a system for administration of ethanol by inhalation modified after Ruwe et al. (1986). Ethanol (95%) is delivered by a solvent metering pump into a 250-ml vaporization chamber. The airtight vaporization chamber is maintained at 37° C by a water bath. Air is delivered into the vaporization chamber with an air pump to provide a flow rate of 2.5 to 4 liters per minute. Animals are exposed to ethanol-vapor concentrations of 7 to 35 mg/liter of air in a Plexiglas experimental chamber. A food tray and water bottle are securely affixed to the experimental chamber, giving the animal free access to food and water. The sample port allows air within the experimental chamber to be sampled for determination of ethanol concentrations. (See color insert.)
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abstinence during the chronic ethanol feeding), which potentiates the severity of withdrawal seizures (Becker and Hale, 1993; Pinel, 1980; Ripley et al., 2002). Blood Sampling and Measurement of Blood Alcohol Concentrations Blood alcohol concentrations are typically measured during intoxication, at the onset of withdrawal symptoms, and during the fully developed withdrawal syndrome. Blood samples are usually collected from the tail vein in mice or by intracardiac sampling in deeply anesthetized rats using large-bore (21-gauge) needles to prevent hemolysis. Blood samples are stored in tubes containing heparin or the anticoagulant potassium oxalate and sodium fluoride (Becton Dickinson Vacutainer Systems, Rutherford, NJ). Blood ethyl alcohol concentrations are measured using gas chromatography (Brown and Long, 1988) or determined in the plasma using the alcohol dehydrogenase method, which requires a spectrophotometer to measure the absorbance at 340 nm (Pointe Scientific, Canton, MI). Seizure Monitoring Following Alcohol Withdrawal
The animal’s behavior is recorded on videotape for subsequent review. The incidence and type of seizures (wild running, bouncing clonus, tonic) are noted, and the overall severity is scored according to the scale of Jobe et al. (1973) (Table 2) and also Chapter 20). Audiogenic seizures can be measured repeatedly in the same animal with high interrater reliability. Handling-Induced Convulsions Mice are observed for seizure activity in response to various degrees of stimulation. Animals that exhibit spontaneous seizures or a tonic-clonic seizure within seconds of being handled during removal from their home cages receive the highest seizure scores. If no seizure occurs immediately with routine handling, animals are picked up by the tail and observed for an additional 2 seconds. If they still fail to exhibit a seizure, they are gently spun by the tail through a 180- to 360-degree arc. A score is assigned as defined in Table 3 based on the degree of stimulation required to elicit the seizure (handling alone, tail lift, or spinning) and the type of seizure (clonic, tonic, tonic-clonic) (Becker et al., 1997; Crabbe and Kosobud, 1990; Goldstein and Pal, 1971). HIC can be measured repeatedly in the same animal with high interrater reliability.
Spontaneous Seizures On administration of the last dose of ethanol, the animals are placed in a Plexiglas observation chamber located in a sound-attenuated room (Gonzalez et al., 1989). The behavior of each animal is recorded on videotape at 30 frames per second for about 84 hours from the time of ethanol discontinuation. The videotapes are scanned at high speed to identify segments with evidence of seizure-like behavior and then at slower speeds for scoring. The incidence and severity of the following behaviors are recorded: (1) myoclonic jerks of the head or trunk; (2) jumping episodes; (3) generalized tonic-clonic seizures consisting of loss of upright posture and repetitive extension and retraction of the limbs and trunk; and (4) rigid, tonic extension of the limbs. Myoclonic jerks occur most frequently during the first 6 hours following alcohol withdrawal. The incidence of generalized tonic-clonic seizures is maximal between 24 and 60 hours after alcohol withdrawal; tonic seizures are most frequent at 60 to 84 hours following withdrawal.
TABLE 2 Behavioral Rating Scale for Audiogenic Seizures Score 0
No response
1
Running only; no convulsion
2
Two running phases separated by a refractory period; generalized clonus involving forelimbs and hindlimbs
3
One running phase; generalized clonus involving forelimbs and hindlimbs
4
Two running phases separated by a refractory period; tonic flexion of neck, trunk and forelimbs with clonus of hindlimbs
5
One running phase and no refractory period; tonic flexion of neck, trunk and forelimbs with clonus of hindlimbs
6
Two running phases separated by a refractory period; tonic flexion of neck, trunk and forelimbs with hindlimbs in partial tonic extension (i.e., tonic extension of thighs and legs with clonus of feet)
7
One running phase and no refractory period; tonic flexion of neck, trunk and forelimbs with hindlimbs in partial tonic extension (i.e., tonic extension of thighs and legs with clonus of feet)
8
Two running phases separated by a refractory period; tonic flexion of neck, trunk and forelimbs with hindlimbs in complete tonic extension (maximal convulsion)
9
One running phase and no refractory period; tonic flexion of neck, trunk and forelimbs with hindlimbs in complete tonic extension (maximal convulsion)
Audiogenic Seizures Mice and rats subjected to alcohol withdrawal are susceptible to AGS between 10 and 26 hours following the last doses of alcohol (Freund, 1969; Riaz and Faingold, 1994). AGSs are induced in a sound-attenuating chamber using an electric bell that produces a sound volume of about 122 dB at the middle of the acoustic chamber. The bell tone is presented either until a seizure is triggered or for 60 seconds.
Description of Behavior
From Jobe et al. (1973).
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Selection of an Experimental Model
TABLE 3 Behavioral Rating Scale for Handling-Induced Convulsion Score
Description of Behavior
0
No convulsion or facial grimace on tail lift, or after gentle 180to 360-degree spin
1
Facial grimace after 180- to 360-degree spin
2
Tonic convulsion after 180- to 360-degree spin
3
Tonic-clonic convulsion after 180- to 360-degree spin
4
Tonic convulsion on tail lift
5
Tonic-clonic convulsion on tail lift, onset delayed by 1–2 s
6
Severe tonic-clonic convulsion on tail lift, no delay in onset
7
Severe tonic-clonic convulsion before tail lift Adapted from Becker et al. (1997) and Crabbe and Kosobud (1990).
BEHAVIORAL FEATURES AND BRAIN MECHANISMS Spontaneous Seizures Spontaneous seizures are observed between 24 to 60 hours following cessation of alcohol consumption, and their incidence reaches a peak between hour 36 and 48 of the withdrawal period (Clemmesen et al., 1988; Gonzalez et al., 1989). The behaviors observed include myoclonic jerks, facial and forelimb clonic seizures, rearing, falling, tonicclonic seizures, and tonic seizures. The neuronal networks that generate these behaviors are as yet unknown. However, the similarity to amygdala kindled seizures suggests that they may originate in the limbic system. Interestingly, studies with 2-deoxyglucose autoradiography have implicated amygdala neurons in the generation of spontaneous seizures associated with alcohol withdrawal (Clemmesen et al., 1988).
Handling-Induced Convulsions Handling-induced convulsions are characterized by the occurrence of tonic-clonic seizures with routine handling as well as tonic or tonic-clonic seizures on tail lift or after spinning. The network for HIC is still poorly understood. Seizures elicited after spinning suggest an initial involvement of the vestibular system. The pontine reticular formation and periaqueductal gray are thought to play important roles in the expression of alcohol withdrawal-related clonic and tonic audiogenic seizures (see subsequent discussion). It is likely that these brainstem loci may also play a similar role in HIC.
Audiogenic Seizures The behavioral features of AGS following alcohol withdrawal consist of wild running, bouncing clonus, and tonic
seizures. Wild running is considered a model of partial seizures, whereas bouncing clonus represents generalized tonic-clonic seizures. The neuronal networks for AGS associated with alcohol withdrawal appear to be located in the brainstem, and a large body of evidence suggests that inferior colliculus (IC) neurons are critical in the initiation of these seizures (Faingold et al., 1998). Bilateral lesions of the IC, but not of the medial geniculate body, the first efferent synaptic target of IC neurons, block AGS susceptibility following alcohol withdrawal (Frey et al., 1986). These findings suggest that alcohol withdrawal seizure activity diverges from the classic auditory pathway at the levels of the IC and that these seizures are not of auditory nature per se. Consistent with this idea, epileptic activity was observed in hippocampal neurons, but with significant delay after the onset of auditory-evoked tonic-clonic seizures following alcohol withdrawal, suggesting an involvement of limbic structures in the network for AGS (Hunter et al., 1973). The IC plays an important role in the processing of auditory information and is the gateway to sensorimotor integration for the auditory system (Atkin, 1986). The IC can be subdivided into three main subnuclei: the central nucleus, dorsal cortex, and external cortex. All three subnuclei of the IC have been implicated in alcohol withdrawal seizures, and the IC external cortex appears to be important for the convergence of the outflow from the IC to the neuronal network responsible for producing motor seizures, which include the superior colliculus, periaqueductal gray and reticular formation (Faingold et al., 1998).
SELECTION OF AN EXPERIMENTAL MODEL There are various tradeoffs in the choice between spontaneous seizures or seizures elicited by audiogenic or handling stimulation as the endpoint in studies of alcohol withdrawal seizures. Spontaneous seizures may have greater face validity to human alcohol withdrawal seizures because human alcohol withdrawal seizures occur paroxysmally without an apparent eliciting stimulus. Spontaneous alcohol withdrawal seizures in rodents are observed over a time course similar to that in humans. However, spontaneous seizures in experimental animals are mainly myoclonic seizures, whereas alcohol withdrawal seizures in humans are mostly generalized tonic-clonic seizures, as are vestibular or auditory-evoked seizures in animals. Such reflex seizures may have significantly different underlying pathophysiologic mechanisms from alcohol withdrawal seizures in humans, which are not elicited by audiogenic or vestibular stimulation. (It is notable that some features of the alcohol withdrawal syndrome in humans are, in fact, triggered by external stimuli, including visual, auditory, or tactile
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hallucinations.) From a practical point of view, monitoring spontaneous seizures following alcohol withdrawal is technically more difficult than assessing seizures that are induced under the control of the experimenter; so reflex seizures are used more often in pharmacologic and physiologic studies. In some mouse strains, mild HIC scores are elicited in some animals, even in the absence of alcohol. In these sensitive mouse strains, it may be possible to obtain potentiation of HIC scores after a single dose of alcohol. Withdrawal from alcohol in these strains is associated with higher HIC scores. A test paradigm using a single dose of ethanol has been used extensively in genetic studies to select for mice that are more or less withdrawal-seizure prone (Metten and Crabbe, 1994; 1999). Figure 2 schematically illustrates the results of a typical experiment with mice that are withdrawal-seizure prone (WSP) or withdrawal-seizure resistant (WSR). At baseline, WSP mice exhibit slightly greater HIC scores than WSR mice. A single hypnotic dose of ethanol
(4 g/kg) is administered at zero time, and HIC susceptibility is monitored for the subsequent 12 hours. Both strains show a reduction in HIC score at 2 hours because of the anticonvulsant effects of alcohol. As blood alcohol levels fall and the animals are in a state of alcohol withdrawal, there is an increase in HIC score, which peaks at about 6 hours; WSP mice exhibit greater HIC scores than do WSR mice.
SINGLE-NEURON FIRING DURING CELLULAR ELECTROPHYSIOLOGY OF ALCOHOL WITHDRAWAL SEIZURES Audiogenic Seizures Forebrain During alcohol withdrawal-related AGS, the cortical electroencephalogram typically shows no sign of paroxysmal activity compatible with the idea that the seizures are mediated largely in the brainstem (Hunter et al., 1973; Maxson and Sze, 1976). Nevertheless, epileptiform activity has been observed in the hippocampus, but with a significant delay after the onset of AGS, suggesting a role in the propagation rather than in the initial generation of the seizures (Hunter et al., 1973). Inferior Colliculus
FIGURE 2 Schematic illustration of the time course of handling-induced convulsion (HIC) scores after withdrawal from a single dose of ethanol in withdrawal-seizure prone (WSP) and withdrawal-seizure resistant (WSR) mouse strains, according to the protocol of Metten et al. (1998). A baseline HIC assessment is recorded (see Table 3). Mice are then injected intraperitoneally with a sedating dose of ethanol (4 g/kg, 20% vol/vol in 0.9% saline) at time 0. HIC scores are initially suppressed by the ethanol injection, but a rebound of the HIC response occurs by about 3 to 4 hours after injection as the ethanol is eliminated. Withdrawal is evidenced by exacerbation of HIC severity, typically peaking at around 6 to 7 hours and returning to control levels at about 12 hours after the ethanol injection. Baseline seizure scores and withdrawal responses of WSR mice are reduced in comparison with WSP mice.
Acute alcohol intoxication suppresses spontaneously and acoustically evoked neuronal firing in the IC central nucleus, whereas alcohol withdrawal is accompanied by significant increases of these responses (Faingold and Riaz, 1995). Electrophysiologic studies have revealed that, at the transition to seizure, the IC central nucleus exhibits sustained increases in firing that persist during wild running, the initial phase of AGS (Chakravarty and Faingold, 1998). It has therefore been suggested that the IC central nucleus plays a role in the initiation of these seizures. The IC external nucleus is also a target of alcohol. Indeed IC external nucleus responses were suppressed during both acute alcohol intoxication and alcohol withdrawal (Chakravarty and Faingold, 1998). However, during alcohol withdrawal, IC external cortex neuronal firing is increased before the onset of AGS and persists during the wild running phase. The IC external cortex is believed to amplify and propagate neuronal activity originating in the IC central nucleus. Increased neuronal activity in these systems is then transmitted to nuclei responsible for the generation of convulsive motor behaviors. Superior Colliculus Spontaneous and acoustically evoked responses are suppressed during acute alcohol intoxication and alcohol
Neuropathological Effects of Alcohol Withdrawal
withdrawal in the deep layers of the superior colliculus (SC) (Yang et al., 2001a). However, these neurons exhibit tonic neuronal firing before the onset and during wild running in alcohol withdrawal-related AGS. The tonic firing preceding the onset of wild running suggests that SC neurons may play a role in the generation of this component of AGS. Periaqueductal Gray Acute alcohol intoxication suppresses spontaneous and acoustically evoked periaqueductal gray (PAG) neuronal responses (Yang et al., 2003). During alcohol withdrawal, a significant increase in spontaneous and acoustically evoked PAG responses is observed. PAG neurons exhibit burst firing during wild running and tonic repetitive firing during the tonic-clonic phase of alcohol withdrawal-related AGS. The bursting activity preceding tonic-clonic seizures suggests that PAG may play a role in the generation of these seizures. Pontine Reticular Formation Spontaneous and acoustically-evoked pontine reticular formation (PRF) neuronal firing is suppressed by acute alcohol intoxication, whereas alcohol withdrawal associated with enhanced seizure susceptibility is accompanied by a significant increase in spontaneous and acoustically evoked PRF responses (Faingold and Riaz, 1994). The neuronal firing during alcohol withdrawal-related sound induced seizures has not yet been characterized. Nevertheless, studies using the genetically epilepsy-prone rat, which exhibits an inherited susceptibility to AGS, have found that PRF neurons exhibit tonic firing during the tonic phase of AGS and this pattern of neuronal activity persists during the postictal depression following the seizures (Faingold and Randall, 1995). Such a pattern of neuronal firing could also occur in PRF neurons during alcohol withdrawal seizures. Synaptic Pathways Generating Audiogenic Seizures The evidence to date indicates that IC neurons are critical in the initiation of alcohol withdrawal-related AGS. It is hypothesized that seizure activity propagates from the IC to deep layers of the SC, a major output of the IC, to trigger the wild running phase of the AGS. The deep layers of the SC send projections directly to the spinal cord via the PRF and the periaqueductal gray, which is thought to trigger clonic seizures, and the PRF is implicated in generation of the tonic phase of AGS activity (Faingold, 2004).
Handling-Induced Convulsions The cellular electrophysiologic correlates of alcohol withdrawal-related HIC have not yet been described. Nevertheless, brief spindle episodes (BSEs) are observed
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in hippocampal neurons in animals that exhibit such HIC (Veatch and Becker, 2002). Although the incidence of BSE peaks earlier than HIC in these animals, the discharges could play a role in the seizures. The electrographic patterns of alcohol withdrawal-related BSEs resemble those observed in models of generalized absence seizures (Danober et al., 1998; Snead, 1995). However, because alcohol withdrawal-related HIC are tonic-clonic seizures, there are likely important differences in the underlying mechanisms.
NEUROPATHOLOGICAL EFFECTS OF ALCOHOL WITHDRAWAL In humans, alcohol withdrawal seizures have been associated with ventricular and sulcal enlargement as well as significantly smaller volume of temporal lobe white matter and hippocampal sclerosis (Essardas-Daryanani et al., 1994). In animal models, there is evidence that alcohol intoxication can lead to selective damage to specific brain regions, including the hippocampus (Ikonomidou et al., 2000; Walker et al., 1980). Withdrawal from long-term alcohol consumption can aggravate alcohol-induced neurodegeneration. Indeed alcohol withdrawal is associated with augmented loss of CA1 and CA3 pyramidal neurons, mossy fiber-CA3 synapses, and dentate gyrus granule cells (Cadete-Leite et al., 1989; Paula-Barbosa et al., 1993; Scorza et al., 2003). The mechanisms underlying alcohol withdrawal-induced neurodegeneration are not completely understood. However, alcohol withdrawal, but not alcohol intoxication, is thought to be associated with significant increases in free intracellular calcium in hippocampal neurons (Prendergast et al., 2004). Thus alcohol withdrawal–induced neurotoxicity may result, in part, from enhanced calcium signaling. Indeed there is evidence for increased synaptic activation of calcium channels in alcohol withdrawal seizures (Whittington et al., 1993, 1995). Another key mediator of calcium entry into neurons is the N-methyl-d-aspartate (NMDA) receptor. There is an increasing body of data supporting the view that NMDA receptor function is enhanced in alcohol withdrawal (Krystal et al., 2003). This may result from enhanced activity in glutamatergic systems (Hoffman and Tabakoff, 1996), and, in fact, there is evidence of increased brain extracellular glutamate concentrations following ethanol withdrawal (Rossetti and Carboni, 1995). Moreover, whereas acute ethanol inhibits NMDA receptors, prolonged ethanol exposure may result in a compensatory upregulation of NMDA receptor function (Carpenter-Hyland et al., 2004; Floyd et al., 2003; Gulya et al., 1991; Snell et al., 1996). Overall the excessive activation of NMDA receptors could be a major contributor to the neuropathology of alcohol withdrawal.
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METABOLIC CHANGES FOLLOWING ALCOHOL WITHDRAWAL Alcohol intoxication decreases local cerebral glucose utilization (LCGU) in many areas of the brain, including the limbic system, cerebellum, and motor system (Eckardt et al., 1992). The most striking effects on LCGU are observed in the IC (Grunwald et al., 1993), in accordance with other evidence indicating that this brain site is a major target of alcohol. Alcohol withdrawal is associated with increased brain glucose uptake, oxygen consumption, and blood flow (Eckardt et al., 1992; Hemmingsen et al., 1979; Newman et al., 1985). Significant increases in LCGU with alcohol withdrawal have been reported in motor systems, the auditory system (including IC), and the mammillary bodies-anterior thalamus-cingulate cortex pathway (Eckard et al., 1992), although one study reported decreases with acute withdrawal in most limbic regions and no changes in cerebellum and subcortical structures (Clemmesen et al., 1988). Animals that had experienced spontaneous withdrawal seizures exhibited relatively greater reductions in glucose utilization in the amygdala, whereas those with previous withdrawal-related audiogenic seizures had greater reductions in the auditory system, including the IC.
GENETICS OF ALCOHOL WITHDRAWAL SEIZURES Alcohol-associated seizures tend to run in families. For example, it has been reported that the incidence of seizures in first-degree relatives of individuals who experienced alcohol associated seizures is 2.45-fold that of unaffected individuals, whereas no excess incidence was observed in family members of persons experiencing posttraumatic seizures (Schaumann et al., 1994). An increased incidence of seizures in relatives was found whether the proband had alcohol-related seizures (spontaneously occurring seizures in association with chronic alcohol abuse) or alcohol withdrawal seizures. Thus some people without a history of epilepsy may have inherited genetic susceptibilities that make seizures more likely to occur in the setting of alcohol abuse. One gene implicated in susceptibility to alcohol withdrawal seizures is the dopamine transporter (DAT) gene (SLC6A3), which has a variable-number tandem repeat (VNTR) in exon 15 encoding the 3¢ untranslated region of its messenger RNA (mRNA) (Gorwood et al., 2003; Vandenbergh et al., 1992). In a study of alcoholics who reported histories of withdrawal seizures or delirium, Sander et al. (1997) found an increased prevalence of the nine-repeat (A9) allele compared with nonalcoholic controls. Two issues must be considered in assessing the significance of this
finding. First, the presence of the marker does not influence coding of the DAT protein and is therefore unlikely to have functional relevance itself. However, it has been noted that the repeat is close to the coding region and could be in linkage disequilibrium with a vulnerability-causing mutation that alters gene expression or protein structure. Precisely how alterations in DAT would influence seizure susceptibility is unknown. Alternatively the marker could be linked to other susceptibility alleles within a haplotype. The second issue is that the candidate gene approach, such as used in the Sander et al. study, is inherently susceptible to false positives. False associations can occur because of the large number of potential candidates and the low a priori probability that any particular candidate is a true susceptibility gene. Moreover, there is evidence of ethnic stratification of the DAT VNTR alleles so that uncontrolled differences in ethnicity between the affected and control populations could increase the risk of false positives, particularly in a study with a small sample size as was the case in Sander and colleagues (Uhl, 2004). Although the association of the DAT A9 allele with susceptibility to alcohol withdrawal seizures in humans requires confirmation, studies in animals do indicate the existence of inherited factors that predispose to the symptoms of alcohol withdrawal, including seizures. GoraMaslak et al. (1991) concluded that an aggregate set of genetic markers accounts for up to 62% of the variability in withdrawal syndrome severity. However, candidate gene studies have been disappointing, with no consistent results linking genes for serotonin, g-aminobutyric acid (GABA), endorphin, and dopamine receptors and the serotonin transporter with susceptibility to the withdrawal syndrome (Schmidt and Sander, 2000). The available evidence suggests that multiple genes are involved in various components of the syndrome, each of them contributing only modestly to withdrawal vulnerability. Nevertheless, in the mouse, there is good evidence that allelic variation in Mpdz influences the liability of alcohol withdrawal seizures (Fehr et al., 2004). Mpdz encodes the multiple PDZ domain protein (MPDZ). Mpdz haplotypes in standard mouse strains encode the three distinct protein variants MPDZ1–3. Recently it was reported that MPDZ status cosegregates with withdrawal convulsion severity in lines of mice selectively bred for phenotypic differences in severity of acute withdrawal from alcohol. MPDZ1 strains have significantly less severe acute alcohol withdrawal seizures than strains that express MPDZ2 or the closely related MPDZ3. Severity of pentobarbital withdrawal seizures is similarly correlated with MPDZ status (Fehr et al., 2002). Many ion channels and transporters possess PDZ-binding domains that are important in their trafficking and targeting to synapses. Although the MPDZ allelic variants could participate in such targeting, their precise functional roles are poorly understood and the differences among the variants that
Cellular and Molecular Mechanisms
would confer differences in seizure susceptibility have not yet been defined.
CELLULAR AND MOLECULAR MECHANISMS Although alcohol is the most widely used psychoactive agent, the pharmacologic basis of its intoxicating effects is incompletely understood. Similarly the molecular mechanisms underlying alcohol dependence and withdrawal are obscure. Nevertheless, it is well recognized that alcohol affects the functional activity of many receptors and ion channels, including NMDA (Lovinger et al., 1989, 1990), kainate (Carta et al., 2003), serotonin (Lovinger and White, 1991), GABAA (Davies, 2003) and glycine (Mihic et al., 1997) receptors, and G protein–coupled inwardly rectifying potassium channels (Kobayashi et al., 1999) and calcium channels (Walter and Messing, 1999). In most cases, the effects of alcohol on these targets occur at high concentrations. However, the effects of alcohol on certain GABAA receptor isoforms occur with concentrations within the intoxicating range. Acute alcohol potentiates these GABAA receptor isoforms and therefore enhances GABA-mediated inhibition through allosteric modulation of the receptors. Recent evidence indicates that GABAA receptors containing d-subunits are preferentially affected (Wei et al., 2004). Such d-subunit-containing receptors appear to be located extrasynaptically, where they sense ambient GABA in the extrasynaptic environment and mediate tonic inhibition. The role of GABAA receptor-mediated tonic inhibition is not fully defined. However, the extrasynaptic d-containing receptors that are responsible for tonic inhibition appear to be an important target of alcohol at intoxicating concentrations (Hanchar et al., 2004). It is interesting to speculate that these extrasynaptic receptors may be activated by spillover of GABA when GABAergic interneurons are intensely activated, such as occurs during a seizure discharge. The ability of alcohol to potentiate extrasynaptic GABA receptors could therefore contribute to the anticonvulsant activity of ethanol, including its protective activity against alcohol withdrawal seizures. Alcohol tolerance and dependence have been linked to changes in the function of GABAA receptors that are possibly related to alterations in subunit assembly (Devaud et al., 1997; Kang et al., 1996, 1998; Mahmoudi et al., 1997; Matthews et al., 1998; Mhatre et al., 1993; Morrow et al., 1990). In addition to effects on GABAA receptor isoforms that are located postsynaptically, there is emerging evidence that alcohol also enhances GABA-mediated inhibition via a presynaptic mechanism that involves GABA release from interneurons (Carta et al., 2004). It is hypothesized that there is a compensatory downregulation of GABAA receptors during chronic exposure to alcohol. When alcohol is with-
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drawn and its potentiating effects are no longer present, the reduced functional activity of GABAA receptors would predispose to seizures. Indeed a loss of GABA-mediated inhibition has been associated with enhanced susceptibility to alcohol withdrawal seizures (Faingold et al., 2000; N’Gouemo et al., 1996). Alcohol exposure can also influence excitatory neurotransmission (Dodd et al., 2000). Acute alcohol exposure inhibits NMDA and kainate receptors, whereas chronic alcohol exposure results in upregulation of these receptors (Carta et al., 2002; Costa et al., 2000; Lovinger et al., 1990; Peoples et al., 1997). The ability of ethanol to inhibit NMDA responses appears to be dependent on NR1/NR2A and NR1/NR2B NMDA receptor isoforms (Wirkner et al., 1999). Abrupt cessation of alcohol exposure may result in brain hyperexcitability because inhibition of the upregulated NMDA and kainate receptors is uncovered (Whittington et al., 1995). There is evidence of upregulation of the NR1 and NR2A subunits of the NMDA receptor (Gulya et al., 1991; Snell et al., 1996; Trevisan et al., 1994) and the GluR6/7 kainate receptors (Carta et al., 2002). Alcohol also produces important effects on ion channels mediating intrinsic neuronal excitability, particularly calcium channels. Electrophysiologic studies suggest that increased neuronal firing is critically important for alcohol withdrawal seizures (Chakravarty and Faingold, 1998). However, the shape of action potentials is not significantly altered in brain neurons following alcohol withdrawal (Evans et al., 2000; Yang et al., 2002), indicating that there are not likely to be changes in the voltage-gated sodium and potassium channels that are responsible for action potentials. On the other hand, there is some evidence that ethanol selectively inhibits N-, P-, and Q-type calcium channels that mediate neurotransmitter release (Maldve et al., 2004; Newton et al., 2004). Moreover, increased L- and P-type calcium channel-current density has been observed following alcohol withdrawal associated with enhanced seizure susceptibility (N’Gouemo and Morad, 2003; PerezVelazquez et al., 1994). As previously noted, neurons within the central nucleus of the IC represent an important target of alcohol and seem to play a critical role in the initiation of alcohol withdrawalrelated AGS. Although it is plausible that alcohol withdrawal could be associated with changes in synaptic function and intrinsic excitability within this subnucleus (Evans et al., 2000; Yang et al., 2002), experimental support is not yet available.
TESTING PHARMACOLOGICAL AGENTS IN ANIMAL MODELS OF ALCOHOL WITHDRAWAL SEIZURES Because alcohol withdrawal-related AGS and HIC can be elicited at the will of the experimenter during a defined
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period after cessation of alcohol intake, the anticonvulsant properties of pharmacologic agents can be easily studied (unlike the situation with spontaneous seizure models, where seizures occur unpredictably, requiring sophisticated monitoring systems and less robust trial designs). A limitation of these models is that AGS or HIC do not occur in every animal. However, the incidence (and severity) of AGS can be increased with intermittent ethanol administration (C.L. Faingold, personal communication). The peak incidence of AGS occurs at about 24 hours following alcohol withdrawal. Thus pharmacologic agents are typically tested between hour 20 and 28 following alcohol withdrawal, which encompasses a period of high seizure likelihood. Pharmacologic substances are typically administered 30 to 60 minutes before the time at which the AGS is elicited, but the choice of interval for any specific substance must be based on its pharmacokinetic characteristics. If the time of peak blood-brain levels is not known, a time course study should be carried out. Table 4 compares the potencies of various anticonvulsant substances for protection against alcohol withdrawal seizures (AGS in rats and HIC in mice) and in conventional seizure models used for the evaluation of antiepileptic drugs (White et al., 2002). The values in the diverse models are not directly comparable because of significant experimental differences. However, it is apparent that there is a rough concordance in the effectiveness of substances for protection against seizures in the alcohol withdrawal and nonalcohol–related models, with the possible exception of
the sodium channel–blocking anticonvulsant drugs carbamazepine and phenytoin (Rogawski and Löscher, 2004), which may be less effective against alcohol withdrawal seizures than in conventional seizure models. This corresponds with the lack of effectiveness of these drugs for many forms of generalized seizures (although the drugs are generally believed to be useful for generalized tonic-clonic seizures).
SEIZURES RELATED TO WITHDRAWAL OF OTHER CENTRAL NERVOUS SYSTEM DEPRESSANTS Drugs that increase GABAA-mediated inhibition (e.g., benzodiazepines, barbiturates) are commonly prescribed for their anxiolytic, sedative, hypnotic, muscle relaxant, and anticonvulsant properties. Prolonged administration of benzodiazepines and barbiturates can result in tolerance and physical dependence. The spectrum of behavioral signs and symptoms that occur following withdrawal from barbiturates and benzodiazepines—including hyperexcitability, tremors, and seizures—is similar to that occurring following alcohol withdrawal (Busto et al., 1986; Hillbom et al., 2003). In addition, genetic characteristics that influence susceptibility to alcohol withdrawal also affect susceptibility to barbiturate and benzodiazepine withdrawal (Metten and Crabbe, 1994, 1999). Furthermore, there is cross-tolerance among benzodiazepines, ethanol, and barbiturates, and ben-
TABLE 4 Comparison of the Potencies of Anticonvulsant Substances for Protection Against Alcohol Withdrawal Seizures and in Conventional Seizure Models ED50 (mg/kg) Alcohol withdrawal Substance Carbamazepine Chlormethiazole Diazepam Dizocilpine (MK-801) Gabapentin Phenytoin Valproic acid
Conventional seizure models
AGS (rat)
HIC (mice)
AGS (mice)
150a
NEb ~100d 20h 0.1l
11.2c
NEg 0.33k ~50 (mice)o 50p
NEq 300r
0.04–0.12i 0.047m 91.1c 3.9c 55–300c,i
PTZ (mice) >50c 36e 0.27j 1.17n 47.5c >50c 220c
MES (mice) 7.8c 137.6f 18.7j 0.95n 78.2c 5.6c 263c
ED50, median effective dose; HIC, handling-induced convulsions; PTZ, pentylenetetrazol seizure test; MES, maximal electroshock seizure test; NE, not effective. Conventional AGS testing is performed in strains of mice that are genetically susceptible to AGS, mainly DBA/2 but also Frings. a Chu, 1979; bGrant et al., 1992; cWhite et al., 2002; dGreen et al., 1990; eOgren, 1986; fPilip et al., 1998; gLittle et al., 1986; hCrabbe, 1992; iChapman et al., 1984; jSwinyard and Castellion, 1996; kMorrissett et al., 1990; lGrant et al., 1982; mChapman et al., 1989; nRogawski et al., 1991; oWatson et al., 1997; pChu et al., 1981; qGessner, 1974; rGoldstein, 1979.
Relevance of Alcohol Withdrawal Seizures in Rodents to the Human Condition
zodiazepines and barbiturates can protect against ethanol withdrawal convulsions in humans and rodents (see section on Relevance of Alcohol Withdrawal Seizures in Rodents to the Human Condition for further discussion of benzodiazepines in alcohol withdrawal). These various factors suggest that similar underlying mechanisms mediate the withdrawal syndromes that occur with the different CNS depressants (Kliethermes et al., 2000). Handling-induced convulsions have been used extensively to study the relationship between seizures that occur on withdrawal of alcohol and other CNS depressants such as benzodiazepines, barbiturates, and also inhalation anesthetics. However, pharmacokinetic factors may require alterations in the specific procedures that are used with certain agents. For example, because of its long half-life, diazepam does not produce the waxing and waning pattern of HIC exacerbation after injection that occurs with alcohol withdrawal as discussed previously and illustrated in Figure 2 (Crabbe, 1992). However, injection of the benzodiazepine receptor antagonist flumazanil precipitates a brief, relatively intense withdrawal reaction that lasts several minutes after the injection (Metten and Crabbe, 1994, 1999). In genetic studies in mice, a single dose of diazepam (20 mg/kg) is administered, followed by flumazenil (10 mg/kg) at an interval of 60 minutes. Withdrawal HICs are scored 1, 3, 5, 8, and 12 minutes later. In contrast to diazepam, zolpidem, a benzodiazepine that is selective for GABAA receptors containing a1 subunits, does not require precipitation by an antagonist (Metten et al., 1998). Withdrawal from barbiturates, such as pentobarbital, is also associated with potentiation of HICs. In studies of pentobarbital withdrawal for genetic studies, HICs are assessed at hourly intervals from 1 to 8 hours following injection (Metten and Crabbe, 1994). Nitrous oxide withdrawal is similarly associated with potentiation of HICs (Belknap et al., 1993). In a typical paradigm used for genetic studies, mice are exposed to a mixture of 75% nitrous oxide and 25% oxygen for 1 hour in an inhalation chamber and then returned to room air. HICs are assessed at baseline; immediately on removal from the inhalation chamber; and 5, 10, 15, 20, 40, and 60 minutes later (Metten et al., 1998). In addition to withdrawal of alcohol and GABA-potentiating drugs, seizures can also be induced by withdrawal of GABA administered locally in susceptible brain regions, including the cerebral cortex, amygdala, hippocampus, or IC (Brailowsky et al., 1988; Yang et al., 2001b). GABA solutions have been delivered through an indwelling catheter using a subcutaneously implanted osmotic minipump. In experiments examining IC infusion in rats, 1 M GABA is delivered bilaterally at the rate of 0.25 mliters per hour for 7 days (Yang et al., 2001b). Thirty minutes following abrupt cessation of the GABA infusion, animals exhibited spontaneous seizures (17% of rats tested) and a susceptibility to
171
AGS (39% of rats) that persisted in some animals for as long as 6 months. The sound-induced behaviors following GABA withdrawal consisted of wild running and bouncing clonus, which resemble seizures observed after ethanol withdrawal.
RELEVANCE OF ALCOHOL WITHDRAWAL SEIZURES IN RODENTS TO THE HUMAN CONDITION Although alcohol withdrawal seizures in rodents do not represent a perfect model of human alcohol withdrawal seizures, the available evidence indicates that the animal models are valid in many respects. As noted, most alcohol withdrawal seizures in humans are generalized tonic-clonic seizures. Similarly, the various forms of alcohol withdrawal seizures in rodents represent generalized convulsions. In both humans and rodents, the peak incidence of alcohol withdrawal related generalized seizures occurs between 20 to 24 hours following cessation of alcohol intake. In addition to exhibiting shared behavioral features, the brain systems underlying alcohol withdrawal seizures in humans and rodents are likely to be similar across species. There is no cortical paroxysmal activity in the electroencephalogram during auditory-evoked tonic-clonic alcohol withdrawal seizures in rodents (Hunter et al., 1973; Maxson and Sze, 1976). Epileptiform activity is also rare in the electroencephalogram recorded between episodes of alcohol withdrawal tonic-clonic seizures in humans (Sand et al., 2002; Touchon et al., 1981). The lack of cortical epileptic activity interictally during alcohol withdrawal suggests that the withdrawal seizures may not be initiated by cortical hyperexcitability but instead result from the abnormal function of subcortical neuronal networks that eventually trigger seizure discharges in the cortex. One neuronal network of interest is the brainstem auditory pathway, which has been implicated in rodent AGS (see previous discussion). Indeed significant abnormalities in auditory-evoked potentials have been reported in humans suffering from alcohol withdrawal seizures, including increased latency to wave V, which is unique to individuals suffering from alcohol withdrawal seizures (Neiman et al., 1991; Touchon et al., 1984). IC neurons are the major source of wave V in brainstem auditory-evoked potentials (Hughes and Fino, 1985), suggesting that abnormalities in the function of IC neurons can contribute to the genesis of alcohol withdrawal seizures in humans, as is believed to be the case in rodents. Indeed IC neurons are not only a component of the neuronal network for alcohol withdrawal seizures, but they are also believed to play an important role in other models of epilepsy and are considered a critical site for the genesis of tonic-clonic seizures whatever the underlying etiology (Faingold, 1999).
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Neuronal plasticity mechanisms may play a role in the susceptibility to alcohol withdrawal seizures in humans and rodents. In humans the number of detoxifications, not the absolute amount of alcohol intake, best predicts the likelihood of subsequent alcohol withdrawal seizures (Ballenger and Post, 1978). Similarly studies in rodents have shown that repeated alcohol withdrawal experiences increase the severity and duration of subsequent withdrawal seizures. For example, this was the case in the study of Becker and Hale (1993) in which adult male mice were chronically exposed to ethanol vapor by inhalation. Animals in a multiple withdrawal group experienced three 16-hour exposure periods separated by 8-hour periods of abstinence; a single withdrawal group received a single 16-hour bout of ethanol exposure. The severity of HIC was significantly greater in the multiple withdrawal group than in the single withdrawal group. In additional studies, mice experiencing multiple withdrawal episodes were found to have greater susceptibility to chemoconvulsant-induced seizures (Becker et al., 1998). Furthermore, in rats, multiple withdrawal episodes from chronic alcohol treatment facilitate the rate of the development of IC kindling while at the same time inhibiting the evolution of amygdala and hippocampal kindling (Gonzalez et al., 2001; McCown and Breese, 1990). This observation provides further support for the concept that brainstem systems encompassing the IC are critical to the initiation of alcohol withdrawal seizures, whereas the forebrain mechanisms mediating “limbic” seizures (the equivalent of complex partial seizures in humans) do not play a major role, at least in triggering these seizures. This conclusion is consistent with observations from studies of cerebral glucose metabolism (see previous section entitled Metabolic Changes Following Alcohol Withdrawal). In chronic alcohol abusers, it seems likely that kindling-like effects of multiple detoxifications leads to hyperexcitability in IC neurons, which further predisposes to withdrawal seizures (Duka et al., 2004). Overall the various lines of evidence discussed in this section support the view that the neural mechanisms mediating alcohol-withdrawal tonic-clonic seizures in humans and rodents are similar. Do the animal models represent appropriate test systems for the evaluation of agents useful in the treatment of alcohol withdrawal seizures in humans? The available data suggest that the models can be applied for identification of agents useful in preventing alcohol withdrawal seizures, but there could be limitations, as highlighted by what appears to be poor concordance between the efficacy of benzodiazepines in the models and their use in clinical practice. In the United States, benzodiazepines are considered the drugs of choice to treat alcohol withdrawal and to prevent the occurrence of seizures (D’Onofrio et al., 1999; Mayo-Smith, 1977). In Europe, carbamazepine, chlormethiazole, and valproate are often used. Benzodiazepines have been shown to be protective in some animal
models of alcohol withdrawal seizures (Becker and Veatch, 2002; Mhatre et al., 2001), although they may not exhibit high potency (see Table 4). In fact, benzodiazepines generally have low potency in models of tonic seizures, such as the maximal electroshock test (see Table 4). In animal models, benzodiazepines are modestly effective in preventing the increased withdrawal severity that occurs with repeated withdrawals (Ulrichsen et al., 1995), although the drugs can also produce a paradoxical worsening (Becker and Veatch, 2002), and not all studies have yielded positive results (Mhatre et al., 2001), indicating that caution is warranted in using benzodiazepines for alcohol detoxification. Alcohol withdrawal has been associated with alterations in the subunit composition of GABAA receptors, including an increase in the expression of the a4 subunit that confers benzodiazepine insensitivity (Cagetti et al., 2003; Devaud et al., 1997; Sanna et al., 2003). Clinical experience demonstrates that benzodiazepines do reduce the risk of recurrent seizures in patients who present with an alcohol withdrawal seizure (D’Onofrio et al., 1999), so that in practice there is not complete benzodiazepine resistance. However, GABAA receptor modulators other than benzodiazepines that would not be expected to lose activity might be superior therapeutic agents. In fact, chlormethiazole is a positive modulator of GABAA receptors, which, in contrast to benzodiazepines, has high efficacy in enhancing GABAA receptors containing a4 subunits (Usala et al., 2003). Chlormethiazole has been shown to protect transiently against alcohol withdrawal seizures in mice withdrawn from exposure to inhaled ethanol (Green et al., 1990) and, in Central Europe, the drug represents the standard of care for the acute treatment of alcohol withdrawal (Majumdar, 1990; Morgan, 1995). It is interesting to speculate that chlormethiazole might be superior to benzodiazepines in the treatment of alcohol withdrawal as a result of its activity as a modulator of benzodiazepine-insensitive GABAA receptor isoforms. Carbamazepine may decrease the craving for alcohol after withdrawal, but there is little evidence that it prevents seizures and delirium. In fact, carbamazepine was inactive in blocking alcohol withdrawal-related HIC in mice (Grant et al., 1992), and only very high doses were able to suppress withdrawal-related AGS in rats (Chu, 1979). Interestingly, in humans, phenytoin is not effective in protecting against the recurrence of alcohol withdrawal seizures (Rathlev et al., 1994). The animal model therefore shows a good correspondence with clinical experience. Valproate also has some protective activity against alcohol withdrawal-related HIC in mice (Goldstein, 1979), and topiramate may also protect against enhanced seizure susceptibility in ethanol-dependent rats (Cagetti et al., 2004). There is increasing interest in the potential of gabapentin as a treatment for alcohol withdrawal, inasmuch as encouraging results have been produced in several small clinical studies (Bonnet et al., 1999; Bozikas et al., 2002; Myrick et al., 1998; Rustembegovic et
References
al., 2004; Voris et al., 2003). Animal studies confirm that gabapentin has protective activity against ethanol withdrawal seizures. For example, in mice undergoing alcohol withdrawal, gabapentin at doses of 50 to 100 mg/kg decreased the incidence of AGS (Watson et al., 1997). Vigabatrin may also be of value in alcohol withdrawal, but data from animal studies are not available as yet (Stuppaeck et al., 1996).
CONCLUSIONS It is estimated that two million Americans experience the symptoms of alcohol withdrawal each year (Bayard et al., 2004). Generalized tonic-clonic seizures are the most dramatic and dangerous component of the syndrome. In this chapter we have reviewed rodent models of alcohol withdrawal seizures that are commonly used for mechanistic and genetic studies and that can also be applied in the identification of new treatment approaches. In each of these models, withdrawal from alcohol, administered either chronically or in some instances acutely, leads to enhanced seizure susceptibility and occasionally spontaneous seizures. Interestingly the brain substrates that trigger these seizures are largely distinct from those responsible for other clinically important seizure types, and it is likely that the pathophysiologic mechanisms are different. Therefore it is not surprising that pharmacologic agents effective in other seizure types may not be effective in the treatment of alcohol withdrawal seizures. The alcohol withdrawal models provide unique opportunities to gain insights into the specific cellular mechanisms underlying this distinctive seizure syndrome. They also provide opportunities to optimize the therapy of alcohol withdrawal seizures. Indeed newer agents such as chlormethiazole, gabapentin, or valproate, which are effective in the models, are gaining acceptance clinically. NMDA receptor antagonists are especially active in animal models of alcohol withdrawal seizures, in accordance with the substantial evidence that alterations in NMDA receptor function play a key pathophysiologic role; whether such agents will have a role in clinical practice will require further study. An important challenge is to develop strategies to interdict the development of enhanced seizure susceptibility that occurs with multiple episodes of detoxification. Determining whether NMDA receptor antagonists or other pharmacologic approaches have such antiepileptogenic actions in repeated episodes of withdrawal will represent an important future application of the animal models.
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B
FIGURE 7 - 6 Orthograde transport of biocytin reveals callosal projection in coronal slices. A: A biocytin crystal (-0.5 mm diameter) was placed in layer V of the contralateral cingulate cortex (right side, crystal placement not shown in this figure). The slice was incubated for 3 hours in an interface chamber and then processed to obtain a final horseradish peroxidase reaction product. The arrows in A indicate labeling of the callosal fibers. The square area marked in A is expanded in B. B: A dark-field image showing the callosal fibers coursing upward through the ipsilateral layer V.
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14 Alumina Gel Injection Models of Epilepsy in Monkeys CHARLES E. RIBAK, LEE A. SHAPIRO, LASZLO SERESS, AND ROY A. BAKAY
culline, provides ample time to study these morphologic and electrophysiologic changes. Third, the initial injury caused by the injection of alumina gel into the sensorimotor cortex is well localized and provides the topographic focus for seizure initiation. Such localized foci are essential for analyzing different stages along the time line of seizure development using anatomic methods. Several studies have taken advantage of these features and are reviewed subsequently herein.
Epilepsy is a neurologic disorder that has been modeled in many animals, including the monkey. This chapter deals with two types of alumina gel injections that model two different human epileptic disorders. Where alumina gel is injected into the brain determines what type of epilepsy it models. For example, alumina gel injections into sensorimotor cortical regions produce seizures that have been used as a model for posttraumatic epilepsy. Alternatively, when alumina gel is injected into the temporal lobe of rhesus monkeys, complex partial seizures and neuropathologic changes occur which are analogous to those seen in human temporal lobe epilepsy. This chapter will examine each of these two models, beginning with the sensorimotor cortex model.
Methods of Generation The central sulcus of either adult or adolescent monkeys is exposed through a frontal craniotomy under general anesthetic, taking great care not to damage the cortex. After opening the dura, the hand-face region of the sensorimotor cortex is identified with cortical stimulation and injected with alumina gel. Although different injection patterns have been used, one of the most successful is the Ward modification of the Kopeloff technique (Bakay and Harrris, 1981). In this method, two injections are made 4 mm apart in the precentral gyrus and two injections in the postcentral gyrus, paralleling each other. A 27-gauge needle is used for the injection, which is placed into the cortex to a depth of approximately 4 mm. A volume of 0.1 ml of aluminum hydroxide (fully saturated solution) in the form of alumina gel is injected at each site. Each injection is made quite slowly to allow time for volume equilibrium to be reached before removing the needle. In general an injection under the pia is avoided because greater scarring will occur in this situation. The epidural space is covered with Gelfoam, and perioperative steroids are used to avoid brain swelling because a watertight dura closure is extremely difficult to
SENSORIMOTOR CORTEX MODEL The advantages of the alumina gel model of cortical focal epilepsy are several. First, it is a model that is focal in its initiation and then proceeds with time to produce spontaneous focal seizures that often become generalized. Focal motor seizures in the human are well characterized, and this model produces seizures that are extremely similar to those in regard to their behavior, electrophysiology, biochemistry, neuropathological, and pharmacologic responses (Lockard, 1980). The second advantage of this model is the time course of the development of seizures: They usually begin 4 to 8 weeks following the application of the alumina gel, allowing time to analyze electroencephalographic (EEG) and neuropathological changes that may underlie the cause of the seizures. This delay in seizure development, as opposed to the use of fast-acting convulsants like bicu-
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perform and marked brain swelling could introduce injury to the cortex. Anticonvulsant drugs should be avoided. In general within 2 to 4 weeks sharp waves can be detected over the injection site. Within 1 to 2 months, epileptiform activity and clinical seizures will begin to be observed. These are initially focal and frequently progress to Jacksonian-type marches. Secondary generalized seizures are not uncommon.
Characteristics and Defining Features Behavioral and Clinical Features Monkeys that received alumina gel applications to the left cerebral hemispheres to produce seizure foci were analyzed for behavioral and EEG changes. Injections into the precentral and postcentral gyri did not cause any abnormalities in the EEG tracings initially. Within 4 to 8 weeks, however, abnormalities appeared on the EEG tracings and focal seizures began in the contralateral upper extremity corresponding to the alumina gel–injected area of the sensorimotor cortex that is the homologous region representing that extremity. Seizure activity was observed in most monkeys after 4 weeks, and the seizure frequency was two or three per day. The duration of each seizure ranged from a few seconds on electrocorticography (ECoG), up to 1 to 2 minutes behaviorally when the monkey’s focal seizure generalized. The epileptic animals demonstrated an ECoG consistent with spike-and-wave discharges in the area immediately adjacent to the alumina gel injection. As indicated by previous studies, the seizures begin with a tonicclonic movement of the hand contralateral to the cortical injection site, followed by tonic-clonic movements of the ipsilateral upper extremity. These tonic-clonic events often generalize to the lower extremity and the face, leading to the monkey’s falling in the cage and to unconsciousness. Although seizure activity spreads to both upper extremities, there is no evidence of a mirror focus in the contralateral homotopic cortex in monkeys; Harris and Lockard (1981) did not show this region as having independent seizure activity. Electroencephalographic Features The ECoG showed spike-and-wave activity around the electrodes overlying the injected sensorimotor cortex. This spike-and-wave activity corresponded to the behavioral manifestations described in the preceding section. Neuropathology Cell Loss Blocks of tissue from epileptic cortex and a homologous area in the contralateral nonepileptic cortex were analyzed in light and electron microscopic preparations. As a result of the injections into the sensorimotor cortex, the area
bordered by the injection site often shows a disruption in the laminar pattern in Nissl-stained sections. It is remarkable that adjacent to the injection site, normal-looking pyramidal neurons are seen throughout the cortical layers, suggesting that the amount of neuronal loss is minimal outside the injection site. In monkeys with chronic seizures a significant loss of neurons was observed that ranged from 11 to 61% at the focus compared with the normal side (Ribak et al., 1989). Details about the loss of g-aminobutyric acid (GABA)ergic neurons are provided later in a separate section of this chapter. Reactive Gliosis The glial changes consist of thick pial-glial membranes (Harris, 1980), hypertrophied neuroglial cell processes containing increased numbers of filaments, and increased numbers of intracellular glial connections of both the gap junction and desmosome type. In layers IV and V of cortex, hypertrophied astrocytes are seen in organized bands where high-potassium ion fluxes have been demonstrated to be associated with seizures (Harris, 1980). Furthermore, analysis of the surface of pyramidal cell bodies in layer V showed a statistically significant increase in apposition by astrocytic processes (Ribak et al., 1982) (Figure 1). For example, at the focus, 50% of the soma of pyramidal cells was apposed by glia, whereas 22% in the contralateral normal cortex were apposed by glia (see Figure 1). In addition, the area in the neuropil occupied by glial profiles showed a 50% increase at the focus relative to the normal cortex (Ribak et al., 1982). Therefore reactive gliosis is a well-documented neuropathological finding in this model of focal epilepsy. Loss of GABAergic Neurons To test the hypothesis that a decrease in the number of inhibitory GABAergic neurons could lead to seizure activity, several studies were performed in this model of epilepsy, beginning in the late 1970s (Ribak et al. 1979). Initially light microscopic analysis of glutamate decarboxylase (GAD) immunocytochemistry indicated a significant decrease in the number of GAD-positive terminals at the epileptic focus compared with the contralateral normal cortex. Because many of these terminals are apposed to the surfaces of layer V pyramidal cells, it was suggested that these pyramidal neurons were inhibited less and therefore were more likely to be engaged in excitatory circuitry and bursting (Ribak et al., 1979). Subsequently, Houser et al. (1986) analyzed GABAergic axon terminals in monkeys before seizure onset and reported a decrease in GABAergic terminals ranging from 14 to 22%, whereas the initial studies of chronic monkeys reported a loss of 58 to 62% (Ribak et al., 1979). The GABAergic somata were analyzed (Ribak et al. 1986) using an antibody that recognizes GAD within somata (Oertel et al., 1981). The results indicated a 35 to 52% significant loss of small GABAergic somata at the foci of
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FIGURE 1 Bar graphs showing the number of synapses per 10 mm-length of somal surface of 90 layer V pyramidal somata (left) and the amount of glial apposition to these layer V pyramidal somata (right). The number of axosomatic synapses was greatest in the nonepileptic cortex (N) and least at the focus (F). The parafocus (P) displayed an intermediate number. The opposite result was found for glial apposition. The error bars represent standard error of the mean (SEM). (Reprinted with permission from Ribak et al., 1982.)
chronic epileptic monkeys. These data suggested that the loss of GABAergic terminals at epileptic foci is due to the loss of neuronal somata that give rise to these axons. Similar to the study by Houser et al. (1986), Ribak et al. (1989) analyzed the cortex next to the alumina gel application for a loss of GABAergic somata before onset of epileptic activity in monkeys. They observed a 23 to 44% reduction in the number of GABAergic neurons at the site of the developing focus. Nissl preparations were also used to determine the selectivity of this loss, and statistical tests showed no differences in the number of total neurons between this and the other site. In contrast, chronically epileptic monkeys had a significant loss of neurons that ranged from 11 to 61% at the focus, compared with the normal side. To determine whether the loss of GABAergic terminals was due to an actual degeneration of these terminals or simply to a loss of GAD immunostaining within the terminals, electron microscopy was utilized. GABAergic axon terminals were identified in electron microscopic preparations by the fact they form symmetric synapses. In two studies (Ribak, 1985; Ribak et al., 1982), pyramidal neurons were analyzed for a loss of GABAergic synapses on both their cell bodies and axon initial segments (Figure 2). The results showed an 80% loss of such axon terminals on the cell body (see Figure 1) and a more severe loss of axon terminals forming symmetric synapses on the axon initial segment. These findings indicated that the previous reported loss of GAD-positive axon terminals was due to their degeneration and that two GABAergic cell types are particularly
affected at the epileptic focus: the basket and chandelier cells. Biochemical Findings The biochemical data for alumina gel–treated epileptic monkeys are consistent with the immunocytochemical results stated previously herein. Bakay and Harris (1981) reported decreased GABA receptor binding, GABA concentration, and GAD activity at the epileptic focus. They suggested that these alterations in presynaptic GABA indices were probably due to the degeneration of GABAergic axon terminals at the epileptic focus. Furthermore, they reported that seizure frequency was correlated with the loss of GAD activity. These results support the GABA hypothesis for focal epilepsy. Response to Antiepileptic Drugs Anticonvulsant drugs have been screened using the alumina gel–injected monkeys. Two of these drugs, phenytoin and phenobarbital, have been shown to protect alumina gel-injected monkeys from developing secondary generalized tonic clonic seizures. In addition both drugs decrease seizure frequency and severity relative to placebo-treated animals (Lockard et al., 1976a, b). However, if cessation of drug treatment occurs in the monkeys injected with alumina gel, seizure frequency and severity are increased relative to placebo-treated monkeys (Lockard et al., 1976b).
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FIGURE 2 Electron micrographs of axon initial segments from layer III pyramidal cells from normal cortex (a) and the epileptic focus (b). The normal axon initial segment in (a) displays two symmetrical synapses (arrows) formed with axon terminals. Two characteristic features of axon initial segments are shown: a fasciculation of microtubules (M) and a dense undercoating that is continuous with the coating over a pinocytotic vesicle (V). In the focus, reactive astrocytes (A) were seen where axon terminals were found in the normal cortex. The initial segment showed normal features including microtubules (M) and a dense undercoating (arrow). Bar = 1 mm. (Reprinted with permission from Ribak, 1985.)
Other drugs that have been screened in the alumina gel injected monkeys include valproic acid. Results from these studies indicate that the efficacy of valproic acid in decreasing seizure frequency and duration correlates with plasma levels and that plasma levels show drastic fluctuations. When plasma levels are high, relatively few EEG interictal spikes are seen, and when they are low, relatively high numbers of interictal spikes are observed (Lockard et al., 1977). Thus valproic acid plasma levels inversely correlate with EEG interictal spike activity. In these monkeys, there is a high correlation between the number of interictal spikes and occurence seizures (Lockard, 1980). Benzodiazepines and their derivatives are common antiepileptic treatments in humans. One of these, clonazepam, has been evaluated in the alumina gel–injected monkey. When administered to achieve plasma levels above
29.9 ng/ml and 59.9 ng/ml, clonazapam caused seizures to decrease in severity and frequency or disappear, respectively (Lockard et al., 1979). It is pertinent that while being treated with clonazapam, the monkeys exhibited improved performance on a lever-pressing behavioral task. However, if drug treatment was stopped, seizure activity increased and a deficit in lever pressing behavior was seen (Lockard et al., 1979).
Limitations In general the alumina gel model can be introduced into any nonhuman primate species, sex, or age. Seizures almost always develop when the appropriate injection technique is used, and in the rare incidences when they do not develop, repeat injections are usually successful. A properly prepared animal will produce stable spontaneous seizures for many
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years. However, in an occasional subject, status epilepticus will develop and anticonvulsants are needed to maintain viability. This is more likely to occur with large injections and subpial spread. There is a relatively high variability as to the seizure rates, with rates as low as one per month or as high as 10 to 20 per day, even though the same injection technique, species, age, and sex of monkey are used. To get an accurate seizure frequency, 24-hour video monitoring is required. A movement-detection device or ECoG monitoring technique allows for the videotape review to be streamlined (Lockard and Ward, 1980). Like the human condition, seizures in monkeys may cluster in relation to estrus and may increase with behavioral or physical stress. These features are particularly useful in a model of focal neocortical epilepsy. Although seizures can be generated in neocortical areas other than sensorimotor cortex, they are more difficult to document and far more variable in their development and maintenance. Such seizures may model a variety of epileptic activities, which might result from dysgenesis, vascular abnormality, or a lesion or tremor. The disadvantages of this method are that it is very expensive and the number of subjects that can be involved in any one project is relatively small; the monitoring techniques are complex and the studies require long-term commitment of resources. The advantages are that the model involves a nonhuman primate model with anatomic, biological, pharmacologic, and electrophysiologic properties much closer to the human than those found in rodent models.
Insights into Human Disorders The neuropathological and biochemical results from the analysis of the sensorimotor cortex of monkeys injected with alumina gel indicate a causal role for GABAergic neuronal loss. This model best replicates most of the features of human focal epilepsy. This model is also a chronic one and follows a time course in its development similar to posttraumatic epilepsy in humans (Wyler and Ward, 1984). That is, the EEG abnormalities and clinical seizures develop at a similar time in this model and in humans with posttraumatic epilepsy. Based on the data from this experimental model, we suggest that people with this type of epilepsy probably display a reduction in the number of GABAergic neurons and synapses at the epileptic focus. This hypothesis is supported by the pharmacologic data showing that GABAergic agonists are effective to varying degrees at limiting the frequency, duration, and severity of seizures in monkeys injected with alumina gel.
TEMPORAL LOBE MODEL Alumina gel injections into the temporal lobe of rhesus monkeys were used in an attempt to mimic complex partial
seizures associated with temporal lobe epilepsy in humans (Ribak et al., 1998). Our analysis is based on injections of alumina gel into several regions of the temporal lobe in rhesus monkeys. Thus structures that were injected include the hippocampus, entorhinal and perirhinal cortices, and the amygdala. In all cases, complex partial and secondarily generalized seizures were observed. They had many similarities to the ictal symptoms of human temporal lobe epilepsy.
Methods of Generation Adult or adolescent monkeys are placed under general anesthesia, and stereotactic injections are made into the mesial temporal structures. In the past these injections have been performed through an approach from above that enters the ventricle. The disadvantage of that approach is that the alumina gel can back-track into the ventricles and spread beyond the desired location. With an updated lateral approach, the tract is below the ventricle so the needle can approach the amygdala, hippocampus, or entorhinal cortex (Ribak et al., 1998). This approach requires a specially designed right-angle needle of approximately 23-gauge (higher gauges tend to deflect in variable directions as a result of the lack of rigidity). The musculus temporalis must be split and the zygoma removed along the path to the target. A small craniotomy is made in the temporal bone, and the dura opened along the needle trajectory. Targets have been selected in the past with stereotactic atlases. However, with current imaging technology, the monkey can be placed in a magnetic resonance imaging (MRI)-compatible stereotactic frame, and the needle directed to its target based on the anatomy of the individual animal. Injections consist of 0.1 to 0.2 ml of alumina gel. The most reliable seizure activity occurs with injections into the entorhinal cortex, 2 to 3 weeks after injection. With hippocampal or amygdala injections, sometimes the seizures evolve so rapidly that after the first several seizures a secondary generalization occurs. A high rate of seizure frequency can endanger the monkey’s health. A somewhat slower, more stable pattern is generally observed with entorhinal injections or mesial temporal cortical injections. Injected subjects develop electrophysiologic and behavioral characteristics of complex partial seizures. Chronic spontaneous seizures have been observed with this technique, and electrographic seizures can be recorded using epidural electrode strips (Figure 3).
Characteristics and Defining Features Behavioral and Clinical Features Alumina gel injections into the hippocampus result in clinical seizure activity that typically develops 12 to 14 days after the injections (Ribak et al., 1998). This seizure activity is characterized as complex partial seizures with secondary generalization. Alumina gel injections into the
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FIGURE 3 X-rays of the anterior-posterior (A) and lateral (B) views of a monkey implanted with epidural electrodes. Lead 1 is in the mesial-inferior temporal region, lead 2 is over the inferior temporal region, lead 3 is over the superior temporal region, and lead 4 is over the parietal cortex. The electrical connections are buried subcuticularly and emerge through a superior head plug (arrows). (Reprinted with permission from Ribak et al., 1998.)
amygdala develop more slowly, typically over a 3- to 6week period (Ribak et al., 1998). Complex partial seizures result from these injections, and they persist over a long period. Alumina gel injections into entorhinal and perirhinal cortices result in similarly characterized complex partial seizures within 2 to 3 weeks (Ribak et al., 1998). Overall, alumina gel injections into mesial temporal lobe structures of monkeys result in subclinical, complex partial and secondarily generalized seizures that have many similarities to the ictal symptoms of human temporal lobe epilepsy (Sharbrough, 1987). Injections of the inferior temporal gyrus showed no behavioral abnormalities and served as a control for the other injected regions of the temporal lobe that are responsible for causing complex partial seizures (Ribak et al., 1998). When complex partial seizures developed in these monkeys, the behavioral pattern was remarkably similar (Ribak et al., 1998). In all instances the seizures started with blank stares followed by head turning. In monkeys injected into the hippocampus, this behavior rapidly became secondarily generalized. Other symptoms included smelling of the fingers, followed by an arrest of movement with a motionless stare for up to a minute. As the seizure generalized, these behaviors evolved to include vocalization, drooling, open-mouth oral-facial automatisms, chewing motions, and head turning with extremity automatisms. Infrequently the secondary generalization would lead to tonic-clonic Jacksonian march.
Ammon’s horn and dentate gyrus all initially displayed focal pathological EEG slowing limited to the injection site. After clinical seizures developed, widespread pathological EEG slowing over both hemispheres followed. In addition, the monkeys displayed the cardinal ictal and interictal epileptiform EEG abnormalities limited to the mesial-inferior temporal lobe on the side ipsilateral to the injection site (Figure 4). Furthermore, other ipsilateral and contralateral structures were observed to have different degrees of spread. These electrophysiologic abnormalities are very similar to those observed in human temporal lobe epilepsy (Ribak et al., 1998).
Electrographic and Electroencephalographic Features
Reactive Gliosis
Electroencephalography can be used to localize the origin of abnormal activity before seizures. Alumina gel injections in the amygdala, perirhinal and entorhinal cortices, or
Increased numbers of glial cells occurred in various regions of the temporal lobe, where cell loss occurred following injections (Ribak et al., 1998). The increased gliosis
Neuropathology Cell Loss Blocks of tissue from the temporal lobe and homologous area in the contralateral nonepileptic temporal lobe were analyzed in light and electron microscopic preparations (Ribak et al., 1998). Nissl-stained sections were used to determine the full extent of the alumina gel injection sites into the different regions of the temporal lobe. Similar to results in the sensorimotor cortex, neurons were lost at injections sites. In addition, one injection site was associated with distant cell loss (Ribak et al., 1998). Thus alumina gel injections into the amygdala were accompanied by cell loss in the CA1 region of the hippocampus, hilar region of the dentate gyrus, and layer III of entorhinal and perirhinal cortex.
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FIGURE 4 Interictal EEG activites (during the first 5 seconds of this epoch) demonstrate asynchronous pathological delta slowing over both hemispheres, with greater delta slowing over the more medial left-sided electrodes. Fast activities (beta and alpha activities) are consistently of higher amplitude over the right side (contralateral to alumina gel injection) interictally. The recording instrument was turned off for about 5 seconds for pen adjustment (indicated by a gap after the interictal sample), and the electrographic seizure had begun when recording resumed. The electrographic seizure consisted initially of high amplitude, shaply contoured, rhythmic theta activities at LtSD1 with lower amplitude representation at the adjacent LtSD2 electrode, and with irregular slowing at other sites; at this time the animal responded to voice by orienting its head and gaze. Twelve seconds later, the ictal discharge had evolved into bursting polyspike activities and had spread to involve LtSD3 and LtSD1-2. By this time, the contralateral electrodes (RtSD1-4) are recording ictal activities of predominantly 2.5–3 Hz rhythmic slowing with superimposed spikes. The animal failed to respond to voice (Call) and gradually assumed a fixed posture of rightward ocular, cephalic, and truncal version. This posturing was maintained throughout the remainder of the electrographic seizure, which lasted 110 sec. The electrographic seizure did not spread to suprasylvian electrodes and the animal did not convulse. The recording was performed using published parameters (Ribak et al. 1998). Note: The wire of the right suprasylvian subdural electrode (RtSD4) was broken, so this channel was turned off during the recording. LtSD, left subdural contacts; RtSD, right subdural contacts; A1, left ear; A2, right ear. Reprinted with permission from Ribak et al. (1998).
was also observed in the molecular layer of the dentate gyrus. Electron microscopy confirmed the presence of reactive astrocytes in the hippocampus and dentate gyrus (Ribak et al., 1998). Plasticity and Mossy Fiber Sprouting In Timm-stained light microscopic preparations, the dentate granule cells exhibited aberrant mossy fiber sprouting into the inner molecular layer (Ribak et al. 1998). Electron microscopy showed that many of these mossy fibers formed synapses with the proximal dendrites and dendritic spines of granule cells (Figure 5). The presence of these mossy fibers on identified granule cells indicates increased recurrent excitatory circuitry in the brains from these monkeys. Light microscopy also showed dispersion of the granule cell layer (Ribak et al., 1998). Thus the granule cells were radially organized (Figure 6). Electron microscopy of these orthogonal rows of granule cells showed intervening radially oriented processes of reactive astrocytes. These processes arose from hypertrophied astrocytes in the subgranular zone (Ribak et al., 1998). Thus some of the morphologic features observed following injections of alumina gel into temporal lobe structures are similar to the neuropathologic changes found in human temporal lobe epilepsy.
Limitations Development of complex partial seizure activity is reasonably reliable to produce. The seizure pattern is frequently extremely stereotypic, but it requires electrophysiologic monitoring because the complex partial seizure may be easily misinterpreted as normal monkey behavior. There is high variability in the rate of seizure activity, and this variability tends to be site-specific. Seizures often develop with such severity that long-term studies are not possible. The temporal lobe alumina gel injection most likely models a mesial temporal lesion, but how effectively it mimics mesial temporal sclerosis remains to be determined. Nevertheless, it is a method of developing mesial temporal epilepsy in nonhuman primates that are anatomically far closer to human hippocampal structure than is the rat. The disadvantages of the model are similar to those of the sensorimotor focal epilepsy model. In addition, however, our experience is extremely limited with this model. Nevertheless it offers great promise for the future and needs to be evaluated further.
Insights into Human Disorders This temporal lobe epilepsy model in monkeys has the potential to provide useful information about the patho-
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FIGURE 5 Electron micrographs of a granule cell obtained from the dentate gyrus ipsilateral to the amygdala injected with alumina gel. A: The nucleus (N) and proximal apical dendrite (d) of the granule cell. Apposed to the ruffled surface of the dendrite is a large axon terminal that has the features of a mossy fiber. Also note the processes of astrocytes (as) with glial filaments. B: An enlargement of the mossy fiber in (A) showing that this mossy fiber forms asymmetrical synapses (arrows) with the proximal dendrite. A spine (s) appears within this mossy fiber. Scale bars = 1 mm. (Reprinted with permission from Ribak et al., 1998.)
physiologic mechanism of human temporal lobe epilepsy. It is well known that chronic seizures may cause hippocampal neuronal loss and behavioral changes in human temporal lobe epilepsy. Serial chronologic studies of this alumina gel model in monkeys might address this question and others. For example, demonstration of interictal disturbances of learning and other behaviors in this model could be used to develop therapies for the interictal impairments that occur in human temporal lobe epilepsy. Also, this model may be useful in testing new medical and surgical therapies for complex partial seizures in humans. This monkey model of temporal lobe epilepsy has the potential to provide valuable
information about the cellular mechanisms of temporal lobe epilepsy in humans because it is more like human temporal lobe epilepsy than existing rodent models.
Acknowledgments The authors gratefully acknowledge the technical assistance of previous technicians and the cooperation of previous collaborators. This work was supported by the Medical Research Service of the Department of Veterans Affairs, the Yerkes Regional Primate Research Center, National Institutes of Health Core Grant RR-00165 (R.A.E.B), National Institutes of Health Grants NS-15669 and NS-38331 (C.E.R.), and National Institutes of Health Training Grant T32-NS045540 (supporting L.A.S.).
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FIGURE 6 Photomicrographs of Nissl-stained sections through the hippocampus from the control hemisphere (A) and the amygdala injected (B) side of a monkey. In (A) the granule cell layer (G) shows a normal appearance. In contrast, the granule cell layer (G) in B is dispersed. Scale bar = 200 mm. (Reprinted with permission from Ribak et al., 1998.)
References Bakay, R.A., and Harris, A.B. 1981. Neurotransmitter, receptor and biochemical changes in monkey cortical epileptic foci. Brain Res 206: 387–404. Harris, A.B. 1980. Structural and chemical changes in experimental epileptic foci. In Epilepsy: A Window to Brain Mechanisms Eds. J.S. Lockard and A.A. Ward Jr. pp. 11–49. New York: Raven Press. Harris, A.B., and Lockard, J.S. 1981. Absence of deizures or mirror foci in experimental epilepsy after excision of alumina and astrogliotic scar. Epilepsia 22: 107–122. Houser, C.R., Harris, A.B., and Vaughn, J.E. 1986. Time course of the reduction of GABA terminals in a model of focal epilepsy: a glutamic acid decarboxylase immunocytochemical study. Brain Res 383: 129–145. Lockard, J.S. 1980. A primate model of clinical epilepsy: mechanisms of action through quantification of therapeutic effects. In Epilepsy: A Window to Brain Mechanisms Eds. J.S. Lockard and A.A. Ward Jr. pp. 11–49. New York: Raven Press. Lockard, J.S., and Ward, A.A. Jr. 1980. Epilepsy: A Window to Brain Mechanisms. New York: Raven Press. Lockard, J.S., Congdon, W.C., DuCharme, L.L., and Huntsman, B.J. 1976a. Prophylaxis with diphenylhydantoin and phenobarbital in alumina-gel monkey model I. Twelve months of treatment: Seizure, EEG, blood, and behavioral data. Epilepsia 17: 37–47. Lockard, J.S., DuCharme, L.L., Congdon, W.C., and Franklin, S.C. 1976b. Prophylaxis with diphenylhydantoin and phenobarbital in alumina-gel monkey model II. Four month period of post-treatment: Seizure, EEG, blood, and behavioral data. Epilepsia 17: 49–57. Lockard, J.S., Levy, R.H., Congdon, W.C., DuCharme, L.L., and Patel, I.H. 1977. Efficacy testing of valproic acid compared to ethosuximide in
monkey model II. Seizure, EEG and diurnal variation. Epilepsia 18: 205–224. Lockard, J.S , Levy, R.H., Congdon, W.C., DuCharme, L.L., Salonen, L.D. 1979. Clonazepam in a focal-motor monkey model: efficacy, tolerance, toxicity, withdrawal, and management. Epilepsia 20: 683–695. Oertel, W.H., Schmechel D.E., Tappaz M.L., and Kopin I.J. 1981. Production of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neuroscience 6: 2689–2700. Ribak, C.E. 1985. Axon terminals of GABAergic chandelier cells are lost at epileptic foci. Brain Res. 326: 251–260. Ribak, C.E., Bradburne, R.M., and Harris, A.B. 1982. A preferential loss of GABAergic inhibitory synapses in epileptic foci: a quantitative ultrastructural analysis of monkey neocortex. J. Neurosci 2: 1725–1735. Ribak, C.E., Harris, A.B., Vaughn, J.E., and Roberts, E. 1979. Inhibitory, GABAergic nerve terminals decrease at sites of focal epilepsy. Science 205: 211–214. Ribak, C.E., Hunt, C.A., Bakay, R.A.E., and Oertel, W.H. 1986. A decrease in the number of GABAergic somata is associated with the preferential loss of GABAergic terminals at epileptic foci. Brain Res 363: 78–90. Ribak, C.E., Joubran, C., Kesslak, J.P., and Bakay, R.A.E. 1989. A selective decrease in the number of GABAergic somata occurs in pre-seizing monkeys with alumina gel granuloma. Epilepsy Res. 4: 126–138. Ribak, C.E., Seress, L., Weber, P., Epstein, C.M., Henry, T.R., and Bakay, R.A.E. 1998. Alumina gel injections into the temporal lobe of rhesus monkeys cause complex partial seizures and morphological changes found in human temporal lobe epilepsy. J Comp Neurol 401: 266–290. Sharbrough, F.W. 1987. Complex partial seizures. In Epilepsy: Electroclinical Syndromes. Eds. H. Luders and R.P. Lesser. pp. 279-302. London: Springer-Verlag. Wyler, A.R., and Ward, A.A.Jr. 1986. Neuronal firing patterns from epileptogenic foci of monkey and human. Adv Neurol 44: 967–989.
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15 Modeling Epilepsy and Seizures in Developing Zebrafish Larvae SCOTT C. BARABAN
relevant seizure disorder is unclear. Each of these studies hints at the possibility of using simple organisms for epilepsy research. However, in none of these simple species has a systematic effort to develop and characterize a seizure model been presented. This chapter discusses a simple alternative to rodents—larval zebrafish—and describes some of the salient features of a pediatric zebrafish epilepsy model. Our early efforts to induce seizures in zebrafish larvae were largely “trial and error.” Nonetheless, we would like to recognize seminal comparative species studies done by Zdenek Servít, a Polish scientist working with turtles, frogs, and fish in the early 1970s. Using curarized adult tenches, Servít described the genesis and pattern of electrographic spikewave discharge elicited in the adult fish forebrain on topical exposure to penicillin or pentylenetetrazol (PTZ) (Servít, 1970, 1972; Servít and Strejcˇková, 1970). These lower vertebrate studies predate the use of zebrafish in neurobiology but are prescient in recommending that “one of the uses of an experimental model is to simplify a complex situation.”
Pediatric epilepsy models are confined primarily to rodents. Although using a rodent model of a human neurologic disorder has distinct advantages, there is no rationale to support our almost exclusive reliance on this species. Indeed research questions related to genetic modifiers of pediatric epilepsy syndromes, high-throughput anticonvulsant drug screening, and “rapid” genetic manipulations aimed at analysis of basic cellular mechanisms of epileptogenesis could be better suited to a simple vertebrate system. Exciting new discoveries in the general field of neurobiology exploit the experimental advantages of simpler organisms such as Caenorhabditis elegans (worms), Drosophila melanogaster (fruit flies), and Danio rerio (zebrafish). Analysis of Parkinson (parkin) gene function in flies (Pesah et al., 2004) and discovery of daf genes regulating the aging process in worms (Hsin and Kenyon, 1999; Lin et al., 1997) are just two examples. Similar discoveries are possible in the epilepsy field, but there is currently little evidence that mammalian-like seizures can be induced in any of these species. For example, hyperexcitable Shaker flies featuring a potassium channel mutation are characterized by a legshaking behavior that most closely resembles human episodic ataxia (Tempel et al., 1987), and it is unclear whether this motor behavior represents a human seizure disorder. In a second group of Drosophila mutants termed “bang-sensitive”, potential seizure suppressor genes were recently identified using a model of evoked electrical afterdischarge in the giant muscle fiber (Kuebler and Tanouye, 2000; Kuebler et al., 2001). In C. elegans a lissencephaly (LIS1) gene mutant was recently described, and these worms exhibited a rapid “head-bobbing” behavior on exposure to a convulsant agent (Williams et al., 2004); again, whether this putatively convulsive behavior resembles a clinically
Models of Seizures and Epilepsy
What Can One Model in Zebrafish Larvae? In choosing zebrafish for pediatric epilepsy studies, as in any animal model choice, it is of utmost importance to consider the type(s) of experimental question one seeks to address. Our laboratory interest in zebrafish larvae stems from a desire to understand better the genetic factors that modify seizure genesis, propagation, and termination in the developing animal. For example, are there gene mutations that render an organism resistant to the generation of seizures? Does introduction of a human epilepsy gene mutation in zebrafish yield a model in which to study the basic
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cellular elements required for generation of seizures? Can we use a zebrafish pediatric epilepsy model to uncover novel antiepileptic drug targets? As a well-accepted model of development, and a species with tremendous potential for genetic modification and forward-genetic screening strategies, zebrafish larvae are an ideal choice for these types of studies. In the premature infant, seizures often mimic the normal behavioral repertoire of infants; infantile spasms are one such example. As infants mature, pediatric epilepsy syndromes (benign rolandic epilepsy, for example) with generalized tonic-clonic behaviors can be observed. Rodent pediatric epilepsy models also exhibit age-dependent behavioral, electrical, and anatomic manifestations (Schwartzkroin et al., 1995). Over time, rodent models have been developed to mimic a wide spectrum of these human pediatric epilepsy disorders, for example, febrile seizures, limbic status epilepticus, hypoxia-induced seizures, malformation-associated epilepsies, and ion channelopathies. In each of these examples, as is evident elsewhere in this volume, specific behavioral, electrical, anatomical or molecular changes occur; in many cases these changes closely mimic the human condition. If it can be demonstrated that zebrafish also exhibit behavioral, electrical, and anatomic features of epilepsy resembling those seen clinically, then it is not too far-fetched an idea that we can begin to model human seizure syndromes in zebrafish. What to model? One suggestion is to start with the development of zebrafish models mimicking well-established rodent epilepsy models. After all, there is a wealth of evidence suggesting that rodent animal models provide important insights into the human condition. Here we describe one such model—generalized tonic-clonic seizures in immature zebrafish induced by exposure to a common convulsant agent. Methods Zebrafish (Danio rerio) are freshwater teleosts, and they have the salient features of being small-sized lower vertebrates with a relatively short life cycle (Nüsslein-Volhard and Dahm, 2002). A single pair of adult zebrafish can produce hundreds of offspring, and zebrafish enjoy a genetic tractability rivaling Drosophila. A further advantage of zebrafish larvae is that they develop ex utero in a transparent egg, making them the current species of choice for a large number of developmental studies (see reviews in Kimmel, 1989; Lewis and Eisen, 2003; Moorman, 2001). Larval life in zebrafish begins around 72 hours post fertilization, when the fish are nearly free-swimming, and proceeds over an approximately 27-day period. Maturation is highly asynchronous and commonly defined by changes in body length. The larval zebrafish brain shows signs of an everted telencephalic organization as early as 5 days post fertilization (DPF), with the ventricular region lying exter-
nally and olfactory bulbs that are well developed. In the larval diencephalon, all the major adult subdivisions are present and optic tectum, the largest visible brain structure in zebrafish, begins to take on a layered “cortical” organization at 5 DPF (Nüsslein-Volhard and Dahm, 2002). How these maturational stages compare with developing infants is unclear, but it is worth noting that genes identified as controlling development in zebrafish (hedgehog, cadherin, notch, Wnt, etc.) often have similar roles in higher vertebrates. The scientific study of chemical convulsant agents in experimental animals has its origins in the late 1800s (Wiedemann, 1877) and has come to include a wide variety of compounds. Some of these compounds have long since fallen out of favor (absinth and strychnine), and others were replaced as our knowledge of underlying mechanism advanced (penicillin), and finally a few remain as widely used models in the present day (picrotoxin and pentylenetetrazol). Because rodent pediatric models often begin by adapting what has been successful in adult models (Holmes and Thompson, 1988; Moshe and Albala, 1982; Nehlig et al., 1996; Priel et al., 1996), a similar strategy was applied. Using larval zebrafish, preliminary studies were made to test several commonly used convulsants, such as picrotoxin, PTZ (Metrazol), pilocarpine, and kainic acid. In general these drugs work by interfering with normal synaptic transmission (Kupferberg, 2001; White, 1997). Because glutamate, GABA, and acetylcholinergic receptors are highly conserved across species, it is likely that potential mechanisms of action in zebrafish are similar to those reported for higher vertebrates. An initial concern, therefore, was not the mechanism of convulsant action but rather a more practical consideration: how to administer drugs to zebrafish larvae. Convulsant administration in immature rodents requires invasive intraperitoneal, subcutaneous, or intraventricular drug injections, and compounds are not always evenly distributed (or accessible) to central nervous system (CNS) structures, such as compounds that can not cross the bloodbrain barrier. Drug microinjections in zebrafish are possible (Oulmi and Braunbeck, 1996) but not a desirable alternative; thus our experimental strategy was to add drugs directly to the normal bathing medium. Surprisingly, and in direct contrast with rodents, zebrafish larvae enjoy a distinct pharmacologic advantage in that drugs dissolved in bathing medium were readily absorbed through the zebrafish skin or gills. We have not observed drug penetration issues with any of the convulsant or anticonvulsant compounds tested to date. Of course a limitation of this approach is that virtually no information is currently available regarding the pharmacokinetic distribution of convulsant drugs in larval zebrafish, and when a drug fails, it is difficult to conclude that drug penetration was not a factor. For all the pharmacologic studies performed in our laboratory, drug concentrations are determined on an empiric basis; then those showing the most
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Description of the Model
promise are further analyzed in concentration-response studies. The protocol described here is primarily focused on PTZ, a convulsive agent first described in 1926 (Hildebrandt, 1926), but it is worth noting that robust seizure activity can be elicited with any of the convulsant agents tested. Wildtype larval zebrafish (4 to 8 DPF) of the Tubingen line (TL) strain were used for all studies. Our procedure for seizure induction in zebrafish larvae is fairly simple and remarkably reproducible. PTZ was dissolved in normal bathing medium at drug concentrations between 2.5 and 15 mM. PTZ solutions were then adjusted to a pH of 7.0 before administration. Freely swimming fish were exposed to PTZ solution in individual 96-well Falcon plates (one fish per plate) or as groups of 20 to 50 fish in a Petri dish. For electrophysiology studies, a static bath recording chamber containing one or two agarembedded zebrafish was filled with about 0.5 ml of PTZ solution. Freely behaving zebrafish larvae respond to PTZ exposure in about 5 to 10 minutes; agar-embedded fish exhibit a slightly slower response time (15–45 minutes). Anesthetics can change the pattern of epileptiform activity produced by a chemical convulsant agent (Bowyer and Winters, 1981; Tuttle and Elliott, 1969) and are unavoidable problems with some types of rodent pediatric epilepsy models, for example, kindling. Because zebrafish embedded in agar (for electrophysiology) are nearly completely immobilized, additional anesthetic exposure was not necessary. All zebrafish larvae exposed to high concentrations of PTZ exhibited seizure activity. The various manifestations of seizures in developing zebrafish are described in the following section, with an emphasis on how each of these features mimic what has been observed in higher vertebrate systems.
DESCRIPTION OF THE MODEL Behavioral Manifestations The clinical significance of any experimental model of epilepsy is limited unless, like humans, a stereotyped set of behavioral changes are observed concomitant with a seizure. Because a wealth of behavioral information is available from the rodent literature, including a detailed and wellaccepted set of behavioral seizure stages (Racine, 1972), we began by investigating freely swimming zebrafish larvae exposed to PTZ. One approach to quantify seizure behaviors was to videotape the zebrafish and then review the recordings at a slower speed (and with the benefit of tape replay). For these studies, individual zebrafish were placed in 96-well Falcon plates and the solution was exchanged for normal bathing medium containing 15 mM PTZ. Fish were monitored for 30 to 60 minutes using a digital video camera. From these videotapes, three distinct stages of behavior were noted. In stage I, zebrafish larvae showed a general
increase in swim activity. This increase occurred in the first few minutes of PTZ exposure and was not seen on exposure to fresh bathing medium. Subsequently, stage II behaviors characterized by a rapid “whirlpool-like” circling around the outer edge of the well were observed. Finally, in stage III, zebrafish larvae exhibited brief head-to-tail convulsions followed by a loss of posture (e.g., fish floating on its side). Stage III convulsions, associated with very rapid movement across the dish (see dashed lines in Figure 1), lasted between 1 and 3 seconds and persisted as long as the fish were exposed to PTZ. Once established as distinct seizure stages, we compared the latency and prevalence of these episodes at varying concentrations of PTZ. As expected, seizure behaviors were elicited in a concentration-dependent fashion. These findings are not terribly surprising and are entirely consistent with well-established rodent pediatric epilepsy models (Jensen et al., 1991; Priel et al., 1996; Sankar et al., 1999). Visual observation of freely behaving animals is a reasonable and commonly used method to document seizure behaviors. However, automated observation systems can provide several distinct advantages. In particular, behaviors can be recorded more reliably because computer algorithms do not suffer from observer fatigue or interobserver variability. Further, computer automated detection is open to a wide array of measurements, for example, distance traveled, duration of movement, and velocity. For automated detection of zebrafish seizure behavior, we set up a computer-based locomotion tracking system. Using a stereomicroscope and high-speed charge-coupled device (CCD) camera acquiring images at 30 Hz, we can record the behavior of single zebrafish larvae during exposure to PTZ. Subsequently video output is digitized by a frame grabber and passed directly to a computer running EthoVision software (Noldus Information Technology, Wageningen, The Netherlands). Image-processing algorithms are then applied to analyze each frame and to distinguish the zebrafish against a background image and, on the basis of user-defined gray-scale values, track the position of the zebrafish in one well (Noldus et al., 2002). From these types of recordings, we obtain locomotion plots as shown in Figure 1. Note that each of the seizure stages defined initially by direct visual observation can be reproduced and quantified using this locomotion tracking system. Of many potential uses for an automated seizure detection system, one could be a comparison between epileptic phenotypes in various wild-type and mutant zebrafish, thus facilitating phenotype-genotype strategies currently in vogue (Lewis et al., 2004; Zumkeller et al., 2004).
Electrophysiologic Manifestations By definition, epilepsy models must exhibit some form of abnormal electrical discharge in a CNS structure, and the
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FIGURE 1 Behavioral seizure stages. a: Frame-grabber image of a 7 days post fertilization zebrafish larvae in one well of a 96-well Falcon plate. b: Sample locomotion tracking plots are shown for individual zebrafish in normal Ringer’s medium (baseline), during stage I (increased swimming activity), stage II (circling), and stage III (clonus-like convulsions). Solid lines indicate movement; dashed lines indicate rapid convulsive seizure activity (this fish exhibited >25 convulsive episodes). Plots were obtained from recording epochs 2 minutes in duration. (See color insert.)
pattern of electrographic activity can be used to classify the model in clinical terms (Engel and Pedley, 1997). To meet this “gold standard,” we sought a means to document PTZ-induced electrographic seizure activity in developing zebrafish. The obvious problem with this model criterion is technical: how to record electrical events in a larval zebrafish brain. A standard experimental strategy in rodent pediatric epilepsy models is to implant indwelling electroencephalographic (EEG) recording electrodes in “complex” brain structures such as neocortex or hippocampus. At first glance, one might consider zebrafish larvae incapable of generating EEG-like discharge as their brains lack a highly ordered laminar cortical structure (Wulliman et al., 1996). Because larval zebrafish are only 3 to 4 mm long, implantation of a chronic EEG monitoring device in a freely swimming fish is not possible. As an alternative we developed a procedure to immobilize zebrafish larvae in a low-melting-point agar block. The principal advantage of this procedure is that invasive surgical techniques and anesthetic drugs (with their undesirable potential for interfering with CNS function) are not necessary. The limitation of this protocol is that we cannot simultaneous monitor electrical activity and behavior. Agar-embedded zebrafish can be placed on the recording chamber of an upright or stereo microscope, which allows for perfusion with various convulsant (or anticonvulsant) solutions. Because zebrafish larvae are translucent, we employed a relatively simple procedure to place glass microelectrodes in visualized central brain structures using a three-dimensional micromanipula-
tor. In our laboratory, successful field recordings have been obtained from the optic tectum, telencephalon, and cerebellum in zebrafish as young as 4 DPF. In 6 to 7 DPF zebrafish, bath application of 15 mM PTZ induced a progressive increase of tectal electrical activity, resulting in population bursts with typical features of interictal discharges, for example, abrupt in onset, primarily monophasic, and brief duration. Interictal-like events were observed within 10 to 20 minutes of drug exposure. Continuous application of PTZ resulted in interictal activity followed by ictal-like discharges lasting 4 to 6 seconds and composed of large-amplitude population spikes. Ictal-like discharge was usually followed by a period of suppressed electrical activity or postictal depression (Figure 2a). Simultaneous field recordings in optic tectum and telencephalon indicate a seizure pattern that could be classified as “generalized” epilepsy; epileptic discharge appeared nearly simultaneously in all brain regions (Figure 2c). Electrographic seizure events generated in CNS structures (optic tectum or telencephalon) preceded convulsive activity monitored as an electromyogram in the fish-tail muscle (Figure 2c). Electrographic activity was similar in fish exposed to curare (10–250 mM, a neuromuscular blocking agent), further demonstrating that agar-embedding is a sufficient method to immobilize zebrafish larvae for electrophysiology. Spontaneous discharges were never observed in fish exposed to normal bathing medium. Of course, convulsant agents effective in eliciting epileptiform-like activity in immature animals are not limited to PTZ, and this review
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Description of the Model
FIGURE 2 Epileptiform-like electrographic activity. a: Representative tectal field recording from a zebrafish larva exposed to 15 mM pentylenetetrazol (PTZ) for 45 minutes. Note the presence of interictal, ictal, and postictal phases of the electrical recording. b: Schematic of the configuration used to obtain electrophysiologic recordings from agar-embedded zebrafish larvae. c: Representative dual field recordings from a zebrafish exposed to 15 mM PTZ. In one set of recordings (left), a nearly simultaneous epileptiform burst discharge is observed in the zebrafish forebrain and optic tectum. In a second set of recordings (right), an epileptiform burst discharge is seen to originate in the optic tectum prior to its appearance in a distal section of the zebrafish tail muscle. EMG, electromyelograph.
highlights one of what are potentially many methods to induce epilepsy in zebrafish. As a brief example of the possibilities, we also found that electrographic seizures were elicited on exposure to high potassium, picrotoxin, or pilocarpine (e.g., manipulations used to elicit seizure-like activity in rodent models) (Figure 3). In each case, a distinct pattern of abnormal electrical discharge was observed. These findings demonstrate the broad nature of using a zebrafish model and suggest that epilepsy research in this species is not limited to PTZ-induced seizures. A further application could be the introduction of gene mutations previously shown to elicit epileptic phenotypes (e.g., generation of “epileptic” zebrafish). In general, field potentials recorded in larval zebrafish are remarkably similar in waveform to those reported in chronic seizure models in vivo (Bragin et al., 1999a), acute seizure models in vitro (Dzhala et al., 2003), and in the epileptogenic region of patients with temporal lobe epilepsy (Bragin et al., 1999b). Research into the basic mechanisms of seizure genesis and propagation has provided evidence consistent with a critical role for excitatory glutamate-mediated synaptic transmission. In the context of pediatric epilepsy models, antagonists to the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor subtype block hypoxia-induced seizures in the immature rat (Jensen et al., 1995), and 3-(RS)-2-carboxypiperazine-4-yl)-propyl1-phosphonic acid (CPP) blocks 4-aminopyridine-induced spontaneous episodes of spreading depression in hippocam-
pal slices from immature rats (Psarropoulou and Avoli, 1996). As such, agents that block presynaptic glutamate release or activation of postsynaptic ionotropic glutamate receptors should be effective at terminating chemically induced seizures in larval zebrafish. Because ionotropic glutamate receptors are expressed in the larval zebrafish brain (Edwards and Michel, 2003), we predicted a role for glutamate-mediated synaptic transmission in the generation or propagation of electrographic seizure discharge in our model. Pharmacologic studies performed on zebrafish larvae at 7 DPF confirmed this hypothesis. First, bath application of tetrodotoxin (TTX) to block Na-dependent action potentials, and therefore synaptic transmission, abolished PTZ-induced electrographic seizures. Second, application of either a nonspecific glutamate receptor antagonist (kynurenate) or a “cocktail” of AMPA/N-methyl-D-aspartate (NMDA) receptor antagonists 6-cyano-7-introquinoxaline2,3-dione-2amino-5-phosphonovalerate (CNQX–APV) abolished PTZ-induced electrographic seizures. These types of studies further support the general concept that chemically induced seizure activity in zebrafish larvae is similar to that observed in higher vertebrates.
Molecular Alterations Revolutionary advances in molecular biology have contributed to our nascent understanding of the many changes in gene expression that can occur prior to, during, and after
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FIGURE 3 Electrographic seizure activity in zebrafish larvae with various convulsants. a: Representative 1-minute field recording traces are shown following 45 to 60 minutes of bath exposure to convulsant agents: 25 mM KCl or “highpotassium” model; 100 mM picrotoxin, a g-aminobutyric acid (GABA) receptor antagonist; and 120 mM pilocarpine, a muscarinic acetylcholine receptor agonist. b: Isolated burst discharge shown at a faster time resolution as indicated by the asterisk in (a). Note that different type of burst discharge waveforms are elicited with the different mechanisms of seizure initiation.
a seizure episode. These changes in gene expression presumably reflect (1) molecular mechanisms that contribute to an acute hyperexcitable state (e.g., upregulation of immediate early gene expression) (Morgan et al., 1987); (2) compensatory alterations in genes that may limit the spread of epileptic activity (e.g., increased neuropeptide Y expression) (Rizzi et al., 1993); or (3) molecular alterations that underlie a process of epileptogenesis (e.g., changes in GABAA receptor subunit expression) (Brooks-Kayal et al., 1998). Like most research in the epilepsy field, findings were first reported in adult seizure models (or tissue obtained from patients with intractable forms of epilepsy) and have only recently been studied in pediatric epilepsy models. Because zebrafish larvae represent a simple vertebrate system for “reductionist” questions related to how seizures develop in an immature nervous system, we were interested in reproducing at least one of the molecular alterations that would be expected in an established seizure model. Because one of the most robust and widely reproduced examples of a seizure-induced change in gene expression is the dramatic upregulation of the immediate early gene (IEG) c-fos in brain regions participating in seizure genesis, we began with this IEG (Dragunow and Robertson, 1987; Morgan et al., 1987). It is important to emphasize that c-fos experiments are discussed as “proof-of-principle” and are not intended to diminish the importance of other molecular alterations in epileptogenesis.
Because we observed robust electrographic seizure discharge and stage III seizure behavior in zebrafish larvae exposed to 15 mM PTZ, our molecular studies were performed at this concentration. Freely swimming zebrafish were exposed to PTZ and monitored for seizures. At 0, 15, 30, and 60 minutes following continuous PTZ exposure, zebrafish were removed and total RNA was isolated. From larval RNA samples, cDNA was synthesized and serial dilutions were amplified by reverse transcriptase (RT)-polymerase chain reaction (PCR). As expected, PTZ-induced seizures resulted in a significant upregulation of c-fos mRNA expression in all zebrafish. Levels of c-fos mRNA expression in untreated zebrafish larvae were fairly low. Further confirmation of a seizure-induced upregulation of cfos mRNA expression in the zebrafish CNS was obtained using whole-mount in situ hybridization techniques; an increase in expression was seen in the optic tectum, forebrain, and cerebellum. At present, antibodies raised against zebrafish c-fos are not available; thus we have not yet confirmed these results using immunohistochemistry. However, as additional zebrafish antibodies are developed, studies to localize the expression of IEGs in neurons participating in seizure genesis in the larval zebrafish brain will be mapped in greater detail. Analysis of postsynaptic GABA receptor subunits, neuropeptides, protein kinases, and the many genes whose expression levels are regulated by seizure activity remains to be performed. It is also likely that this
Advantages and Limitations of the Zebrafish Model
simple model of induced seizures can be combined with gene microarray analysis (or other molecular methodologies) to study an even wider range of known genes, expressed sequence tagging (EST), etc.
Antiepileptic Drugs It is a well-accepted maxim that testing of new therapeutic agents in humans follows on the preliminary screening and evaluation of these compounds in an experimental animal model. Discovery and screening of antiepileptic drugs have long followed this paradigm, and development of “ideal” animal models closely approximating human epilepsy is critical to this process. What, however, are the important criteria in the development of new animal models? Dixon Woodbury, a pioneer in antiepileptic drug (AED) screening, stated that an experimental model for evaluation of anticonvulsant drugs must meet two criteria: (1) electrographic evidence of epileptic-like activity and (2) clinical seizure-like behaviors manifest as tonic-clonic motor movements (Woodbury 1969). PTZ-induced seizure activity in rodents (commonly referred to as the Metrazol test) is a well-established experimental model fitting these criteria and has been successfully used to test anticonvulsant compounds. As we show here, PTZ-induced seizure activity in larval zebrafish also satisfies both of the Woodbury criteria. Because our zebrafish work is based on rodent PTZ data, and because a substantial literature exists on AEDs that suppress (or do not suppress) PTZ-induced seizures (Ferrendelli et al., 1989; Krall et al., 1978), we proposed an additional criterion: demonstration of an AED pharmacologic profile identical to that observed in rodents. For these studies we chose suppression of tectal electrographic epileptiform discharge as a sensitive outcome measure of AED efficacy in
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zebrafish. In all electrophysiologic studies, agar-embedded zebrafish (7 DPF) were exposed to 15 mM PTZ until a stable level of baseline bursting was established (40–65 minutes; interictal + ictal activity). Next AEDs previously demonstrated to suppress (or abolish) PTZ-induced seizure in rodents were tested e.g., benzodiazepines (clonazepam and diazepam) or valproic acid (Figure 4). Both these agents suppressed PTZ-induced epileptiform discharge in a concentration-dependent manner. Reduction of ictal burst discharge amplitude and frequency could be observed within 30 to 45 minutes of drug exposure. Unlike in vitro slice studies, it was not possible to remove or “wash” the compounds efficiently from our in vivo preparation; thus recovery experiments were not performed. In separate control studies, we also tested AEDs previously shown to have little (or no) effect on PTZ-induced seizures in rodents (e.g., carbamazepine, phenobarbital, ethosuximide, phenytoin). As expected, these AEDs did not suppress electrographic discharge in zebrafish, and we present these findings as a critical validation of our model. Indeed, because rodent PTZ model successfully predicts drugs effective against generalized seizures of the absence (or petit mal) type, we anticipate that our chemically-induced zebrafish seizure model could also identify compounds with therapeutic potential in humans.
ADVANTAGES AND LIMITATIONS OF THE ZEBRAFISH MODEL The application of our larval zebrafish model to epilepsy research is probably limited only by one’s imagination. As discussed in the previous section, a simple application of this model would be to evaluate drugs useful in generalized epilepsy syndromes. One might also consider forward- and
FIGURE 4 Response to antiepileptic drugs. a: Representative tectal field recordings obtained from a zebrafish bathed in normal Ringer’s medium plus 15 mM pentylenetetrazol (PTZ) (baseline) and 45 minutes after application of 100 mM diazepam. b: Representative tectal field recordings obtained from a zebrafish bathed in normal Ringer’s medium plus 15 mM PTZ (baseline) and 45 minutes after application of 5 mM valproic acid.
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chemical-genetic screening, commonly used methods to identify gene mutations related to a specific behavior (or phenotype) in an unbiased manner (Malicki, 2000; Patton and Zon, 2001; Specht and Shokat, 2002). Such screening methods provide a strategy to test available AEDs on mutagenized zebrafish larvae, and perhaps will lead to a better understanding of why some patients are refractory to drug treatment. Pharmacogenic strategies (Weinshilboum and Wang, 2004) designed to identify gene mutations that underlie inter-individual variations in AED responsiveness are also possible. When designing zebrafish pharmacologic studies, it is important to consider an empirical testing of drug concentrations; as mentioned above, drug availability and distribution data are not available. Of course this model need not be limited to AED discovery; and not unlike many of the animal models described in this volume, it can be used to study directly how an epileptic brain functions. Epilepsy, particularly pediatric forms of epilepsy, is a neurologic disease that is defined by abnormal electrical discharge, and insights into the basis of this activity often follow from experimental animal model research. In particular the now widespread use of extracellular and intracellular recording techniques in rodent seizure models has provided evidence for intrinsic cellular and synaptic dysfunction that underlie generation of interictal and ictal epileptic seizures. Such research has led, for example, to identification of fuctional changes in H-channels in a febrile seizure model (Bender et al., 2004), and insight into how M-type potassium channel mutations lead to benign familial neonatal convulsions (BFNC) in mutant mice (Watanabe et al., 2000) have benefited from electrophysiology research. Extracellular recording techniques were demonstrated to be feasible (and relatively easy to obtain) in our zebrafish model, and it is not difficult to imagine that intracellular approaches are also possible. Because larval zebrafish possess far fewer neurons and synaptic circuits than immature rodents (but exhibit similar types of complex epileptiform discharge; see Figure 2), analysis of cellular mechanisms in this model could be a fruitful experimental strategy to identify the basic elements required for generation of epileptic seizures. Indeed, owing to the high degree of homology between zebrafish and human genomes, it is likely that identification of genes involved in the basic processes of seizure genesis, seizureinduced neurogenesis, and epileptogenesis will lead directly to insights into the human condition. If one combines this model with morpholino antisense oligonucleotides (Corey and Abrams, 2001) or transgenic methods (Stuart et al., 1990) to alter gene expression in developing zebrafish, a still wider set of experimental questions can be addressed. Perhaps the most significant application of our model, and one that our laboratory currently employs, is to use the zebrafish model to examine the genetic basis of epilepsy. Several types of applications can be envisioned, and we have chosen a forward-genetic screening strategy to identify
“seizure resistant” zebrafish mutants. This research program is based on a fundamental question of how to identify genes that prevent, or protect, an individual from developing epilepsy. Because larval zebrafish are a pediatric epilepsy model, perhaps these types of studies could lead to new therapies for the large number of children suffering with medically intractable forms of epilepsy. Although it is too early to speculate on how data from zebrafish will be extrapolated to human pediatric epilepsy disorders, especially considering the vast complexity of the human brain in relation to the relative simplicity of the zebrafish CNS, it is safe to predict that the process of discovery will lead to novel insights.
CONCLUSIONS Foremost, the potential usefulness of a zebrafish model of pediatric epilepsy should be evaluated with respect to the type of problem to be solved. In the preceding sections, I have tried to provide examples of the features of this model that are clinically relevant. These features include distinct stages of seizure behavior, evidence of abnormal electrical discharge in a CNS structure, seizure-induced expression of c-fos, and AED sensitivity. There are many more features of this model that need to be evaluated, and it is premature to propose that larval zebrafish replace existing rodent seizure models. However, it is not too soon to consider how this simple vertebrate, with striking similarities to commonly used rodent models, may help to open new research directions. Our work was initiated to allow more rapid investigation of genetic modifiers of epilepsy in the developing brain, a fundamental unsolved problem. This goal required establishment of a reliable method to induce and monitor seizures in immature zebrafish. Our methods are described here in detail for the first time, with the intent of encouraging a wider utilization of simple vertebrate systems in pediatric epilepsy research.
Acknowledgments I am grateful for the devotion and effort of my laboratory technicians, graduate students, and postdoctoral fellows, P.A. Castro, S. Guyenet, D.K. Takahashi, D. Botello, J. Greenwood, J. Hsu, M. Dinday, and M.A. Taylor, who have shared in the development of this project. Moral and conceptual support from trusted colleagues, N.M. Barbaro and D.H. Lowenstein, is also gratefully appreciated. I would also like to thank a local zebrafish expert and highly valued collaborator, H. Baier. My research efforts in creating this line of research were supported by grants from the NIH/NINDS, Klingenstein Fund, Epilepsy Foundation of America, and UCSF Innovations in Basic Sciences Fund.
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References Bowyer, J.F., and Winters, W.D. 1981. The effects of various anesthetics on amygdaloid kindled seizures. Neuropharmacology 20: 199–209. Bragin, A., Engel, J. Jr., Wilson, C.L., Vizentin, E., and Mathern, G.W. 1999a. Electrophysiologic analysis of a chronic seizure model after unilateral hippocampal KA injection. Epilepsia 40: 1210–1221. Bragin, A., Engel, J. Jr., Wilson, C.L., Fried, I., and Mathern, G.W. 1999b. Hippocampal and entorhinal cortex high-frequency oscillations (100–500 Hz) in human epileptic brain and in kainic acid–treated rats with chronic seizures. Epilepsia 40: 127–137. Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Rikhter, T.Y.M., and Coulter, D.A. 1998. Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy. Nat Med 4: 1166–1172. Corey, D.R., and Abrams, J.M. 2001. Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol 2: 1015. Dragunow, M., and Robertson, H.A. 1987. Kindling stimulation induces cfos protein(s) in granule cells of the rat dentate gyrus. Nature 329: 441–442. Dzhala, V.I., and Staley, K. J. 2003. Transition from interictal to ictal activity in limbic networks in vitro. J Neurosci 23: 7873–7880. Edwards, J.G., and Michel, W. C. 2003. Pharmacological characterization of ionotropic glutamate receptors in the zebrafish olfactory bulb. Neuroscience 122: 1037–1047. Engel, J. Jr., and Pedley, T.A. 1997. In Epilepsy: A Comprehensive Textbook. Vols. 1, 2 and 3. Philadelphia: Lippincott-Raven Publishers. Ferrendelli, J.A., Holland, K.D., McKeon, A.C., and Covey, D.F. 1989. Comparison of the anticonvulsant activities of ethosuximide, valproate , and a new anticonvulsant, thiobutyrolactone. Epilepsia 30: 617–622. Hildebrandt, F. 1926. Pentamethylenetetrazol (Cardiazol®). Archiv für experimentelle Pathologie und Pharmakologie 116: 100–109. Holmes, G.L., and Thompson, J.L. 1988. Effects of kainic acid on seizure susceptibility in the developing brain. Brain Res 467: 51–59. Hsin, H., and Kenyon, C. 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399: 362–366. Jensen, F.E., Applegate, C.D., Holtzman, D., Belin, T.R., and Burchfiel, J.L. 1991. Epileptogenic effect of hypoxia in the immature rodent brain. Ann Neurol 29: 629–637. Jensen, F.E., Blume, H., Alvarado, S., Firkusny, I., and Geary, C. 1995. NBQX blocks acute and late epileptogenic effects of perinatal hypoxia. Epilepsia 36: 966–972. Kimmel, C.B. 1989. Genetics and early development of zebrafish. Trends Genet 5: 283–288. Krall, R.L., Penry, J.K., White, B.G., Kupferberg, H.J., and Swinyard, E.A. 1978. Antiepileptic drug development: II. Anticonvulsant drug screening. Epilepsia 19: 409–428. Kuebler, D., and Tanouye, M.A. 2000. Modifications of seizure susceptibility in Drosophila. J Neurophysiol 83: 998–1009. Kuebler, D., Zhang, H., Ren, X., and Tanouye, M.A. 2001. Genetic suppression of seizure susceptibility in Drosophila. J Neurophysiol 86: 1211-1225. Kupferberg, H. 2001. Animal models used in the screening of antiepileptic drugs. Epilepsia 42(Suppl 4): 7–12. Lewis, K.E., and Eisen, J.S. 2003. From cells to circuits: development of the zebrafish spinal cord. Prog Neurobiol 69: 419–449. Lewis, J.C., Thomas, H.V., Murphy, K.C., and Sampson, J.R. 2004. Genotype and psychological phenotype in tuberous sclerosis. J Med Genet 41: 203–207. Lin, K., Dorman, J.B., Rodan, A., and Kenyon, C. 1997. daf-16: An HNF3/forkhead family member that can function to double the life-span of Caenorhabditis elegans Science 278: 1319–1322. Malicki, J. 2000. Harnessing the power of forward genetics—analysis of neuronal diversity and patterning in the zebrafish retina. Trends Neurosci 23: 531–541.
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Moorman, S.J. 2001. Development of sensory systems in zebrafish (Danio rerio). Ilar J 42: 292–298. Morgan, J.I., Cohen, D.R., Hempstead, J.L., and Curran, T. 1987. Mapping patterns of c-fos expression in the central nervous system after seizure. Science. 237: 192-197. Moshe, S.L., and Albala, B.J. 1982. Kindling in developing rats: persistence of seizures into adulthood. Brain Res 256: 67–71. Nehlig, A., and Pereira de Vasconcelos, A. 1996. The model of pentylenetetrazol-induced status epilepticus in the immature rat: short- and longterm effects. Epilepsy Res 26: 93–103. Noldus, L.P.J.J., Spink, A.J., and Tegelenbosch, R.A.J. 2002. Computerised video tracking, movement analysis and behaviour recognition in insects. Comput Electron Agric 35: 201–227. Nüsslein-Volhard, C., and Dahm, R. 2002. Zebrafish. Oxford: Oxford University Press. Oulmi, Y., and Braunbeck, T. 1996 Toxicity of 4-chloroaniline in early lifestages of zebrafish (Brachydanio rerio): I. cytopathology of liver and kidney after microinjection. Arch Environ Contam Toxicol 30: 390–402. Patton, E.E., and Zon, L.I. 2001. The art and design of genetic screens: zebrafish. Nat Rev Genet 2: 956–966. Pesah, Y., Pham, T., Burgess, H., Middlebrooks, B., Verstreken, P., Zhou, Y., Harding, M. et al. 2004. Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress. Development 131: 2183–2194. Priel, M.R., dos Santos, N.F., and Cavalheiro, E.A. 1996. Developmental aspects of the pilocarpine model of epilepsy. Epilepsy Res 26: 115–121. Psarropoulou, C., and Avoli, M. 1996. Developmental features of 4aminopyridine induced epileptogenesis. Dev Brain Res 94: 52–59. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysio 32: 281–294. Rizzi, M., Monno, A., Samanin, R., Sperk, G., and Vezzani, A. 1993. Electrical kindling of the hippocampus is associated with functional activation of neuropeptide Y-containing neurons. Eur J Neurosci 5: 1534–1538. Sankar, R., Shin, D., Mazarati, A.M., Liu, H., and Wasterlain, C.G. 1999. Ontogeny of self-sustaining status epilepticus. Dev Neurosci 21: 345–351. Schwartzkroin, P.A., Moshe, S.L., Noebels, J.L., and Swann, J.W. 1995. Brain development and epilepsy. Oxford: Oxford University Press. Servít, Z. 1970. Focal epileptic activity and its spread in the brain of lower vertebrates. A comparative electrophysiological study. Epilepsia 11: 227–240. Servít, Z., and Strejckova, A. 1970. An electrographic epileptic focus in the fish forebrain. Conditions and pathways of propagation of focal and paroxysmal activity. Brain Res 17: 103–113. Servít, Z. 1972. Phylogenetic correlations. In Experimental Models of Epilepsy—A Manual for the Laboratory Worker. Eds. D.P. Purpura, J.K. Penry, D. Tower, D.M. Woodbury, and R. Walter. pp. 509–530. New York: Raven Press. Stuart, G.W., Vielkind, J.R., McMurray, J.V., and Westerfield, M. 1990. Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development 109: 577–584. Tempel, B.L., Papazian, D.M., Schwarz, T.L., Jan, Y.N., and Jan, L.Y. 1987. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science 237: 770–775. Tuttle, W.W., and Elliott, H.W. 1969. Electrographic and behavioral study of convulsants in the cat. Anesthesiology 30: 48–64. Weinshilboum, R., and Wang, L. 2004. Pharmacogenomics: bench to bedside. Nat Rev Drug Discov 3: 739–748. White, H.S. 1997. Clinical significance of animal seizure models and mechanism of action studies of potential antiepileptic drugs. Epilepsia. 38(Suppl 1): S9–S17. Wiedeman, C. 1877. Beiträge zur Pharmakologie des Camphers. Archiv für experimentelle Pathologie und Pharmakologie 6: 216–232.
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Williams, S.N., Locke, C.J., Braden, A.L., Caldwell, K.A., and Caldwell, G.A. 2004. Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans. Hum Mol Genet 13: 2043–2059. Woodbury, D.M. 1969. Role of pharmacological factors in the evaluation of anticonvulsant drugs. Epilepsia 10: 121–144.
Wullimann, M.F., Rupp, B., and Reichert, H. 1996. Neuroanatomy of the Zebrafish Brain. A Topological Atlas. Basel: Birkhäuser Verlag. Zumkeller, W., Volleth, M., Muschke, P., Tonnies, H., Heller, A., Liehr, T., Wieacker, P. et al. 2004. Genotype/phenotype analysis in a patient with pure and complete trisomy 12p. Am J Med Genet 129A(3): 261–2644.
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16 Transgenic and Gene Replacement Models of Epilepsy: Targeting Ion Channel and Neurotransmission Pathways in Mice DANIEL L. BURGESS
spontaneously (see Chapter 17) without any deliberate mutagenesis by humans. Second, in each case, altered function of a single gene was alone sufficient to cause the phenotype (i.e., the mutations produced highly-penetrant “monogenic” disorders). Indeed, because genes involved with polygenic disorders are, for biological and statistical reasons, substantially more difficult to map and identify, virtually all the mutant genes isolated from epileptic mice to date have been associated with single gene disorders. Significant advances in the development of technical tools have made identification of mouse monogenic disease mutations a fairly straightforward proposition today, but researchers no longer need to wait for these mutations to occur naturally and then spend months or years identifying the defective gene. The development and refinement of two powerful molecular techniques—transgenesis and gene replacement by homologous recombination in embryonic stem cells— has taken the waiting out of mouse model development and yielded a wealth of knowledge about the basic molecular mechanisms of epilepsy in just a short time. By identifying all the genes that specify the development and function of mouse and human, the respective genome projects have supplied all the necessary raw material to fuel a revolution in the genetic modeling of human disorders.
“MOUSEQERADING” AS HUMAN The availability of numerous existing inbred mouse strains and the practicality of maintaining large breeding colonies have made the mouse—because of its small size, rapid generation time, and low cost—an easy choice for biomedical researchers seeking animal models. These mouse strains exhibit wide differences in susceptibility to disorders that are important to humans: cancer, diabetes, obesity, cardiovascular and hematologic defects, developmental abnormalities, infertility, endocrine dysfunction, blindness, behavioral abnormalities, and epilepsy, among others. The cumulative health benefits that have been derived over the past century from studies using mice as models of human disease—to learn about pathologic mechanisms and develop new therapies—are incalculable, but they certainly surpass billions of health care dollars and millions of lives saved. Over the most recent decade, we witnessed what may be the most important advance yet for the study of mouse models of disease: the nearly simultaneous completion of the human and the mouse genome sequencing projects. Comparison of the two genomes indicates a remarkable degree of similarity; very few genes are present in one species that are not represented by orthologs in the other. Thus, one reason the mouse has provided such good models of human diseases is that the mouse genome is such a good model of the human genome!
MODELING GENOTYPES VERSUS MODELING PHENOTYPES Many research approaches might drive the development of a mouse model of inherited epilepsy. Most can be characterized as either an attempt to model a phenotype or an attempt to model a genotype. These concepts are illustrated
WAITING FOR THE RIGHT MUTATION The earliest epilepsy-associated mutations isolated from mice shared two characteristics. First, these mutations arose
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by considering some common questions an investigator might pose in the course of planning a study:
Question 1: What Does this Gene Do? An investigator beginning to study a recently identified gene (let us call it Xyz) may not have any initial clue about the gene’s function—and no interest at all in seizures or epilepsy. Because an increasingly popular approach to probing gene function is to inactivate the gene in the mouse germline (by homologous recombination in embryonic stem cells, a gene “knockout”) and then observe the resulting phenotype, our researcher decides to knock out Xyz. The possible outcomes of a mutated Xyz gene might include anything: a new coat color, extra toes, early embryonic lethality, or seizures. Although the investigator may not have had any initial interest in epilepsy research, this scenario could lead—and has led—to the development of an animal model that is useful for the epilepsy field. For example, the Beta2 (NeuroD1) gene, expressed in pancreatic endocrine cells, intestine, and brain, was originally knocked out to investigate the importance of this gene in pancreatic endocrine cell differentiation (Naya et al., 1997). It was subsequently discovered that loss of this gene causes both malformation of the dentate gyrus and seizures, and produces a good model of developmental brain malformation-associated epilepsy (Liu et al., 2000).
Question 2: Is this Gene Involved in Epilepsy? Alternatively, a researcher might begin with the hypothesis that gene Xyz is involved in epilepsy but have no direct evidence. The experimental approach taken to address this hypothesis might be identical to that following question 1 (i.e., targeted inactivation of Xyz in mouse ES cells). However, in this case, the investigator predicts a specific outcome: a seizure phenotype. For example, genes involved in the inhibitory GABA (g-aminobutyric acid) neurotransmitter pathway had long been considered excellent candidates for mutations in epilepsy, but no spontaneous mutations had ever been found to support this belief. In 1997 Kash et al. knocked out the mouse Gad2 (glutamic acid decarboxylase 2) gene (which catalyzes the formation of GABA) and observed spontaneous seizures. This outcome provided some of the first direct evidence supporting a role for GABA pathway genes in epilepsy. Seizures in some mice can be subtle or infrequent, and if this phenotype is not expected or screened for appropriately (e.g., by electroencephalographic [EEG] analysis), even a relevant phenotype can be easily overlooked. Several knockout mice may exist with yet unrecognized neuronal excitability defects.
Question 3: Is this Human Mutation Involved in Epilepsy? Occasionally the first indication that gene Xyz might be associated with epilepsy is identification of a sequence difference that is present in human epilepsy patients but not in unaffected controls. Perhaps Xyz itself had never before been associated with epilepsy; alternatively, maybe other epilepsy mutations had been found in Xyz, but this particular sequence variant had not yet been described. In either case, how can we determine whether the human sequence variant (often a missense change) is a causative epilepsy mutation or whether it is only a rare, but functionally neutral, polymorphism? To help address this question, an investigator might create a mouse model in which the candidate human Xyz mutation is inserted (by homologous recombination) into the identical molecular context in the orthologous mouse gene. This method (termed a gene knock-in) allows the variant mRNA and protein to be expressed and is thus a better reflection of the human mutation than obliteration of the mouse Xyz gene would be. Because this method is labor intensive and time consuming, it has not been widely used to investigate whether a genetic variant might be a causative mutation rather than simply a polymorphism. More frequently, knock-in mutations are created to produce a viable mouse model when the complete inactivation of a gene results in a lethal phenotype. A good example of this approach has focused on the human DKPQ mutation in SCN5A, which causes long-QT syndrome type 3 (LQT3). A complete knockout mutation of the mouse Scn5a gene results in embryonic lethality in homozygotes, but mice heterozygous for a knock-in of the human DKPQ deletion survive and exhibit all the essential features of LQT3, including ventricular arrhythmias (Nuyens et al., 2001).
Question 4: How Can I Produce a Mouse Model of a Particular Epilepsy Phenotype? In some cases, an investigator may desire to study a mouse model of epilepsy with a particular seizure phenotype but not particularly care whether or how the mouse genome is altered to produce it. Application of convulsant drugs has often been used to generate such models. However, an inherited phenotype may produce a seizure-relevant phenomenon that is unique, more consistent among individuals, or present at ages earlier than can be produced with convulsants. As in the preceding scenarios, gene replacement mice can be useful in some cases. In other situations, creation of a transgenic mouse might result in the optimal model. For example, Kearney and colleagues (2001) used a transgene to overexpress in mouse neurons a mutant Scn2a protein with slow inactivation and increased persistent current. These transgenic mice displayed seizures beginning at 2 months of age that were accompanied by
Genome-Environment Interactions in Mouse Epilepsy Models
behavioral arrest and other abnormal behaviors. Transgenes can be designed to overexpress a gene that is endogenous to the mouse, from a different species, or even an artificial gene. They can be used to express genes ectopically (i.e., in a cell type or at a developmental stage where they are not normally expressed). Transgenes can even be designed to be turned on and off simply by adding chemicals to the mouse’s drinking water, providing much more flexibility in manipulating gene expression than is possible with gene replacement strategies. The preceding scenarios provide some general examples of how or why a mouse model of epilepsy might be developed in the first place. In the initial three cases, we are primarily modeling a genotype and are particularly interested in the role of the gene product or genetic pathway in epilepsy. In the fourth example, the mouse genome may be altered in such a way that could not occur naturally in humans but nevertheless produces a mouse model of a phenotype. Whether it is the goal of an investigator to produce a mouse model from scratch or to study an available model, an understanding of the conceptual pathway that leads to development of the model is important for guiding the way that model is studied and in deciding how we interpret the resulting data in reference to the human epileptic condition.
GENOME-ENVIRONMENT INTERACTIONS IN MOUSE EPILEPSY MODELS Although this chapter is concerned specifically with transgenic and gene replacement mouse models of epilepsy, a preliminary general discussion of the genome-environment interaction as it relates to mouse seizure phenotypes provides a useful framework for subsequent discussions. Let us begin by considering the commonly (mis)applied distinction between wild-type and mutant mice. If all mice of a given strain exhibited an epilepsy phenotype, and genetic analysis indicated that the phenotype was linked to a single chromosome locus, we would probably feel comfortable referring to that mouse strain as a mutant strain. But then imagine that we moved these mice to a different animal facility (perhaps one with a different temperature, light-dark cycle, or brand of mouse food) and the mice were no longer observed to have seizures. No change in the genome of this mouse strain has occurred, so something in the environment must be responsible for the change in phenotype. Are these mice still mutant? Another example makes a similar point: Multiple studies have now demonstrated that some inbred strains of so-called wild-type mice, even if they never show spontaneous seizures, can be much more easily induced to have seizures than other wild-type strains. Given this information, is it really accurate to imply that the seizuresusceptible and seizure-resistant mouse strains are all equally wild type? Clearly mice are only wild type with
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respect to a specific genetic locus and according to a specific user-defined assay for the structure or function of that locus in a controlled laboratory environment. Every mouse carries multiple genetic differences (mutations or polymorphisms) that distinguish it from mice of other strains, although only some of these differences produce phenotypes that are obvious to a biologist. It can be argued that the terms mutant and wild type refer primarily to the interaction of that mouse’s genome and its environment rather than the genome alone. Further, this interaction can be very dynamic. The practical implications of these observations may be vital for anyone planning to generate or analyze targeted gene models of epilepsy. Figure 1 provides a visual schematic that helps to explain the interaction of the genome and the environment as it relates to seizure susceptibility and epilepsy. Six hypothetical mouse strains (A–F) exhibit a low to high risk (left to right) of genetically determined epilepsy within environment 1 (Figure 1A). In this environment, the sum of seizure triggers (theoretical constructs that may occur in the external environment, such as flashing light, loud sound, or in the internal environment, such as hormone levels, blood pressure) present at any one time may range from a level of 0 to about 2.5 (on an arbitrary scale of 0 to 5). This same scale that measures the sum level of seizure triggers can be thought of as measuring an animal’s genetic resistance to seizures in that environment. In environment 1, mouse strains D–F (but not strains A–C) exhibit seizures. Figure 1B examines temporal and quantitative aspects of seizures among mouse strains D–F in environment 1. Genetically determined resistance to seizures in each animal (highest in D, lowest in F) is stable over time (dashed lines) because the genome of each animal is stable over the lifespan of that animal, but the level of seizure triggers in environment 1 is highly variable (shaded area). Mouse strain F has such a low intrinsic resistance to seizures (about 0.4) that the sum of seizure triggers is always greater than the threshold for seizures, so that strain F animals would persist in status epilepticus. Strain E has a moderate intrinsic genetic resistance to seizures (~1.3), so that the sum of seizure triggers only occasionally surpasses this threshold; in this example, a mouse from strain E would have a total of five seizures over the period shown (i.e., recurrent seizures, or “epilepsy”). Mice from strain D have a higher intrinsic genetic resistance to seizures (about 2.2). In this example, the seizure triggers in environment 1 surpass the strain D seizure threshold only once over the indicated time period. Figure 1C represents the propensity of an individual mouse (or an entire mouse strain) to appear epileptic in one environment and nonepileptic in another. Here the genetic resistance to seizures across strains remains constant, but environments with different seizure trigger ranges are shown. Mice from strain A (with the highest genetic seizure resistance) do not exhibit seizures in any of the three
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environments. Mice from strain B also have a relatively high intrinsic seizure resistance (~4.0) but would be expected to have occasional seizures in environment 2. The other strains (C–F) show corresponding environment-dependent shifts in seizure and epilepsy risk. Now consider a scenario in which a researcher selects strain A mice for creating a gene knockout, with the intention of creating a model of epilepsy. After mutating the Xyz gene in strain A, no seizure phenotype is observed. Is this result strong evidence that gene Xyz is not involved in epilepsy? No, the targeted mutation in Xyz might have actually lowered the intrinsic resistance of strain A, but a relatively moderate environmental seizure trigger range “prevented” the appearance of spontaneous seizures. Introduction of the Xyz-mutated strain A mouse into a different environment might expose the latent seizure phenotype and thus the potential role of gene Xyz in epilepsy. The exposure of gene-modified and control animals to electrical or chemical convulsants to examine changes in latent hyperexcitability phenotypes is theoretically equivalent to moving animals to an environment with a higher average sum seizure trigger level. Creation of the Xyz gene knockout using strain B or C originally, rather than strain A, would have accomplished a similar goal. In summary, a genetic epilepsy phenotype cannot be evaluated in terms of only a single gene of interest but should be viewed instead as a dynamic interaction of gene, genome, and environment.
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FIGURE 1 A model of genome-environment interaction in mouse epilepsy models. All mice, whether described as mutant or wild-type, can be considered to have some genetically determined risk of epilepsy in any given environment. Because epilepsy is defined as recurrent, spontaneous seizures, this risk of epilepsy is directly correlated with the animal’s genetic resistance to seizure triggers in that environment. This model assumes that all seizures have some proximal trigger, either endogenous or exogenous (the precise nature of which is unimportant for the model). Genetic resistance to seizure triggers should be stable at any point during its life as long as the animal’s genome remains unchanged. A: The range of seizure triggers in environment 1 has a maximum and minimum level, and any individual mouse or mouse strain (e.g., A, B, or C) whose genetic resistance is stronger than the maximum seizure trigger level will never exhibit seizures in that environment. Any mouse (e.g., D, E, or F) with a genetic resistance within the seizure trigger range of environment 1 will eventually have one or more seizures. B: Seizure trigger levels are not constant within their range in a given environment. Whereas the genetic resistances of mouse strains D–F are all within the seizure trigger range for environment 1, seizure frequency can vary depending on temporal fluctuations in actual seizure trigger levels. C: The range of seizure triggers can vary among different environments. Thus, a mouse may be normal in one environment but experience seizures if moved to another. This model provides a useful framework for interpreting the genotype-phenotype relationship for mouse epilepsy models.
TRANSGENIC APPROACHES TO MODELING EPILEPSY The Basic Concept The term transgenesis most broadly refers to the introduction of genetic material into the germline of a recipient organism; the donated genetic material may come from either the same or different species, or the introduced DNA may be artificial (i.e., generated by DNA synthesis and not present in nature). Technically speaking, the process of carrying out “gene replacement” by homologous recombination can also be considered transgenesis because some introduced foreign DNA is always left at the recombination site. However, for the purpose of clarity, we will refer to homologous recombination as the process of introducing exogenous DNA into a specific site in the genome of a recipient animal (this topic will be discussed later in this chapter) and to transgenesis as the introduction of DNA into a random site. Transgenes can be introduced into mice for a variety of purposes, including to study and define gene promoters, to introduce biomarkers (e.g., green fluorescent protein, bgalactosidase) into specific cell types to distinguish those cells for subsequent in vivo analysis, to selectively “rescue” a gene’s function when endogenous copies of that gene have
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been mutated, to explore the effect of expressing a mutant protein in vivo, or to produce an animal model with a very specific phenotype (such as epilepsy). When considering a transgenic approach to developing a mouse epilepsy model, three questions must be addressed: (1) What sort of transgene would cause a mouse to have seizures? (2) Where in the mouse would it need to be expressed? (3) When during the animal’s life should it be expressed? Because there are so many different possible transgenes (and a similarly wide range of mechanisms) that could cause seizures or epilepsy in a mouse, the choice of transgene depends on the specific needs or interests of the investigator. In this chapter, we discuss primarily ion channel genes. Two general categories of ion channel genes can be used to create a transgenic mouse model of epilepsy: wild-type and mutant. A wild-type transgene can be expressed in the same cell type as the endogenous version of that gene if the appropriate promoter is available, or it can be expressed in a cell type that does not normally express the endogenous gene. It might seem intuitive that overexpressing a gene that increases membrane excitability (e.g., a voltage-gated Na+ channel) would favor the development of seizures and epilepsy, whereas overexpressing a gene that decreases membrane excitability (e.g. a K+ channel) would inhibit seizures. However, this scenario is not always realized. In some cases the genome can employ feedback regulation to alter the expression of endogenous genes to compensate for the effects of a transgene. Transgenic expression of a mutant ion channel gene can also be used to create an epilepsy model, but not all ion channel mutations associated with human epilepsy are good candidates for this purpose. Because a transgenic mouse already has two functioning endogenous copies of every gene, only those mutant ion channel transgenes that carry gain-of-function mutations (not those that carry loss-of-function mutations) might be expected to replicate a human seizure phenotype.
Methods The technique of transgenesis via microinjection into mouse embryos was described more than 25 years ago (Gordon et al., 1980). An overview of that process is shown in Figure 2. Most transgenes consist of the same elements as endogenous genes and include a promoter, a region of transcribed sequences that encode a protein, and a poly(A)addition signal. The protein coding segment usually consists of an intronless cDNA fragment rather than a fragment of genomic DNA containing the gene because the latter tends to be too large for simple cloning procedures (notable exceptions include transgenes consisting of entire bacterial artificial chromosomes, or BACs). A transgene is constructed by standard recombinant DNA techniques in a plasmid vector and is amplified by growth in Escherichia coli. The vector portion (which can interfere with transgene expression) is
Promoter
Poly(A)
Gene
Transgene plasmid
1.
Promoter
E. coli
Gene
Poly(A)
2. male pronucleus
3.
female pronucleus
4.
Mouse zygote
Transgene array inserted into chromosome
FIGURE 2 Construction of a transgenic mouse model. A typical transgene plasmid contains a promoter, structural gene, and poly(A) addition signal. This is amplified in Escherichia coli (1), linearized to remove vector sequences (2), and purified. The purified transgene fragment is microinjected into one pronucleus of a recently fertilized mouse zygote (3). The transgene usually inserts as a multicopy tandem array at a single site in the genome (bottom). The transgenic zygote is implanted into a surrogate female (4) to develop until birth about 20 days later.
removed, and the purified transgene fragment is diluted in a physiologically buffered solution. This transgene-containing solution is then microinjected into one of the two pronuclei of a newly fertilized zygote that has been surgically removed from a recently mated female donor mouse (the female is usually treated with hormones before mating to cause “superovulation,” the production of a greater number of mature ova than generated during a normal cycle). Following fertilization, the haploid egg and sperm pronuclei do not fuse immediately. Because the male pronucleus is usually larger than the female pronucleus, it is generally an easier target for the microinjection needle. Thus, most transgenes actually insert into the paternal genome. It is also interesting to note that certain mouse strains produce zygotes with larger pronuclei than others or that can survive the process of microinjection better and therefore are technically easier to access. These practical concerns are often balanced by a desire to create the transgenic mouse on a specific background strain. Following microinjection, multiple copies of the transgene concatemerize (head-to-tail, head-to-head, tail-to-tail, or a combination of these) and insert into a single site in the genome. More rarely multiple transgene arrays insert into different sites in the genome. It remains unclear whether the resulting copy number in the transgene array is correlated with the concentration of transgene DNA in the microinjection solution. The insertion site in the genome is believed to
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be essentially random. In fact, 3 to 5% of transgenes insert into an endogenous gene, resulting in an insertional mutation that can confound analysis of the transgenic mouse phenotype (Meisler, 1992). The microinjected embryos are then implanted into a pseudopregnant surrogate female that had been previously treated with hormones to facilitate implantation. The mice that are born about 20 days later (referred to as “founders,” or F0-generation pups) may or may not carry the transgene; they must be screened, for example, by carrying out polymerase chain reaction (PCR) genotyping of genomic DNA extracted from a blood sample, a tail clipping, or an ear-punch tissue sample. An alternate approach to creating transgenic mice is to infect mouse embryos with a retrovirus engineered to express a transgene, which then inserts itself into the genome using natural viral mechanisms (Ivics and Izsvak, 2004). This approach is used less frequently than embryo microinjection. However, because retroviral transgenes tend to insert as a single unit rather than as a variably sized tandem array, this approach may provide the investigator with some control over transgene copy
Basic transgene:
Promoter
number. It is unclear whether retroviral transgenes insert at random into the target genome or preferentially at certain sites (Wu and Burgess, 2004). Variations of the Basic Transgene Although a basic transgene contains a promoter, a protein-coding gene segment, and a poly(A)-addition signal, there are variations that can be useful (Figure 3). Some studies have shown that inclusion of a single intron in the transgene can increase expression significantly, perhaps by increasing pre-mRNA stability or mRNA nuclear export. This intron does not need to be derived from the same gene that is being expressed and is usually positioned between the promoter and the translation start site (first ATG codon) of the transcribed gene. Another variation is the addition of chromatin scaffold attachment regions, or SARs (also known as matrix attachment regions, MARs, or insulators). These are fragments of DNA that are thought to regulate local chromatin structure (Bode et al., 2003; McKnight et
Gene
Poly(A)
(~ 2 - 10 kb)
Intron added:
Promoter
Gene
Poly(A)
intron
SARs added:
SAR
Promoter
Epitope added:
Promoter
IRES vector:
Promoter
BAC transgene: (~ 80 - 300 kb)
Native promoter
Gene
Gene 1
Gene
Poly(A)
SAR
Epitope tag Poly(A)
IRES
Gene 2
Poly(A)
Native Poly(A)
FIGURE 3 Alternative transgene designs. Modifications (gray boxes) to the basic transgene design include the addition of introns to increase mRNA stability and nuclear export, chromatin scaffold attachment regions (SARs) to insulate the transgene from position-dependent effects on transcription, epitope tags (including GFP or LacZ) for identification or localization in vivo and an internal ribosome entry site (IRES) to allow the translation of two independent proteins from a single mRNA molecule. The bottom panel indicates that an entire bacterial artificial chromosomes (~80–300 kb) can be used as a transgene, with no additional modification, to preserve native regulatory features that might be lost when using cDNAs for transgenes.
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al., 1992). By insulating the transgene and its promoter from the DNA environment into which the transgene inserts, flanking SARs can help to ensure that the transgene is expressed in the intended tissue and not influenced by nearby endogenous enhancer or repressor elements. This process can improve concordance between different mouse lines carrying the same transgene. Transgenes may also incorporate small (£20 amino acids) epitope tags at the nterminus, c-terminus, or inside the transgene. These tags are translated as an integral part of the transgene protein and allow the use of existing antibodies to the epitope tag (myc, HA, and His tags are popular) to detect or immunoprecipitate the protein. Larger proteins can also be fused directly to the target gene, such as green fluorescent protein (GFP) or b-galactosidase (LacZ), which allows visualization of transgene expression without antibodies. However, the addition of these larger proteins may interfere with the functions of the gene under study. Finally some transgene vectors allow the simultaneous expression of two genes separated by an internal ribosome entry site (IRES). With this system, a single mRNA is transcribed, but then two separate proteins are translated from that transcript. This process can be useful for expressing a protein with a separate visualization label. The choice of genes that can be expressed transgenically is practically unlimited. Currently the major constraint on transgene design is the availability of well-characterized promoters to drive tissue- or developmental stage-specific transgene expression. Promoters can be cloned from endogenous genes in an attempt to express the transgene in its native pattern; alternatively, promoters can be obtained from viral, bacterial, or even plant genes to drive expression in the mouse. With regard to expression in the central nervous system (CNS), a review of the literature reveals transgenes that incorporate Purkinje or granule neuron-specific, GABAergic neuron-specific, hippocampal, or pan-neuronal promotors. However, for a large number of interesting and functionally discrete brain regions or cell types, there are no currently available transgene promoters. It is extremely time consuming to clone and characterize new gene promoters, and some promoters are simply too complex or large to incorporate into a standard transgene construct. An increasingly popular approach is to express a gene from its native promoter via a BAC transgene (Gong et al., 2003; Heintz, 2000). The large size of most BACs (80–300 kb) increases the likelihood that the complete promoter, intronic, and 3¢ regulatory sequences will also be contained within that clone. Unlike a standard transgene construct, a BAC does not need to be linearized or separated from its relatively small vector segments, but it can simply be purified, resuspended in the appropriate buffer, and directly microinjected into the zygote pronucleus. The technology to modify genes within a BAC clone (e.g., to introduce epitope tags, mutations, or other complex alterations)
is rapidly advancing (Giraldo and Montoliu, 2001; Testa et al., 2004). In addition to autonomous promoters that either direct constitutive transgene expression or direct transgene expression in response to developmental cascades or natural cues, significant progress has been made toward developing transgene promoters that are truly inducible (Yamamoto et al., 2001). Much of this work has been driven by gene therapy and agricultural genetic research. The most commonly utilized technique for development of mouse transgenic models is the tetracycline (tet)-inducible promoter. Two versions are commercially available so that an investigator can select a transgene promoter that is always activated until tet is added, or always inactivated until tet is added (Backman et al., 2004). Tetracycline (or the analogue doxycycline) can simply be added to the transgenic mouse’s water supply to induce the promoter to express the transgene or turn it off. Another benefit of this technique is its capacity to regulate the activity of the promoter quantitatively, that is, by adding more or less tet to the water. Inducible promoters provide a powerful opportunity to address cause-and-effect relationships between transgene expression and resulting mouse phenotypes, including the roles of development and plasticity. Additional resources describing the theory and methodology of transgenesis, including more detailed protocols, include Jackson and Abbott (2000), Hofker and Deursen (2002), Nagy et al. (2002), Pinkert (2002), and Houdebine (2003).
Examples Compared with homologous recombination-mediated gene knockouts, only a relatively small number of studies have used transgenically expressed ion channels to study epilepsy and neuronal excitability in mice (Table 1). Some examples include the following: AKv1.1a In 1999 Sutherland et al. created three transgenic mouse lines with a human HPRT promoter driving the CNSspecific overexpression of an Aplysia shaker-type K+ channel cDNA with a small 11-amino acid epitope tag. Transgene expression was detected in neurons, but not glia, in several brain areas, including the neocortex, hippocampus, and cerebellum. Electroencephalographic (EEG) activity from adult neocortex showed spontaneous cortical discharges similar to those seen in spike-wave epilepsy. Because overexpression of shaker K+ channel currents in Aplysia neurons shortened action potential duration, enhanced the hyperpolarizing afterpotential, and depressed transmitter release from terminals (Kaang et al., 1992), the presence of an epilepsy phenotype in transgenic mice expressing the same gene was surprising. Further analysis revealed extensively
TABLE 1 Transgenic and Targeted Gene Knockout Models of Ion Channels, Transporters, and Neurotransmission Pathways Associated with Seizure Phenotypes Gene symbol
Speciesa
Phenotype or syndromeb
Abcc8
Mouse Mouse Human
KO Transgene overexpression Inherited
No seizures reported. Failure of insulin secretion in response to glucose Suppressed kainic acid-induced seizures and excitotoxic neuron damage Persistent hyperinsulinemic hypoglycemia of infancy (PHHI); occasional seizures
AKv1.1a
Mouse
Aplysia K+ channel transgene
Spontaneous cortical discharges similar to those seen in spike-wave epilepsy
Atp1a2
Mouse
KO
Mouse Human
KI Inherited
No seizures reported, but homozygotes show perinatal lethality and selective neural damage consistent with excitotoxicity No seizures reported Familial hemiplegic migraine-2 (FHM) ± benign familial infantile convulsions (BFIC)
Aqp4
Mouse
KO
Reduced susceptibility and increased latency to PTZ-induced seizures
Cacna1a
Mouse Mouse
KO KI
Mouse Human Human Human
Spontaneous Inherited Inherited Inherited
Lethality ~30 days; behavioral absences Reduced threshold for, and increased velocity of, cortical spreading depression Absence epilepsy, ataxia Familial hemiplegic migraine (FHM); episodic ataxia type-2 (EA-2) Spinocerebellar ataxia type 6 (SCA6). Primary generalized epilepsy, ataxia, mild learning difficulties; absence epilepsy
Cacna1g
Mouse
KO
Thalamus was resistant to the generation of spike-and-wave discharges in response to GABA-B receptor activation
Cacna1h
Mouse Human
KO Inherited
No seizures reported CAE
Chrna4
Mouse Mouse Mouse Human
KO KI Spontaneous deletion (Szt1) Inherited
No seizures reported Reduced threshold to nicotine induced seizures Low electroconvulsive threshold Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)
Chrna5
Mouse Mouse
KO KO
Shorter latency time to induced seizures Resistant to nicotine-induced seizures
Chrnb2
Mouse Human
KO Inherited
No seizures reported Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)
Chrnb4
Mouse Mouse
KO KO
No seizures reported Resistant to nicotine-induced seizures
Clcn2
Mouse Human
KO Inherited
Abnormalities of eye and testis Multiple seizure types (CAE, JAE, JME, EGMA)
Gabbr1
Mouse Mouse
KO (gene trap) KO
Mouse
KO
Homozygotes are neonatal lethal Spontaneous seizures (clonic, tonic-clonic, absence), hyperalgesia, memory impairment. Growth retardation, generalized epilepsy, premature death
Gabra1
Mouse Mouse
KO KI
Human
Inherited
Gabrb3
Mouse Mouse Mouse Human
KO KI Spontaneous (cleft palate, Cp1) Rearrangements of 15q11-q13
Human
Inherited
Myoclonus and occasional epileptic seizures; cleft palate Reduced sensitivity to some anesthetics Cleft palate Angelman syndrome (AS) and Prader-Willi syndrome (PWS)-associated seizures Chronic insomnia
Gabrg2
Mouse Mouse Mouse Human Human
KO (partial) KO KO (conditional) Inherited Inherited
No seizures reported Perinatal lethality Epilepsy, lethality around 4 wk Generalized epilepsy with febrile seizures plus (GEFS+) CAE; FS
Gad2
Mouse Mouse Mouse
KO KO KO
Spontaneous seizures (genetic strain-dependent), increased mortality Spontaneous seizures, increased mortality No spontaneous seizures, but seizures more easily induced by PTZ, picrotoxin
Mutation type
No seizures reported No spontaneous seizures but diazepam was less effective against PTZinduced tonic convulsions in mutants Juvenile myoclonic epilepsy
(continues)
TABLE 1 (continued) Gene symbol
Speciesa
Mutation type
Phenotype or syndromeb
Glra1
Mouse Mouse Mouse Human
KI Spontaneous (spasmodic, spd) Spontaneous (oscillator, spd-ot) Inherited
Seizures and lethality by 3 wk; hyperekplexia Tremors, stiff posture, difficulty in righting Seizures and early death Hyperekplexia; no seizures reported
Gria2
Mouse Mouse Mouse
KO KI KI; disruption of intronic mRNA editing site
No seizures reported No seizures reported Seizures; premature lethality
Hcn2
Mouse
KO
Spontaneous absence seizures
Htr2c
Mouse
KO
Spontaneous death from seizures; susceptible to audiogenic seizures
Kcna1
Mouse Mouse
KO KI
Mouse Human Human
Spontaneous (mceph) Inherited Inherited
Homozygotes viable; frequent spontaneous seizures through adult life Homozygous lethality; higher frequency of spontaneous IPSCs in heterozygotes Megencephaly; recurrent behavioral seizures Episodic ataxia/myokymia syndrome; no seizures reported; normal EEG Tonic-clonic and simple partial seizures with myokymia and no ataxia
Kcnc2
Mouse
KO
Spontaneous tonic-clonic convulsions
Kcnj6
Mouse Mouse
KO Spontaneous (weaver, wv)
Spontaneous and stress-induced seizures; reduced threshold to PTZ Spontaneous seizures
Kcnj11
Mouse Human
KO Inherited
No seizures reported. Failure of insulin secretion in response to glucose Persistent hyperinsulinemic hypoglycemia of infancy (PHHI); occasional seizures
Kcnn2
Mouse
Viral transgene
Kcnn2 (SK2) protected neurons against excitotoxic insult
Kcnq2
Mouse
KO
Mouse Mouse Human
Spontaneous deletion (Szt1) Conditional dominant-negative Kcnq2 transgene Inherited
Homozygous lethality; heterozygotes show decreased PTZ seizure threshold Low electroconvulsive threshold Spontaneous seizures, behavioral hyperactivity and morphologic changes in the hippocampus Benign familial neonatal convulsions (BFNC)
Scn1b
Mouse Human
KO Inherited
Spontaneous generalized seizures Generalized epilepsy with febrile seizures plus (GEFS+)
Scn2a
Mouse Mouse Human Human Human
KO Transgenic Inherited Inherited Deletion including SCN1A and SCN2A
Perinatal lethal; no seizures reported Spontaneous seizures Generalized epilepsy with febrile seizures plus (GEFS+) Benign familial neonatal-infantile seizures (BFNIS) Severe epilepsy, retardation, and dysmorphic features
Scn2b
Mouse
KO
Increased susceptibility to pilocarpine-induced seizures
Slc2a1
Mouse
Antisense transgene
Rat Human Human
Viral transgene Inherited Inherited
No seizures reported; growth retardation and developmental malformations Protection against kainic acid seizure-induced neuron loss Infantile seizures, acquired microcephaly, developmental delay Mild to severe seizures, developmental delay, ataxia
Slc9a1
Mouse Mouse
K.O. Spontaneous (slow-wave epilepsy, swe)
Unspecified seizures 3-Hz absence seizures; tonic-clonic seizures
Slc12a5
Mouse
KO
Mouse
KO
No seizures reported; homozygotes mutants died immediately after birth due to severe motor and respiratory deficits Frequent seizures, death between P7–P17; heterozygotes show increased susceptibility for PTZ-induced seizures
Mouse
Deletion [Df(11)17] or duplication [Dp(11)17] of the mouse SMS region
Smith-Magenis syndrome (SMS)
a
Df(11)17/+ mice exhibit seizures, obesity, craniofacial abnormalities, male-specific reduced fertility. Dp(11)17/+ animals are underweight, and have no seizures, craniofacial defects, or reduced fertility.
The corresponding spontaneous mouse and human mutations are listed if known. Data are taken from the following databases, which contain full literature references: Mouse Genome Informatics (MGI) at The Jackson Laboratory (URL: http://www.jax.org/), and Online Mendelian Inheritance in Man (OMIM) and LocusLink/Entrez Gene at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). CAE, childhood absence epilepsy; EGMA, epilepsy with grand mal seizures on awakening; FS, febrile seizures; JAE, juvenile absence epilepsy; JME, juvenile myoclonic epilepsy; KO, gene “knockout” (generally refers to a functionally null allele); KI, gene “knock-in” (generally refers to an incompletely inactivated allele); PTZ, pentylenetetrazol. b
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altered regulation of endogenous ion channel genes within the CNS secondary to expression of the Aplysia K+ channel transgene, which could explain the phenotype. This study provides an excellent example of how brain plasticity can complicate a transgene-based approach to studying ion channel gene effects in the brain. Scn2a In 2001 Kearney et al. developed three transgenic mouse lines with a neuron-specific enolase promoter driving overexpression of a mutant rat brain sodium channel (Scn2a; NaV1.2). The three-amino-acid substitution (879–881 GAL>QQQ) was not based on any spontaneous human or mouse epilepsy mutation but was originally generated for use in basic structure-function studies, where the mutation resulted in slowed channel inactivation and increased persistent Na+ current when expressed in Xenopus oocytes. These channel properties would be predicted to cause a gainof-function hyperexcitability phenotype if expressed in mouse neurons. The transgenic mice did exhibit seizures beginning at 2 months of age. Clinically the seizures were characterized by a combination of behavioral arrest and repetitive behaviors. EEG monitoring detected focal seizure activity in the hippocampus, which sometimes generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic transgenic mice showed increased persistent sodium current. Seizure onset was followed morphologically by progressive cell loss and gliosis within CA1, CA2, CA3, and hilar regions. Four independent transgenic lines expressing the wild-type sodium channel, an important control for such studies, did not exhibit any such abnormalities. It is relevant to note that mutations in the Scn2a gene have been associated with epilepsy in humans (Heron et al., 2002; Pereira et al., 2004), but a functionally null knockout of the endogenous Scn2a gene in mice results in perinatal lethality (with no seizures reported) (Planells-Cases et al., 2000).
the mutant Kcnq2 protein (which retained the ability to bind other channel subunits but was otherwise dysfunctional) interfered with the production of normal M-channels. Depending on when the transgene was expressed, the transgenic mice exhibited seizures, hyperactivity, cognitive deficits, or changes in brain morphology. The creative approach used in this study to examine the role of Kcnq2 in brain development and function demonstrates that transgenes can be used in certain circumstances to produce loss-of-function, or hypomorph, phenotypes through the dominant-negative suppression of endogenous proteins. Furthermore, the use of transgenes for this purpose can be implemented much more quickly and cost-effectively than production of a gene replacement model using homologous recombination approaches.
Limitations and Pitfalls Transgene Lethality The expression of a particular transgene might result in the death of the transgenic animals. Depending on the pathophysiologic insult, this lethality could occur at any age and could be dose dependent. This pitfall may be unavoidable if the hypothesis being addressed requires overexpression of this transgene at a certain level or in a certain cell type. Chimeric Founders Occasionally an F0 (founder) pup will be born “chimeric” for the transgene and carry it in some cells but not others. This phenomenon occurs when the microinjected transgene remains in the zygotic nucleoplasm and does not insert into a chromosome until after one or more cell divisions have occurred. To verify that a mouse carries the transgene in every cell, it is necessary to cross the F0 animal with a wild-type mouse. All the transgenic offspring from this cross (the number should be 50%) will be transgenic in their germline.
Kcnq2 Mutations in human KCNQ2, which coassembles with KCNQ3 to produce the neuronal M-type K+ current, result in benign familial neonatal convulsions (BFNCs). Complete inactivation of the mouse Kcnq2 gene by gene replacement results in perinatal lethality (Watanabe et al., 2000), which makes functional analysis of this gene impossible in older animals. In 2005 Peters and colleagues created transgenic mice that conditionally expressed—using the inducible TetOff promoter system—dominant-negative mutant Kcnq2 subunits in the brain (Backman et al., 2004). These mice survived early postnatal development, during which time the endogenous Kcnq2 genes were free to function normally. When expression of the transgene was turned on in later life,
Insertional Mutation The insertion of transgene DNA at random into the genome can cause an unintended loss of function if it interrupts or disturbs the expression of an endogenous gene. This is believed to occur in as many as 5% of all transgenic lines (Meisler, 1992). The potential phenotype produced by this insertional mutation is unpredictable, but it can clearly interfere with interpretation of any phenotype produced by expression of the transgene itself. This pitfall can best be avoided through comparative analysis of multiple independent transgenic lines (≥3) containing the same construct.
Gene Replacement (Knockout) Approaches to Modeling Epilepsy
209
Insertion Site and Copy Number Variability
Transgene Instability
The phenotype may vary considerably among multiple independent transgenic lines produced with the same construct as a result of differences in the level or site of transgene expression. Differences in expression levels among transgenic lines are likely due to either transgene copy number variation (transgenes seldom insert as a single copy; rather, they tend to concatemerize before integration as an array) or are due to insertion site variability (i.e., positiondependent expression). Insertion site variability is believed to occur when transgenes insert near a strong gene enhancer (possibly a component of a nearby gene) that overrides the transgene’s own promoter and alters the transgene levels or site of expression. Insertion site variability could also be caused by insertion of a transgene into a site that is transcriptionally suppressed as a result of the general chromatin structure of the region. These pitfalls may be minimized by analyzing multiple transgenic lines for each construct or by designing transgenes flanked by insulator elements (SARs).
Transgenes are exogenous fragments of DNA introduced into the genome at random sites. They may also be quite large, especially when concatemerized into a high copynumber tandem array. Furthermore, when maintained in the hemizygous state, there is no balancing transgene on the paired chromosome during meiosis. This situation may cause the transgene to become unstable and rearrange, perhaps progressively to lose copy number either within somatic cells of an individual animal or in the germline from generation to generation. As a direct result the phenotype of transgenic mice could vary from animal to animal. To monitor for instability, DNA from transgenic individuals, especially those from progressive generations, should be sampled occasionally by Southern blot analysis to determine whether the transgene copy number or the restriction pattern exhibits any variability compared with DNA from the original founder animal.
GENE REPLACEMENT (KNOCKOUT) APPROACHES TO MODELING EPILEPSY Multiple Insertion Sites Although transgenes usually concatemerize and insert into a single site in the genome of the founder zygote as a transgene array, occasionally two or more independent insertions occur. When such a founder animal is bred, the transgene arrays assort independently (as long as the insertions are unlinked on different chromosomes), and the resulting progeny may inherit zero, one, or more of the transgene arrays. To detect this event, Southern blot analysis should be conducted on tail DNA from all the offspring of the founder to see whether the restriction pattern or copy number varies. PCR, which is usually used to identify transgene-carrying animals, will not distinguish between different arrays at different insertion sites.
Gender-Dependent Transgenes In some instances either transgene inheritance or transgene expression is gender dependent. In the first case, the transgene may insert into the Y chromosome and therefore be carried only by males. This may limit subsequent analysis in some cases, depending on the purpose of the study. In the second case, a transgene may be carried on an autosome but only be expressed in some of the heterozygous transgenic animals. One possible explanation for this pattern is the phenomenon of “imprinting,” in which a gene will be expressed if it is inherited from the paternal side and suppressed if inherited from the maternal side or vice-versa. Imprinting of transgenes is rarely observed, but it does occur (Surani, 1991).
The Basic Concept Whereas transgenesis introduces extra copies of a gene into the mouse germline, gene knockout strategies (also known as gene replacement) alter the mouse’s endogenous genes. Because a transgenic mouse always carries at least two functioning copies of each gene, transgenic approaches are generally limited to producing gain-of-function phenotypes (however, see preceding discussion for an example of “dominant negative” loss of function). Gene replacement, on the other hand, can be employed to study either gain-offunction or loss-of-function phenotypes. It is therefore a more versatile technique, albeit more expensive and time consuming. Gene replacement was developed beginning in the late 1980s (Capecchi, 1989a, 1989b), and technical improvements continue. The basic concept is that a piece of DNA, when introduced into a cell nucleus, will find its matching sequence in the host genome and “trade places” through a mechanism called homologous recombination. As a result, researchers are able to replace a specific target gene with a completely inactive copy (i.e., a “knockout”) or perhaps a more subtly mutated version and study the resulting phenotype. In a conventional gene knockout, one or both alleles are replaced, and the alterations are present in every cell of the organism from the very beginning of embryonic development. As a result many gene knockouts produce lethal phenotypes that provide little information on gene function. A recent improvement of the method is the “conditional” (or inducible) knockout. When this technique is used, the replaced allele functions normally, but small DNA elements are left in place that allow the gene to be
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inactivated at a later stage, or in a subset of cell types, at a time and place determined by the researcher. Thus a gene that would have produced a lethal phenotype if knocked out from the single-cell stage can be left intact; the animal is born and matures normally, and only then is the gene knocked out (perhaps only in a single tissue type) to study the phenotype.
1
Methods The basic approach for mouse gene replacement to create a functionally null mutant allele is outlined in Figure 4. The first step is to define the genomic target (including the intron/exon organization) and to identify some part of the gene that is critical for gene function. This may include an
3
2
4 Genomic Target
Homology Arm 1
1.
Homology Arm 2
2r neo
2
tk
Targeting Plasmid
Embryonic Stem (ES) cells
2.
3.
4. 2
neo r
tk
1
Incorrect Genomic Targeting
2
neo r
7.
tk
2
neo r
2
3
tk
4
Correct Genomic Targeting
1
6.
2
neo r
4
Blastocyst
5.
FIGURE 4 Construction of a gene replacement mouse model. A basic gene replacement construct consists of two genomic homology arms (~1–5 kb) derived from sequences flanking the exon to be deleted (1), an intervening marker (e.g., neomycin resistance, neor) for positive selection in embryonic stem (ES) cell culture, and a distal marker (e.g., thymidine kinase, TK) for negative selection in ES cells. The construct is transfected into ES cells, and the cells are grown in media containing neomycin (positive selection) and gancyclovir (negative selection). Cells with random insertions (3) retain both the neor marker and the TK marker and are killed by the conversion of gancylovir to a toxic product by TK. Correct homologous recombinants (4) retain the neor marker but lose the TK marker and thus survive the selection. Clones with correctly targeted genes are injected into donor blastocysts (5), which are then implanted into surrogates to give birth to mice chimeric for the mutation. The chimeric mice are bred to produce mice carrying the mutation in their germline.
Gene Replacement (Knockout) Approaches to Modeling Epilepsy
enzymatic active site, an ion channel pore-forming region, the DNA-binding region of a transcription factor, among others. In some cases, the promoter can be targeted to disrupt gene function; however, because cryptic promoters may be present that can take over and drive expression even after the core promoter is deleted, protein coding regions of genes are generally considered better targets. Next a gene replacement plasmid is constructed that contains four basic elements: a 5¢ homology arm, a 3¢ homology arm, a positive selection cassette, and a negative selection cassette. The homology arms, which are the critical component regulating homologous recombination, flank the exon(s) targeted for deletion. The size of these arms may vary, but both are usually larger than 1 kilobase (kb). A typical construct might contain one arm that is 1 kb long and the other of 3 to 5 kb long. Larger homology arms may not improve targeting efficiency appreciably, but it may be helpful to select arms that do not contain repetitive elements such as L1, B1, or B2 retrotransposons. Homology arms may contain exons or other functional elements in the gene that are not targeted for deletion. It is arguable whether or not the DNA cloned into the targeting construct should be isogenic with the strain of embryonic stem cell line, but this may improve efficiency. The selection cassettes are meant to function in cultured embryonic stem cells and usually consist of a strong, ubiquitous promoter, such as the phosphoglycerate kinase (PGK) or herpes simplex virus (HSV) promoter, driving expression of the selectable marker. The purpose of both markers is to allow identification of embryonic stem cell clones that have undergone successful homologous recombination. The positive selection marker (usually the neomycin resistance gene, neor) allows for survival of embryonic stem cells that have integrated the targeting construct (by either homologous or nonhomologous recombination) when the cells are grown in media containing neomycin, or G418. The positive selection marker is cloned between the two homology arms. The negative selection marker enriches for embryonic stem cells that have incorporated the targeting construct by homologous recombination, and it eliminates those that integrated the targeting construct by random, nonhomologous insertion. A common negative selection marker is thymidine kinase (TK), which converts gancyclovir added to the embryonic stem cell culture medium into a toxic product. The negative selection marker is cloned at one end of the targeting vector. On random, nonhomologous insertion (like a typical transgene), the entire vector is inserted, including both the neor and TK selectable markers. In cells that have undergone homologous recombination, only the positive selection marker (neor) is inserted; the negative selection marker (TK) lies outside the region of homology and does not integrate into the chromosome. Many investigators, however, do not use negative selection cassettes in their targeting construct, arguing that it does not significantly
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increase the percent of embryonic stem cell clones that are accurately targeted. The gene replacement plasmid is transfected into embryonic stem cells by electroporation or lipofection, and (in our example) the cells are selected in media containing G418 (positive selection) and gancyclovir (negative selection). Genetically altered mice can exhibit variable phenotypes as a result of differences in genetic background, and this feature is partly controlled by the choice of embryonic stem cell strain. Historically most mouse strains proved refractory to the derivation of embryonic stem cell lines (Gardner and Brook, 1997), and investigators were limited to using only one or two strains, such as 129/SvJ or C57BL/6J. However, recent advances in cell culture technology are overcoming the biological barriers and permitting the efficient derivation of embryonic stem cell lines from additional mouse strains (Schoonjans et al., 2003). Following selection, the genomic DNA of surviving targeted ES cell clones is tested by PCR or Southern blot analysis to verify that the correct homologous recombination event has occurred. This assay is a critical step; sometimes only a small fraction of clones (<5%) will be accurately targeted even though those cells survived the selection process. Some investigators perform a karyotype analysis of targeted clones to identify chromosomal rearrangements or polyploidy-aneuploidy events that may have occurred in culture. Correctly targeted embryonic stem cells are microinjected into normal donor mouse blastocysts, where they mix with the population of normal embryonic stem cells that constitute the inner cell mass of the early embryo. The injected blastocysts are implanted into surrogate females, where they implant in the uterus and develop until birth about 18 to 20 days later. These mice will be chimeric; that is, their bodies will be composed of a mixture of cells derived from the targeted embryonic stem cells and from the normal recipient blastocyst. Usually the targeted embryonic stem cells and the recipient blastocysts are derived from mouse strains with different coat colors so that when the chimeric mice are born, they are visibly chimeric (e.g., black and white patched coats). These chimeric mice may or may not show a phenotype, depending on the function of the targeted gene, whether or not it acts in a cell-autonomous fashion, the overall percentage of cells in the chimeric animal that are targeted, and which tissues these cells constitute. It is also important to remember that the targeted cells in a chimeric animal are heterozygous for the mutation. However, methods do exist to knockout both alleles of a gene during the embryonic stem cell culture phase, utilizing two different positive selection markers and a sequential transfection-selection approach so that targeted cells in the chimeric mice are homozygous for the mutation. The chimeric mice are then bred with wild-type mice. For those chimeras where the targeted embryonic stem cells had populated the germ tissue, 50% of the offspring will be
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heterozygous for the mutation in every cell. These heterozygotes are then bred to produce wild-type offspring that are heterozygous for the targeted alteration or homozygous for the alteration (with a 1 : 2 : 1 ratio). Alterations from this ratio might be observed if the targeted gene affects prenatal viability. Variations of the Basic Gene Replacement Strategy Numerous variations of the basic gene replacement strategy have been developed to add efficiency, flexibility, and power to the approach (Figure 5). Many of these rely on use of the Cre-loxP or flipase-frt site-specific recombinase (SSR) systems. SSRs are enzymes that recognize specific DNA sequence motifs and recombine the DNA between them. If two recombinase recognition sites are placed on the same DNA strand in the same orientation, the recombinase will delete the sequences between them. If the sites are oriented in the opposite directions, the recombinase will invert the sequences between them. The distance between the two recognition sites may be very short (<100 base pairs [bp]) or very large (>100 kb). If the sites are placed on different DNA fragments, even on different chromosomes, the recombinase will mediate a translocation event. Cre recombinase is a native component of the P1 bacteriophage, while Flp recombinase (flipase) was isolated from Saccharomyces cerevisiae (yeast). The Cre and Flp recombinases recognize 34-bp target sites, called loxP and frt, respectively. Their small size allows loxP and frt sites to be placed into a gene intron, promoter, or untranslated region without noticeably disrupting gene function. One use of the SSR system is to create an excisable selection cassette (see Figure 5). The presence of a positive selection marker (e.g., neor) is required for selection in embryonic stem cells, but is often undesirable after the knockout is established. If the selection cassette is flanked by loxP sites, it can be deleted (after a viable targeted embryonic stem cell clone is identified but before they are injected into blastocysts) by the transient expression of Cre recombinase in those cells. All that remains at the site formerly occupied by the selection marker is one copy of the 34-bp recognition site. Another important use for an SSR system is to produce a gene knock-in. With a knockout, the goal is to obliterate gene function completely, so it is largely irrelevant whether large fragments of foreign DNA are incorporated into the locus in the homologous recombination process. However, in a gene knock-in, the goal is to replace an endogenous exon with a mutant version of the same exon and to allow the gene to continue to function so that the effect of that mutation on protein expression can be assessed. In this case removal of the positive selection cassette is absolutely critical because even if it is located in an intron, it will significantly disrupt either or both expression and splicing patterns of the gene. Positioned correctly, so as not to inter-
fere with intronic enhancer elements or splicing donor or acceptor sites, the residual 34-bp recognition motif should not interfere with gene function. One benefit of producing a knock-in construct is that once a correctly targeted embryonic stem cell clone is identified, it can be used to produce both a mild knock-in and a severe knockout allele by either expressing Cre or not before injection of the cells into blastocysts. The SSR systems can also be used to create inducible (i.e., “conditional”) gene knockout models. The goal with an inducible knockout is to produce a targeted allele where a critical exon(s) is flanked by two loxP (or frt) recognition sites that do not interfere with normal gene function. This requires a much more complicated series of manipulations than necessary for a basic gene replacement, including the employment of two distinct SSR systems. First a targeted allele must be produced and the selectable marker removed (e.g., using the flipase-frt system) while the targeted exon remains intact but flanked with different SSR recognition sites (e.g., loxP sites). These embryonic stem cells are microinjected to produce a mouse with a normally functioning, but silently targeted, allele. A gene with two loxP sites in place, waiting to be recombined to delete an exon, is said to be floxed. A floxed gene can be disrupted, or “turned off,” by later expressing the second recombinase (e.g., Cre) in the mouse. The power of the inducible knockout is that the Cre can be expressed in only some of the cells of the floxed mouse, or only at a specific stage of development, if desired. There are two general strategies to express Cre in a floxed mouse: either as a transgene or, less commonly, as delivered by a viral vector. In either case, the approach is limited only by the availability of well-characterized tissue-specific promoters that can be used to drive Cre expression. Alternatively Cre can be expressed under the control of an inducible promoter, such as the tetracycline-inducible promoter described previously, to add an additional level of spatiotemporal control over recombination of the floxed gene. One of the drawbacks of conditional knockouts is that 100% recombination of the targeted gene is rarely achieved (depending on what method or promoter is used to drive Cre expression), but the levels achieved are usually acceptable for most purposes. Additional resources on the theory and method of gene replacement, including detailed protocols, include the following: Joyner (2000), Turksen (2001), Tymms and Kola (2001), and Nagy et al. (2002).
Examples As of this writing, 26 genes encoding ion channels, transporters, or other critical components of neurotransmission pathways have been altered by gene replacement in the mouse (see Table 1). Of these, spontaneous seizures were reported in 15 cases, a reduced threshold to chemically or
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Gene Replacement (Knockout) Approaches to Modeling Epilepsy
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FIGURE 5 Alternative gene replacement designs. The use of site-specific recombination (SSR) systems (gray arrows) permits useful modifications to the basic knockout construct design. An excisable selection cassette flanked by 34-bp loxP sites allows added Cre recombinase to delete the marker after positive selection in ES cells but before introduction into mice, thus preventing read-through transcription by the selectable marker promoter from transcribing downstream gene elements or interfering with the expression of neighboring genes. A knock-in is similar, but it replaces an endogenous exon with a mutant exon (asterisk), leaving only a single 34-bp loxP recognition site within an intron. Rather than obliterating gene function completely, a gene knock-in allows the gene to continue expressing in a normal pattern and thus allows for analysis of more subtle or gain-of-function mutations. An inducible, or conditional, knockout utilizes two different SSR systems (e.g., Cre-loxP and flipase-frt) to allow the targeted allele to function normally (b) until the investigator decides to induce deletion of the “floxed” exon (c). This permits mutations that would normally result in embryonic lethality to be studied in adult mice or limits the mutation to a specific subset of cells. The diagonal lines represent recombination events; a, b, and c represent sequential steps in the modification of targeting constructs following homologous recombination in embryonic stem cells.
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audiogenically induced seizures in 6 cases, and increased resistance to induced seizures in 2 cases. A mouse model of Smith-Magenis syndrome (SMS), which relied on gene replacement to alter an entire chromosome region rather than just a single gene, also exhibited spontaneous seizures (Walz et al., 2003). In some mutated strains, the seizure or excitability phenotype was not investigated (or at least not reported), presumably because the model was generated for purposes other than epilepsy research. In other cases, independent models were created by targeting the same gene; in these cases differences in targeting strategies, mouse genetic strains, or subsequent methods of analyses led to discordant reported phenotypes. Finally, in some cases the mouse phenotype was similar (i.e., obviously relevant) to the clinical phenotype of human epilepsy patients carrying mutations in the same gene; in other cases, there was no apparent correspondence between the mouse and human phenotypes. A few of these cases are considered in greater detail to illustrate particular points regarding the development and analysis of mouse gene replacement models of epilepsy: Cacna1a Spontaneous mutations in the voltage-gated Ca2+ gene, Cacna1a, are responsible for a phenotype that includes ataxia and generalized absence-like seizures (Fletcher et al., 1996) as well as an altered threshold for cortical spreading depression (Ayata et al., 2000) in the tottering mouse. A functionally null knockout of mouse Cacna1a also produces ataxia and absence-like seizures but results in lethality by postnatal day 30. Mutations in human Cacna1a are associated with several distinct disorders, including familial hemiplegic migraine (FHM), episodic ataxia type 2 (EA-2), and spinocerebellar ataxia type 6 (SCA-6) (Ophoff et al., 1996; Zhuchenko et al., 1997). Given these phenotypes, the spontaneous and knockout mutations in mouse Cacna1a originally appeared to be more accurate models of human ataxia and FHM caused by mutations in this gene. Recently, however, Jouvenceau et al. (2001) and Imbrici et al. (2004) identified human mutations that were associated with inherited ataxia plus primary generalized epilepsy. Thus Cacna1a provides an example of a mouse model of epilepsy where the complex phenotype (ataxia plus seizures) corresponds to only a small subset of human patients with mutations in this gene. Cacna1g Synchronized thalamocortical spike-wave discharges are a basic pathophysiologic finding in absence epilepsy and are believed to depend strongly on the activity of the lowvoltage-activated T-type Ca2+ channel. Three genes can encode a pore-forming alpha subunit capable of mediating T-type Ca2+ currents: Cacna1g, Cacna1h, and Cacna1i. To
examine the role of one of these genes in absence seizures, Kim et al. (2001) inactivated mouse Cacna1g by gene replacement. These authors reported a lack of burst firing by thalamocortical relay neurons and resistance to the generation of spike-and-wave discharges in response to GABAB receptor activation. Subsequent analysis of double-mutant mice (Cacna1g -/- and Cacna1a -/-) extended these findings to show a complete loss of T-type Ca2+ currents in thalamocortical neurons and no evidence of the spike-wave discharges seen in Cacna1a single-mutant mice. Mutations in human Cacna1g have not yet been identified. Whereas the Cacna1g knockout mouse cannot be considered a model of epilepsy in the strictest sense (because it does not exhibit spontaneous seizures), it nonetheless demonstrates the importance of the a1G T-type Ca2+ channel for mediating absence seizures in the thalamocortical pathway and the value of the gene replacement approach for elucidating seizure mechanisms. Clcn2 Haug et al. (2003) reported mutations in the human voltage-dependent Cl- channel gene CLCN2 in patients with a variety of seizure types, including childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA). Bosl et al. (2001) previously described the phenotype of mice with targeted inactivation of the Clcn2 gene but reported the conspicuous absence of expected seizures. Instead, Clcn2-null mice exhibited a severe degeneration of cells in the retina and testes, suggesting a possible role for Clcn2 in controlling the ionic environment of these cells. The basis for this dramatic interspecies difference in mutant phenotypes is unclear. Gabbr1, Gabra1, Gabrb3, Gabrg2 Genes encoding subunits of the ionotropic GABAA receptors and metabotropic GABAB receptors are excellent candidates for involvement in epilepsy, given the central role of GABA in mediating inhibition in brain. Mutations in two of these genes have been associated with epilepsy in humans: GABRA1 with juvenile myoclonic epilepsy (Cossette et al., 2002), and GABRG2 with generalized epilepsy with febrile seizures plus (GEFS+), childhood absence epilepsy, and febrile seizures (Baulac et al., 2001; Wallace et al., 2001). Mice carrying knockouts in these genes and two other GABA receptor genes (Gabbr1 and Gabrb3) have been produced. Null mutations in Gabbr1 resulted in spontaneous clonic, tonic-clonic, and absence seizures as well as hyperalgesia, memory impairment and premature death (Prosser et al., 2001; Schuler et al., 2001). Seizures were not reported to result from inactivation of Gabra1 (Sur et al., 2001; Vicini et al., 2001). A knockout of Gabrb3 caused fre-
Gene Replacement (Knockout) Approaches to Modeling Epilepsy
quent myoclonus and occasional epileptic seizures in addition to cleft palate (Homanics et al., 1997). It is possible that the Gabrb3-null mouse seizures are a model of the seizures observed in some Angelman and Prader-Willi syndrome patients because this gene is contained within the region (15q11–q13) affected by the large deletions and translocations that are associated with these two syndromes (Dooley et al., 1981; Veenema et al., 1984; Wagstaff et al., 1991). However, mutations in human GABRB3 were also recently found in patients with chronic insomnia (Buhr et al., 2002). Finally, a knockout mutation of mouse Gabrg2 was shown to result in perinatal lethality in one study (Gunther et al., 1995), but to cause epilepsy with lethality around 4 weeks in another (Schweizer et al., 2003). The reason for this age disparity in mortality between the two knockouts is unclear, but it might relate to differences in the genetic background of the mice. Gad2 No mutations in the human GAD2 (glutamate decarboxylase 2) gene have yet been associated with seizures, although this gene is believed to be critical for mediating inhibition in brain through production of the inhibitory neurotransmitter, GABA. Three knockout studies of mouse Gad2 have been reported. Interestingly, the phenotypes varied. In one case, the Gad2-null mice exhibited no spontaneous seizures, but seizures were more easily induced by PTZ or picrotoxin (Asada et al., 1996). In the other two studies, spontaneous seizures and increased mortality were observed (Kash et al., 1997; Yamamoto et al., 2004). Interestingly, Kash et al. (1997) reported that the seizures were genetic strain-dependent—which might explain the phenotypic variability observed in the different studies. Gria2 AMPA-type ionotropic glutamate receptors, composed of four related subunits (GRIA1-4) in various combinations, mediate the fast component of excitatory postsynaptic currents in CNS neurons. Inclusion of the GRIA2 subunit in AMPA receptors decreases Ca2+ permeability significantly (Hollmann et al., 1991). The human and mouse GRIA2 genes both undergo functionally important mRNA editing at a site corresponding to the second transmembrane domain of the receptor. No human mutations in GRIA2 have yet been associated with epilepsy and, consistent with this, both knock-out and knock-in mutations of mouse Gria2 protein coding sequences do not result in seizure phenotypes (Jia et al., 1996; Kask et al., 1998; Sans et al., 2003). Interestingly, a knock-in mutation that alters the intronic mRNA editing site of Gria2 resulted in seizures and early lethality (Brusa et al., 1995; Feldmeyer et al., 1999). This example demonstrates a potential role for mRNA editing in epilepsy, and shows that gene replacement approaches can be useful for
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studying non-coding gene regions as well as coding sequences. Kcna1 Mutations in human KCNA1 usually result in episodic ataxia-myokymia syndrome without any seizures or EEG abnormalities (Browne et al., 1994), but rare mutant alleles have been identified that result in tonic-clonic and simple partial seizures with myokymia and no ataxia (Eunson et al., 2000). A spontaneous mutation in mouse Kcna1 is associated with megencephaly and recurrent behavioral seizures (Donahue et al., 1996; Petersson et al., 2003). Complete inactivation of mouse Kcna1 via gene replacement resulted in frequent spontaneous seizures through adult life as well as abnormal axonal action potential conduction in peripheral nerve which may be related to the pathophysiology of episodic ataxia-myokymia (Smart et al., 1998). Kcnq2 The protein products of the KCNQ2 and KCNQ3 genes co-assemble into a channel to produce the M-type K+ current in neurons, a slowly activating and deactivating current important for regulating intrinsic membrane excitability and response to synaptic input. Mutation of either of these genes in humans results in benign familial neonatal convulsions (BFNC) (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998). Complete inactivation of Kcnq2 by gene replacement in mice resulted in homozygous perinatal lethality. Mice heterozygous for the mutation did not exhibit spontaneous seizures but showed a decreased threshold for seizures induced by the convulsant PTZ (Watanabe et al., 2000). Peters et al. (2005) used a novel transgenic approach to conditionally suppress M-channels in mouse brain and demonstrated their function in neuronal excitability and behavior. In this way these investigators obtained information similar to what might have been obtained by a knockin mutation with incomplete loss of function but in only a fraction of the time. Scn1b, Scn2a, Scn2b Mutations in human SCN1B are associated with generalized epilepsy with febrile seizures plus (GEFS+) (Wallace et al., 1998). A complete knock-out of the orthologous mouse gene, Scn1b, also results in spontaneous generalized seizures and should be a good model for studying the human disorder (Chen et al., 2004). Targeted inactivation of the closely related mouse Scn2b gene did not cause spontaneous seizures but did lead to increased susceptibility to pilocarpine-induced seizures (Chen et al., 2002). Mutations in human SCN2A are also associated with generalized epilepsy with febrile seizures plus (GEFS+) (Sugawara et al., 2001) but cause benign familial neonatal-infantile seizures
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(BFNIS) in other cases (Heron et al., 2002). While a transgenic mouse overexpresing a mutant form of Scn2a does exhibit spontaneous seizures (Kearney et al., 2001), the functionally null knockout of Scn2a causes recessive perinatal lethality with no seizures reported (Planells-Cases et al., 2000). A wide variety of different human Na+ channel mutations have been reported, associated with a range of seizure phenotypes; it seems likely that these genes will continue to provide a rich source of mutations to be modeled by knock-in approaches in the future.
Limitations and Pitfalls
partial function or gain a novel function. A targeted allele might skip a deleted region by alternative pre-mRNA splicing, use alternative or cryptic promoters to produce a novel protein fragment, or transcribe straight through an inserted selection cassette (allowing initiation of translation from downstream start codons). Even a small protein fragment could retain the ability to interact with other proteins in the original pathway or multi-subunit complex and produce a dominant-negative effect. This pitfall can be minimized by careful initial design of the targeting construct and by post knockout screening for residual mRNA and protein products expressed from the targeted locus.
Expense and Time
Selection Cassette Interference
Gene knockout models are at least an order of magnitude more costly to produce, on average, than standard transgenic models. Much of this extra cost is due to the extensive embryonic stem cell culturing and analysis that must be carried out to produce a knockout model. Knockout models also require significantly more time to generate than transgenics. Hemizygous transgenic mice (the founder generation) can be born as few as about 20 days after the transgene construct is microinjected into donor zygotes. Knockout mice, on the other hand, typically require several months after the targeting construct is built just to obtain a clonal embryonic stem cell line with an appropriately targeted gene. The embryonic stem cells must then be microinjected into blastocysts and implanted into surrogate mothers to wait another 20 days before heterozygous chimeric mice can be born. These heterozygous chimeric mice must then be raised to breeding age and interbred at least once to produce homozygous germline mutants (total time from construct to mutant is 1 to 2 years).
The selection cassettes used in standard replacement-type constructs usually contain very strong promoters. Transcription from these promoters may lead to transcription of any remaining parts of a target gene or interference with the normal expression pattern of a neighboring gene and thus confuse interpretation of the experiment. The solution to this problem is to remove the selection cassette following confirmation of homologous recombination in embryonic stem cells using one of the available recombinase systems, such as CRE or FLP (see preceding discussion).
Genomic Redundancy and Cooperative Pathways One of the problems that complicates evaluation of mouse gene knockout phenotypes is the relatively common phenomenon of redundant or interacting genetic pathways (for an example of how this can occur in a mouse epilepsy model, see Burgess et al., 1999). Thus the absence of an abnormal phenotype in a knock-out mouse does not necessarily indicate that the targeted gene is not important for regulating neuronal excitability or synchronization. Elucidating its role might only be revealed by inactivating a compensating gene or gene pathway. Incomplete Inactivation When the goal of a gene knockout project is to obliterate gene function to create an epilepsy phenotype, but a targeting strategy cannot be designed that will completely delete all coding sequences of the target gene, the targeted allele might express a truncated form of the protein and thus retain
Disruption of Overlapping or Adjacent Genes The creation of a significant alteration in a target gene could also lead to the unintentional mutation of as yet unidentified genes, for example, genes residing in introns of the target gene or overlapping the target gene but encoded by the opposite strand. It is also conceivable that the target gene contains within its structure one or more regulatory elements important for controlling the expression of unrelated nearby genes. A related possibility is that any alteration in chromatin structure in a knocked-out gene, regardless of how minor, could be propagated to affect one or more adjacent genes. If any of these situations occurred, the resulting phenotype would not accurately reflect alterations in the target gene alone. A brief analysis of the expression function of adjacent genes is recommended to detect this potential problem.
ANALYZING MOUSE GENETIC EPILEPSY MODELS AND INTERPRETING PHENOTYPES Creating a mouse strain with a targeted alteration of one or more endogenous genes in its genome, or carrying an added transgene, is only the first step in the process of developing a seizure or epilepsy model. An exhaustive characterization of the phenotype must then be performed. Typically the first question asked is, Do the mice exhibit seizures? What is usually meant by this question, however, is, Do the
Analyzing Mouse Genetic Epilepsy Models and Interpreting Phenotypes
mice exhibit spontaneous seizures? This question is more difficult to answer than it might initially appear.
Spontaneous Versus Induced Seizures As a platform for launching investigations into the causes, pathophysiologies, and treatments of seizures and epilepsy, mouse models are typically placed into either of two categories: induced models or spontaneous models. Induced Models Mice carrying various mutations may have altered thresholds for seizure induction even if they do not exhibit spontaneous seizures. Depending on the specific stimulating paradigm, induced seizures may also lead to epilepsy (defined as recurrent unprovoked seizures) in which the seizures no longer occur only as an immediate and one-time response to the stimulus but instead persist long after the stimulus is applied (perhaps for the lifetime of the animal). In this case the brain structure and function presumably have been altered by the induction process or, secondarily, by the induced seizures themselves in a more significant and permanent way. Spontaneous Models Spontaneous seizures are often defined as “seizures that the investigator did not intentionally provoke,” a definition that applies equally well to the seizures of both spontaneously occurring mutants and gene-targeted strains of epileptic mice. However, because we do not yet really understand what triggers individual seizures in most cases (an exception being true “reflex seizures”), the word spontaneous is not especially informative. Largely because of the difficulty in precisely defining spontaneous seizures, there appear to be no clear biological criteria with which to separate mice with spontaneous seizures epilepsy from mice with induced seizures-epilepsy. As an alternative to a binary “spontaneous or induced” system of classification, a more realistic model postulates that all mice (strains of mice as well as individuals within a strain) exist along a continuum of high-to-low intrinsic seizure susceptibility (i.e., determined genetically) and that fluctuations in the internal or external environment of the animal are responsible for triggering seizures as well as determining seizure frequency and possibly modulating duration and severity (see Figure 1).
Analyses of Model Phenotypes The following outline offers some minimal analyses that should be performed to characterize a potential mouse seizure or epilepsy model.
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Visual Observation Does the animal’s size and weight and other gross morphology appear normal compared with controls? Autopsy of a few heterozygous and homozygous animals should be performed to inspect internal organs for gross abnormalities. Does the mouse’s diurnal and nocturnal behavior appear normal? Are there any “spontaneous” behavioral seizures? Are there signs of lethargy or hyperactivity? Do the animals interact normally with littermates? Viability-Mortality and Fertility-Fecundity Do colony records indicate any change in average lifespan or number of offspring (compared with wild-type controls of the identical background strain)? Video Electroencephalography Behavioral convulsions and seizures and other abnormal motor activities do not always correlate with atypical electrical activity in brain, and abnormal electrical activity in brain does not always produce aberrant motor behaviors in the behaving animal. Video-EEG (V-EEG) should be performed to evaluate the correlation of behavioral and electrical phenotypes and allow spatiotemporal correlation of any behavioral and electrical activity. V-EEG should be carried out at different times of night and day and in mice of different ages and genders. When possible, depth electrodes should also be used to detect limited focal abnormalities. Seizure Thresholds In conjunction with visual observation and V-EEG, several methods can be used to determine whether a mouse has a lowered threshold for seizures, even when no spontaneous seizures are detected. These include testing the threshold for seizure activity (time to onset, duration, frequency, severity, and refractory period) after treatment with chemical convulsants (e.g., pilocarpine, kainic acid, pentylenetetrazole), electroconvulsive stimulation, physical handling, loud noises, and temperature extremes. Pathology The brain should be examined for major structural abnormalities (e.g., dysplasia, heterotopia) and changes in the onset and frequency of necrotic and apoptotic cell death. Are average cell counts normal for different brain regions? Is gliosis apparent? The microanatomy of various neuron populations should be examined and compared with normal controls. Is axonal sprouting evident? Are dendritic branching patterns normal? Are dendrites hyperspiny or hypospiny?
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Electrophysiologic Analyses The electrophysiologic properties of cells expected to express the targeted defect should be analyzed by appropriate techniques. For example, if the model is a Ca2+ channel gene knockout in hippocampal CA1 neurons, studies should be carried to determine whether these neurons show abnormal Ca2+ currents, abnormal transmitter release, and other abnormalities. Genomic Analyses One form of molecular analysis that is becoming common is to screen for global changes in gene expression by hybridization to cDNA microarrays. A targeted mutation might result in no obvious seizure phenotype, but there may be evidence from gene expression studies that the genome of that organism is compensating by changing the expression of a few (or many) other genes. Such information can be very valuable for elucidating activity-dependent regulatory pathways that could lead to clues about seizure prevention. Genetic Analyses An attempt to create a mouse model of epilepsy by any form of genomic manipulation should not be considered a failure until the alteration is studied on the genetic background of several different mouse strains. From the very beginning, if possible, a targeted mutation or transgene should be bred onto at least two genetically distinct strains. It is quite clear that different mouse strains can have dramatically different seizure thresholds; a gene knockout on one strain may cause frequent seizures while the same mutation on a different strain could cause no obvious change in phenotype. Furthermore, a strain that is exquisitely sensitive to one kind of seizure (or seizure-induction method) is not necessarily the most sensitive to another method of seizure induction.
CURRENT MOUSE MODELS OF SEIZURE AND EPILEPSY TARGETING ION CHANNELS AND NEUROTRANSMISSION PATHWAYS A current summary of mouse models of seizures and epilepsy resulting from the targeting of genes for ion channels, transporters, and neurotransmission pathways is presented in Table 1. Several of the mutations in this table were discussed in more detail in preceding sections of this chapter. Thirty-three individual genes are represented as well as a large targeted mouse chromosomal rearrangement
associated with seizures (a model of SMS). The corresponding spontaneous mouse and human mutations are also listed, if they are known, for comparison. In some cases the mouse model appears to recapitulate faithfully a human seizure disorder caused by mutation of the orthologous gene (e.g., Scn1b), but in other cases it does not (e.g., Clcn2). Unfortunately, most mouse models shown belong to the latter category. As discussed previously, such results should not lead to dismissing that model out of hand but rather to studying it further, such as via introduction of a different mutation into that same mouse gene, selection of a different genetic strain background to carry the mutation, a change in the habitat-environment of the mouse, or more extensive phenotyping.
OUTSOURCING MOUSE MODEL PRODUCTION Maximizing Efficiency Two of the most important questions an investigator should ask before committing the significant resources needed to develop a mouse model are, Is the mouse I want to make already available? and Should I do some or all of the work in my own laboratory? The first question can be much more difficult to answer than it first appears. Although most scientists are familiar with the literature in their own areas of expertise and would probably be aware of a published model, many transgenic and gene knockout mice are in various stages of production in academic laboratories or have been commercially developed and not yet been publicized. Thus it is always a good idea first to contact prominent researchers in the field who might have heard that a mouse already exists, is being made, or is about to be published. Commercial mouse vendors can also be good sources of this information. A small amount of preliminary detective work could save months or years of bench work that otherwise would have been spent duplicating an existing resource. The second question is usually much easier to answer. Most laboratories that intend to develop a new animal model, including those focused on epilepsy research, are not at all equipped to carry out each of the steps involved in making either transgenic or gene replacement models. Typically the individual laboratory builds the transgene or genetargeting construct, which requires only the most basic molecular biology skills and equipment. The subsequent steps—microinjection into fertilized eggs (transgenics) or embryonic cell transfection-selection or microinjection into blastocysts (gene replacement)—are usually performed through either an academic core facility or a commercial animal production service.
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Academic and Commercial Transgenic and Knockout Facilities Access to on-campus transgenic and knockout core facilities at most major research universities and medical centers provides a significant advantage to scientists who wish to create genetic animal disease models. These facilities usually provide staff that can assist researchers with designing transgene and knockout constructs, providing a wide range of vectors and protocols, engineering the constructs, creating the mice, genotyping and housing them, and arranging maintenance and test-breeding schemes (all for a fee). Many academic facilities will also provide at least some forfee services to investigators outside of the institution, which can prove an invaluable resource for small college laboratories and clinician-investigators associated with hospitals. Finally, several academic core facilities provide Internetbased learning resources and specific protocols that can help guide investigators through every technical aspect of mouse model development. A perusal of these diverse resources is highly recommended for anyone planning to work with transgenic or knockout mouse models, for example, the University of Michigan Transgenic Animal Model Core (URL: http://www.med.umich.edu/tamc/) and the Darwin Transgenic Mouse Core Facility at Baylor College of Medicine (URL: http://imgen.bcm.tmc.edu/dtmc/). Several commercial and nonprofit entities either sell existing mouse disease models or work with investigators to develop custom transgenic and gene knockout mice. A custom-made, conditional gene knockout can be made commercially from start to finish, but the prices are more than most academic researchers can afford. The world’s largest current supplier of different mouse strains, including transgenic mice, spontaneous mutants, and gene knockout mice, is The Jackson Laboratory, a nonprofit institution located in Bar Harbor, Maine. The Jackson Laboratory also maintains extensive public databases with information on practically every aspect of mouse genetics and bioinformatics. Other significant mouse model resources, several of which offer custom transgenic, gene knockout, and model development services, are Taconic, Harlan, Charles River Laboratories, Lexicon Genetics, B&K Universal Ltd, Xenogen, Deltagen, Ozgene, and Genoway.
phenotypes (Austin et al., 2004; Auwerx et al., 2004). Complementary projects will study the expression patterns of all these genes (Su et al., 2002; Zhang et al., 2004) and begin looking at the proteome produced after this genome is translated (Marcuset et al., 2004). From the phenotype and genotype starting lines, analyses of mechanistic pathways will proceed in both directions, each toward the other, and one day meet in the middle (Ashburner et al., 2000). If successful, epilepsy research will benefit tremendously. Of course, not everybody is enthusiastic about the prospects (or wisdom) of such grandiose biology projects (Accili, 2004; Huber, 2003). In the meantime, several techniques are emerging that will be of much more immediate use for facilitating the creation of novel mouse models of epilepsy. In particular the ongoing development of RNA-interference (RNAi) techniques promises to add a versatile tool to the kit of molecular biologists (Dykxhoorn et al., 2003; Novina and Sharp, 2004). RNAi is a form of posttranscriptional gene silencing that was originally recognized in plants but is now being discovered in a wide range of different organisms (Cogoni and Macino, 2000). Very briefly, expression of a small, doublestranded fragment of RNA (dsRNA), homologous to a target gene, is able to suppress the expression of that gene in that cell. Thus RNAi is similar in effect to a gene knockout by homologous recombination but with the following differences: (1) it is cheaper, (2) it is faster, (3) it is rapidly reversible, (4) it allows for the generation of a graded series of “knock-down” phenotypes rather than only an all-or-none knockout, and (5) multiple genes can be targeted simultaneously in the same animal. On the downside, RNAi will not permit evaluation of most gain-of-function mutations. Further, the methods for delivering the dsRNA payload to target cells in mammals are yet very crude, but these shortcomings seem to be minor in light of the tremendous advantages RNAi promises. The RNAi technique has already been applied to study seizure and epilepsy phenotypes in nonmammalian model organisms (Williams et al., 2004; Zhang et al., 2002), and extension of the methods to mouse and rat models is on the horizon (Hasuwa et al., 2002; Kunath et al., 2003; Kuwabara et al., 2004; Prawitt et al., 2004; Xia et al., 2004).
References SUMMARY AND FUTURE PROSPECTS The use of gene targeting (transgenesis, gene knock-outs and knock-ins) to generate mouse models of epilepsy is expanding rapidly. In the years ahead projects will be initiated that aim to generate a wide variety of mutant alleles for each of the estimated approximately 20,000 to 25,000 genes in the mouse genome and to measure all of the associated
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Walz, K., Caratini-Rivera, S., Bi, W., Fonseca, P., Mansouri, D.L., Lynch, J., Vogel, H., et al. 2003. Modeling del(17)(p11. 2p11. 2) and dup(17)(p11. 2p11. 2) contiguous gene syndromes by chromosome engineering in mice: phenotypic consequences of gene dosage imbalance. Mol Cell Biol 23: 3646–3655. Watanabe, H., Nagata, E., Kosakai, A., Nakamura, M., Yokoyama, M., Tanaka, K., and Sasai, H. 2000. Disruption of the epilepsy KCNQ2 gene results in neural hyperexcitability. J Neurochem 75: 28–33. Williams, S.N., Locke, C.J., Braden, A.L., Caldwell, K.A., and Caldwell, G.A. 2004. Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans. Hum Mol Genet 13: 2043–2059. Wu, X., and Burgess, S.M. 2004. Integration target site selection for retroviruses and transposable elements. Cell Mol Life Sci 61: 2588–2596. Xia, H., Mao, Q., Eliason, S.L., Harper, S.Q., Martins, I.H., Orr, H.T., Paulson, H.L., et al. 2004. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med 10: 816–820. Yamamoto, A., Hen, R., and Dauer, W.T. 2001. The ons and offs of inducible transgenic technology: a review. Neurobiol Dis 8: 923–932. Yamamoto, T., Yamato, E., Tashiro, F., Sato, T., Noso, S., Ikegami, H., Tamura, S., et al. 2004. Development of autoimmune diabetes in glutamic acid decarboxylase 65 (GAD65) knockout NOD mice. Diabetologia 47: 221–224. Zhang, H., Tan, J., Reynolds, E., Kuebler, D., Faulhaber, S., and Tanouye, M. 2002. The Drosophila slamdance gene: a mutation in an aminopeptidase can cause seizure, paralysis and neuronal failure. Genetics 162: 1283–1299. Zhang, W., Morris, Q.D., Chang, R., Shai, O., Bakowski, M.A., Mitsakakis, N., Mohammad, N., et al. 2004. The functional landscape of mouse gene expression. J Biol 3: 21. Zhuchenko, O., Bailey, J., Bonnen, P., Ashizawa, T., Stockton, D.W., Amos, C., Dobyns, W.B., et al. 1997. Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 15: 62–69.
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17 Spontaneous Epileptic Mutations in the Mouse JEFFREY L. NOEBELS
nization, it is worth beginning this discussion with a reminder that it was the entire group of spontaneous mouse mutants that played the groundbreaking role in the neurogenetic analysis of seizure disorders: this model system was the first to demonstrate the significance of single genes in the hereditary transmission of epilepsy in the mammalian nervous system.
Spontaneous mutations in mice, along with those generated by genetic engineering approaches, are essential tools to understand the pathogenesis of human epilepsies. For every inherited seizure disorder in man, a genetically defined mouse mutant can help identify the key molecular mechanisms of excitability and plasticity in the developing epileptic brain, uncover new molecular targets for therapy, and serve as a reproducible biological test system for experimental strategies to prevent or reverse the onset of seizures. This chapter reviews some basic considerations for those investigators in search of a naturally occurring mouse model and provides a coherent experimental framework to analyze a specific pattern of epilepsy. Even for this somewhat limited purpose, the individual models are hardly an end to themselves; in fact, certain questions that arise in their analysis may be better answered using entirely different approaches in other model systems. Furthermore, because species differences may distort the molecular and cellular patterns of a gene defect, the physiologic findings in mouse models must be continuously validated using direct comparisons with previously known or emerging information from the human disease. In many cases the mismatches between human clinical disorders and mouse models are equally as important as the similarities in tracing the molecular pathways leading to epilepsy; in other cases, the differences may point to functionally irrelevant reactive changes. Nevertheless the developmental analysis of these mouse mutants offers an unparalleled opportunity to understand precisely how a single inherited molecular defect alters excitability to give rise to an episodic seizure phenotype. Whereas experimentally engineered epileptic mouse mutants are favored models in neurobiology and neuropharmacology for the study of abnormal brain synchro-
Models of Seizures and Epilepsy
THE MOUSE AND MENDELIAN INHERITANCE OF EPILEPSY Early human genetic studies of epilepsy developed clear evidence for the heritability of idiopathic epilepsy through twin studies. Yet multigenerational pedigrees of epilepsy that demonstrate the influence of single “major” genes are difficult to find, and until relatively recently relied largely on models of complex polygenic inheritance to explain the heredity of idiopathic seizure disorders. One decade before the first of many reports of family studies revealing single loci for human idiopathic generalized epilepsies appeared (Leppert et al., 1989; Scheffer and Berkovic, 2003), several fundamental principles governing the hereditary transmission of epilepsy were derived from the study of single locus spontaneous neurological mutations in the mouse (Noebels, 1979). The first stated that epileptic disorders featuring spontaneous age-dependent seizures resembling those in human generalized epilepsy can be inherited as individual Mendelian traits. At the neurobiological level, this tenet translated directly into both the search for hereditary defects in single molecules that could alter the firing properties of neurons, and the overall strategy of exploring how the expression of these single genetic errors could trigger
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abnormal excitability changes within a restricted set of neuronal pathways at a specific developmental stage. The second principle was derived from the genetic mapping information then available in the mouse, namely, that both convulsive and generalized absence seizure phenotypes observed in spontaneous mouse mutants were genetically heterogeneous. Even without knowing the genes responsible, this evidence for locus heterogeneity foretold the extensive biochemical diversity of inherited epilepsies and predicted both the wide range of clinical variation and the pharmacologic sensitivity of molecular lesions in the human brain that share epilepsy as a common manifestation. Today, now that Mendelian inheritance of human idiopathic epilepsy has been demonstrated many times, the genetics derived from single-locus mutant mice remain of strategic importance in deciphering nonfamilial cases of epilepsy, which constitute the largest fraction of the human patient population. Knowing that a dominant mutation at only a single locus may be sufficient to produce a seizure disorder means that de novo mutation is a far more likely cause of common sporadic cases than if the seizure phenotype required simultaneous mutations in multiple genes. In addition, the recognition that inherited epilepsy is genetically heterogeneous, that is, that many different single genes give rise to the disorder, is a key factor in designing surrogate markers for epilepsy—markers that can be used in epidemiologic surveys, clinical diagnostic screening and even drug discovery. Genetic heterogeneity implies that any single biochemical marker associated with the primary cause (rather than the effect) of cerebral seizure activity will a priori detect only a subset of the population at risk for epilepsy.
A RATIONALE FOR ANALYZING BOTH SPONTANEOUS AND ENGINEERED SINGLE-GENE MODELS Spontaneous mutants with epilepsy arise in both large and small mouse breeding colonies and have been available since the 1950s. Their numbers are limited only by the mutation rate and by the practical difficulties of detecting seizures, an episodic and often subtle neurologic phenotype. Both convulsive and absence seizure phenotypes were represented in the first group of mice analyzed, and at first glance these few models seemed sufficient for basic epilepsy research, particularly once the gene in question was identified. However, by the mid-1990s, genetic engineering technology added an entirely new dimension to epilepsy gene discovery and model building. This “reverse genetic” strategy was built around the premise that if a preselected gene was inactivated and epilepsy was the resulting phenotype, the gene would serve as an excellent candidate for human disease. The first gene deletion study that produced epilepsy
in a mouse, which targeted the protooncogene BMI-1, described seizures as an incidental finding (van der Lugt et al., 1994). This report was rapidly followed by a dozen others demonstrating the molecular diversity of brain synchronization defects (Noebels, 1996). Now, genetically engineered mice with spontaneous seizure phenotypes currently number more than 50, with additional genotypes being continually reported. Genetic access to the brain in these models allows the design and introduction of specific molecular defects into identified cellular pathways, and places every synaptic network within experimental reach (see Chapter 16). In addition, engineered models enable a “gene-forward” approach, with enormous advantages in defining new candidate gene families and signaling pathways for de novo epilepsy model building; these approaches have proven extremely informative in deducing convergent biological pathways for epileptogenesis (Noebels, 2003). Given these experimental advantages, the transgenic animal must be considered an attractive alternative to spontaneous mutants as strategic analytic tools in epilepsy research. Both approaches have provided important insights.
ORTHOLOGOUS VERSUS ORPHAN GENE MODELS OF EPILEPSY In addition to the preceding issue—whether to use a gene-driven or phenotype-driven modeling approach—there is a parallel question that the genetic modeler must address: Which should come first, the human disease or the disease model? Despite the attraction of the transgenic approach, if the goal is to obtain a mouse model of human epilepsy, there is significant risk in selecting an engineered model of epilepsy for study. If a gene is deleted and no seizures result in the animal, has the involvement of that gene in epilepsy been excluded? The answer to this question is clearly no. A null mutation is only one of many potential perturbations of the gene; because epilepsies begin (and end) at different ages and may be present only when the mutant gene is expressed on certain genetic backgrounds, substantial effort must be expended in phenotyping the mutant before it can be assumed that the loss of a particular gene does not produce epilepsy. In fact, a firm conclusion that a given null mutation is not involved in epilepsy is almost impossible to support (the difficulty of “proving” the negative). Thus, although the engineered models can tell us what can happen (if epilepsy is found in the transgenic mouse), they offer no assurance that it actually does or ever will (in humans). This distinction is irrelevant if the goal of the study is strictly to understand the mechanisms of cortical synchronization of the nervous system, but it is critical if the purpose is to obtain models of real human epilepsy, as encountered in the clinic.
Orthologous Versus Orphan Gene Models of Epilepsy
Thus, in asking which genetic model system—spontaneous or transgenic—to select for further study, the answer depends primarily on the purpose of the model. Both spontaneous mouse mutations and transgenic mutants, created without foreknowledge of an epileptic phenotype, are examples of “orphan’ ” epilepsy models, that is, an epileptic lesion with no human counterpart. These models join those created by other nongenetic strategies (chemical-induced, electrical stimulation, focal lesions) in teaching us something important about epileptogenesis. However, they do not precisely recapitulate a clinically relevant molecular mechanism, and thus they cannot be considered an exact model of a human disease until a similar molecular lesion is described in human. In contrast either spontaneous or engineered mutation in a gene shared by both human and mouse, even if the mutation is extremely rare, constitutes an inherently valid orthologous gene model pairing. Such gene pairs are highly prized as intrinsically valid models of human disease. Fortunately, although only a few of the spontaneous mouse mutants are known to have human counterparts, it is safe to predict such genes will be found in the future. One recent example is the report of human absence seizures in a family bearing a mutation of CACNAA1A, the gene encoding the alpha subunit of a P/Q type calcium ion channel. This is the same gene that is mutated in the tottering mouse, the first absence seizure model in mouse, reported 25 years ago (Imbrici et al., 2004; Noebels and Sidman, 1979). A more recent example is the finding of febrile seizures in a family bearing nonsense mutations in MASS1, a gene that was discovered in the Frings audiogenic seizure model only a year earlier (Nakayama et al., 2002; Skradski et al., 2001). The reverse scenario can also be expected. For example, a mouse strain with a decreased seizure threshold was determined to contain a deletion of two contiguous genes previously implicated in human epilepsy syndromes: KCNQ2, a voltage-gated potassium channel that contributes to the hyperpolarizing m-current, and CHRNA4, a nicotinic cholinergic receptor alpha subunit (Yang et al., 2003). Inherited defects in these channel genes had been found a few years earlier to cause BNE1, a benign neonatal epilepsy syndrome, and ADNFLE1, an autosomal-dominant frontal lobe epilepsy (Biervert et al., 1998; Singh et al., 1998; Steinlein et al., 1995). Finally, although certain attributes of both types of genetic models may seem obvious, they are worth restating clearly at the outset. The major virtues of a genetic lesion to an experimentalist are its innate reproducibility and the opportunity it affords the investigator to study the natural history of the disease from embryonic stages through adulthood. The major drawbacks of these genetic models are similar to those encountered when directly studying human patients themselves, namely, the need for comprehensive seizure monitoring (to detect and characterize unprovoked
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and therefore unpredictable seizure events), the difficulty in designing “before and after” studies (it is not currently possible to know conclusively whether an animal has ever had a seizure before analysis commences, and thus the brain can never be considered truly “preclinical” or “naïve”), and the significant time required to breed adequate numbers of affected mice of a specified age (a significant practical problem confronting investigators planning neurophysiologic or neuropharmacologic studies).
Classic Mouse Mutants with Epilepsy: Finding a Spontaneous Mutant With these considerations in mind, the initial step is to identify and obtain an interesting model for study. The first listing of epileptic phenotypes in mouse mutants appeared in 1965 in the slender volume, Catalog of the Neurological Mutants of the Mouse, by Richard L. Sidman, Margaret Green, and Stanley H. Appel. This list was based on purely behavioral observations of “seizures” in mutant mice that arose at The Jackson Laboratory (TJL) in Bar Harbor, Maine. Because of the large mouse breeding colonies in this production facility, behavioral deviants were (and are) relatively common. To this day, FLMs (“funny looking mice”) are collected weekly by the mouse handlers at TJL and displayed for potential selection and further characterization of the phenotype. Once selected, the deviant trait is genetically mapped to a chromosomal linkage region and evaluated for allelism by complementation testing with other known mutants mapped at or near the locus. Remutation of previously known genes is not uncommon, and when available, multiple alleles (an “allelic series”) of the same gene can provide an extremely important resource for genotypephenotype correlations. Such studies can clarify how different alterations in a protein are linked to the pathogenesis and variable phenotypes of the disorder. The initial list of neurologic mutants comprised 92 mice, from agitans to zigzag. Of these, 15 mice were considered to display behavioral “convulsions,” although in retrospect, some of these assumed seizures proved to be nonepileptic paroxysmal movement disorders. At the time, the mice were assigned to a genetic linkage group on a particular chromosome. Since that first listing, almost all of these mutated genes have been molecularly defined, and a few more of special interest to epilepsy research have been added. At least 19 models of epilepsy arising from spontaneous mutations in the mouse are currently known (Table 1). The genes identified in these models constitute only a small fraction of the total number that have been so far linked to human and murine epilepsy (Noebels, 2003). Yet they show a similar distribution in gene function, namely, a majority of genes associated with ion channels compared with other genes of unrelated function.
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TABLE 1 Spontaneous Single-Locus Mouse Mutants with Epileptic Phenotypes Mutant Spike wave Tottering (tg) Lethargic (lh) Ducky (du) Stargazer (stg) SWE (swe) Mocha2j (mh) Coloboma (Cm) Convulsions Dilute lethal (dl) Jimpy (jp) Jittery (ji) Megencephaly (mceph) Quaking (qk) Staggerer (sg) Torpid (td) Varitint waddler (Va) Wabbler-lethal (wl) Weaver (wv) Writher (wh)
Chr.
Gene
Protein
Ref
8 2 9 15 4 10 2
Cacnala Cacnb4 Cacna2d2 Cacng2 NHE1 AP3d Cm
P/Q type calcium channel alpha subunit Calcium channel beta 4 subunit Alpha2delta2 subunit Gamma2 subunit Sodium hydrogen exchanger Delta subunit AP3 adaptor protein Microdeletion including SNAP25 and phospholipase C isoform b1
Fletcher et al., 1996 Burgess et al., 1997 Brodbeck et al., 2002 Letts et al., 1998 Cox et al., 1997 Kantheti et al., 2003 Zhang, Hess et al., 1994
9 X 10 6 17 9
Myo5a plp
Myosin Va Proteolipid protein Novel protein Potassium ion channel Microdeletion, including parkin Nuclear receptor Unknown TRP ion channel Unknown G-protein-coupled potassium channel
Mercer et al., 1991 Dautigny et al., 1986 Bomar et al., 2003 Peterson, 2003 Lorenzetti, 2004 Matysiak-Scholze, 1997
Novel protein Glutamate Receptor delta subunit
Skratski et al., 2001 Zuo et al., 1997
3 14 16
Kv1.1 RORa mcoln3 GIRK2
Convulsions evoked by sensory stimuli Frings 13 Mass1 Lurcher (Lu) 6 GluR d2
Periodically new spontaneous mutants are found in both large and smaller breeding colonies in laboratories around the world. When this occurs, the founder animal is backcrossed to determine whether the deviant trait arises from a germline, rather than somatic, mutation and can be propagated for further study. Traditionally a nickname evocative of some behavioral trait is chosen for the new mutant, although the reader should be aware that there is a committee responsible for adherence to the “Standardized Genetic Nomenclature for Mice,” which asks that the newly discovered mutation be shown through appropriate genetic testing not to be allelic with other mutants—or if it is, that if differs in phenotype from other allelic mutations at that locus. The final name assigned is that of the mutated gene. For example, the mutant mouse rocker is allelic to tottering. Both are mutations of the calcium channel alpha subunit Cacnala, and both display ataxia and absence epilepsy. However, dendritic arbors of rocker Purkinje cells display a distinct “weeping willow” appearance, whereas those of tottering are unaffected (Fletcher et al., 1996; Zwingman et al., 2001). Strains with mutant and wild-type alleles on the same genetic background (congenic strains) are developed by repeated crossing of the mutant to an inbred strain or by brother-sister mating with forced heterozygosis of the mutant. Proper maintenance of strains requires vigilance and a well-defined breeding scheme. Difficulties with viability and fertility are often encountered with neurologic mutants, and propagation of the mutant may be assisted in various
DiPalma, 2002 Slesinger et al., 1996 unknown
ways (Green, 1975); however, the strain designation must reflect any changes in this background. Because mutants originate from many different colonies, the designation of strain origin can be very informative.
Gene Identification and Induced Mutagenesis Strategies Early mutant genes were isolated in a lengthy process by mapping the disease locus, followed by laborious strategies to identify genes contained within the region, allowing the final evaluation of each gene for a mutation. With the application of comparative and functional genomic identification strategies, informative marker strains, and publication of the entire sequence of the mouse genome (Waterston et al., 2002), the initial mapping step narrows the disease locus to a precise region containing a small number of genes (positional candidate gene cloning) that can all be sequenced. The process can now lead to rapid identification of the mutation. Once the fine structure of the single locus mutation has been characterized, mutant alleles are generally found to fall into several different categories. Traits that are transmitted in a Mendelian pattern are often point mutations (e.g., single amino acid substitutions, sequence inversions, or repetitive elements) that lead to gain or loss of function in a single gene. However, there are other possibilities, including duplications or small deletions that encompass several closely linked genes, similar to small contiguous gene deletion syn-
Orthologous Versus Orphan Gene Models of Epilepsy
dromes in man. The pink eye dilution (p) mutation, for example, has seizures and is missing three closely linked subunit genes for GABAA receptors on chromosome 7 (Culiat et al., 1994), and more than 36 variants at this locus have been studied (Johnson et al., 1995). Although the discovery of rare epileptic mutant phenotypes depends on relatively infrequent spontaneous mutation rates, ancillary strategies have been recently employed to accelerate the pace. Induced (but untargeted) point mutations can be obtained by ethylnitrosourea (ENU) mutagenesis in mice, followed by phenotypic screening for excitability variants (Balling, 2001; Bult et al., 2004; O’Brien and Frankel, 2004). Accelerating the mutation rate by the introduction of random single- base-pair changes is a valuable strategy to generate novel phenotypes. However, the phenotype of either epilepsy or altered seizure threshold must still be ascertained in the mutagenized mice, which then require the same detailed gene isolation procedure as spontaneous mutants. Nevertheless, there are two advantages to this approach: first, the acquisition of novel phenotypes arising from random mutation of as yet unstudied genes; and second, the possibility of generating an expanded series of gain of function alleles that differ from those obtained by targeted deletion strategies. Advanced regional chromosomal saturation methods and “oligogenic chromosomal lesions” are also within the reach of gene targeting technology if required to model a human mutation (van der Weyden et al., 2002).
Validating the Epileptic Phenotype Electroencephalographic Recording To satisfy the currently accepted definition of an epileptic seizure, the model must display abnormal synchronous electroencephalographic (EEG) activity as an electrocortical correlate of any paroxysmal behavior. Not uncommonly a spontaneous epileptiform behavioral disturbance originates as a subcortical event, as evidenced by the lack of change in the EEG. This is the case for the sudden myoclonic jerks in spasmodic mice (mutation of the alpha1 subunit of the glycine receptor), which is therefore considered a model of the movement disorder, hyperekplexia, or “startle disease,” rather than epileptic myoclonus (Buckwalter et al., 1993). Some mice may display both an epileptic seizure as well as nonepileptic paroxysmal dyskinesia, two separable disorders arising in independent brain networks. For example, tottering mice display spike-wave seizures with behavioral arrest involving the thalamocortical circuitry; they also exhibit paroxysmal dyskinesias with dystonic features involving pontocerebellar circuitry that are unaccompanied by EEG discharges (Campbell and Hess, 1998; Noebels and Sidman, 1979). To validate the mutant as a model of epilepsy, repeated spontaneous seizures must be recorded
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from neocortex or hippocampus in multiple mutants and compared with control recordings in mice of the same strain that are wild-type for the mutant allele. We have carried out an EEG/photic screen of a large number of mutants in the Jackson Laboratory Mutant Mouse Resource over the years (J.L. Noebels, X. Qiao, and M. Davisson, unpublished). Although the screen validated epilepsy in several mutant strains, more than 100 others, many with neurologic abnormalities, failed to show epilepsy during the EEG monitoring process. Although it is not possible to state categorically that these mice do not have epilepsy at some point in their development, seizures were sufficiently rare to escape detection during the screen (in this case, typically restricted to three affected adult mice for periods from 2 to 10 hours on two or more separate recording days). With that significant caveat, this experience substantially confirms what is well known to clinical neurologists: that seizure disorders, though common, are not an inevitable result of aberrant circuitry or cortical damage and hence represent a specific subset of latent excitability defects in neural networks. Phenotypic Discovery: Surrogate Markers for Screening Because episodic seizure disorders require an abnormal EEG to identify correctly the phenotype, many mutant models of epilepsy have gone unrecognized. Although both video monitoring and EEG seizure-detection algorithms are available to facilitate this step, prolonged monitoring may be required to detect rare events, making alternative screening strategies desirable. Unfortunately only a few reliable or specific biochemical indicators of past seizure history are currently available for this purpose. Regional anatomic rearrangements and molecular plasticity of varying longevity are seen in both human and rodent models of limbic epilepsy and can serve as useful (if not specific) indicators of prior seizure events. One such marker is granule cell axonal sprouting, readily visualized by Timm or dynorphin staining of mossy fibers in the inner molecular layer of the dentate gyrus. Dispersion of granule cell bodies and aberrant axonal reorganization appear to be essentially permanent network changes closely linked to prolonged hippocampal seizures (Houser, 1990; Sutula et al., 1989). Molecular markers induced by seizure-induced changes in gene expression and cell death are also abundant in the hippocampus, but they tend to be shorter lasting (Borges et al., 2003) and are drastically modified by genetic background (Morrison et al., 1996; Schauwecker, 2002). Several other molecules that may be useful for indirect detection of recent seizure activity in adult (Frankel et al., 1994; Storey et al., 2002), but not immature (Storey et al., 2002), brains include the reactive transcription factors C-Fos and JunB and the ectopic expression of neuropeptide Y (NPY) in granule
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cell axons (Tonder et al., 1994). Both mossy fiber sprouting and ectopic axonal NPY expression have been observed in spontaneous epileptic mutants, such as stargazer (Chafetz et al., 1995; Qiao and Noebels, 1993). Identification of seizureinduced neurogenesis among cells located in the infragranular layer of the dentate hilus is not a useful screening tool because the cell division must be labeled at the time of the seizure (Parent et al., 1997). C-Fos activation distinguishes between convulsive (Frankel et al., 1994; Morgan et al., 1987) and absence epilepsy models (Morgan et al., 1987). This observation reinforces the genetic evidence for fundamental differences not only in the circuitry but also in the biology of neuronal synchronization among major EEG and clinical seizure types in the brain. An alternative screening method for detecting epileptic mutants involves acute testing for a lowered seizure threshold induced by auditory activation, convulsant drugs, or maximal electroshock (White et al., 1995). The validity of this approach relies on the assumption that epilepsies arise from circuits that are disinhibited or otherwise more likely to engage in rhythmic bursting; however, this relationship may not always be true. The “seizure threshold” is a separable trait from spontaneous seizures, and mutant mice (like humans) with latent hyperexcitable neural circuits may never display epilepsy; in turn, spontaneously epileptic mice may show only a reduced threshold for triggering seizures if the correct circuit or pattern of activation is applied. For example, P/Q-type calcium ion channel mutants such as tottering and leaner show thalamocortical spike-wave epilepsies; yet cortical excitability is reduced as a result of decreased calcium currents and impaired neurotransmitter release (Ayata et al., 2000; Qian and Noebels, 2000). Mutant mice with a lowered threshold for seizures evoked by one modality may demonstrate an elevated threshold through another pathway. Finally, significant differences in the convulsive threshold exist among inbred strains, which may confound the interpretation of mutations on different backgrounds (Frankel et al., 2001). Nevertheless, the threshold method, though not infallible, has been valuable in identifying certain gene mutations of interest (Yang et al., 2003).
MATCH AND MISMATCH WITH HUMAN SEIZURE DISORDERS: WHAT DOES THE MUTATION MODEL AND HOW WELL? Inherited lesions in the same gene may show widely disparate phenotypes in mice and humans. The correct explanation, most of the time, is that two different functional alleles are being compared. For example, in the Kv1.1 potassium ion channel gene, dominant human point mutations may alter the kinetics of pore inactivation, thereby prolonging depolarization; such mutations are associated with myokymia, episodic ataxia, or a temporal lobe epilepsy phe-
notype (Browne et al., 1994; Zuberi et al., 1999). In the mouse, a deletion of the same channel leads to a more severe convulsive phenotype with cold-induced neuromyotonia (Smart et al., 1998; Zhou et al., 1998); a truncation of the same channel is linked to a dominant negative syndrome of epilepsy and megencephaly (Petersson et al., 2003). It is noteworthy that insertion of a human ataxia mutation in mouse produces the expected phenotype (Herson et al., 2003). It should also be recalled that many of the human disorders are clinically apparent in heteroygotes, whereas many of the mouse models are studied in their more severe homozygous forms. However, in a variety of murine disorders, an apparently identical functional change may still create dissimilar phenotypes. When this occurs, what conclusions can be drawn about the similarity of the intervening mechanisms? Reconciling species-specific phenotypic differences requires an ability to compare key elements of the disorder in mice with those assembled for human epilepsy syndromes, beginning with the electrographic and behavioral semiology of the seizure phenotype (Blume et al., 2001) and then working backward toward the precise mutation of the gene. The cortical EEG, which is readily but subjectively described, exhibits important differences in background rhythmicity even between normal rodent and human brains. For example, mice and rats lack spontaneous alpha (7– 12 Hz) rhythms and other complex discharges and sleep stages normally present in the human EEG. Would these background oscillatory differences alter the synchronization patterns of an otherwise identical epileptogenic lesion? The behavioral correlate of the seizure is also often difficult to compare because the semiology of spontaneous seizures in rodents has not been formalized, and many model descriptions are incomplete. Terms such as epileptic seizures, behavioral arrest, convulsions, wild running, and Straub tail phenomenon are typically the only seizure descriptions reported. Ictal durations are rarely recorded, and the changing behavior pattern throughout an ictal episode, well described for the kindling model (Engel et al., 1978), is usually overlooked. Finally, the pharmacological sensitivity and natural history (onset and duration of seizures over the life span), two key descriptors of an epilepsy syndrome, are difficult to determine precisely and rarely mentioned in descriptions of murine epilepsies. At the neuropathological level, the assumption that an orthologous gene model should show molecular or cellular lesions in the mouse that are identical to those in human may also be premature (at least in some cases). Several basic explanations for potential differences are routinely overlooked. For example, murine genes may be spliced differently than those in human, leading to proteins that differ in functional properties or interaction domains with other molecules. Anatomically, the cellular context and neurobiology of the mouse and human brain vary; for example, some
Spontaneous Mutants with Absence Epilepsy Phenotypes
mouse brain regions lack cellular components present in humans. Additional differences may reside in the temporal and regional expression of brain genes, in compensatory gene family members, or directly interacting partners such as channel regulatory subunits, transmitter receptors, or other members of the molecular pathway. Finally, the mouse or human may coexpress an unknown number of mutations in modifying genes that mask the penetrance of the phenotype.
ANALYSIS OF PATHOGENIC MECHANISMS IN SPONTANEOUS MOUSE MODELS Working Backward from the Phenotype In the absence of an identified gene, pathological analysis of the nervous system in the spontaneous mutant models was historically descriptive and phenotype-driven using hypotheses generated from acute experimental models of epilepsy rather than driven by any inherently logical approach. Routine neuropathological studies of major neurotransmitter systems, using available antibodies or other functional assays, were typically followed by neurophysiologic analyses of firing properties in well-studied neural circuits (such as the hippocampus). This approach leads to the discovery of a wide range of interesting, yet seemingly unconnected, “endophenotypes;” it has largely been replaced by a more focused strategy based upon knowledge of the mutant gene.
The Gene-Forward Approach At the subcellular level, once the gene has been identified, a direct strategy working forward from the gene is productive. Molecular genetic studies can define the precise effects of the mutation on the sequence of the transcribed protein, and cell biology methods can describe its heteromeric interactions and longevity. At the cellular level, molecular anatomic techniques are required to determine where and when the mutant gene and related members of the gene family are expressed and the cell types and subcellular compartments in which the various isoforms are localized. Many mutations impair protein trafficking, and the levels and distribution of the mutant protein must be carefully examined, along with its physiologic or biochemical behavior, to understand how overall function has been impaired. If the hereditary transmission pattern suggests a dominant trait, evaluation must include the possible negative effects of the mutant protein on the function of its wildtype allele, which can explain defective neural activity in the heterozygous animal. At the network level of analysis, a priority list of questions includes the following: How do mutant neurons inter-
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act with cells not expressing the mutation? Does the mutation result in a gain or loss of function (e.g., of connectivity) within the circuit, and how does this interaction impact overall network signaling properties? What developmental plasticity changes add to, or compensate for, the abnormal signaling? Finally, how does this pattern fit with what is known about existing mechanisms of epileptogenesis derived from other acute and chronic models? At each level of analysis, once defects are identified in the brain of the mutant, the major challenge is to determine whether these changes are the cause of the epilepsy, the result, or functionally unrelated. Age-specific studies may help untangle this complex lesion. Is there significant damage in the brain before seizures occur? What pathological changes follow a prolonged history of seizures? Serial developmental studies may be required to visualize pathogenic changes before they are obscured by a misleading sequence of seizure-induced events.
SPONTANEOUS MUTANTS WITH ABSENCE EPILEPSY PHENOTYPES Summarizing the experimental analysis of spontaneous mutants with an absence epilepsy phenotype is a useful way of comparing the relative contributions of the two-directional approaches. Spontaneous mutations in the past provided the only model of this seizure type; however, two transgenic models have recently been reported (Ludwig et al., 2003; Song et al., 2004). Before the causative genes were identified by positional cloning, EEG recordings were employed to validate spike wave absence epilepsy in four neurologic mutants with ataxia, including tottering, lethargic, ducky, and stargazer (Noebels, 1999). Following the phenotype-driven approach, analysis of tottering forebrain identified multiple abnormalities relating to network synchronization: noradrenergic hyperinnervation accompanied by cortical norepinephrine (NE) subsensitivity (Levitt and Noebels, 1981; Magistretti et al., 1987), elevated cortical cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) levels (Tehrani and Barnes, 1990, 1995), decreased synaptosomal sodium channels, and muscarinic acetylcholine (ACh) receptor density (Liles et al., 1986; Willow et al., 1986), increased hippocampal network excitability in response to convulsants (Noebels and Rutecki, 1990; Psarropoulou and Kostopoulos, 1990), depressed cellular inhibition with prolonged depolarizations, and reduced hippocampal pyramidal cell afterhyperpolarization (Helekar and Noebels, 1991, 1992, 1994). Hippocampal mossy fiber sprouting was also detected (Stanfield, 1989). These changes generally favor hypersynchronization within tottering circuits, and at least one of these abnormalities was present at the onset of epilepsy (Helekar and Noebels, 1991). Once the mutation in
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the Cacna1a gene had been identified (Fletcher et al., 1996), gene-driven hypotheses refocused on a variety of direct alterations in mutant channel behavior: decreased currents through neuronal P/Q type calcium channels (Wakamori 1998), decreased excitatory transmission in thalamus (Caddick et al., 1999), and impaired neurotransmitter release (43, 72Ayata et al., 2000; Leenders et al., 2002; Qian and Noebels, 2000). Recent analysis of thalamocortical relay neurons involved in spike-wave electrogenesis has uncovered a secondary elevation of low-voltage activated (T-type) calcium currents (Zhang et al., 2002) known to favor rebound bursting in this cell type (Crunelli and Leresche, 2002). Taken together, the data from both backward and forward approaches provide several candidate mechanisms for epileptogenesis in calcium channelopathy mice. Interestingly, the impaired calcium currents that result directly from the primary inherited defect are unlikely to elevate membrane excitability. However, many of the secondary developmental effects of reduced transmitter release are strong contenders for enhanced neuronal bursting and network oscillations. Downstream plasticity therefore appears to be a critical step in this model of epileptogenesis. Not surprisingly, the evidence for this component is seen first by testing network hypotheses rather than by working forward from the mutant gene. Our insight into the seizure disorder associated with calcium channelopathy suggests that a bidirectional approach may prove the most efficient strategy to outline the key steps of the disease pathway. This example also emphasizes the need to search for selective vulnerability mechanisms that create excitability defects in thalamocortical circuits at specific developmental ages while sparing other central synaptic pathways also expressing the mutant gene. Although calcium channelopathy represents only one of several molecular pathways defined by spontaneous mouse mutants that lead to the expression of spike-wave epilepsy, the results obtained from studies of this model clearly illustrate the promise—as well as the remaining challenges—for gaining a full understanding about how single gene defects lead to epileptic phenotypes.
Acknowledgments We are indebted to the dedicated mouse handlers of the Jackson Laboratory for their careful observations and genetic analyses of mutants maintained over the years in the Neurological Mutant Resource and funded by the National Institutes of Health. I also thank Muriel Davisson, Ken Johnson, Carol Spencer, Sue Cook, and Wayne Frankel as well as the many members of the Developmental Neurogenetics Laboratory for their invaluable collaboration. The laboratory at Baylor is supported by grants from the National Institute of Neurologic Disorders and Stroke (NINDS) and the Blue Bird Circle Foundation for Pediatric Neurology.
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18 Genetic Models of Absence Epilepsy in the Rat Antoine Depaulis and Gilles van Luijtelaar
et al., 1994; Loiseau et al., 1995; Panayiotopoulos, 1999). Because typical absence epilepsies mainly affect children and teenagers and have moderate consequences, studies of their pathophysiological mechanisms cannot be conducted in humans for ethical reasons. Therefore, animal models are mandatory to understand this form of epilepsy and the mechanisms underlying the generation and control of SWD. Models displaying electrical, behavioral and pharmacologic characteristics of absence seizures have been used in rodents, cats or primates by injection of pentylenetetrazol, penicillin, gamma-hydroxybutyrate or GABA agonists (see Chapter 10). However, although these models have contributed to our understanding of SWD generation, the lack of recurrence of the seizures does not allow us to study the development of the disease. About 20 years ago, our two groups reported for the first time the spontaneous occurrence of SWDs evocative of absence seizures in untreated rats, as seen during cortical EEG recordings (Vergnes et al., 1982; van Luijtelaar and Coenen, 1986). These first observations led to the development and characterization of two genetic models of absence epilepsy in rats from two different initial breeding colonies. In Strasbourg (France), a fully inbred strain of rats, with 100% of animals displaying the EEG and behavioral characteristics of absence seizures, was derived from an outbred Wistar colony: the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The Wistar Albino Glaxo strain was inbred in the United Kingdom and then kept in Rijswijk (WAG/Rij) and later at Nijmegen (The Netherlands). It was already fully inbred when it was discovered that these rats had absence seizures. A strain of nonepileptic control animals was also selected in Strasbourg from the same
GENERAL DESCRIPTION OF THE MODELS: WHAT DO THEY MODEL? Absence epilepsy is a particular epileptic syndrome in which patients show generalized nonconvulsive seizures characterized by a brief unresponsiveness to environmental stimuli and cessation of activity, which may be accompanied by automatisms or moderate tonic or clonic components affecting the limbs, the eyeballs, or the eyelids (Loiseau et al., 1995; Panayiotopoulos, 1999). Typical absences seizures are associated on the electroencephalogram (EEG) with bilateral, synchronous and regular three cycles per second (c/s) spike-and-wave discharges (SWDs), which start and end abruptly. In contrast to generalized convulsive or partial seizures, there is no postictal depression or slowing following typical absences. They occur as frequently as several hundred times per day, mainly during quiet wakefulness, inattention, and at transitions between sleep and awakening, and generally last 10 to 20 seconds. The pharmacologic reactivity of absence seizures is also unique: They are suppressed by ethosuximide, which is ineffective in all other forms of seizures, whereas they are aggravated by carbamazepine, phenytoin, and some of the other antiepileptic drugs (AEDs) effective in generalized convulsive and partial seizures. Absences seizures are found in five nonlesional idiopathic generalized epileptic syndromes (Hirsch et al., 1994; Loiseau et al., 1995; Porter, 1993): childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, myoclonic absence epilepsy, and eyelid myoclonia with absences. Besides absence seizures, patients do not present any other neurologic or neuropsychological disorders. In childhood absence epilepsy, remission is observed during adolescence in about 70% of the patients (Hirsch
Models of Seizures and Epilepsy
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initial breeding colony as the GAERS, whereas the Nijmegen group used another inbred strain as controls (August Copenhague Irish or ACI rats): These rats have no or very few absence seizures (Inoue et al., 1990). GAERS and WAG/Rijs have many similarities in the characteristics of their seizures, ontogeny, and pharmacologic reactivity. Yet they seem to differ in some features. Their study using different approaches, from electrophysiology to molecular genetics, has generated an incredible amount of data during the last 20 years on the mechanisms underlying SWD and has led to the publication of more than 400 articles. The present chapter reviews the data obtained in these two models and compares their respective features.
spontaneous SWD are usually associated with other neurologic disorders. For instance, lethargic mice (lh/lh) exhibit spontaneous SWDs together with ataxia and lethargic behavior (Hosford et al., 1992), whereas the tottering mice also display paroxysmal abnormal movements and postures (Noebels and Sidman, 1979) (see also Chapter 17). Spontaneous SWDs have also been reported in transgenic mice (see Chapter 16). A strain of mice that was selected for their sensitivity for a b-carboline was found to display spontaneous SWDs with behavioral and pharmacologic features similar to those of GAERS and WAG/Rij (Rinaldi et al., 2003).
Procedures METHODS OF GENERATION Animal Issues For the GAERS strain, epileptic rats were first selected from Wistar colonies that displayed spontaneous SWDs on routine cortical EEG. Breeding of selected pairs over three or four generations produced a strain with 100% of rats displaying SWDs (Vergnes et al., 1982; for review, see Danober et al., 1998). A control strain, free of SWD, was also selected over five or six generations. Since then more than 50 generations have been bred (see genetic description later). Through serendipity it was discovered in Nijmegen that the already fully inbred WAG/Rij strain of rats showed SWDs in their cortical EEG (van Luijtelaar and Coenen, 1986). The spontaneous occurrence of high-voltage rhythmic activities on cortical EEG of various types of laboratory rodents was also described by many authors (Buszaki et al., 1990; Chocholova, 1983; Inoue et al., 1990; Jando et al., 1995; Klingberg and Pickenhain, 1968; Libouban and Oswaldo-Cruz, 1958; Noebels, 1984; Semba and Komisaruk, 1984; Shaw, 2004; Willoughby and MacKenzie, 1992). The percentage of animals with such activities varies from 0 to 100%, depending on the species and the strain. However, in Wistar rats recorded at old age (i.e., 84 to 94 weeks), 100% of the animals display SWDs that are indistinguishable from those observed in GAERS and WAG/Rij rats (van Luijtelaar et al., 1995). This finding suggests that aging is associated with aggravation of SWDs. In Long Evans rats, several studies reported the occurrence of “highvoltage rhythmic spike discharges” concomitant with immobility and whisker twitching. These discharges have been suggested to be associated with movement disorder such as alpha tremors or a dynamic process of sensory information from vibrissa. Recently a detailed analysis of the EEG, behavioral, and pharmacologic characteristics of theses discharges has demonstrated that they are similar to absencelike SWD (Shaw et al., 2004). Contrary to what is observed with rats, most strains of mice do not present spontaneous SWDs. When observed,
In rats SWDs can be easily recorded through chronically implanted monopolar electrodes positioned over the cortex. Usually stainless steel jewelry screws or epidural wire electrodes are fixed in the skull of the frontal and parietal cortices in both sides of the brain with the rat under generalized anesthesia. A reference electrode is also fixed over the occipital cortex or over the cerebellum to allow referential recording. All electrodes are soldered to a female connector, which is fixed to the cranium of the animals with acrylic dental cement. After surgery the animals are habituated to the recording procedure by placing them in the test cage once or twice for a few hours. Depth electrodes can be also implanted using stereotaxic procedures, and animals can be equipped with multiunit electrodes, microinjection cannulae, or microdialysis probes as described for other models.
Monitoring Once implanted and habituated to the recording conditions, SWDs can be EEG recorded in freely moving animals for several hours or even days. As in humans, the level of vigilance plays a critical role in the occurrence of absence seizures (Coenen et al., 1991; Lannes et al., 1988). When GAERS or WAG/Rij rats are continuously recorded for 24 hours, most SWDs occur during a state of passive wakefulness or drowsiness and often are followed by or preceded slow-wave sleep. On the contrary, SWDs are sporadic during active arousal, deep slow-wave sleep, and paradoxical sleep. In addition, a sudden noise or handling of the animals generally interrupts SWDs. Because of these observations, the recording conditions are critical: Very few SWDs are recorded when the animals are placed in a new cage, when the cage has previously hosted a female or a mouse, or during performance of various motivated tasks. For methods that require a complete immobility of the animals (e.g., in vivo extra and intracellular electrophysiology, functional magnetic resonance imaging [fMRI]), most anesthetics cannot be used because they suppress SWDs. Instead, neuroleptic analgesia and local injections of lido-
Characteristics of Genetic Absence Seizures in the Rat
caine have been used to allow the fixation of the animals into a stereotaxic frame (Pinault et al., 1998). Using this preparation, spontaneous SWDs and extracellular unit activity can be recorded with the same pattern and occurrence as in the awake, freely moving rats (Deransart et al., 2003; Pinault et al., 2001), although an increase in the number of SWDs and a decrease in the frequency of interspikes has been found in WAG/Rij rats after anesthetic doses of Hypnorm (Inoue et al., 1994).
CHARACTERISTICS OF GENETIC ABSENCE SEIZURES IN THE RAT EEG Characteristics In both WAG/Rij and GAERS, SWDs start and end abruptly on a normal cortical desynchronized EEG background (Figure 1). In adult animals the frequency of spikewave complexes within a discharge can range from 7 to 11 c/s; the frequency is always higher at the beginning of the seizures and slows down toward the end (Drinkenburg et al., 1993; Midzianovskaia et al., 2001; Meeren et al., 2002; Slaght et al., 2002) (see Figure 1). The mean frequency is 8.0 ± 1.0 c/s in both strains. Their voltage varies from 300 to 1000 mV, depending on the structure that is recorded (see later discussion). When GAERS are maintained in a state of quiet wakefulness, SWDs generally last for about 25 ± 8
235
seconds, a duration that varies from one animal to the next because some animals can display SWDs lasting up to 60 seconds. In this strain, absence seizures occur about every minute on average, depending on the time of the day and the recording conditions (Vergnes et al., 1982; Marescaux et al., 1992a, 1984; Vergnes et al., 1982). In undisturbed WAG/Rij rats, SWDs last about 5 seconds. In 6-month-old WAG/Rij rats, the number of SWDs is about 15 to 20 per hour, and the circadian distribution shows few SWDs during the beginning of the sleep period, when rats have the largest amount of deep slow-wave sleep, which seems incompatible with the occurrence of SWDs. The maximum number of SWDs can be found during the dark period of the 24-hour light-dark cycle (van Luijtelaar and Coenen, 1988). Postictal depression of the EEG is never observed in either strain. In WAG/Rij rats two types of SWDs have been described (van Luijtelaar and Coenen, 1986). The first type occurs commonly in all animals and is similar to the SWDs described in GAERS (Figure 2). It is the most often studied absence seizure type. The second type of SWD, which is seen in about 60% of the WAG/Rij rats, is a “local” SWD that is recorded bilaterally at the occipital and parietal cortices (Midzianovskaia et al., 2001). Its frequency is 6 to 7 c/s (i.e., lower than that of the first type), and its duration is 1 to 2 seconds. In addition, the polarity of the spikes is opposite that of the first type (Figure 2). Finally the number of SWDs per hour is three to five, which is also less than that of the first type. The number of this type of SWD and the number of animals with this type of SWD also increase with age.
Cx FP R
Behavioral Characteristics
Cx FP L
500 µV 2s
(Hz) 50 30 10
Cx FP R Cx FP L 500 µV 1s
FIGURE 1 Example of cortical electroencephalographic (EEG) recording of a spike-and-wave discharge in the genetic abscense epilepsy rats from Strasbourg (GAERS) and corresponding spectral analysis. Bottom, lower speed recording of the discharge showing the spike-wave complex. Cx FP R, right frontoparietal derivation; CxFP L, left frontoparietal derivation. (Courtesy of Colin Deransart.) (See color insert.)
The SWDs are always concomitant with behavioral immobility and sometimes rhythmic twitching of the vibrissae and facial muscles. Muscle tone in the neck is generally diminished, inducing a gradual and slight lowering of the head. Simultaneously with the end of the SWD, the animal resumes its behavior and the muscle tone of the neck is restored (Marescaux et al., 1992a, 1984; van Luijtelaar and Coenen, 1986; Vergnes et al., 1982). Comparisons with nonepileptic rats have shown that spontaneous activity, exploration, feeding, social interactions, or learning of positively or negatively reinforced tasks are not impaired in GAERS or WAG/Rij rats. Similarly sexual and reproductive behaviors also appear normal (Coenen et al., 1991; Vergnes et al., 1991). However, recent data reported that WAG/Rij rats show decreased exploration, increased immobility in the forced swim test, and reduced sucrose intake compared with Wistar control rats (Sarkisova et al., 2004). When GAERS were trained to press on a lever to obtain food reward, they always interrupted their conditioned behavior during SWDs and resumed it on cessation of the seizure (Vergnes et al., 1991). The absence of motor responses in a positively moti-
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FIGURE 2 The two types of seizures in the Wistar Albino Glaxo/Rijswijk strain (WAG/Rij) of rat. The first is a type II SWD, which is followed by a type I SWD (similar to that shown in Figure 1). The trace shows a differential electroencephalographic (EEG) recording with two active EEG electrodes, one at the frontal and one at the parietal cortex (courtesy of Ulrich Schridde).
vated discrimination task during the presence of a SWD was also typical for WAG/Rij rats, although the postictal responses were less accurate. In addition, WAG/Rij rats do not evaluate correctly the time that has passed when a SWD occurs, just as in children with absence epilepsy (van Luijtelaar et al., 1991a, b). Together, these findings suggest that the animals are less able to initiate or execute an adequate motor response during or after a SWD and that their perception of elapsed time is disturbed (Drinkenburg et al., 1995). However, this disturbance does not imply that all cognitive functions are impaired during SWDs: When auditory stimuli were presented during a SWD, WAG/Rij rats were able to discriminate between a previously rewarded and a previously nonrewarded tone (Drinkenburg et al., 2003).
Ontogeny In GAERS, SWDs are first detected around 30 days of age, whereas they are observed at around 60 to 80 days in WAG/Rij (Vergnes et al., 1986; Coenen and van Luijtelaar, 1987; Schridde and van Luijtelaar, 2004). At 40 days about 30% of the GAERS are affected, and this percentage increases gradually with age to reach 100% at the age of 3 months. The first SWDs are rare (1 or 2 per hour) and shortlasting (1–3 seconds), with a lower frequency of SWDs during a discharge (4–5 per second). With age, the number, the duration, and the frequency of SWDs increase, whereas their amplitude is not modified. Their number reaches a maximum at around 4 to 6 months. A similar but delayed development can be observed in WAG/Rij rats. At the age of 3 months, 50% of the WAG/Rij display fully developed SWDs, and at 6 months of age 100% of the animals show SWDs (Coenen and van Luijtelaar, 1987). SWDs can be recorded in both strains until the death of the animals (van Luijtelaar et al., 1995; Vergnes et al., 1986).
Neuropathology Depth-recording EEG, intracerebral microinjection, and lesion and in vivo electrophysiologic studies have shown
that the thalamocortical interconnections are critical in the generation of SWDs in both GAERS and WAG/Rij (see later discussion on generating circuits). In these structures, no gross histologic modifications were ever observed. In particular, no neuronal loss was detected in the reticular and ventrolateral-posterior nuclei of the thalamus in GAERS (Sabers et al., 1996). Furthermore the synaptic organization of the reticular nucleus of WAG/Rij appears very similar to that of controls (van de Bovenkamp-Janssen et al., 2004b). An increase of glial fibrillary acidic protein (GFAP) expression has been reported in the cortex and the thalamus in both adult and young GAERS, suggesting that reactive astrocytes are already present before the onset of absence seizures (Dutuit et al., 2000). Recently, detailed morphometric studies of the upper granular layer showed that pyramidal cells in layer II-III of the somatosensory cortex in WAG/Rij rats exhibit aberrant dendritic arborization, increased length of dendritic segments, and differences in the number of free terminations of apical dendrites compared with control rats (Karpova et al., 2005). How these dendritic changes are related to increased excitability or capacity to synchronize remains to be established.
Imaging and Metabolic Changes Local cerebral metabolic rates for glucose (LCMRglc) have been measured in several brain structures using the [14C]-2-deoxyglucose method in adult GAERS to map the neuronal circuits involved in the generation and control of SWDs (Nehlig et al., 1991, 1994) (see also Chapter 47). An overall increase in LCMRglc was observed in most structures whether they exhibit SWDs (i.e., neocortex and thalamus) or not (e.g., limbic and brainstem structures). However, because of the long duration of a 2-deoxyglucose experiment (i.e., 45 minutes), the cerebral metabolic level recorded represents a combination of ictal and interictal phases. These results suggest that GAERS may have a higher glucose metabolism than nonepileptic controls, which seems to reflect primarily the interictal periods (see Chapter 47).
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Characteristics of Genetic Absence Seizures in the Rat
Using laser-Doppler flowmetry, it was shown that SWD in GAERS were associated with a decrease in the level of cerebral blood flow at the surface of the parietal cortex, which started 2 to 7 seconds after the onset of the cortical SWDs. At the end of SWDs, cerebral blood flow returned to baseline level (Nehlig et al., 1996). In a recent study using an intracerebral probe that combines laser-Doppler flowmetry and extracellular microelectrode recording, a parallel increase of cerebral blood flow and neuronal activity was observed in the somatosensory cortex of WAG/Rij (Nersesyan et al., 2004). Whether these opposite data are due to the difference in the techniques used, to the difference between the two strains, or to the differences in localization remains to be further examined. Two studies used functional magnetic resonance imaging (fMRI) to characterize more fully, in a noninvasive way, the neural circuits involved in the generation of absence seizures in the WAG/Rij (Nersesyan et al., 2004; Tenney et al., 2004). Using T2-weighted echo planar imaging, the bloodoxygenation-level dependent (BOLD) signal coupled with EEG was analyzed (1) in awake rats previously habituated to the experimental conditions (Tenney et al., 2004) or (2) in fentanyl-haloperidol anesthetized animals (Nersesyan et al., 2004). Comparisons of images during spontaneous SWDs and interictal activity showed in both studies an increase of more than 6% of the BOLD signal in the sensory, parietal, and temporal cortices as well as in the reticular, mediodorsal, ventroposterior, and posterior nuclei of the thalamus. No significant changes were seen in temporal or limbic structures (e.g., hippocampus), and no significant negative BOLD signal was observed for any seizures. These data are thus in agreement with neurophysiologic data showing the role of the sensorimotor cortex and relay thalamic nuclei in the generation of SWD (see later). When seven Tesla MRI was used, however, an increase in BOLD signal was also observed in the hippocampus, the basal ganglia nuclei, and the tectum and tegmental nuclei. The local changes in BOLD signal recorded with a 7 Tesla magnet are also in agreement with increases in LCMRglc recorded in 21-day-old GAERS before the occurrence of SWD in this strain (Nehlig et al., 1998). These changes confirm that although SWDs are recorded only in the thalamocortical circuit, the mutations allowing the expression of absence epilepsy are ubiquitously expressed across the whole nervous system and most likely affect also the functional activity of structures located outside the generating circuit. It must be noted that no significant or consistent SWD-related decreases of fMRI have been observed in both studies using WAG/Rij. This contrasts with other fMRI/EEG studies performed on either other animal models (Tenney et al., 2003) or in patients (Archer et al., 2003; Salek-Haddadi et al., 2003). In these studies, numerous cortical areas have shown a decrease in BOLD signal following SWD. However, more work is necessary to draw any conclusion
about the functional significance of the distinction between increase and decrease in the BOLD signal. fMRI/EEG is a promising technique that will certainly be of great help in understanding the organization of the different circuits involved in the generation, propagation, and control of SWD. It should bring further insights in epileptic mechanisms when it becomes possible to disentangle neuronal processes from fMRI data by using detailed generative model of fMRI signals (Aubert and Costalat, 2002; Friston et al., 2003).
Response to Antiepileptic and Proconvulsant Drugs Classic AEDs have been tested on GAERS and WAG/Rij rats to check how pharmacologically predictable models are. Indeed, SWDs are suppressed by the four main AEDs that are effective against human absence seizures (ethosuximide, trimethadione, valproate, and benzodiazepines). On the contrary, they are worsened by drugs that are either ineffective or aggravating in humans (carbamazepine, phenytoin) (Table 1) (Micheletti et al., 1985). Phenobarbital evokes biphasic effects: it is suppressive at 2.5 to 10 mg/kg, but not at 20 mg/kg (Micheletti et al., 1985; Peeters et al., 1988). Almost all recently developed AEDs have been tested in either GAERS or WAG/Rij to evaluate their possible efficacy on absence seizures (see Table 1). Drugs like vigabatrin, tiagabine, and gabapentin have been shown
TABLE 1 Effects of Antiepileptic Drugs on Spike-Wave Discharges in Humans with Typical Absence Epilepsy and in Rat Models of Absence Epilepsy Antiepileptic drugs
Humans*
Rat models
Benzodiazepines
Suppression
Barbiturates
Biphasic effects
Suppression Biphasic effects
Valproate
Suppression
Suppression
Ethosuximide
Suppression
Suppression
Trimethadione
Suppression
Suppression
Carbamazepine
Aggravation
Aggravation
Phenytoin
Aggravation
Aggravation
Gabapentin
Aggravation
Aggravation
Lamotrigine
Suppression
No effects
Levetiracetam
Suppression
Suppression
Pregabalin
Suppression
Suppression
Progabide
No effects
No effects
Tiagabine
Aggravation
Aggravation
Topiramate
Suppression
Suppression
Vigabatrin
Aggravation
Aggravation
* From Panayiotopoulos 1999.
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to aggravate SWDs, whereas progabide is ineffective (Bouwman et al., 2004; Coenen et al., 1995; Marescaux et al., 1992a, b; van Luijtelaar et al., 2002). On the contrary, topiramate, levetiracetam, and pregabalin suppress SWDs (Gower et al., 1995; Rigoulot et al., 2003). Together, the data in the two animal models show a great similarity with the pharmacologic reactivity of typical absence epilepsy in human (Panayiatopoulos, 1999; Schlumberger et al., 1994). The pharmacologic predictability of these models is further confirmed by the aggravation of SWDs induced by drugs like pentylenetetrazol, gamma-hydroxybutyrate, tetrahydroisoxazolopyridinol (THIP), or penicillin that are known to induce “absence-like” discharges in normal rats (Marescaux et al., 1984, 1992a; Snead, 1988, 1994). More generally the reactivity of seizures to g-aminobutyric acid (GABA)ergic compounds differs from what is generally observed in models of convulsive seizures. In GAERS, intraperitoneal administration of GABAA agonists (muscimol and THIP), GABA transaminase inhibitors (gammavinyl GABA and l-cycloserine), or GABA reuptake inhibitors (SKF 89976 and tiagabine) induces a dosedependent increase in the duration of SWDs (Coenen et al., 1995; Marescaux et al., 1992b; Vergnes et al., 1984). Similarly the injection of R-baclofen, a GABAB agonist, increased SWDs in GAERS or in old Wistar rats, whereas administration of CGP 35348 and other GABAB antagonists suppresses seizures (Marescaux et al., 1992c; Puigcerver et al., 1996). GAERS and WAG/Rij also appear more sensitive to the convulsive effects of systemic injections of GABAA antagonists (picrotoxin, bicuculline, or pentylenetetrazol [PTZ]) and benzodiazepine inverse agonists compared with rats of the nonepileptic strain (Klioueva et al., 2001; Vergnes et al., 2001). This sensitivity suggests a possible dysfunction of GABAA receptors in absence epilepsy (see later discussion). It is interesting to note that SWDs also appear to be modulated by ligands of the dopamine receptors, as in human patients (see Starr, 1996). Systemic injections of agonists of the D1 and D2 receptors were shown to suppress absence seizures in both GAERS and WAG/Rij (van Luijtelaar et al., 1996; Warter et al., 1988). On the contrary, antagonists of these receptors such as haloperidol significantly increase the number and duration of SWD and may lead to an absence status. However, no seizures are induced by these compounds in nonepileptic animals. This pharmacologic reactivity to dopaminergic ligands may result from their effects on the basal ganglia circuits known to modulate SWDs (De Bruin et al., 2001; Deransart et al., 2000). It is interesting to note that, in the WAG/Rij, an inverse reactivity to dopaminergic agonists is observed for type II SWDs, which are suppressed by haloperidol but aggravated by the DA agonist apomorphine (Midzianovskaia et al., 2001). However, AEDs like tiagabine or vigabatrin increased both type I and type II SWDs (Bouwman et al., 2004; Coenen et al., 1995).
Many other ligands of receptors influence SWDs. The best studied are the effects of glutamatergic and cholinergic drugs and opioid peptides (e.g., Danober et al., 1998; Lason et al., 1995; Peeters et al., 1990a). However, it is beyond the scope of this review to discuss the effects of these compounds.
Genetic Transmission and Chromosomal Mapping WAG/Rij are fully inbred rats, which means that they are homozygous for all autosomal genes. All individuals display SWDs on their EEG. In the initial colonies of Wistar rats in Strasbourg, 30% of the animals showed spontaneous SWDs. Breeding of selected parents over three or four generations produced a strain in which 100% of the rats were affected. Indeed, both data sets demonstrate that transmission of SWDs is inherited (Marescaux et al., 1992a; Peeters et al., 1990b, 1992). In epileptic ¥ nonepileptic F1 generations, more than 95% of the animals showed SWDs after 6 months, suggesting a dominant transmission, and similar SWDs were recorded in male and female animals, indicating that the transmission is autosomal. Interindividual variability for age of appearance and duration of SWDs is extremely high, supporting the view that the inheritance of SWD is probably not due to a single gene locus or that environmental effects might play a role. This mode of inheritance was confirmed in F2 (F1 ¥ F1) and backcross (F1 ¥ control) generations in both GAERS and WAG/Rij (Marescaux et al., 1992a; Peeters et al., 1992). Recent efforts to manipulate characteristics (e.g., the number, incidence, mean duration) of type I and type II SWDs by housing WAG/Rij in an enriched environment demonstrated that type I SWDs were less sensitive to environmental factors such as housing. This result demonstrates that genetic factors are more important than the contribution of the environment to the phenotypical expression of SWD. Type II SWD, however, appear more easily affected by the environment (Schridde and van Luijtelaar, 2004). After EEG recordings in an F2 population of rats resulting from the breeding of GAERS and Brown Norway rats, the polygenic inheritance of SWD-related phenotypes was demonstrated (Rudolf et al., 2004). Three quantitative trait loci (QTLs) were identified on chromosomes 4, 7, and 8, which appear to control different variables of SWD (e.g., frequency, amplitude, severity). In this study, age was a major factor influencing the detection of genetic linkage to the various components of the SWDs. Using a similar method, two different QTLs were characterized in a WAG/Rij ¥ ACI F2 population, which are located in chromosomes 5 and 9 (Gauguier et al., 2004). Each of these QTLs appear to control independently the two types of SWDs which have been described in this model. Although some differences in the experimental designs may account for different QTLs for GAERS and WAG/Rij rats, the
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Insights Into Human Disorders
identification of distinct QTLs suggests that SWDs have been fixed in these two strains (Gauguier et al., 2004).
VALIDITY OF THE MODELS AND THEIR LIMITATIONS In both GAERS and WAG/Rij, the behavioral and EEG patterns during SWD share several points of similarity with those observed in humans during absence seizures (Loiseau, 1992; Loiseau and Cohadon, 1970; Panayiatopoulos, 1999). Furthermore, as in humans, SWDs occur within restricted limits of vigilance state, corresponding to quiet wakefulness or somnolence (Mirsky et al., 1986; Niedermeyer, 1996). Both genetic models have an almost identical pharmacologic reactivity to human absence epilepsy. However, several discrepancies exist with human absence epilepsy. First, in patients with typical absence epilepsy, the frequency of SWDs is around 3 c/s, whereas it varies from 7 to 11 c/s in the two rat models. A frequency of 3 c/s appears difficult, if not impossible, to observe in rodents (Avoli, 1980; McQueen and Woodbury, 1975), and SWDs with such a rhythmicity have been observed only in primates during pharmacologically induced absence seizures. This finding suggests that the frequency of SWDs, like other oscillations, is species dependent (Snead, 1978). Second, absence epilepsies are known to be associated with brain development and maturation (see Crunelli and Leresche, 2002). In humans, typical childhood absence epilepsy occurs between the ages of 2 and 8 years and remissions occur around puberty in most patients (Loiseau, 1992; Loiseau and Cohadon, 1970; Loiseau et al., 1995). In GAERS and WAG/Rij, SWDs first occur around 30 or 60 days, that is, when brain maturation is finished in the rat. Furthermore, unlike in humans, they persist throughout the life of the animals. The mechanisms underlying the remission of SWD in human remain unknown, but it is very likely that they belong to brain maturation processes that may not exist in rodents. Definite evidence to validate these strains as models of absence epilepsy will come from our knowledge of the genetic mechanisms at the origin of SWD generation. Indeed, human absence epilepsies appear to be genetically determined. In homozygous twins, concordance rates of 84% for EEG discharges and 75% for absence seizures were found, whereas heterozygous twins showed no concordance (Lennox and Lennox, 1960). In some families, the high incidence of 3 c/s SWDs during scalp EEG performed in firstdegree relatives suggests a monogenic autosomal-dominant mode of inheritance (Gloor et al., 1982). In addition, evidence of genetic linkage to human 8q24 has been found in absence epilepsy associated with tonic-clonic seizures (Sugimoto et al., 2000). Furthermore, an association was found between absence epilepsy and the genetic variants in the GABAA receptor subunit b-3 located on human
15q11-q13 (Feucht et al., 1999). Finally, a mutation of the GABAA receptor g-2 subunit gene located on chromosome 5 was found in a family with childhood absence epilepsy and febrile seizures (Marini et al., 2003; Wallace et al., 2001). In this latter case, the mutation seems to be associated with febrile seizure rather than with absence epilepsy. In summary, absence epilepsy appear to result from a polygenic control in both rodents and humans. It is quite possible that the occurrence of SWDs is the result of a combination of several genes.
INSIGHTS INTO HUMAN DISORDERS Because most characteristics of absence seizures in GAERS and WAG/Rij are highly reminiscent of human absence epilepsy, these two strains have become models of reference for studying the mechanisms underlying SWDs. Several articles have addressed this issue, which is still under debate. Only a general overview is presented in the following sections of this chapter.
Underlying Mechanisms Neurophysiologic experiments in either the GAERS or the WAG/Rij animal models have clearly implicated the coupling of thalamic oscillations and cortical rhythms as the cause of SWDs. This is partly in agreement with the previous hypothesis proposed from data collected from cats, in which SWDs were elicited by injection of penicillin (see Gloor, 1968). Early accumulated data suggest a predominant role of the thalamic neurons in the generation of SWD (Danober et al., 1998). In addition, multisite recordings have provided evidence for a critical role of the sensorimotor cortex (Meeren et al., 2002). Role of Thalamic Hypersynchronization Depth EEG recordings performed with bipolar electrodes, as well as electrolytic or chemical lesions, have shown that the relay nuclei of the thalamus are critical in the generation of SWD. Indeed, in addition to the lateral frontoparietal cortex, where they were first recorded, highamplitude SWDs were also collected in the posterolateral thalamus. On the contrary, SWDs are strongly reduced and delayed in the anterior and midline nuclei of the thalamus, the striatum, the lateral hypothalamus, and the ventral tegmentum (Marescaux et al., 1992a; Vergnes et al., 1990). The importance of the relay nuclei was confirmed by lesion studies in which they abolished SWDs (Vergnes and Marescaux, 1992, 1995). It is noteworthy that SWDs have never been recorded in the hippocampus or in any limbic structures (i.e., septum, amygdala, cingular and piriform cortex) in GAERS (Marescaux et al., 1992a; Vergnes et al.,
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Chapter 18/Genetic Models of Absence Epilepsy in the Rat Cortex
I
Glutamatergic synapse γA GABAA
II-III
γB GABAB Electrical coupling
IV γA γB
V VI
γA γB Thalamic relay nucleus
nRT γA
Pre-thalamic afferent
FIGURE 3 A schematic representation of the thalamocortical interactions. NRT, nucleus reticularis of the thalamus. (Courtesy of Pierre-Olivier Polack.)
1987, 1990) or in the WAG/Rij model (Inoue et al., 1993; Kandel et al., 1996). Organization of the Thalamocortical Circuit The thalamus is the major source of subcortical inputs to the cerebral cortex. Most of the thalamocortical axons terminate in cortical layers IV and III (Alloway et al., 1993; Jones, 1985; Lu and Lin, 1993). Reciprocally, pyramidal cells from the cortical layers V and VI provide an important innervation to the thalamus (Boussara et al., 1995; Burkhaler and Charles, 1990; Chmielowska et al., 1989; Jones, 1985), and thalamocortical and corticothalamic connections are mainly glutamatergic (Deschênes and Hu, 1990; Fonnum et al., 1981; Kanedo and Mizumo, 1988; Kharazia and Weinberg, 1994; Ottersen et al., 1983; Zilles et al., 1990). In addition, all thalamic relay nuclei receive a massive GABAergic inhibitory projection from the reticular thalamic nucleus, the primary source of GABA in the rat thalamus (Cox et al.,
1996; De Biasi et al., 1988; Jones, 1985; Pinault et al., 1995). Reticular thalamic neurons do not project directly to the cerebral cortex; rather, they receive excitatory collaterals from thalamocortical and corticothalamic neurons (Bourassa et al., 1995; Contreras et al., 1993; de Curtis et al., 1989; Frassoni et al., 1984; Harris, 1987; Jones, 1985; Luebke, 1993; Spreafico et al., 1991; Warren et al., 1994) (Figure 3) (see also Chapter 7). Electrophysiologic in vitro and in vivo studies have shown that thalamocortical neurons spontaneously exhibit two different modes of intrinsic activity during various states of the wakefulness-sleep cycle (Llinas, 1988; McCormick, 1992; McCormick and Bal, 1997; Steriade, 1993; Steriade and Deschênes, 1984; Steriade et al., 1993). Periods of wakefulness and attentiveness are associated with a tonic mode of discharge of fast, sodium- and potassiummediated action potentials, allowing transmission of information to the cortex (Llinas, 1988; McCormick, 1992). On
Insights Into Human Disorders
the contrary, during stages of drowsiness and slow-wave sleep, thalamocortical neurons generate bursts of spikes triggered by rhythmic low-threshold calcium spikes (Leresche et al., 1991; Llinas, 1988; McCormick, 1992; McCormick and Pape, 1990; Steriade, 1993; Steriade and Deschênes, 1984; Steriade et al., 1993). For more information about the organization and functions of the reticular nucleus, see the review by Pinault (2004). Neurons of the thalamocortical and reticular nuclei have the capability of generating spindles, a typical oscillatory activity (Crunelli and Leresche, 1991; Gloor et al., 1990; McCormick, 1992; McCormick and Bal, 1997; Steriade, 1990; Steriade et al., 1993). Spindles consist of rhythmic oscillations of 7 to 14 c/s that occur during the initial phase of slow-wave sleep (Steriade, 1993; Steriade and Deschênes, 1984). In vivo and in vitro recordings have demonstrated that these oscillations are generated by GABAergic inhibitory neurons of the reticular thalamic nucleus, imposing synchronous inhibitory postsynaptic potentials (ipsps) in a rhythmic manner to thalamocortical neurons (Bal and McCormick, 1993; Destexhe et al., 1996; Steriade, 1993; Steriade and Deschênes, 1984; Steriade et al., 1993; Von Krosigk et al., 1993). Recent in vivo studies indicate that corticothalamic glutamatergic projections to the reticular and relay thalamic neurons strongly reinforce the synchronization of spindles throughout the thalamocortical circuit (Contreras and Steriade, 1996; Contreras et al., 1996). Activity of Thalamocortical and Reticular Neurons During SWDs and Their Relationship with Spindles In GAERS models, in vivo extracellular and intracellular recordings showed that thalamocortical neurons fire during the spike component of the SWD (Pinault et al., 1998). Similarly, extracellular recordings showed that thalamic neurons located in the ventrolateral and ventroposterior nuclei fire in phase with the spike component of the SWDs in WAG/Rij rats (Inoue et al., 1993). In these neurons, the phasic discharges are generated by a rhythmic excitatory postsynaptic potential/GABAA inhibitory postsynaptic potential sequence superimposed on a tonic hyperpolarization (Pinault et al., 1998). The GABAA-mediated inhibition appears to result from reticular inputs. Similarly, a large-amplitude hyperpolarization was recorded in the reticular neurons at the start of a SWD and a short interruption of burst firing was shown to occur during the early phase of a SWD, which is mediated by a slowly decaying depolarization (Slaght et al., 2002). During the full development of SWD, thalamocortical and reticular neurons progressively discharge in synchrony about 12 ms before the spike component of the SWD complex, suggesting that both types of neurons are driven by a common input (Pinault et al., 2001). It has been suggested that thalamic circuits underlying SWDs are the same as those generating spindles (van
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Luijtelaar, 1997), and the reticular nucleus has been proposed to be a pacemaker for both kinds of oscillations (Avanzini et al., 1992, 1993, 2000; Seidenbecher et al., 1998). However, this hypothesis has never been conclusively demonstrated. In particular, SWDs do not always occur during the same state of vigilance as spindles, and this hypothesis does not explain the bilateral and widespread generalization of SWDs. Furthermore, short-lasting (<3 seconds) bursts of 5- to 9-c/s oscillations of medium voltage were recorded interictally in the GAERS but also in nonepileptic rats and are concomitant with immobility (Pinault et al., 2001). These bursts occur during quiet wakefulness associated with desynchronized EEG and are distinct from spindles, which are observed during sleep, with a higher frequency (10 to 16 c/s) and for a shorter duration. Such 5- to 9-c/s bursts often precede SWDs in GAERS, and the extracellular patterns of discharges of thalamocortical and reticular neurons in both GAERS and nonepileptic rats are very similar to what is recorded during SWDs. These observations suggest that SWDs and medium-voltage 5- to 9-c/s oscillations share similar cellular mechanisms and that SWD may evolve from 5- to 9-c/s oscillations in GAERS, probably as a result of some genetically mediated dysfunctions in the thalamocortical network (Pinault et al., 2001). Role of GABAergic Neurotransmission The possibility that the thalamus, and more particularly the GABAergic projections coming from the reticular thalamic nucleus, play a critical role in the generation of SWD was raised by the key role of the GABAergic receptors in the capability of the thalamic neurons to generate rhythmic oscillations (Crunelli and Leresche, 1991; McCormick, 1992; Steriade et al., 1993). The aggravating effect observed following microinjections of GABAA and GABAB agonists into relay nuclei, as well as the suppression of SWD induced by injection of GABAB antagonists into the same sites (Liu et al., 1991; 1993; Marescaux et al., 1992c), suggested that a thalamic enhancement of GABAergic neurotransmission could underlie absence seizures by increasing thalamic synchronizing mechanisms. Indeed, an increase in the extracellular concentration of GABA in thalamic relay nuclei was shown in GAERS by microdialysis (Richards et al., 1995). This increase appears to result from a lower uptake of GABA that is probably mediated by the GABA transporter-1 in the thalamus of GAERS (Sutch et al., 1999). However, no differences in the number of GABAergic neurons that are immunoreactive to GABA and to its synthesis enzyme glutamic acid decarboxylase (GAD) (Spreafico et al., 1993) were observed in the thalamic reticular nucleus between adult GAERS and nonepileptic control rats. Likewise autoradiographic and immunocytochemical studies have reported that the number, affinity, and expression of GABAA receptor subtypes were identical in the reticular and relay thalamic nuclei between adult GAERS and control nonepileptic
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rats (Knight and Bowery, 1992; Snead et al., 1992; Spreafico et al., 1993). Nevertheless GAERS were reported to be more sensitive to the convulsive effects of antagonists of the GABAA receptor as well as to benzodiazepine inverse agonists, whereas no differences were found for convulsions induced by glutamate analogues (Vergnes et al., 2000). In particular, the b-carbolines (FG 7142 and DMCM) and the imidazobenzodiazepines (RO 19-4603) were several times more convulsant in GAERS than in nonepileptic rats (Vergnes et al., 2001). These convulsions mainly involve the cortex and the hippocampus and suggest that, in these structures, the number of GABAA receptors and their intrinsic binding properties are different in GAERS. However, no differences were found in the number and sensitivity of the GABAA receptors in the two strains (Vergnes et al., 2001); these findings are in agreement with previous studies. Furthermore the inhibitory response to clonazepam was not significantly modified in the nucleus reticularis in GAERS (Badiu, 2004). It is presently difficult to explain, at the cellular level, the increased sensitivity of rats with absence epilepsy to drugs reducing the GABAA mediated neurotransmission. GABAB receptor activation mediates a late and longlasting inhibitory postsynaptic potential, which produces the hyperpolarization necessary to elicit rhythmic low-threshold calcium currents (IT). Such de-inactivation of these calcium channels results in repeated bursts of action potentials in thalamocortical neurons and may be involved in the generation of SWDs (Crunelli and Leresche, 1991). In particular, intracellular recordings of nRT neurons suggested that the large-amplitude hyperpolarization of these neurons during SWD is mediated by GABAB (Slaght et al., 2002). Using an autoradiographic method, no significant difference in [3H]GABA binding to GABAB receptor in the thalamus was found between adult GAERS and control nonepileptic rats (Knight and Bowery, 1992). Similarly, the density and affinity of GABAB receptors, measured with GABAB agonist and antagonist radioligands [3H]-CGP 27492 and [3H]-CGP 54626, respectively, on thalamic membranes (Mathivet et al., 1994; 1996) appeared similar in adult GAERS and in control rats. However, a recent study using in situ hybridization and immunohistochemistry showed an increase of mRNA coding for the GABAB1 subunit in the somatosensory cortex in GAERS, whereas a decrease was observed in the ventrobasal nuclei (Princivalle et al., 2003). On the contrary, an increase in both B1 and B2 subunits was observed in all regions, suggesting the existence of an up-regulation of the GABAB receptor in GAERS. These data were obtained in adult rats and may result from reiteration of SWD. It is thus difficult to determine whether an overexpression of GABAB receptor is a key factor in SWD ontogeny. In GAERS, the amplitude of the IT calcium current was found to be increased in the reticular neurons,
which could strengthen the synchronizing mechanisms in thalamocortical circuits (Tsakiridou et al., 1995). Although this finding remains controversial (Guyon et al., 1993), a significant increase in mRNA levels of alpha-1G in ventrobasal nuclei and of alpha 1H in the nRT was found using in situ hybridization (Talley et al., 2000). The increase in alpha-1H was also found in juvenile GAERS before they express SWD. This suggests that this modest but significant increase could facilitate IT current and thus participate in the occurrence of SWDs. Other calcium-channel changes were found in WAG/Rij rats. Quantification of channel expression indicated that the development of SWDs in WAG/Rij rats is concomitant with an increased expression of the P/Q type in the nRT. These channels are mainly presynaptic, as revealed by double immunofluorescence involving the presynapse marker syntaxin (van de Bovenkamp-Janssen et al., 2004a). Dysfunction of the P/Q type of calcium channels (i.e., reshuffling of the beta subunit in this type of channel) was proposed to underlie the pathological phenotype of lethargic mice (Burgess and Noebels, 2000). Role of the Cortex The first mapping data in GAERS showed that SWDs of maximal amplitude were recorded primarily recorded in the sensorimotor cortex (Vergnes et al., 1990). In addition, they suggested that SWDs occur simultaneously in the thalamus and cortex but that thalamus was sometimes leading, whereas the reverse never seems to occur. Using multisite electrode recordings in WAG/Rij and nonlinear association analysis of the signals, a consistent cortical focus was found within the perioral subregion of the somatosensory cortex (Meeren et al., 2002). Coupling between this region and connected thalamic sites in the ventrobasal nuclei was found to change direction during the course of the seizure. During the first 500 msec of a SWD, the cortical focus appears to lead the thalamus. This is further supported by the observation that cortical SWDs can sometimes occur without concomitant thalamic discharge, whereas the reverse was never observed (Meeren et al., 2002). Furthermore, it is consistent with the fact that rhythmic unit firing was reported to start a few cycles earlier in the cortex than in the thalamus in GAERS (Seidenbecher et al., 1998). This study also suggested a fast intracortical spread of the SWDs, with the intrahemispheric spread being slower than the interhemispheric one. Although the existence of the cortical perioral focus remains to be confirmed in GAERS, the intrahemispheric versus the interhemispheric spread could explain the discrepancies with the previous studies suggesting the leading role of the thalamic nuclei. Indeed, in the first mapping studies, the cortical electrode was never located in the perioral area and was generally placed in a more posterior region. In this region, cortical activity occurs a few
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Acknowledgments
cycles later according to the study of Meeren et al. (2002). These findings led this group of investigators to suggest that absence seizures have a focal origin. The primary driving source for the rhythmic activity could be the cortex (or at least a specific region of the cortex), not the thalamus. However, once the seizure is initiated, the cortex and the thalamus form an oscillatory network in which both structures drive each other (Meeren et al., 2002). The generalized and synchronous aspects of these seizures could be due to the very fast cortical spread of seizure activity. The focal cortical origin of SWDs is in agreement with the recent finding that application of ethosuximide at the somatosensory cortex suppresses absence seizures in GAERS, whereas no or weak effects were observed when this compound was injected in the thalamus. Moreover, local injections at other cortical areas were not effective (Manning et al., 2004). This finding is also in agreement with the fact that focal unilateral injection of lidocaine into the perioral region of the cortex in WAG/Rij caused a general decrease in the number of SWDs recorded in other cortical areas in both hemispheres (Sitnikova and van Luijtelaar, 2004). This finding is consistent with clinical data suggesting that generalized SWD tend to begin in the cortex (see Sitnikova and van Luijtelaar, 2004). In addition, the somatosensory region of the cortex is known to produce physiologic oscillations in the frequency domain of 7 to 12 c/s (Ritz et al., 1997). This “somatosensory rhythm” is supposed to be due to the synchronous functioning of the cortical pyramidal cells and inhibitory interneurons (Silva et al., 1991). Because SWD share several similarities with somatosensory rhythms, it has been suggested that SWD could derive from these cortical physiologic oscillations (Sitnikova and van Luijtelaar, 2004). Several data are also in agreement with the existence of a cortical and eventually “focal” generator of SWD. In the cerebral cortex, the main source of inhibition comes from local nonpyramidal GABAergic interneurons (basket cells, chandelier cells, double bouquet cells, and bipolar cells), which strongly control the activity of cortical pyramidal neurons (Beaulieu, 1993; Defelipe, 1993; Meinecke and Peters, 1987; Somogyi and Cowey, 1981). In WAG/Rij rats, extracellularly and intracellularly recorded synaptic responses revealed an intracortical hyperexcitability accompanied by a reduction of the efficiency of GABA-ergic inhibition in slices of the frontal cortex (Luhmann et al., 1995). In addition, immunocytochemical studies on the WAG/RiJ cortex showed that some areas lacked the calcium-binding protein parvalbumin, which is co-localized with GABA (van de Bovenkamp-Janssen et al., 2004b). Finally, it was shown that voltage-gated sodium channels, as measured by quantitative polymerase chain reaction (PCR) and immuno-cytochemistry, are upregulated selectively at the facial somatosensory cortex in WAG/Rij rats (Klein et al., 2004). Further experiments are required to determine the cellular mechanisms underlying SWD in the
focal cortical generator and whether such a focus also exists in the GAERS and can be assessed in the human patients.
Usefulness for Treatment Assessment Both genetic models, GAERS and WAG/Rij rats, offer a very high predictability for an antiabsence effect of an AED under development (see preceding discussion). In addition, both models have been useful in predicting adverse effects of some AEDs, such as vigabatrin and tiagabine, which should not be prescribed in cases of childhood absence epilepsy or in other cases of generalized absence seizures. In general, GAERS and WAG/Rij are very useful models to estimate the potential antiepileptic or adverse effect of a new drug candidate for the central nervous system (van Luijtelaar et al., 2002). Designing new drugs dedicated to suppressing oscillatory rhythms in the cortex or the thalamus may lead to important cognitive or vigilance side effects.
GENERAL CONCLUSIONS During the last 20 years, the GAERS and the WAG/Rij strains of Wistar rats have proven to be valid and predictive models of human absence epilepsy. Although some features of this form of epilepsy appear species specific (e.g., frequency, ontogeny, and aging), the pharmacologic reactivity and the underlying circuits described in both strains appear very similar to the human pathology. Differences between the two strains and recent identification of QTLs suggest that several genetic mechanisms can lead to the generation of SWDs. However, in both models the thalamocortical circuits clearly appear as the critical generator of absence seizures. Multidisciplinary studies of these two strains, and also other strains and species, will improve our understanding of the role of the cortex and the thalamus, and probably other subcortical circuits, in the generation of SWDs.
Acknowledgments The authors thank L. Danober, O. David, C. Deransart, I. Guillemain, P. Kahane, A. Nehlig, and B. Martin for their help in the preparation of the manuscript. A.D. wishes to thank Any Boehrer, Christian Marescaux, and Marguerite Vergnes for their fruitful collaboration during the past 20 years.
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Vergnes, M., Marescaux, C., Depaulis, A., Micheletti, G., and Warter, J.M. 1986. Ontogeny of spontaneous petit mal-like seizures in Wistar rats. Dev. Brain Res 30: 85–87. Vergnes, M., Marescaux, C., Depaulis, A., Micheletti, G., and Warter, J.M. 1987. Spontaneous spike and wave discharges in thalamus and cortex in a rat model of genetic petit mal-like seizures. Exp Neurol 96: 127–136. Vergnes, M., Marescaux, C., Micheletti, G., Depaulis, A., Rumbach, L., and Warter, J.M. 1984. Enhancement of spike and wave discharges by GABAmimetics drugs in rats with spontaneous petit mal-like epilepsy. Neurosci Lett 44: 91–94. Vergnes, M., Marescaux, C., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L., and Warter, J.M. 1982. Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized non-convulsive epilepsy. Neurosci Lett 33: 97–101. Von Krosigk, M., Bal, T., and McCormick, D.A. 1993. Cellular mechanisms of a synchronized oscillation in the thalamus. Science 261: 361– 364. Wallace, R.H., Marini, C., Petrou, S., Harkin, L.A., Bowser, D.N., Panchal, R.G., Williams, D.A. et al. 2001. Mutant GABA(A) receptor gamma2subunit in childhood absence epilepsy and febrile seizures. Nat Genet 28: 49–52. Warren, R.A., Agmon, A., and Jones, E.G. 1994. Oscillatory synaptic interactions between ventroposterior reticular neurons in mouse thalamus in vitro. J Neurophysiol 72: 1993–2003. Warter, J.M., Vergnes, M., Depaulis, A., Tranchant, C., Rumbach, L., Micheletti, G., and Marescaux, C. 1988. Effect of drugs affecting dopaminergic neurotransmission in rats with spontaneous petit mal-like seizures. Neuropharmacology 27: 269–274. Willoughby, J.O., and Mackenzie, L. 1992. Nonconvulsive electrocorticographic paroxysms (absence epilepsy) in rat strains. Lab Anim Sci 42: 551–554. Zilles, Z., Wree, A., and Dausch, N.D. 1990. Anatomy of the neocortex: neurochemical organization. In The Cerebral Cortex of the Rat Ed. B. Kolb and R.C. Tees. pp 113–150. Cambridge, MA: MIT Press.
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19 Models with Spontaneous Seizures and Developmental Disruption of Genetic Etiology RADDY L. RAMOS AND JOSEPH J. LOTURCO
The p35 mutant mouse has a less severe spontaneous seizure phenotype than the others (Chae et al., 1997; Wenzel et al., 2001) but has a cellular neocortical migration defect similar to human lissencephaly and shares anatomic and physiologic correlates in the hippocampus with other animal seizure models that are not associated with developmental disruption. The cellular changes that have been described in these four models are diverse and suggest that there may be many cellular routes to over-excitability in malformed cerebral cortex.
GENERAL DESCRIPTION OF MODELS Malformation of the cerebral cortex is a significant risk factor for epilepsy in humans. Several genes have now been identified in both rodents and humans that, when mutated, result in malformed cerebral cortex and seizure susceptibility. Although the genes mutated in the models described below do not involve genes currently known to be associated with epilepsy in humans, they do reveal a set of underlying mechanisms that may lead to a clearer understanding of epileptogenesis in the malformed cerebral cortex. Observations on animal models displaying cortical malformations—induced by trauma, teratogen, or genetic mutation—reveal several cellular changes associated with altered excitability in the malformed brain. As over-excitability can arise from many types of pathophysiologic changes, analysis of multiple models is necessary to determine the mechanisms underlying over-excitability in the malformed brain. Genetic models have well-known advantages that make them particularly useful for epilepsy studies. In this chapter, we will review data from the four best-studied rodent mutants that display spontaneous seizures and are associated with defined cortical malformations. The four models display a range of malformation type and seizure severity (Table 1). The two models with the most severe seizure phenotype, the flathead mutant rat (Sarkisian et al., 1999) and the Otx-/- mouse (Acampora et al., 1996), share the morphological phenotype of severe microencephaly and reduced thickness of layers of neocortex, without significant neuronal migration disruption. The tish mutant rat, which also presents with regular spontaneous seizures, displays a remarkable anatomic phenotype which models subcortical band heterotopia or “double cortex” syndrome in humans.
Models of Seizures and Epilepsy
GENERAL CLINICAL FEATURES Flathead Mutant Rat The flathead rat is a spontaneous mutant that was discovered in 1995 within a breeding colony of Wistar rats at the University of Connecticut, Storrs (Roberts et al., 2000). Affected individuals display marked neurologic impairment, with frequent seizures and premature death (Sarkisian et al., 1999). Breeding experiments and subsequent genetic mapping studies identified the causative mutant allele as an autosomal recessive mutation in the Citron Kinase gene (CitK) (Cogswell et al., 1998; Sarkisian et al., 2002). In addition to robust generalized seizures, homozygous mutants display motor disruptions, including resting tremor, severe ataxia, dystonia, atonia, and astatic episodes. Several neurobiological abnormalities have been identified in the flathead mutant, including microencephaly, abnormal cell death, neuronal and glial cytomegaly, GABAergic interneuron loss, and neurogenic cytokinesis failure (Roberts et al., 2000; Sarkisian et al., 2001; LoTurco et al., 2003). A strik-
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Chapter 19/Models with Spontaneous Seizures and Developmental Disruption of Genetic Etiology
TABLE 1 Summary of Rodent Models Seizure occurence frequency
Onset
Seizure type(s)
Neuropathology
Primary developmental disruption
flathead
100%, 1–4/h, status
2nd postnatal week
Generalized tonic-clonic
Microencephaly, dentate granule layer hypoplasia, interneuron reduction
Cytokinesis defect and apoptosis
tish
100%, 2–15/week
1st postnatal month
Generalized tonicclonic & partial
Subcortical band heterotopia
Heterotopic neurogenesis
Otx1-/-
100%, 1–5/day
1st postnatal month
Generalized tonicclonic & partial
Microencephaly, disrupted layer V projections
Cell differentiation and patterning
p35-/-
25%, generalized; 50%, partial
?
Generalized tonicclonic, partial
Migration disruption in neocortex, dentate granule cell dysplasia, and sprouting
Neuronal migration
ing feature of the mutant is the regularity of generalized seizures from the first through second week after birth (Sarkisian et al., 1999). The early onset, reliability, and regularity of the spontaneous seizures make the flathead mutant a valuable resource for exploring cellular changes associated with seizure progression in epilepsy syndromes associated with severe developmental disruption. The flathead phenotype is lethal within the first 3 weeks after birth, and therefore homozygous mutants must be generated by breeding heterozygotes. Heterozygous animals have no defined phenotype and therefore must be identified either with genetic markers or by proven breeding. The flathead mutation is a completely penetrant autosomal recessive, so 25% of offspring from matings between proven heterozygous animals show the mutant phenotype. Twothirds of unaffected offspring with no phenotype are heterozygous and can be used for further propagation. The flathead mutant phenotype can be easily identified in newborn rat pups by marked microcephaly, flat skull, and tremor. Through the first postnatal week, the phenotype becomes more noticeable: normal littermates increase in size and develop smooth coordinated movement, while the flathead develops tremor and then seizure activity. There is currently no commercial source for the mutant, but a colony is maintained at the University of Connecticut and delivery and transfer of proven breeding pairs can be arranged for research purposes. In addition, detailed protocols for genotyping the rats for identification of heterozygous offspring are available. Tish Mutant Rat The tish rat is a spontaneous mutant first discovered after histologic examination of brains from Sprague-Dawley rats used in unrelated studies at the University of Virginia (Lee et al., 1997). The tish mutant, similarly to the flathead rat, is an autosomal recessive mutation. However, unlike the flathead mutation, the tish mutation is not lethal, and homozy-
gous animals appear healthy and breed normally. The most distinguishing anatomic feature of the tish mutant is the presence of well-developed bilateral subcortical band heterotopias which can be detected by MRI or by routine histologic examination. The homozygous mutants also exhibit spontaneous seizures, starting from approximately 1 month of age to at least 6 months of age (K. Lee, personal communication). The gene mutation responsible for the tish phenotype is not yet identified. The autosomal recessive nature of the mutation, however, makes it unlikely that the causative mutation is in the same gene that gives rise to the majority of double cortex syndromes in humans—which are typically caused by mutations in the X-linked gene DCX, and to a lesser extent to mutations of Lis1 (Pilz et al., 1998; D’Agostino et al., 2002). Since there is a significant number of double cortex cases in humans that do not involve DCX or LIS1 mutations, identification of the causative gene in the tish mutation could help identify such mutations in human double cortex syndromes. The well-formed heterotopia in tish, and presence of spontaneous seizures in these rats, make tish an ideal model in which to determine the mechanisms of epileptogenesis in double cortex syndrome. OTX1-/- Mouse The Otx1 gene, related to the drosophila othodentical gene, was initially inactivated by homologous recombination to study its role in patterning of the forebrain (Acampora et al., 1998; Boyl et al., 2001). The homozygous mutants have a pronounced spontaneous seizure disorder, with 100% of homozygous mutants displaying generalized tonic-clonic seizures. Homozygous mutants are smaller than heterozygous animals, which are fertile and healthy (Avanzini et al., 2000). Thirty percent of Otx-/- mice die in the first postnatal month. The primary neurodevelopmental defect is microencephaly, with pronounced reduction in
Characteristic Features
thickness of neocortex. In addition, subsets of cortical layer V neurons are missing, and therefore the cortex does not provide appropriate output to subcortical regions (Acampora et al., 1996; Weimann et al., 1999). To date, there is no known human developmental disorder associated with mutations of the OTX1 gene. Nevertheless, the relatively high frequency of seizures in this mutant, and the mix of both behavioral and electrographic signs of focal and generalized seizures, make this animal a unique and potentially important model to determine the mechanisms of seizure progression in the malformed brain. p35-/- Mouse The p35 protein is a neuron-specific activator of the cyclin-dependent kinase 5, Cdk5. Deletion of p35 by homologous recombination in mice causes migration disruption in neocortex, dysplasia in hippocampus, spontaneous seizures in a percentage of mutants, and reduced threshold to convulsants (Chae et al., 1997; Wenzel et al., 2001). The developmental progression of the spontaneous seizures has not yet been fully described, although 75% of mice from 3 to 5 months of age show electrographic signs of epileptiform activity in neocortex and hippocampus, and 25% display spontaneous tonic-clonic seizures. The dentate granule cells show sprouting characteristic of other temporal lobe seizure models, and the dentate granule layer is also more diffuse than that in the wild type. This model provides for an interesting mix of features that may help to identify mechanisms of over-excitability that are common to both models containing developmental disruption and models in which pharmacologic stimulation leads to dentate granule cell sprouting and epileptogenesis.
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As early as postnatal day 8 (P8), flathead mutants display intermittent episodes of tail flexion and extension (strub tail) and tremor of the limbs and head. These observations are the earliest signs of spontaneous generalized seizures of any of the models described here. Alternating forelimb clonus becomes more prevalent between P15 and P19. During clonic episodes, animals frequently ambulate around the home cage, propelling themselves by their forelimbs. Bursts of forelimb movements often result in forward lunges. Clonus of the neck musculature produces rhythmic head movements. Flathead rats display episodes of tonus that can involve one or more limbs or the entire body; tonus persists for approximately 1 minute and is sometimes preceded by loud vocalizations. Behavioral seizures are rare after P21, although single episodes of loud vocalization, jumping, and tonus have been observed in animals as late as P25—near the time of premature death, which is thought to be caused by a lethal seizure. Tish Mutant Rat Convulsive seizures become apparent in the tish mutant approximately 1 month after birth (K. Lee, personal communication). Otherwise the behavior of tish mutants, assessed by routine observation, is indistinguishable from unaffected littermates or wildtype Sprague-Dawley rats. Behavioral seizures occur simultaneously with high amplitude voltage oscillations in the EEG. The rat’s behavior during seizures is more variable than the electrographic seizure activity and is characterized by facial and forelimb twitching that can progress to convulsive tonic-clonic seizures. The average frequency of such seizures is 48 seconds, and the frequency of the events (assessed in 4–6 months of monitoring) is between 1.5 to 15 events/week (Chen et al., 2000).
CHARACTERISTIC FEATURES Behavioral and Electrographic Features Flathead Mutant Rat In the first postnatal week, flathead pups display severe generalized seizures and motor deficits. During ambulation, astatic episodes can be observed and are characterized by the splaying of limbs. Mutant rats also often fall to one side during walking but are able to right themselves quickly. The falling and astatic episodes in flathead mutants are likely related to general muscle weakness, since flathead mutants cannot grip a small metal bar and are unable to rear onto their hind limbs. Seizures begin reliably toward the end of the first postnatal week and increase in duration and frequency until the third postnatal week (Figure 1). Seizures subsequently become more rare, but more severe, as identified by behavioral observations and electrophysiologic recordings.
Otx1-/- Mouse Seizures in Otx-/- mice range from short episodes of approximately 30 seconds, to longer lasting, 60-second episodes. The shorter episodes are characterized by automatisms, including head bobbing and teeth chattering. The longer seizures are characterized by upper limb clonus, rearing, and falling. Similarly to the flathead mutant, seizure frequency in Otx-/- mice is reduced in older animals, although there is no indication of an overall cessation or remission of seizures (Acampora et al., 1996). During the shorter episodes, electrographic recordings indicate large amplitude oscillations in hippocampus with occasional and asynchronous spikes in the neocortex. This pattern is consistent with focal generation of epileptiform events. The longer convulsive seizures, in contrast, are characterized by synchronized activity in both the neocortex and hippocampus (Avanzini et al., 2000).
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FIGURE 1 Flathead seizures are generalized and increase in duration with age. A: Representative six-channel, electrographic recordings of a P16 flathead rat. Electrodes were placed across the rostrocaudal extent of the cortical surface (top to bottom: anterior left hemisphere; anterior right hemisphere; middle left hemisphere; middle right hemisphere; posterior left hemisphere; posterior right hemisphere). B: The average duration of seizures increases with age in flathead. C: There were no significant differences in average interval between seizures across age. D: Representative in vitro extracellular recording from a flathead hippocampal slice (electrode positioned in CA1 pyramidal layer). Ictal and interictal periods are identifiable in the upper trace (10-minute recording). Burst-type spiking can be seen in the expanded (lower) trace. Scale bars: A, 1000 ms/200 mV; D, upper trace, 2 min/70 mV; lower trace, 8 sec/70 mV.
P35 -/- Mouse Both partial and generalized seizures occur in p35 -/mice (Wenzel 2001). Approximately 75% of mutants display some sign of electrographic abnormality, and 25% display both behavioral and electrographic features of generalized
seizure episodes (Wenzel et al., 2001). Seizures typically start from a state of quiet rest or sleep and initiate upon a high-amplitude spike in the EEG. Behaviorally, generalized seizures begin with back and forth head movements that progress to a period characterized by a rigidly up-turned head and extended forelimbs. In many animals this behav-
Characteristic Features
FIGURE 2 The flathead phenotype is associated with a decrease in brain size but preserved patterning. A: Saggital Nissl-stained sections from a P1 (left column) and P14 (right column) flathead brain (top row) and wildtype brain (bottom row). B: Coronal Nissl-stained section of a P21 flathead brain (left) and wildtype brain (right). Note lack of the infrapyramidal blade of the dentate gyrus but normal lamination of neocortex. Scalebars: A & B, 1 mm.
ioral pattern progresses to a period of vigorous hind limb thrusts. The behavioral manifestations last 1 to 1.5 minutes (Wenzel et al., 2001). During seizures, high-amplitude electrographic spiking is observed bilaterally in both hippocampus and neocortex. Epileptiform spiking can continue longer in the hippocampus than in the neocortex, and ictal periods are followed by a 3 to 5 minute postictal period characterized by a quiet EEG and behavioral inactivity (Wenzel et al., 2001).
Neuropathology Flathead Mutant Although the primary developmental disruptions in flathead mutants are restricted to the CNS (Roberts et al., 2000), disruptions have been reported in testes and liver (Liu et al., 2003; Naim et al., 2004). The brain of flathead mutants is approximately 50% the size of unaffected littermates (Figure 2). This difference emerges in late embryogenesis and is not detectable at embryonic day 16 (E16). By E18, mutant brains weigh 26% less than unaffected littermates, and by
253
E19 weigh 51% less. Reduction in size involves most brain areas, although the tectum is least affected (Figure 2). In addition to general reduction in brain size, some neuronal populations are disproportionally reduced in number. The most affected cell populations include granule cells in cerebellum, dentate granule cells of the hippocampus, and layer 2/3 pyramidal neurons of neocortex. This pattern of loss is consistent with a model of progenitor depletion that would preferentially target the latest born populations of neurons. In flathead mutants, the number of inhibitory interneurons—as a proportion of the total number of neurons in the cerebral cortex—is also greatly reduced (Figure 3). Quantitative analysis in both somatosensory cortex (SS) and entorhinal cortex (EC) indicates that the percentage of GABA-positive neurons is reduced in mutants relative to littermates. In upper layers of EC, 30% of neurons in normal littermates are GABA-positive, compared to only 9% of neurons in mutants; in deeper layers of EC, 15% of neurons in normal littermates are GABA-positive compared to only 6% of neurons in flathead rats. In deeper layers of SS cortex, 15% of neurons in unaffected littermates, compared to only 4% of neurons in mutants, are GABA-positive. In contrast, in upper layers of SS there is only a slight decrease in the percentage of GABA-positive neurons: ~11% of neurons in littermates compared to 9% of neurons in mutants. The calcium-binding proteins calretinin (CR), parvalbumin (PARV), and calbindin (CAL) are specifically expressed in largely nonoverlapping populations of GABA-positive interneurons in the cerebral cortex and correspond to different functional and morphological classes of inhibitory interneurons. The flathead mutation results in differential loss of these different classes (Alcantara et al., 1993; Alcantara et al., 1996); the CR-positive interneurons are not significantly decreased in number, but the PARV and CAL-positive cells are reduced by 70% (Sarkisian et al., 2001). Therefore, the flathead mutation causes a widespread and differential decrease in the relative number of inhibitory interneurons across neocortex. Tish Mutant Rat The tish mutant has large bilateral secondary cortices that form below the normal neocortical layers. The heterotopias are variable in size from animal to animal, but typically extend from the frontal to occipital poles (Lee et al., 1997). The normotopic cortex above the heterotopic cortex is thinner than normal, although all neocortical layers and major projection neurons are present. The heterotopic cortex, in contrast, is not laminated. Cellular birth-dating experiments with BrdU indicate that cells in normotopic cortex are born in a normal inside-to-outside pattern, while the cells in the nonlaminated heterotopic cortex do not show a clear birth-dating pattern (Lee et al., 1998). In spite of the lack of lamination, the heterotopic cortex (like the
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Chapter 19/Models with Spontaneous Seizures and Developmental Disruption of Genetic Etiology
FIGURE 3 The flathead phenotype is associated with cytokinesis failure of neocortical neurons and a reduction of GABAergic interneurons. A: Electron micrograph of a neocortical neuron where two nuclei are visible within a single cell membrane. B: An embryonic BrdU pulse results in labeling of multiple nuclei within single Nissl-stained neocortical neurons. C: Quantification of the relative number of GABAergic interneurons in the upper and deeper layers of entorhinal cortex and somatosensory cortex in flathead and wildtype brains at P0 and P14. D: Representative example of a calbindin-positive neocortical interneuron in flathead, containing two nuclei (arrows). E: Representative example of a parvalbumin-positive neocortical interneuron in the flathead rat, containing two nuclei (arrows). F: Relative percentage of GABAergic interneuron subtypes in the flathead (closed bars) and wildtype (open bars) neocortex (entorhinal and somatosensory). G: Percentage of GABAergic and DiI-labeled pyramidal neurons in flathead neocortex that contains multiple nuclei. Scale bars: A, 5 mm; B, 10 mm; D, 15 mm; E, 20 mm.
Characteristic Features
normotopic cortex) contains neurons that project to appropiate subcortical and cortical targets. Similarly, the heterotopic cortex receives patterned thalamic input from somatosensory thalamus; cortical barrels form in both heterotopic and normotopic cortex (Schottler et al., 2001). Otx1-/- Mouse The brains of Otx-/- mice are significantly smaller than the brains of wildtype or heterozygous animals. The neocortex is most reduced in size, while the midbrain and hind brain are relatively unaffected. The neocortical lamina are reduced in thickness by approximately 25%, and there is a significant reduction in cellular density in neocortical lamina ranging from 4 to 35% (depending on the cortical area examined) (Cipelletti et al., 2002; Panto et al., 2004). This pattern of microencephaly is similar to, although less severe than, that in the flathead mutant. There is also a decrease in the density of some interneuron populations within the neocortex of Otx1-/- mice. The density of GAD67-positive inhibitory interneurons in the Otx1-/- neocortex, as well as parvalbumin- and calbindin-positve neurons, shows reductions. Parvalbumin interneurons are reduced by approximately 60% and calbindin-positive interneurons are reduced by approximately 52%, whereas the total decrease in neurons is approximately 30% (Cipelletti et al., 2002). Thus, similar to the flathead mutant cortex, the Otx-/- mutant cortex has an overall reduction in neurons and a disproportionate decrease in interneurons. p35-/- Mouse Both neocortex and hippocampus show developmental disruption in p35-/- mice. The lamination of neocortex is disrupted and the laminar position of pyramidal cell populations is generally inverted relative to control neocortex (Chae et al., 1997). There has been no report of significant/disproportionate interneuron loss. The normally compact dentate granule cell layer in hippocampus is more diffuse in the p35-/- mutants. In addition, the dentate granule cells exhibit ectopic sprouting. This sprouting has not been observed in the other three developmental models discussed here but is a common feature of many other models of epilepsy. Such axonal sprouting results in recurrent feedback excitation that may create hyperexcitable circuits in hippocampus (Patel et al., 2004).
Neurophysiology Flathead Mutant Flathead electrographic seizures occur at a high enough frequency, and with sufficient reliability, to allow for quan-
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titative measurements of seizure duration and interval across postnatal development. Figure 1 shows the changes in seizure frequency and duration from animals from P7 to P18. The interval between seizures does not change significantly; however, the seizure duration increases with age. By P20, electrographic and behavioral seizures are rare; however, severe lethal seizures have been noted in several animals from P21 to P27. Long-duration seizures increase in number from P7 to P18, and this increase accounts for the increase in mean seizure duration. Simultaneous multi-site surface recordings, as well as recordings from brain slices, demonstrate that epileptiform activity in the flathead can be generated from multiple sites within cerebral cortex. Following callosal transection, both hemispheres can show independent and isolated electrographic seizure activity. Similarly, spontaneous 20 to 60 second self-regenerating epileptiform discharges are present in brain slices prepared from neocortex or hippocampus. Tish Mutant In vivo depth electrode recordings and slice recordings from tish rats indicate that epileptiform activity is present in both the normotopic and heterotopic cortices (Chen et al., 2000). Epileptiform activity in normotopic cortex appears to precede activity in the heterotopic cortex. In experiments on brain slices, when normotopic cortex was isolated from heterotopic cortex (either pharmacologically or surgically), epileptiform activity persisted in normotopic but not in heterotopic cortex (Chen et al., 2000). Thus, the driver of epileptiform activity in the tish rat appears to be normotopic, not heterotopic, cortex. Otx-/- Mouse A thorough electrophysiologic analysis has been carried out in brain slices prepared from Otx-/- mice. Stimulation of white matter can initiate polysynaptic bursts in the Otx-/- cortex at stimuli strengths that fail to elicit bursts in normal controls (Sancini et al., 2001). These polysynaptic bursts are characterized by prominent slow inhibitory postsynaptic potentials (IPSPs) and delayed large excitatory PSPs (EPSPs). The polysynaptic burst activity is blocked by NMDA receptor antagonists but not by AMPA receptor antagonists. Intrinsic excitability of pyramidal cells is not altered in the mutant. However, a redistribution of electrophysiologic cell types is apparent in layer V, apparently resulting from a loss of a subpopulation of layer V pyramidal neurons. It is likely that the combination of increased synchrony of synaptic inhibition, along with increases in NMDA-mediated excitation, results in the aberrant polysynaptic activity described in Otx-/- cortex (Sancini et al., 2001). The enhanced synchrony of inhibition may seem paradoxical considering the relative loss of inhibitory interneu-
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Chapter 19/Models with Spontaneous Seizures and Developmental Disruption of Genetic Etiology
rons. However, it is possible that a decrease in interneuron number may result in increased synchrony because fewer inhibitory neuronal elements contribute to the IPSPs. p35 -/- Mouse Both in vivo recordings and in vitro brain slice experiments have been carried out in p35 -/- mutant mice. In vivo depth electrode recordings from hippocampus and neocortex show that intermittent epileptiform spikes typically occur in the hippocampus, and sustained ictal discharges occur synchronously in hippocampus and neocortex (Wenzel et al., 2001). Field EPSP recordings from the dentate gyrus further indicate an aberrant response in the granule cell layer that is likely due to a dispersed “somal” current sink (Patel et al., 2004). Intrinsic properties of dentate granule cells are similar between mutants and controls, but hyperexcitability of dentate granule cell responses, relative to controls, is observed in low concentrations of the GABA receptor antagonist bicuculline methiodide (BMI) (Patel et al., 2004). In addition, consistent with an increase in granule cell sprouting, antidromic activation of granule cells in the p35 -/- hippocampus results in enhanced excitatory synaptic responses in granule cells (Patel et al., 2004).
Response to AEDs To date, only the flathead rat has been used in published studies to test the effectiveness of different antiepileptic drugs (AEDs) (Sarkisian et al., 1999). Three AEDs have been evaluated for their effects on the duration and frequency of electrographic seizures in flathead mutants. Phenobarbital (PB), ethosuximide (ESM), and valproate (VPA) induce sedation for at least 2 hours in neonatal rats (PB, 40mg/kg; ESM, 600mg/kg; VPA, 400mg/kg), and suppress behavioral manifestations of the seizures. However, electrographic seizures are still observed after AED treatment, and occur at rates of at least 2 per hour. Seizure occurrence is differently affected by the different AEDs. PB significantly increases the interval between seizures in mutants but does not significantly change the duration of seizures. VPA, in contrast, does not significantly alter the interval between seizures but shortens the duration of seizures in the flathead mutant. ESM has no effect on seizure interval or duration in the flathead mutant (Sarkisian et al., 1999).
MECHANISMS OF DEVELOPMENTAL DISRUPTION Flathead Mutant Rat An advantage of genetic models is the potential for tracking the cause(s) of the malformation from a single molecu-
lar defect. A positional cloning strategy was used to identify the genetic location and mutation responsible for the flathead phenotype. The flathead gene was initially localized to a small region of rat chromosome 12 that is syngenic to a region of human chromosome 12 between Nos-1 and TCF1 (Cogswell et al., 1998; Sarkisian et al., 2002). Sequencing of the gene Citk in mutants indicated that the flathead mutation is a single base deletion (G) in the kinase domain of a gene essential to neurogenetic cytokinesis (Sarkisian et al., 2002; Bai et al., 2003). The single base deletion causes a non-sense mutation within the first exon that terminates translation prematurely, 33 amino acids into translation. The Citk protein is absent from homozygous flathead mutants; this protein is expressed only in the normal developing brain at cytokinesis furrows in neurogenic regions (Sarkisian et al., 2002). The presence of multinucleate GABAergic neurons in the flathead indicates failed neurogenic cytokinesis during the generation of neocortical GABAergic neurons. Moreover, a six-fold increase in pyknotic cells in the medial ganglionic eminence indicates that cell death may be responsible for the interneuron reduction. Similar to interneuronal progenitors, pyramidal neuronal progenitors fail to undergo appropriate mitoses, resulting in both cell death and cytokinesis failure. The progressive loss of neuronal progenitors from apoptosis and ineffective cell division results in a depletion of neuronal progenitors and premature cessation of cell production in both neocortex and hippocampus. Tish Mutant Rat Although the mutation responsible for the tish phenotype has not been identified, the nature of the developmental defect has been determined. Early in corticogenesis (E12) in the tish mutant, there is an increased number of displaced cell divisions away from the ventricular surface (Lee et al., 1998). This heterotopic neurogenesis then leads to the expansion of a secondary cortex that fails to laminate. Interestingly, the normotopic and heterotopic cortices both receive cells generated from both the heterotopic and normotopic neurogenic zones. The development of subcortical band heterotopia in the tish mutant is therefore due to ectopic proliferation and not due to a disruption in the migration of postmitotic neurons, as is thought to be responsible for human double cortex syndrome associated with mutations in DCX. Otx1-/- Mouse Otx1 is a member of the homeobox gene family of transcription factors well-known to regulate developmental patterning (Acampora et al., 1998). Central to the function of homeobox genes is a highly restricted pattern of expression in the developing embryo. Otx1 is initially expressed in the
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neuroepithelial cells of embryonic cortex; as neurogenesis procedes, Otx1-expressing cells move to layers V and VI of mature cortex (Frantz et al., 1994). Mutation of Otx1 results in a proportional decrease in all cortical layers, as well as an aberration in the normal pruning of exuberant connections of layer V projection neurons (Weimann et al., 1999; Panto et al., 2004). The timing of Otx1-dependent pruning correlates in time with translocation of otx1 from the cytoplasm to the nucleus (Frantz et al., 1994; Weimann et al., 1999; Zhang et al., 2002). p35-/- Mouse The mechanisms underlying the developmental disruption in the neocortex of the p35-/- mice have been well studied and reviewed elsewhere (Gupta and Tsai, 2003). Briefly, p35 acts as the neuron-specific activator of Cdk5 and is essential to normal neuronal migration of neocortical neurons. Cdk5 has many substrates that include other proteins (e.g., DCX, NUDEL, and DAB1) shown to be essential to neuronal migration. Cdk5 appears to function in the migrating neuron by connecting extracellular signals to cytoskeletal restructuring necessary for neuronal motility (Gupta and Tsai, 2003).
INSIGHTS INTO HUMAN DISORDERS The models discussed here provide avenues for further insights into epileptogenesis in the malformed brain. Several features of the seizures and underlying developmental disruption in the flathead mutant are similar to those described in human syndromes, including Ohtahara syndrome and Lennox-Gastaut syndrome. The primary microencephaly phenotype has not been generally associated with epilepsy in humans; however, many migration disorders, including those arising from ARX and Lis1 mutations, can also be associated with epilepsy and microencephaly. Microencephaly may simply be a gross phenotype that suggests the possibility of differential cell loss. In addition, cellular features in neocortex of flathead resemble some human cortical dysplasias associated with epilepsy. The neocortices of flathead contain cytomegalic neurons, binucleate cells, and reductions in the number of GABAergic interneurons similar to that observed in human dysplasias associated with epilepsy (Spreafico et al., 1998). Although it is not currently known which, if any, of these cellular features are causal to the seizures, the flathead model may provide an opportunity to dissect the relative contributions of a variety of developmental disruptions. Of the rodent models that display spontaneous seizures, the tish mutant most closely models the anatomic features of a known human cortical malformation. Interestingly, the development of the secondary cortex in this model is not
likely to be the same as in most cases of double cortex in humans. The most common genetic cause of subcortical band heterotopia in humans is a mutation in the DCX gene. DCX is expressed in postmitotic neurons, and knockdown of DCX by RNAi creates subcortical band heterotopia in rats by blocking radial migration without formation of a secondary neurogenic zone (Bai et al., 2003). In contrast, the second cortex in tish arises from a heterotopic neurogenic zone. These observations indicate that there are at least two different mechanisms for the development of double cortex and that each of them can lead to seizures. There are currently no known human syndromes associated with mutations in Otx-/-. Two other homeobox genes that function in forebrain development, ARX and EMX2, have been recently linked to cortical malformation and seizures. EMX2 mutations have been described in schizencephaly, a malformation type for which there is yet no animal model. Mutations in ARX cause a spectrum of developmental disorders, including infantile spasms and mental retardation. One of the CNS malformations associated with ARX mutation is microcephaly, similar to that seen in flathead and Otx1-/-. Microencephaly alone is unlikely to be a cause of seizure syndromes, as primary microencephaly is not sufficient to cause epilepsy in humans (Mochida and Walsh, 2001; Woods, 2004). Other disruptions in development—perhaps a differential loss of certain cell types as occurs in flathead and Otx-/-—may be necessary for seizure development. Mutations in p35 have not been associated with epilepsy in humans. However, the Cdk5 signaling pathway has been associated with the function of several genes connected to cortical malformation and epilepsy: DCX, Lis1, and 14-3-3 epsilon pathways. Specifically, Cdk5 phosphorylates DCX and lowers its affinity for microtubules. Cdk5 also phosphorylates NUDEl protein, which functions in the pathway of the two Miller-Dieker lissencephaly genes, LIS1 and 14-3-3 epsilon. There are several other mouse mutations in other migration control genes that create lamination defects in neocortex; however, none of these other mice have been reported to show spontaneous seizures. This raises the possibility that a difference between the migration defects in the p35 mutant and the other mouse migration mutants— perhaps the alteration in hippocampal development—is a key to epileptogenesis.
CONCLUSIONS The flathead mutant and the Otx1-/- mutant show severe seizure phenotypes, and both models are associated with microcephaly and a change in the relative proportion of cell types. Since microencephaly alone is not thought to be a cause of seizure syndromes, it is the change in relative proportion of different neurons that is likely to be critical to
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epileptogenesis in these models. The secondary cortex in tish does not appear to be necessary for epileptogenesis, and therefore alterations in the development of normotopic cortex are likely to be responsible for the seizures. The thinning of normotopic neocortex in tish and the finding that neurons in normotopic cortex arise from both heterotopic and normotopic neurogenic zones further suggest the possibility of alterations in the proportion of cell types in tish normotopic cortex. The molecular defect in the p35-/- mouse is directly connected to signaling pathways implicated in seizure syndromes in humans. The findings in the p35-/model, and their similarity to other models of epilepsy, indicate the possibility that the hippocampus may play a significant role in seizures in these animals, even though there is a primary malformation and developmental disruption within the neocortex. Finally, there is a significant need to develop additional models that more precisely model both the genetic and malformation types seen in human syndromes associated with seizures and developmental disruption. Mutations in genes that cause epilepsy in humans, such as LIS1, DCX, and ARX, have yet to produce viable animal models that show spontaneous seizures—because of both embryonic lethality and lack of a robust spontaneous seizure phenotypes. Lethality may be avoided in future models that are generated with temporally specific knockouts or alternative molecular manipulations such as RNAi (Bai et al., 2003). Alternatively, the causal chain from specific genetic defect through malformation type to spontaneous seizures may be speciesspecific. In this case, models such as the widely-used pharmacologic models for idiopathic epilepsies—that model seizure phenotype but not the exact molecular causes—may also be suitable for models of developmental disruption. In either case, careful characterization of spontaneous seizures and the underlying mechanisms in several genetic models may be necessary to understand the complicated etiology of seizures in the malformed brain.
References Acampora, D., Avantaggiato, V., Tuorto, F., Barone, P., Reichert, H., Finkelstein, R., and Simeone, A. 1998. Murine Otx1 and Drosophila otd genes share conserved genetic functions required in invertebrate and vertebrate brain development. Development 125: 1691– 1702. Acampora, D., Mazan, S., Avantaggiato, V., Barone, P., Tuorto, F., Lallemand, Y., Brulet, P. et al. 1996. Epilepsy and brain abnormalities in mice lacking the Otx1 gene. Nat Genet 14: 218–222. Alcantara, S., Ferrer, I., and Soriano, E. 1993. Postnatal development of parvalbumin and calbindin D28K immunoreactivities in the cerebral cortex of the rat. Anat Embryol Berl 188: 63–73. Alcantara, S., de Lecea, L., Del Rio, J.A., Ferrer, I., and Soriano, E. 1996. Transient colocalization of parvalbumin and calbindin D28k in the postnatal cerebral cortex: evidence for a phenotypic shift in developing nonpyramidal neurons. Eur J Neurosci 8: 1329–1339. Avanzini, G., Spreafico, R., Cipelletti, B., Sancini, G., Frassoni, C., Franceschetti, S., Lavazza T. et al. 2000. Synaptic properties of neo-
cortical neurons in epileptic mice lacking the Otx1 gene. Epilepsia 41: (Suppl 6), S200–205. Bai, J., Ramos, R.L., Ackman, J.B., Thomas, A.M., Lee, R.V., and LoTurco, J.J. 2003. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci 6: 1277–1283. Boyl, P.P., Signore, M., Annino, A., Barbera, J.P., Acampora, D., and Simeone, A. 2001. Otx genes in the development and evolution of the vertebrate brain. Int J Dev Neurosci 19: 353–363. Chae, T., Kwon, Y.T., Bronson, R., Dikkes, P., Li, E., and Tsai, L.H. 1997. Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron 18: 29–42. Chen, Z.F., Schottler, F., Bertram, E., Gall, C.M., Anzivino, M.J., and Lee, K.S. 2000. Distribution and initiation of seizure activity in a rat brain with subcortical band heterotopia. Epilepsia 41: 493–501. Cipelletti, B., Avanzini, G., Vitellaro-Zuccarello, L., Franceschetti, S., Sancini, G., Lavazza, T., Acampora, D. et al. 2002. Morphological organization of somatosensory cortex in Otx1(-/-) mice. Neuroscience 115: 657–667. Cogswell, C.A., Sarkisian, M.R., Leung, V., Patel, R., D’Mello, S.R., and LoTurco, J.J. 1998. A gene essential to brain growth and development maps to the distal arm of rat chromosome 12. Neurosci Lett 251: 5–8. D’Agostino, M.D., Bernasconi, A., Das, S., Bastos, A., Valerio, R.M., Palmini, A., Costa da Costa, J. et al. 2002. Subcortical band heterotopia (SBH) in males: clinical, imaging and genetic findings in comparison with females. Brain 125: 2507–2522. Frantz, G.D., Weimann, J.M., Levin, M.E., and McConnell, S.K. 1994. Otx1 and Otx2 define layers and regions in developing cerebral cortex and cerebellum. J Neurosci 14: 5725–5740. Gupta, A., and Tsai, L.H. 2003. Cyclin-dependent kinase 5 and neuronal migration in the neocortex. Neurosignals 12: 173–179. Lee, K.S., Collins, J.L., Anzivino, M.J., Frankel, E.A., and Schottler, F. 1998. Heterotopic neurogenesis in a rat with cortical heterotopia. J Neurosci 18: 9365–9375. Lee, K.S., Schottler, F., Collins, J.L., Lanzino, G., Couture, D., Rao, A., Hiramatsu, K. et al. 1997. A genetic animal model of human neocortical heterotopia associated with seizures. J Neurosci 17: 6236–6242. Liu, H., Di Cunto, F., Imarisio, S., and Reid, L.M. 2003. Citron kinase is a cell cycle-dependent, nuclear protein required for G2/M transition of hepatocytes. J Biol Chem 278: 2541–2548. Mochida, G.H., and Walsh, C.A. 2001. Molecular genetics of human microcephaly. Curr Opin Neurol 14: 151–156. Naim, V., Imarisio, S., Di Cunto, F., Gatti, M., and Bonaccorsi, S. 2004. Drosophila citron kinase is required for the final steps of cytokinesis. Mol Biol Cell 15: 5053–5063. Panto, M.R., Zappala, A., Tuorto, F., and Cicirata, F. 2004. Role of the Otx1 gene in cell differentiation of mammalian cortex. Eur J Neurosci 19: 2893–2902. Patel, L.S., Wenzel, H.J., and Schwartzkroin, P.A. 2004. Physiological and morphological characterization of dentate granule cells in the p35 knock-out mouse hippocampus: evidence for an epileptic circuit. J Neurosci 24: 9005–9014. Pilz, D.T., Matsumoto, N., Minnerath, S., Mills, P., Gleeson, J.G., Allen, K.M., Walsh, C.A. et al. 1998. LIS1 and XLIS (DCX) mutations cause most classical lissencephaly, but different patterns of malformation. Hum Mol Genet 7: 2029–2037. Roberts, M.R., Bittman, K., Li, W.W., French, R., Mitchell, B., LoTurco, J.J., and D’Mello, S.R. 2000. The flathead mutation causes CNSspecific developmental abnormalities and apoptosis. J Neurosci 20: 2295–2306. Sancini, G., Franceschetti, S., Lavazza, T., Panzica, F., Cipelletti, B., Frassoni, C., Spreafico, R. et al. 2001. Potentially epileptogenic dysfunction of cortical NMDA- and GABA-mediated neurotransmission in Otx1-/- mice. Eur J Neurosci 14: 1065–1074.
References Sarkisian, M.R., Rattan, S., D’Mello, S.R., and LoTurco, J.J. 1999. Characterization of seizures in the flathead rat: a new genetic model of epilepsy in early postnatal development. Epilepsia 40: 394–400. Sarkisian, M.R., Frenkel, M., Li, W., Oborski, J.A., and LoTurco, J.J. 2001. Altered interneuron development in the cerebral cortex of the flathead mutant. Cereb Cortex 11: 734–743. Sarkisian, M.R., Li, W., Di Cunto, F., D’Mello, S.R., and LoTurco, J.J. 2002. Citron-kinase, a protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant rat. J Neurosci 22: RC217. Schottler, F., Fabiato, H., Leland, J.M., Chang, L.Y., Lotfi, P., Getachew, F., and Lee, K.S. 2001. Normotopic and heterotopic cortical representations of mystacial vibrissae in rats with subcortical band heterotopia. Neuroscience 108: 217–235.
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Spreafico, R., Battaglia, G., Arcelli, P., Andermann, F., Dubeau, F., Palmini, A., Olivier, A. et al. 1998. Cortical dysplasia: an immunocytochemical study of three patients. Neurology 50: 27–36. Weimann, J.M., Zhang, Y.A., Levin, M.E., Devine, W.P., Brulet, P., and McConnell, S.K. 1999. Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 24: 819–831. Wenzel, H.J., Robbins, C.A., Tsai, L.H., and Schwartzkroin, P.A. 2001. Abnormal morphological and functional organization of the hippocampus in a p35 mutant model of cortical dysplasia associated with spontaneous seizures. J Neurosci 21: 983–998. Woods, C. 2004. Human microcephaly. Curr Opin Neurobiol 14: 112–117. Zhang, Y.A., Okada, A., Lew, C.H., and McConnell, S.K. 2002. Regulated nuclear trafficking of the homeodomain protein otx1 in cortical neurons. Mol Cell Neurosci 19: 430–446.
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20 Mammalian Models of Genetic Epilepsy Characterized by Sensory-Evoked Seizures and Generalized Seizure Susceptibility PHILLIP C. JOBE AND RONALD A. BROWNING
Some people with epilepsy are known to have seizures induced by flickering light, thinking, music, contemplation of a tune, reading, and eating. Similarly, some animals exhibit seizures in response to conditions that do not cause seizures in normal mammals–conditions such as sound, flickering light, proprioception, touch, postural changes and emotional factors (Buchhalter, 1993; Jobe et al., 1991; King and LaMotte, 1989; Loscher, 1984; Seyfried et al., 1986; Suzuki and Nakamoto, 1977). By selectively breeding individual animals (with either convulsive or nonconvulsive epilepsies) within a large population, investigators can produce colonies of animals with specific epileptic traits, including seizure predisposition and expression (Jobe et al., 1991). Selective breeding has resulted in essentially 100% of the progeny with one or multiple indices of epilepsy (Mishra, et al., 1988, 1989; Noebels et al., 1998). Despite the use of breeding protocols designed to select for a specific type of epilepsy, these breeding efforts commonly result in strains characterized by a multiplicity of epilepsy-related phenotypes, with seizure predisposition represented throughout the rostral-caudal extent of the brain. The Genetically EpilepsyProne Rats (GEPRs) exemplify mammals in this category. In contrast, some breeding protocols have produced colonies in which the indices of epilepsy are seemingly restricted to a single system of the brain (e.g., the audiogenic seizuresusceptible rat of Strasbourg (Kiesmann et al., 1988) appears to be restricted to the ascending auditory system). Among primates living in the wild, natural selection appears to have resulted in analogous epileptic states. In the band of Papio papio from the Casamance region of Senegal, 80% or more of the baboons show seizure predisposition (Killam and Killam, 1984; Loscher, 1984). Other Papio species, such as
Models of Seizures and Epilepsy
Papio cynocephalus, from parts of Ethiopia, have also been observed to exhibit these neurologic abnormalities (Corcoran et al., 1979). However, this striking epileptic trait is not believed to be a characteristic of the genus or species; seizure predisposition and expression occur in a small number of baboons in the general population of these primates. This chapter focuses on a selected pair of animal models of the genetic epilepsies, with special reference to sensoryinduced seizures and general seizure predisposition. First, the epileptic baboon, P. papio (Killam et al., 1966c, d; Loscher, 1984) offers a model of convulsive epilepsy that was derived by natural selection in the wild. Second, the GEPR (Reigel et al., 1986) provides a model of convulsive epilepsy derived from inbreeding for selected epilepsy traits. Although not covered in detail in this chapter, other sensory-sensitive seizure-epilepsy models have been described, including the DBA/2 mouse (Vicari, 1951; Schreiber and Graham, 1976), the EL mouse (Seyfried et al., 1986; Seyfried and Glaser, 1985), the audiogenic seizuresusceptible rats of Strasbourg (Kiesmann et al., 1988), and the Mongolian gerbil (Kaplan and Miezejeski, 1972; Loskota and Lomax, 1975; Paul et al., 1981; Schonfeld and Glick, 1981).
GENETICALLY EPILEPTIC BABOONS Clinical Applicability: Characteristics and Background The susceptibility of Senegalese P. papio to flickering light-induced seizures was first reported by Killam and
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colleagues (Killam, et al., 1966a, b, d). With continuing investigation, it became apparent that the epileptic condition of these animals was broadly represented throughout the brain and that seizure predisposition in the epileptic P. papio included the expression of spontaneous seizures (Wada et al., 1972) and an accelerated rate of electrical limbic kindling (Wada and Osawa, 1976; Wada et al., 1975). Another important development of the model has been a limited exploration of the anticonvulsant properties of pharmacologic agents (Loscher, 1983; Meldrum et al., 1975). The high prevalence of epileptic P. papio in Senegal, Africa, with susceptibility to light-induced and spontaneous seizures, plus the absence of known morphologic defects, supports the concept of genetic determinants for this disorder. Nevertheless genetic studies have not yet been undertaken (Szabo, et al., 2004). Experimental investigation of the genetically epileptic baboon has provided information regarding the phenomenology and mechanisms of neuroanatomically pervasive predisposition to seizures. In particular, the seizurerelated phenomenology in these baboons provides potential insights into a number of features of human epileptic activity.
Focal Seizures Some flickering light-induced seizures in the epileptic P. papio appear to begin (bilaterally) as localized events in the frontal rolandic cortex (Naquet and Valin, 1990; SilvaBarrat and Menini, 1990). Flickering light is also known to produce focal interictal discharges and focal seizures in humans (Zifkin and Andermann, 2001). Focal light-induced epileptic seizures in baboons predominate in the two frontorolandic areas, whereas those in the human are typically localized in the occipital cortex (even though the occipital cortex in the baboon and in the human has been proposed to play the same role in the induction of paroxysmal discharges in the frontorolandic cortex) (Naquet and Valin, 1990). Focal Seizures Secondarily Generalized to Forebrain Seizure Circuitry Electrographic seizures appear bilaterally in the frontorolandic cortex before their appearance in other components of forebrain seizure circuitry (Naquet and Valin, 1990). Clinically these seizures correspond to the known behaviors driven by forebrain circuitry. Because flickering light-induced seizures in these animals appear to begin bilaterally as localized events in the central rolandic cortex, they can be expected to provide a means for discovery of mechanisms that underlie focal seizure ignition, continuation, and termination.
Focal Seizures Secondarily Generalized to Brainstem Seizure Circuitry to Produce Generalized Tonic-Clonic Seizures Electrographic seizures appear in the brainstem following those in the frontorolandic cortex and other components of the forebrain seizure circuitry (Naquet and Valin, 1990). These caudal seizures correspond to those driven by brainstem circuitry (Wada et al., 1972). Because in some instances the focal seizures become secondarily generalized to the forebrain seizure circuitry and tertiarily generalized to the brainstem seizure circuitry, epileptic baboons have potential as a model for studies of the genetic determinants of such complex events.
Primary Generalized Tonic-Clonic Seizures The behaviors driven by these seizures correspond to behaviors determined by known brainstem circuitry (Wada et al., 1972). Generalized Seizure Predisposition Humans with epilepsy characterized by focal and generalized seizures exhibit a marked predisposition to seizures induced by agents such as pentylenetetrazol (Cure et al., 1948). The epileptic P. papio also exhibits this trait (Killam et al., 1967). Because some epileptic baboons exhibit only focal interictal spiking and focal seizures, comparison of these particular subjects to counterparts that exhibit secondary generalization to tonic-clonic seizures provides a means to determine the mechanisms of secondary ignition. Perhaps isolated spikes are of little or no consequence in the nonepileptic brain, wherein the two major sets of seizure circuitry, as well as the local areas involved in the spiking, are adequately protected against ictal onset. Because some epileptic P. papio and other genetic variants among baboons have been observed to exhibit spontaneous seizures (Szabo et al., 2004; Wada et al., 1982), these primates may provide a means for advancing our knowledge of the mechanisms of spontaneous seizures. They may also provide a means for advancing the scientific understanding of the underlying mechanistic commonalities between evoked and spontaneous seizures. Because epileptic P. papio exhibits a predisposition to cortical and limbic kindling (Goddard et al., 1969; Wada et al., 1975; Wada and Osawa, 1976), these animals may provide insights into the mechanistic interactions between genetically determined and stimulusdependent seizure predisposition. Because P. papio exhibits a predisposition to other seizure-inducing modalities as well as to spontaneous seizures, these primates may provide insight into the mechanisms of pervasive seizure predisposition (Killam et al., 1967; Wada et al., 1972).
Genetically Epileptic Baboons
How well does Papio papio seizure activity—particularly photic-flickering—induced seizure activity–model a human syndrome? Existing data indicate that the epileptic P. papio is an excellent model of idiopathic, secondarily generalized human seizure predisposition and expression. Seizure predisposition is a condition of many epilepsies, and it is part of the syndrome in that this condition exists in addition to the actual seizure that might expressed. Thus the different seizure types, plus the underlying seizure predisposition of the epileptic baboon, represent part of the epilepsy syndrome of the epileptic P. papio. However, if one focuses exclusively on the neuroanatomic mechanisms of flickering light-induced interictal spikes, there are some model weaknesses. In humans most such spiking occurs in the occipital cortex, whereas in baboons these events are prominent in the frontorolandic cortex. Nevertheless comparative information derived from studies of lightinduced interictal paroxysmal activity and seizures in the frontorolandic cortex may enable invaluable advances in framing improved hypotheses, treatments, and preventions.
How Are Seizures Triggered? The experimental protocols designed to induce seizures in P. papio via flickering light have been described by many investigators (Fischer-Williams et al., 1968; Killam and Killam Jr, 1984; Killam et al., 1967; Meldrum and Horton, 1979; Meldrum et al., 1975b; Pedley et al., 1979; Szabo et al., 2004; Weinberger and Killam, 1978). Indeed the original published discovery of the epileptic condition of these animals was based on this approach (Killam et al., 1966a, d). The baboon is prepared for electroencephalographic (EEG) recordings and seated in a primate chair, facing a stroboscopic light source. Flashes of light are delivered (over a broad range of flash rates) for up to 5 mintues at hourly intervals; a rate of 25 Hz appears to be optimal. In the early work by Killam et al. (1967), the light source was either a white xenon gas discharge tube or a red neon tube. Intermittent light stimulation may induce focal paroxysmal discharges in the frontorolandic cortex (FischerWilliams, et al., 1968), or it may produce diffuse initial EEG evidence of a seizure (Wada, et al., 1972). During interictal periods in P. papio, spikes or spike-wave discharges occur most prominently in the central rolandic region of the cerebral cortex (Wada et al., 1972). This specific type of interictal electrographic activity reportedly does not occur as often in subcortical regions (Fischer-Williams, et al., 1968). “Sharp wave-and-spike activity” has been detected in the posterior pons (Wada et al., 1972), and spike and wave discharges (SWDs) occasionally become evident in the lateral hypothalamus (Fischer-Williams et al., 1968). These caudal events apparently occur with some level of temporal independence from paroxysmal electrographic activity in the cortex.
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In many or perhaps all of the epileptic P. papio, seizure predisposition is evident throughout the neuroanatomic extent of the brain (Wada et al., 1972). Animals that exhibit focal discharges may ultimately exhibit secondarily generalized tonic-clonic seizures (Naquet and Valin, 1990; SilvaBarrat and Menini, 1990). Localized interictal epileptiform discharges of the brain may be of little dysfunctional consequence if they (or some closely related process) do not ignite a seizure, especially if they do not activate either the rostral or caudal seizure circuitry of the brain. Although interictal epileptiform discharges may contribute to the process of diagnosing epilepsy, such discharges have been detected in normal individuals (Walczak and Jayakar, 1998). Moreover, most interictal discharges appear immediately following a seizure, a time when the major sets of seizure circuitry of the forebrain and of the brainstem are in refractory modes. Interictal spikes are characteristic of the epileptic P. papio as well as the nonepileptic rhesus monkey. In the epileptic P. papio, these spikes may give rise to “spontaneous” seizures because the animals have an innate seizure predisposition, whereas the lack of such predisposition in the rhesus monkey “protects” the animals against the onset of seizures (in response to interictal spikes or other events that might trigger discharge synchrony among neurons). In the epileptic baboon, temporally dependent variations in the level of endogenous “protection” determine whether the frequently occurring localized interictal spikes ignite a seizure. It seems likely that most interictal spikes are unassociated with sensory or motor consequences because during most intervals endogenous anticonvulsant properties provide sufficient levels of protection.
Clinical Features and Electroencephalographic Characteristics of Seizure Behavior At the beginning of flickering light stimulation, the number of action potentials per 100 ms detected by extracellular recordings in the frontal rolandic cortex of P. papio is approximately equivalent to that observed in the absence of the light stimulus (Silva-Barrat and Menini, 1990). In the subsequent phase of frontorolandic activity, the action potentials are grouped. With each of these bursts, the action potential rate is between 150 and 300 Hz, but the overall rate across approximately 100 ms remains approximately equal to that before application of the stimulus. Simultaneous field potential recordings demonstrate that electrographic paroxysmal discharges are not detectable when the internal frequency within each burst is below 300 per second. With extension of flickering light stimulation, the individual bursts become shorter in duration, and the intraburst frequency of action potentials rises to a new range of 300 to 800 Hz. This sharp rise in discharge rate coincides with the appearance of electrographic paroxysmal discharges.
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In epileptic baboons that exhibit focal seizures with secondary generalization or that exhibit primary generalized seizures, light-induced spike-and-wave complexes may occur as isolated EEG events or as a burst of such events (Naquet and Valin, 1990). When occurring as an isolated event, a spike-wave is not usually associated with behavioral manifestations. However, when these discharges occur in bursts with sufficient intraburst frequency, an impairment of consciousness may become evident. Bursts of polyspikes and waves may sequentially give rise to behaviors that include eyelid, facial, head, and limb clonus that may become “generalized and violent.” If the seizure behaviors progress beyond this point, loud vocalization with maximal opening of the mouth occurs just before the rhythmic EEG discharges are displaced by an electrodecremental interval (low-amplitude fast activity) and generalized tonic convulsion (Fischer-Williams et al., 1968). It is at the onset of these last behavioral manifestations that intracerebral EEG recordings indicate the beginning of a brainstem determined convulsion (Silva-Barrat and Menini, 1990). Within seconds, repetitive spike and sharp wave complexes of temporally increasing amplitude become evident as generalized clonus displaces tonus. The convulsion ends with an EEG of rapidly decreasing amplitude. The events are followed by postictal generalized loss of muscle tone and a period of confusion and unresponsiveness to stimuli. Some seizures in the epileptic P. Papio are merely focal so that generalized activation of the forebrain seizure circuitry does not occur. In instances wherein this rostral circuitry is ignited (e.g., by kindling), the convulsions largely conform to those described for limbic kindling in rats. This correspondence occurs whether the seizures are kindled from the amygdala (Wada and Osawa, 1976) or from the prefrontal cortex (Wada et al., 1975). However, behavioral differences may also exist. GTCS and partial (forebrain seizures) in epileptic P. papio can be induced by electrical kindling stimuli, or they may occur spontaneously during the post-kindling interval (Wada and Osawa, 1976). An important finding is that spontaneous GTCSs have also been noted in nonkindled epileptic baboons. Finally a preliminary study of several baboon species (i.e., other than P. papio) found that the EEG morphology of interictal epileptic discharges were different in younger baboons compared with those in older animals (Szabo et al., 2004). These investigators suggest that the interictal epileptic discharges in younger subjects represent an ontogenetically less mature form of the epileptic phenotype.
Cognitive and Biological Issues Behavioral and motor dysfunction has been observed in P. papio with recurrent spontaneous seizures or with prominent susceptibility to light-induced seizures (Wada et al., 1972). These behaviors include a lack of aggressive behav-
ior coupled with clumsiness and occasional tremor of the hands, head, and trunk tremor. Because P. papio expresses seizure predisposition even in the absence of seizure, this model should be useful in assessing the role of predisposing factors in learning and memory deficits. These baboons would also provide a model for testing the interactions on learning and memory that may occur from effects between seizure predisposition, seizure sensitization paradigms (kindling), and anticonvulsant medications (Killam, 1979; Weinberger and Killam, 1977, 1979). Only a few studies have been undertaken to determine the underlying neurobiological factors that might be responsible for the epileptic condition of the Senegalese P. papio. A review in the mid 1980s concluded that possible abnormalities in catecholaminergic and perhaps serotonergic transmission might contribute to seizure predisposition in these animals (Killam and Killam, 1984). However, concrete evidence of an intrinsic deficit in any of the neurotransmitter systems does not exist. Although some progress has been made in more recent years (Brailowsky et al., 1989; Meldrum and Balzamo, 1972; Meldrum and Horton, 1978; Meldrum et al., 1983; Menini and Silva-Barrat, 1990; Suzdak and Jansen, 1995; Valin et al., 1991; Zhang, et al., 1990), an understanding of the biological basis for epilepsy in the Senegalese P. papio remains largely unknown.
Advantages and Limitations There are obvious disadvantages in considering the experimental use of the epileptic P. papio. These subjects are primates–and difficult to maintain in the laboratory and vivarium–for both technical and ethical reasons. The cost of each baboon (and the per diem charges for appropriate housing and feeding) is often prohibitive, and many facilities do not have the appropriate space, equipment, or personnel. However, the overriding issue is justification for primate use in research. Investigators must make a persuasive case that potential improvements in human health obtained via investigations with P. papio cannot be reasonably expected from appropriately planned and executed scientific studies with subprimate mammals. Considerable variability in seizure predisposition and expression has been found in studies of P. papio. If the model were to be optimized, a substantial investment would be needed to study the population genetics and then to support selective breeding to develop nonepileptic and epileptic strains of P. papio. Goals should probably include the development of a strain that has a high incidence of spontaneous seizures as well as strains that have defined levels of seizure predisposition but essentially no spontaneous seizures. A separation is also needed between baboon strains that exhibit spontaneous forebrain seizures and those that display brainstem seizures. Additionally, there is a need for a strain that is characterized by focal forebrain seizures
The Genetically Epilepsy-Prone Rat
that ignite generalized forebrain seizures that ignite tertiary brainstem seizures. Finally, it should be noted that the epileptic P. papio is not suitable for routine screening of drugs for antiepileptic activity. Nevertheless, with sufficient generation of data, it may be suitable for assessments of specific and narrowly defined biological activities after the candidate treatment has proven suitable in less time-consuming and costly models.
THE GENETICALLY EPILEPSY-PRONE RAT Clinical Applicability: Characteristics and Background Two independently derived inbred strains of genetically epilepsy-prone rats (GEPRs) have been developed: the moderately epileptic GEPR-3 and the more severely epileptic GEPR-9. Details for the development of the GEPR-3 and GEPR-9 strains have been previously described (Reigel et al., 1986). The GEPR strains have been developed via inbreeding of naturally occurring epilepsy occasionally seen in Sprague-Dawley rats. At this point in the development of GEPR strains, essentially 100% of the progeny exhibit epilepsy-relevant traits (Mishra et al., 1988, 1989). The GEPR strains exhibit a broad range of seizure traits and are applicable to studies of the two major sets of convulsive seizure circuitry. Both the GEPR-3 and the GEPR-9 are characterized by a prominent level of seizure predisposition in the forebrain seizure circuitry and in the brainstem seizure circuitry. As would be anticipated under such conditions, either a focal or a generalized forebrain seizure in the GEPR9 will ignite brainstem seizures (Coffey et al., 1996). This trait is of potential clinical relevance in that it appears to mimic a partial seizure secondarily generalized to tonicclonic seizures. Both the GEPR-3 and GEPR-9 strains are characterized by a complex interaction between multiple genes (Kurtz et al., 2001). Both strains show incomplete penetrance and variable expressivity of underlying genetic factors. Compared with GEPR-9 females, GEPR-9 males have greater differences in expressivity and penetrance. GEPR-3 also exhibits sex-associated variable penetrance and expressivity of the epileptic phenotype. Although GEPRs are known to exhibit GTCSs as well as seizures expressed by the forebrain seizure circuitry, the underlying causes of seizure predisposition exist even in the absence of seizures. Seizure predisposition is exacerbated by seizure repetition at weekly intervals (Mishra et al., 1988, 1989). Seizure-induced seizure exacerbation has been identified as a characteristic of the seizure circuitry of the forebrain and also of the brainstem. The term seizure predisposition in GEPRs—as well as in humans and several other genetically epileptic mammals—is probably too
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limited in scope. Two underlying components of seizure predisposition—noradrenergic and serotonergic deficits—also appear to give rise to a marked predisposition to the expression of affective disorder (Jobe, 2003, 2004a–c; Jobe et al., 1999a). Thus a single individual experiences a predilection to a major neurologic and to a common psychiatric disorder. As appears to be true in humans, this comorbidity is apparent in GEPR-3s and to a lesser extent in GEPR-9s. Partially because of the underlying noradrenergic and serotonergic deficits characteristic of the GEPR-3, these animals are prone to express not only limbic seizures but also GTCSs of the brainstem circuitry. As has been noted in humans with depressive disorder, the GEPR-3 exhibits serotonergic and noradrenergic deficiencies, glucocorticoid elevation, growth hormone deficiency, anhedonia, behavioral despair, sleep disturbances, and anticonvulsant and antidepressive responses to antidepressive drugs (Jobe, 2004a, c). Three types of seizure predisposition are exhibited by GEPRs (Jobe et al., 1994): (1) seizures that are triggered by endogenous and exogenous stimuli that do not ignite seizures in normal mammals, (2) exaggerated seizure responsiveness to stimuli that also cause seizures in nonepileptic mammals, and (3) abnormally low thresholds to many convulsant agents. With regard to seizures that do not occur in normal mammals, infrequent spontaneous seizures have been observed in GEPRs (Dailey et al., 1989). Although longterm continuous EEG monitoring (weeks and months) in GEPRs has not been undertaken, interictal spikes or spikewave complexes are believed to occur as they do in epileptic baboons and humans. Because the spontaneous seizures in GEPRs are not known to be caused by identifiable exogenous stimuli or focal lesions, they are thought to be ignited by endogenous variations in brain activity-excitability that would not be capable of producing seizures in normal animals. Both GEPR-3s and GEPR-9s are susceptible to seizures induced by sound and hyperthermia. In addition a few young GEPRs have been observed to exhibit GTCSs in response to postural changes or handling. Finally, several examples of predisposition in GEPRs, characterized by abnormally low thresholds (or exaggerated seizure responsiveness), have been discovered. Seizures are induced in GEPRs by low-level exposures to aminophylline (DeSarro and DeSarro, 1991), electroshock (Browning et al., 1990), flurothyl (Franck et al., 1989), hyperbaric conditions (Millan et al., 1991), pentylenetetrazol (Browning et al., 1990), and barbiturate withdrawal (Bourn and Garrett, 1983). An accelerated rate of limbic kindling represents another aspect of seizure predisposition in GEPRs (Coffey et al., 1996; Savage, et al., 1986a, b). These observations support the concept that the limbic system and other components of the forebrain seizure circuitry of the GEPR-3 and GEPR-9 contribute to the overall seizure predisposition of these mammals.
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Both GEPR-3s and GEPR-9s are far past the 20 generations of brother-sister inbreeding needed to develop inbred strains. Seizure predisposition is present in all progeny, even in those occasional individuals in which susceptibility to sound-induced seizures is absent. GEPRs are reliable models of disorders caused by noradrenergic, serotonergic, and GABAergic deficits. Pioneering work established the role of GABAergic deficits as primary participants in translating incoming auditory impulses into seizure activity in the inferior colliculus of the GEPR (Evans et al., 1994; Faingold et al., 1986a, 1994). Other investigations have demonstrated that noradrenergic and serotonergic deficits contribute appreciably to the generalized seizure predisposition that characterizes the GEPRs (Browning et al., 1989; Dailey et al., 1991; 1992; Jobe et al., 1973a, b, 1984, 1994; Ryu et al., 1999). The latter deficits appear to play no role in the generation of seizure activity within the inferior colliculus per se. In contrast, they do contribute to the atypical state of seizure predisposition that appears to cause these animals to respond to apparently innocuous stimuli with seizures. Both GEPR-3 and GEPR-9 strains are highly susceptible to sound-induced GTCS. The phenomenon of “audiogenic kindling” occurs in a high percentage of the animals so that the mechanisms of brainstem seizure–induced forebrain seizures are facilitated.
How Are the Seizures Triggered? The GEPRs are characterized by a multiplicity of seizure expressions and seizure triggering mechanisms. Some mechanisms appear to trigger focal seizures, others trigger brainstem-driven tonic-clonic seizures, and others ignite “fully generalized’ ” forebrain seizures. Two examples of GEPR seizure phenomena are described in the following sections. Details of the underlying mechanisms remain to be elucidated. Generalized Tonic/-Clonic Seizures (GTCSs) Although the mechanisms that ignite spontaneous GTCSs have not been fully determined, existing studies (Browning et al., 1991, 1999; Garcia-Cairasco et al., 1998) have shown that the occurrence of spontaneous GTCSs is facilitated by a precollicular transection in GEPR-9s. This finding provides support for the concept that spontaneous GTCSs do not require neuronal connections with the cerebral cortex or other forebrain structures. Indeed, in GEPR-9s with an intact central nervous system, the rostral brain appears to exert an overall anticonvulsant effect so as to reduce sharply the appearance of spontaneous seizures. These observations are consistent with the concept that GTCSs are triggered and maintained via the brainstem seizure circuitry. A second mechanism for triggering GTCSs—via forebrain after-
discharge initiation—may also exist within GEPR brain. Both unilateral and bilateral forebrain seizures, initiated via a kindling electrode in the amygdala, can trigger GTCSs secondarily in GEPR-9s (Coffey et al., 1996). In instances where these secondary GTCSs occur following the termination of the afterdischarge in the amygdala, the onset of GTCSs may be delayed by up to 60 seconds. A third mechanism for triggering GTCSs in GEPRs exists in the inferior colliculus and its interactions with other components of the brainstem seizure circuitry (Faingold, 2004). This mechanism is activated in response to a sound stimulus, which sharply increases the rate of unit activity within the inferior colliculus. Within this nucleus, incoming impulses from an acoustical stimulus produce a localized seizure. This exceedingly dramatic rise in sound-induced neuronal activation arises, at least partially, from deficient GABAA receptor–mediated inhibition within the inferior colliculus. The excessive output from the inferior colliculus triggers, with very short latency, brainstem seizure circuitry to produce GTCSs. The participating components of this circuitry ultimately include the inferior colliculus. Thus this nucleus plays two roles in sound-induced GTCSs: First, it generates a focal seizure that activates the brainstem seizure circuitry; second, it participates as part of the brainstem seizure circuitry in producing the generalized tonic-clonic seizure.
Forebrain Seizure Ignition The GEPR brain contains mechanisms whereby brainstem seizures trigger forebrain seizures. When an animal experiences a severe brainstem seizure, the forebrain reflects the brainstem-type seizure. However, when repeated brainstem seizures are triggered at sufficiently close intervals, the brainstem seizures become progressively less severe, and the modestly severe brainstem seizure is followed by forebrain seizure behaviors. To account for these changes, some investigators have advanced the concept of “kindling.” Because these studies were undertaken using sound-induced brainstem seizures, the term audiogenic kindling has been applied to the phenomenon. In this scenario, the seizures in the brainstem have been viewed as kindling stimuli applied to the forebrain. Each time the brainstem seizure is applied, the forebrain becomes progressively sensitized so that eventually a forebrain seizure ensues. Another possibility, however, is that as the brainstem circuitry becomes increasingly refractory to a GTCS, its capacity to determine seizure expression in the forebrain is progressively diminished. According to this hypothesis, the brainstem seizure in the forebrain is initially so severe that it leads to seizure refractoriness. Subsequently the brainstem seizure in the forebrain become less severe and seizure refractoriness becomes less pronounced, allowing the forebrain seizure to emerge. Electrophysiologic evidence sup-
The Genetically Epilepsy-Prone Rat
ports this latter viewpoint (Moraes et al., 1998), as do observations made on the developing GEPR-3 (Reigel et al., 1989). In some young animals, sound stimulus triggers relatively mild brainstem seizures, followed by 10 to 15 seconds of quiescence; the pup then spontaneously rights itself and exhibits behaviors typical of class 4 or 5 forebrain seizures. Developing pups do not exhibit forebrain seizure behaviors following severe brainstem seizures. Consistent with this concept that brainstem seizure severity regulates forebrain seizure expression, the anticonvulsant drug phenytoin has been found to allow the expression of secondary forebrain seizures in adult female GEPR-9s, and to allow development of secondary forebrain seizures in response to daily induction of brainstem seizures (Merrill et al., 1998). Moreover, the severity of the forebrain seizures is greater in phenytoin-treated animals. Sound-induced seizures in susceptible rats are evoked by delivering an auditory stimulus, usually in the form of a ringing bell. The most commonly used apparatus consists of a galvanized metal cylindrical chamber (40 cm in diameter, 50 cm in height) encased in a sound-attenuating wooden box (with a hinged lid). On the inside of the lid (in which a glass observation window is embedded) are two fire bells and a light. Faingold and colleagues (1986b) determined that GEPRs are most susceptible to a pure tone of 12 kHz at 100 db; however, a mixed-frequency stimulus (1–20 kHz) generated by a fire bell (90–110 db) is generally considered maximally effective in evoking audiogenic seizures. If no EEG recording is required, the rat is placed in the chamber and allowed 15 seconds to habituate; then the sound is initiated. Latencies to onset of the running and convulsive phases of the seizure are recorded. Seizure behavior is evaluated according to the severity classification of Jobe and colleagues (1973b) to obtain the audiogenic response score (ARS). If no seizure occurs within 90 seconds, the stimulus is terminated. In the early generations of the GEPR colonies, the scores ranged from 0 to 9, with a zero score indicating no response and a score of 9 (seen in GEPR-9s) indicating that the rat had progressed through a running stage and a clonic convulsion, and had achieved a full tonic extensor convulsion. In the present GEPR-9 strain, some rats go immediately into a short series of massive myoclonic thrusts in lieu of a running episode and then exhibit a tonic phase of flexion and extension (high-frequency, low-amplitude clonus superimposed on the dominant tonus). As the convulsion nears an end, there is a diminution of tonic rigidity and an increasing clonic amplitude and decreasing clonic frequency. These animals are given a score of 10 (Jobe and Daily, 2000). To undertake simultaneous EEG recording (and video monitoring), investigators have employed an acrylic chamber (16-inch diameter, 20-inch height) that is shielded and grounded (Moraes et al., 2005).Two tweeters are mounted atop the chamber to deliver 12- to 16-kHz tone sweeps at an average intensity of 90 db.
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Clinical Features and Electroencephalographic Characteristics of Seizure Behavior Because GEPRs are characterized by seizure predisposition both in the forebrain and brainstem seizure circuitry, clinical features and EEG characteristics of both sets of circuitry are pertinent. Several behavioral and EEG studies have been undertaken to describe these features (Jobe and Dailey, 2000; Jobe et al., 1973b; Ludvig and Moshe, 1989). Activation of the brainstem seizure circuitry in the GEPR-9 results in seizures that closely mimic GTCSs in humans (Jobe et al., 1995, 1999b; Moraes, et al., 2005). These events include (1) sudden onset of massive myoclonus coupled with initial high-amplitude polyspikes and waves; (2) a sudden, marked electrodecremental interval in the EEG with tonic rigidity of all muscles; (3) high-amplitude spike-wave complexes with partial reduction and recurrence of tonus, followed by progressive displacement of tonus with clonus of increasing amplitude; and (4) postictal reduction of EEG amplitude with muscular flaccidity (postictal depression). In GEPR-3s, as in GEPR-9s, most investigators have induced brainstem seizures with a sound stimulus. When this method is used for GEPR-3s, a running episode of 10 to 20 seconds typically precedes behaviors and EEG events that are analogous, but not identical, to those exhibited by GEPR-9s. The GTCS of GEPR-3s is less severe and the electrodecremental interval is less prominent than in GEPR9s. Sound-induced brainstem seizures are reduced in severity by treatment of GEPR-9s with an antiepileptic drug (AED) such as carbamazepine or phenytoin. If the dose is sufficient, the convulsive behavior is prevented completely. Another method of producing brainstem convulsions in GEPR-9s is through use of a forebrain seizure, for example, in response to electrical stimulation of the amygdala. Brainstem seizures occur in response to any class of forebrain seizures (1–5) and may occur during the forebrain convulsion or during the postictal period. Forebrain seizures can be produced in GEPRs via the same methods used to induce kindling in nonepileptic rats. However, in GEPRs, limbic kindling is accelerated compared with nonepileptic rats, with GEPR-9s exhibiting a more rapid rate than GEPR-3s. GTCS (convulsions) have been noted to occur spontaneously in GEPR-3s and GEPR-9s, with the higher incidence in GEPR-9s (Dailey et al., 1989). In some instances these convulsions resulted in status epilepticus and death. Ontogenetic data in GEPRs have been obtained from studies of sound-induced (Hjeresen et al., 1987; Ribak et al., 1988; Thompson et al., 1991) and flurothyl-induced (Franck et al., 1989) seizures. These experiments have shown that seizure incidence and severity develop as a function of maturation in GEPR pups. During a narrow temporal window, sound-induced brainstem seizures appear to give rise to secondarily ignited forebrain seizures. Studies with flurothyl show that seizure predisposition in GEPRs becomes evident
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earlier in development (Franck et al., 1989) than does susceptibility to sound-induced seizures (Reigel et al., 1989). The GEPRs dependably survive acute forebrain and brainstem convulsions. Repetitive seizures in immature GEPRs cause significant deficits in measures of learning, memory, and behavior (performance on the T-maze, water maze, open-field activity test, home-cage intruder test and handling test) (Holmes et al., 1990).
Advantages and Limitations of the Model As described in the next section, the GEPR-3 and GEPR9 models are important for assessment of pharmacologic treatments and other therapies associated with seizure propensity. The major drawback associated with GEPRs is that they are not commercially available. Presently the two strains of GEPRs are in jeopardy of extinction because the burden of support falls on the specific laboratories that have studied this model. Animals can be obtained via special arrangements with Dr. Carl L. Faingold at Southern Illinois University School of Medicine in Springfield, Illinois.
Pharmacologic Screening The GEPR models offer the potential of unique opportunities in the future development of AEDs. The GEPR models are already known to detect and accurately separate currently existing AEDs into clinically applicable categories (Dailey and Jobe, 1985; Jobe and Dailey, 2000). The findings suggest that the GEPR model can identify three categories of AEDs: (1) those that are clinically useful in GTCS and partial (forebrain) seizures; (2) those that have a potential to suppress absence seizures in addition to convulsive seizures in humans; and (3) those that are useful in humans against absence seizures, but not against convulsive seizures. No false-positives have occurred through the use of GEPRs. However, most opportunities to develop novel types of antiepileptic treatments in the GEPR have not been exploited. Of special note are the missed opportunities to develop drugs that would work to reverse the specific factors responsible for seizure predisposition coupled with the avoidance of adverse effects (Jobe and Laird, 1987). Also, the GEPR-3 strain is a model of epilepsy and affective disorder comorbidity. Development of treatments or preventions designed specifically for this type of comorbidity has not been attempted.
SUMMARY AND CONCLUSIONS The major advantage of the epileptic Papio and GEPRs models is their clearly defined predisposition to convulsive epilepsies and the heritable nature of this predisposition. As in patients with idiopathic generalized epilepsy, the disorder
in these genetic models is multigenic rather than monogenic. The epileptic P. papio is a good model of idiopathic secondarily generalized focal seizures that are triggered by flickering light. These mammals appear to have a reduced seizure threshold in both the forebrain and brainstem seizure circuitry. Although some pathophysiologic mechanisms have been examined, the underlying genetic abnormalities have not been identified. Clearly these primates could offer a wealth of information if more cellular and molecular studies were undertaken. The two GEPR substrains, with their innate predisposition for both brainstem and forebrain seizures, have already yielded a plethora of information regarding seizure initiation and propagation and neurotransmitter regulation of seizure propensity in both local and projection circuitries. However, because GEPRs can model primary generalized or secondary generalized seizures in either forebrain or brainstem seizure circuitry, they provide a unique opportunity to study the interactions between brainstem and forebrain seizure networks. Because of their innate noradrenergic and serotonergic deficits and some behavioral traits associated with depression, they also offer a unique opportunity to investigate the comorbidity of epilepsy and depression in the same model. Additionally, the GEPRs have proven to be excellent predictive models in drug development.
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References Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E., and Jobe, P.C. 1991. Noradrenergic abnormalities in the central nervous system of seizurenaïve genetically epilepsy-prone rats. Epilepsia 32: 168–173. Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E., and Jobe, P.C. 1992. Serotonergic abnormalities in the central nervous system of seizure-naïve genetically eplepsy-prone rats. Life Sci 50: 319–326. Dailey, J.W., Reigel, C.E., Mishra, P.K., and Jobe, P.C. 1989. Neurobiology of seizure predisposition in the genetically epilepsy-prone rat. Epilepsy Res 3: 3–17. DeSarro, A., and DeSarro, G.B. 1991. Responsiveness of genetically epilspy-prone rats to aminophylline-induced seizures and interactions with quinolones. Neuropharmacology 30: 169–176. Evans, M.S., Biola-McCabe, K..E, Caspary, D.M., and Faingold, C.L. 1994. Loss of synaptic inhibition during repetitive stimulation in geneticall epeilspy-prone rats (GEPR). Epilepsy Res. 18: 97–105. Faingold, C.L. 2004. Emergent properties of CNS neuronal networks as targets for pharmacology: application to anticonvulsant drug action. Prog Neurobiol 72: 55–85. Faingold, C.L., Gehlbach, G., and Caspary, D.M. 1986a. Decreased effectiveness of GABA-mediated inhibition in the inferior colliculus of the genetically epilepsy-prone rat. Exp Neurol 93: 145–159. Faingold, C.L., Travis, M.A., Gehlback, G., Hoffmann, W.E., Jobe, P.C., Laird, H.E., and Caspary, D.M. 1986b. Neuronal response abnormalities in the inferior colliculus of the genetically epilepsy-prone rat. Electroencephalogr Clin Neurophysiol 63: 296–305. Faingold, C.L., Marcinczyk, M.F., Casebeer, D.J., Randall, M.E., and Arneric, S.P., Browning RA. 1994. GABA in the inferior colliculus plays a critical role in control of audiogenic seizures. Brain Res 640: 40–47. Fischer-Williams, M., Poncet, M., Riche, D., and Naquet, R. 1968. Lightinduced epilepsy in the baboon Papio papio: cortical and depth recordings. Electroencephalograph Clin Neurophysiol 25: 557–569. Franck, J.E., Ginter, K.L., and Schwartzkroin, P.A. 1989. Developing genetically epilepsy-prone rats have an abnormal seizure response to flurothyl. Epilepsia 30: 1–6. Garcia-Cairasco, N., Moraes, M.F.D., Browning, R.A., Mishra, P.K., and Jobe, P.C. 1998. Precollicular transection and its effect on brainstem seizure in GEPR-9: role of cortex in brainstem seizure regulation. Soc Neurosci Abst 24: 1211. Goddard, G.V., McIntyre, D.C., and Leech, C.K. 1969. A permanent change in brain functiion resulting from daily electrical stimulation. Exp Neurol 25: 295–330. Hjeresen,, D.L., Franck, J.E., and Amend, D.L. 1987. Ontogeny of seizure incidence, latency, and severity in genetically epilepsy prone rats. Dev Psychobiol 20: 355–363. Holmes, G.L., Thompson, J.L., Marchi T.A., Gabriel, P.S., Hogan, M.A., Carl, G.F., and Feldman, D.S. 1990. Effects of seizures on learning, memory, and behavior in the genetically epilepsy-prone rat. Ann Neurol 27: 24–32. Jobe, P.C. 2003. Common pathogenic mechanisms between depression and epilepsy: an experimental perspective. Epilepsy Behav 4: 14–24. Jobe, P.C. 2004a. Affective disorder and epilepsy comorbidity: Implications for development of treatments, preventions and diagnostic approaches. Clin EEG Neurosci 35: 53–68. Jobe, P.C. 2004b. Current and future therapeutic opportunicties in the comorbidity between the epilepsies and affective disorders. Clin EEG Neurosci 35: 1–3. Jobe, P.C. 2004c. Shared mechanisms of antidepressant and antiepileptic treatments: drugs and devices. Clin EEG Neurosci 35: 25–37. Jobe, P.C., and Dailey, J.W. 2000. Genetically epilepsy-prone rats (GEPRs) in drug research. CNS Drug Rev 6: 241–260. Jobe, P.C., Dailey, J.W., and Wernicke, F.J. 1999a. A noradrenergic and serotonergic hypothesis of the linkage between epilepsy and affective disorders. Crit Rev Neurobiol 13: 217–356.
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Jobe, P.C., Ko, K.H., and Dailey, J.W. 1984. Abnormalities in norepinephrine turnover rate in the central nervous system of the genetically epilepsy-prone rat. Brain Res 290: 357–360. Jobe, P.C., and Laird, H.E. 1987. Neurotransmitter systems and the epilepsy models: distinguishing features and unifying principles. In Neurotransmitters and Epilepsy. Eds. P.C. Jobe and H.E. Laird. pp. 339. Clifton, NJ: Humana Press. Jobe, P.C., Mishra, P.K., Adams-Curtis, L.E. et al. 1995. The genetically epilepsy-prone rat (GEPR). Ital J Neurol Sci 16: 91–99. Jobe, P.C., Mishra, P.K., and Browning, R.A. et al. 1994. Noradrenergic abnormalities in the genetically epilepsy-prone rat. Brain Res Bull 35: 493–504. Jobe, P.C., Mishra, P.K., Ludvig, N., and Dailey, J.W. 1991. Scope and contribution of genetic models to an understanding of the epilepsies. CRC Crit Rev Neurobiol 6: 183–220. Jobe, P.C., Mishra, P.K., Dailey, J.W., Ko, K.H., and Reith, M.E.A. 1999b. Genetic predisposition to partial (focal) seizures and to generalized tonic/clonic seizures: Interactions between seizure circuitry of the forebrain and brainstem. In Genetics of Focal Epilepsies. Eds. S.F. Berkovic, P. Genton, E. Hirsch, and F. Picard. p. 251. Avignon, France: John Libbey & Company. Jobe, P.C., Picchioni, A.L., and Chin, L. 1973b. Role of brain 5-hydroxytryptamine in audiogenic seizure in the rat. Life Sci 13: 1–13. Jobe, P.C., Picchioni, AL, and Chin, L. 1973b. Role of brain norepinephrine in audiogenic seizure in the rat. J Pharmacol Exp Ther 184: 1–10. Kaplan, H., and Miezejeski, C. 1972. Development of seizures in the mongolian gerbil (Meriones unguiculatus). J Comp Physiol Psychol 81: 267–273. Kiesmann, M., Marescaux, C., Vergnes, M., Micheletti, G., Depaulis, A., and Warter, J.M. 1988. Audiogenic seizures in Wistar rats before and after repeated auditory stimuli: clinical, pharmacological, and electroencephalographic studies. J Neural Transm 72: 235–244. Killam, E.K. 1979. Photomyoclonic seizures in the baboon, Papio papio. Fed Proc 38: 2429–2433. Killam, E.K., and Killam, K.F., Jr. 1984. Evidence for neurotransmitter abnormalities related to seizure activity in the epileptic baboon. Fed Proc 43: 2510–2515. Killam, K.F., Killam, E.K., and Naquet, R. 1967. An animal model of light sensitive epilepsy. Electroencephalogr Clin Neurophysiol 22: 497–513. Killam, K.F., Killam, E.K., and Naquet, R. 1966a. Mise en evidence chez certains singes d’un syndrome photomyoclonique. CR Acad Sci 262: 1010–1012. Killam, K.F., Killam, E.K., and Naquet, R. 1966b. Etudes pharmacologiques realisees chez des singes presentant une activite E.E.G. paroxystique particuliere a la stimulation lumineuse intermittente. J Physiol 58: 543–544. Killam, K.F., Killam, E.K., and Naquet, R. 1966c. Study of responses evoked by intermittent light stimulation in monkeys presenting paroxysmal responses to this type of stimulation. Rev Neurol (Paris) 115: 422–423. Killam, K.F., Naquet, R., and Bert, J. 1966d. Paroxysmal responses to intermittent light stimulation in a population of baboons (Papio papio). Epilepsia 7: 215–219. King, J.T. Jr., and LaMotte, C.C. 1989. El mouse as a model of focal epilepsy: a review. Epilepsia 30: 257–265. Kurtz, B., Lehman, J., Garlick, P., Amberg, J., Mishra, P.K., Dailey, J.W., Weber, R.J., and Jobe, P.C. 2001. Penetrance and expressivity of genes involved in the development of epilepsy in the genetically epilepsyprone rat (GEPR). J Neurogenet 15: 233–244. Loskota, W.J., and Lomax, P. 1975. The mongolian gerbil (Meriones unguiculatus) as a model for the study of the epilepsies: EEG records of seizures. Electroencephalogr Clin Neurophysiol 38: 597–604. Loscher, W. 1984. Genetic animal models of epilepsy as a unique resource for the evaluation of anticonvulsant drugs. a review. Methods Find Exp Clin Pharmacol 6: 531–547.
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Ludvig, N., and Moshe, S.L. 1989. Different behavioral and electrographic effects of acoustic stimulation and dibutyryl cyclic AMP injection into the inferior colliculus in normal and in genetically epilepsy-prone rats. Epilepsy Res 3: 185–190. Meldrum, B.S., Anlezark, G.M., Balzamo, E., Horton, R.W., and Trimble, M. 1975a. Photically induced epilepsy in Papio papio as a model for drug studies. Adv Neurol 10: 119–132. Meldrum, B.S., Chir, B., Horton, R.W., and Toseland, P.A. 1975b. A primate model for testing anticonvulsant drugs. Arch Neurol 32: 289–294. Meldrum, B.S., and Balzamo, E. 1972. Epilepsy in the photosensitive baboon, Papio papio, and drugs acting on cerebral GABA and serotonin mechanisms. Med Primatol Part II: 282–288. Meldrum, B.S., Croucher, M.J., Badman, G., and Collins, J.F. 1983. Antiepileptic action of excitatory amino acid antagonists in the photosensitive baboon, Papio papio. Neurosci Lett 39: 101–104. Meldrum, B., and Horton, R. 1978. Blockade of epileptic responses in the photosensitive baboon, Papio papio, by two irreversible inhibitors of GABA-transaminase, gamma-acetylenic GABA (4-amino-hex-5-ynoic acid) and gamma-vinyl GABA (4-amino-hex-5-enoic acid). Psychopharmacology 59: 47–50. Menini, C., and Silva Barrat, C. 1990. Value of the monkey Papio papio for the study of epilepsy. Pathol Biol 38: 205–213. Merrill, M.A., Clough, R.W., Shaw, T., Jobe, P.C., and Browning, R.A. 1998. Brainstem (BS) seizure severity regulates forebrain FB seizure expression in the audiogenic kindling model. Soc Neurosci Abst 24(1): 475.13. Millan, M.H., Wardley-Smith, B., Durmuller, N., and Meldrum, B.S. 1991. The high pressure neurological syndrome in genetically epilepsy prone rats: protective effect of 2-amino-7-phosphono heptanoate. Exp Neurol 112: 317–320. Mishra, P.K., Dailey, J.W., Reigel, C.E., and Jobe, P.C. 1989. Audiogenic convulsions in moderate seizure genetically epilepsy-prone rats. (GEPR-3s). Epilepsy Res 3: 191–198. Mishra, P.K., Dailey, J.W., Reigel, C.E., Tomsic, M.L., and Jobe, P.C. 1988. Sex-specific distinctions in audiogenic convulsions exhibited by severe seizure genetically epilepsy-prone rats (GEPR-9s). Epilepsy Res 2: 309–316. Moraes, M.F., Chavali, M., Mishra, P.K., Jobe, P.C., and Garcia-Cairasco, N. 2005. A comprehensive electrographic and behavioral analysis of generalized tonic-clonic seizures of GEPR-9s. Brain Res 1033: 1–12. Moraes, M.F.D., Moraes, C., Jobe, P.C., Mishra, P.K., and Garcia-Cairasco, N. 1998. Neural substrates correlated with the appearance of limbic seizures during repetitive acoustic stimulation of GEPRs. Soc Neurosci Abst 24:(part 1): 475.14. Naquet, R., and Valin, A. 1990. Focal discharges in photosensitive generalized epilepsy. In Generalized Epilepsy: Neurobiological Approaches. Eds. M. Avoli, P. Gloor, G. Kostopoulos, and R. Naquet. pp 273. Boston: Birkhauser. Noebels, J.L., Fariello, R.G., Jobe, P.C., Lasley, S.M., and Marescaux, C. Genetic models of generalized epilepsy. In Epilepsy, A Comprehensive Textbook 1st ed. Eds. J. Engel Jr. and T.A. Pedley. pp 457–466. Philadelphia: Lippincott—Raven. Paul, L.A., Fried, I., Watanabe, K., Fosythe, A.B., and Scheibel, A.B. 1981.Structural correlates of seizure behavior in the Mongolian gerbil. Science 213: 924–926. Pedley, T.A., Horton, R.W., and Meldrum, B.S. 1979. Electroencephalographic and behavioral effects of a GABA agonist (muscimol) on photosensitive epilepsy in the baboon, Papio papio. Epilepsia 20: 409–416. Reigel, C.E., Dailey, J.W., and Jobe, P.C. 1986. The genetically epilepsyprone rat: an overview of seizure-prone characteristics and responsiveness to anticonvulsant drugs. Life Sci 39: 763–774. Reigel, C.E., Jobe, P.C., Dailey, J.W., and Savage, D.D. 1989. Ontogeny of sound-induced seizures in the genetically epilepsy-prone rat. Epilepsy Res 4: 63–71.
Ribak, C.E., Roberts, R.C., Byun, M.Y., and Kim, H.L. 1988. Anatomical and behavioral analyses of the inheritance of audiogenic seizures in the progeny of genetically epilepsy-prone and Sprague-Dawley rats Epilepsy Res 2: 345–355. Ryu, J.R., Jobe, P.C., Milbrandt, J.C., Mishra, P.C., Clough, R.W., Browning, R.A., Dailey, J.W. et al. 1999. Morphological deficits in noradrenergic neurons in GEPR-9s stem from abnormalities in both the locus coeruleus and its target tissues. Exp Neurol 156: 84–91. Savage, D.D., Reigel C.E., and Jobe P.C. 1986a. Angular bundle kindling is accelerated in rats with a genetic predisposition to acoustic stimulusinduced seizures. Brain Res. 376: 412–415. Savage, D.D., Reigel, C.E., and Jobe, P.C. 1986b. The development of kindled seizures is accelerated in the genetically epilepsy-prone rat. Life Sci 39: 879–886. Schonfeld, A.R., and Glick, S.D. 1981. Handling-induced seizures and rotational behavior in the Mongolian gerbil. Pharmacol Biochem Behav 14: 507–516. Schreiber, R.A., and Graham, J.M. Jr. 1976. Audiogenic priming in DBA/2J and C57BL/6J mice: interactions among age, prime-to-test interval and index of seizure. Dev Psychobiol 9: 57–66. Seyfried, T.N., and Glaser, G.H. 1985. A review of mouse mutants as genetic models of epilepsy. Epilepsia 26: 143–150. Seyfried, T.N., Glaser, G.H., Yu, R.K., and Palayoor, S.T. 1986. Inherited convulsive disorders in mice. Adv Neurol 44: 115–133. Silva-Barrat, C., and Menini, C. 1990. Photosensitive epilepsy of the baboon: A generalized epilepsy with a motor cortical origin. In Generalized Epilepsy: Neurobiological Approaches. Eds. M. Avoli, P. Gloor, G. Kostopoulos, and R. Naquet. p. 286. Boston: Birkhauser. Suzdak, P.D., and Jansen, J.A. 1995. A review of the preclinical pharmacology of tiagabine: a potent and selective anticonvulsant GABA uptake inhibitor. Epilepsia 36: 612–626. Suzuki, J., and Nakamoto, Y. 1977. Seizure patterns and electroencephalograms of El mouse. Electroencephalogr Clin Neurophysiol 43: 299–311. Szabo, C.A., Leland, M.M., Sztonak, L. Restrepo, S., Haines, R., Mahaney, M.A., and Williams, J.T. 2004. Scalp EEG for the diagnosis of epilepsy and photosensitivity in the baboon. Am J Primatol 62: 95–106. Thompson, J.L., Carl, G.F., and Holmes, G.L. 1991. Effect of age on seizure susceptibility in genetically epilepsy-prone rats (GEPR-9s). Epilepsia 32: 161–167. Vicari, E. 1951. Fatal convulsive seizures in the DBA mouse strain. J Psychol 32: 79–97. Valin, A., Bryere, P., and Naquet, R. 1986. Convulsant effect of Ro 5–4864, a peripheral type benzodiazepine, on the baboon (Papio papio). Neurosci Lett 66: 210–214. Valin, A., Voltz, C., Naquet, R., and Lloyd, K.G. 1991. Effects of pharmacological manipulation on neurotransmitter and other amino acid levels in the CSF of the Senegalese baboon Papio papio. Brain Res 538: 15–23. Wada, J.A, and Osawa, T. 1976. Spontaneous recurrent seizure state induced by daily electric amygdaloid stimulation in Senegalese baboons (Papio papio). Neurology 26: 273–286. Wada, J.A., Osawa, T., and Mizoguchi, T. 1975. Recurrent spontaneous seizure state induced by prefrontal kindling in senegalese baboons, Papio papio. Can J Neurol Sci 2: 477–492. Wada, J.A., Terao, A., and Booker, H.E. 1972. Longitudinal correlative analysis of epileptic baboon, Papio papio. Neurology 22: 1272– 1285. Walczak, T.S., and Jayakar, P. Interictal EEG. In Epilepsy: A Comprehensive Textbook. 1st ed. Eds. J. Engel Jr. and T.A. Pedley. pp. 831–848. Philadelphia: Lippincott–Raven. Weinberger, S.B., and Killam, E.K. 1977.A comparison of the effects of chronically administered diazepam and phenobarbital and learning in the Papio papio model of epilepsy. Proc West Pharmacol Soc 20: 173–177.
References Weinberger, S.B., and Killam, E.K. 1979. Learning and behavioral abnormalities in the seizure-prone baboon. Biol Psychiatry 14: 525–535. Zhang, N., Walberg, F., Laake, J.H., Meldrum, B.S., and Ottersen, O.P. 1990. Aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: a light microscopic and semi-
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21 Inherited Epilepsy in Mongolian Gerbils PAUL S. BUCKMASTER
they burrow extensively and live on grain, seeds, roots, and grass. Their head is adapted for burrowing, being shorter and broader than that of rats. Gerbils live in large social groups and are extremely territorial, earning their name “fingernail warrior.” If they are caged together without habituation, adult gerbils fight relentlessly, resulting in high mortality. However, they are easy to handle and make good pets. In some states (California, for example) they are considered a threat to agricultural crops and are illegal, and research institutions must obtain special permits to keep gerbils. Mongolian gerbils were established as laboratory animals in the 1960s (Schwentker, 1963; Marston and Chang, 1965; Rich, 1968; Vincent et al., 1979). Today they are commercially available from laboratory animal vendors. They are used in various aspects of biomedical research, as models for bacteriology (Peek and Blaser, 2001), parisitology (Fenoy et al., 2001; Abraham et al., 2002), and for research on the auditory system (Scheich et al., 1997; Chatterjee and Zwislocki, 1998), aging (Cheal, 1986), behavior (Thiessen and Yahr, 1977), stroke (Traystman, 2003), and epilepsy (Robinson, 1968; Loskota et al., 1974; Jobe et al., 1991; Bertorelli et al., 1995). This chapter reviews the establishment of gerbils as a model of human epilepsy, the ontogeny of epilepsy in gerbils, their seizure characteristics and effects, the use of gerbils for anticonvulsant drug screening, hypotheses on the etiology of epilepsy in gerbils, and the relevance of gerbil epilepsy to human epilepsy.
Epilepsy is common in domestic Mongolian gerbils (Meriones unguiculatus), and seizure-sensitive strains have been developed. Mild seizures begin after one month of age and with further development they typically become severe, generalized tonic-clonic seizures. Electrographic seizure activity has been recorded most consistently in neocortical areas, but the site of seizure onset is unclear. Seizure-sensitive gerbils have been used to evaluate anticonvulsants. Numerous neuroanatomic, neurophysiologic, and neurochemical changes have been identified between epileptic and control gerbils. Epilepsy in gerbils is not consistently associated with any recognized neuropathology, but defects in the GABAergic system have been proposed. The genetic basis of epilepsy in gerbils is unknown. The direct relevance of epilepsy in gerbils to human epileptic syndromes is unclear. Nevertheless, advantages of the gerbil model offer many opportunities to increase understanding of epilepsy. Primary among these are that epilepsy develops without surgical, chemical, or electrical intervention, seizures can be triggered by environmental stimuli that are controlled by the investigator, and epileptogenesis can be evaluated over developmental stages, from preepileptic to epileptic. Identifying the underlying mutation(s), the seizure-triggering mechanisms, and the mechanisms that control the age-dependent expression of epilepsy in gerbils are likely to provide insight into human epilepsy.
INTRODUCTION Mongolian gerbils (Meriones unguiculatus) are a member of the rodent family Muridae. They reach puberty at 65–85 days old, weigh 70–110 g as adults, and live for approximately 3 years (Cheal, 1987b). They are native to desert regions of Mongolia and northeastern China, where
Models of Seizures and Epilepsy
ESTABLISHMENT OF SEIZURE-SENSITIVE AND SEIZURE-RESISTANT GERBIL STRAINS In 1935 twenty pairs of wild gerbils were caught in Eastern Mongolia and sent to the Kitasato Institute in Japan.
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In 1954 eleven pairs of their offspring were imported from the Central Laboratory for Experimental Animals in Japan to the United States. From those, five females and four males bred and established the first colony of gerbils in the United States (Schwentker, 1963). All commercially available gerbils in North America and Europe derived from this stock (Neumann et al., 2001). Soon after gerbils were established as laboratory animals, seizures induced by handling or cage changing were reported (Marston and Chang, 1965; Zeman, 1967), and the conditions that evoke and suppress seizures began to be evaluated (Thiessen et al., 1968). The value of gerbils for epilepsy research quickly became clear to investigators. Selective inbreeding was used to establish epileptic strains and control, nonepileptic strains. Strains were developed at the University of California, Los Angeles (WJL/UC, seizure-sensitive; STR/UC, seizureresistant) (Loskota et al., 1974), the New York State Institute for Basic Research in Developmental Disabilities, Staten Island (IBR/SP, seizure-sensitive; IBR/NSP, seizureresistant) (Kaplan, 1981; Donadio et al., 1982), and the National Institute of Infectious Diseases in Japan at the Institute for Developmental Research (MGS/Idr, seizuresensitive) and the Tokyo Institute of Psychiatry (MGR, seizure-resistant) (Seto-Ohshima et al., 1992, 2003). In addition, without formally establishing strains, other gerbil colonies were selectively inbred for the epilepsy trait at Gödecke Research Institute in Freiburg, Germany (Bartoszyk and Hamer, 1987) and at the University of Washington (Buckmaster et al., 1996). Strain development takes a great deal of time, and maintaining a large colony of gerbils is expensive. Fortunately, it is not necessary to establish and maintain an epileptic strain in order to utilize gerbils for epilepsy research. Nonselectively bred colonies (Löscher and Frey, 1984; Scotti et al., 1998; Kang et al., 2000b) and gerbils obtained directly from commercial vendors can be used. However, when using randomly bred gerbils, individuals must be carefully screened for seizure susceptibility to identify epileptic and control animals, because even if both parents are epileptic, the trait is not necessarily inherited by all of their offspring. An advantage of using established seizure-sensitive strains is that epileptic parents produce offspring that have an extremely high probability of developing epilepsy later in life. Before the age that epilepsy develops, these “pre-epileptic” gerbils provide an opportunity to study a brain that is about to generate chronic spontaneous seizure activity but has not yet been altered by the genetic, morphologic, pharmacologic, and electrophysiologic consequences of repetitive seizures.
ONTOGENY OF EPILEPSY IN GERBILS Most rodents that have been evaluated are maximally susceptible to seizures 2 to 3 weeks postnatally (Moshé et
al., 1996). However, gerbils usually do not begin having seizures until they are more than 1 month old, and seizure susceptibility and severity increases with age, stabilizing at approximately 6 months (Loskota et al., 1974; Löscher and Frey, 1984; Seto-Ohshima et al., 1992; Buckmaster et al., 1996). This progressive age-dependent development of seizure susceptibility in gerbils resembles human absence or myoclonic epilepsies in childhood, which at a later age often proceed to generalized tonic-clonic seizures (Löscher, 1987). To account for the progressive nature of epilepsy in gerbils it has been proposed that at least some individuals undergo a kindling process as they experience repeated, mild seizures (Scotti et al., 1998). However, even after many seizures, chronically epileptic gerbils do not display hilar neuron loss (Mouritzen-Dam et al., 1981; Buckmaster et al., 1996) or granule cell axon reorganization (Peterson and Ribak, 1987; Ribak and Peterson, 1991), which would be expected for kindled animals (Sutula et al., 1988; Cavazos and Sutula, 1990). Another possibility is the gradual expression of an inherited epileptogenic defect. Alternatively, increased seizure susceptibility may be attributable to the gradual developmental reduction of an endogenous anticonvulsant mechanism. Consistent with this hypothesis, pairedpulse depression of evoked responses in the dentate gyrus decreases with development in gerbils (Buckmaster et al., 1996; Buckmaster and Wong, 2002). However, these scenarios do not account for the delayed and progressive expression of seizures that has been reported in some repeatedly tested adult gerbils, whereas a kindling process does (Scotti et al., 1998). Once established, epilepsy usually is permanent in gerbils (Cheal, 1987a; Spangler et al., 1997), but some individuals appear to outgrow their seizure-susceptibility (Löscher, 1987; Buckmaster et al., 1996). This developmental change parallels that seen in some human epilepsies; many children with epileptic disorders outgrow their seizures (Gross-Tsur and Shinnar, 1993). The mechanisms underlying the developmental expression of human epilepsy and its remission are unclear, but gerbils may be useful for studying these phenomena. Environmental conditions during development affect seizure susceptibility. Gerbils reared in isolation beginning at 15 days old are more likely to develop seizures later in life (Berg et al., 1975). On the other hand, when gerbils are placed alone in a novel environment for 3 min/week beginning at 1 week old they do not (or rarely) display seizures when tested later in life (Kaplan and Miezejeski, 1972). Milder stimulation (rocking the home cage for 3 min/day) on postnatal days 1–21 does not affect seizure frequency but prolongs latency to seizure onset (Kaplan and Silverman, 1978). Together, these findings suggest that early experiences have long-lasting effects on seizure susceptibility. Important early experiences include interactions with
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parents, and parental behavior and/or nurturing ability may affect seizure development. If the offspring of epileptic gerbils are fostered at postnatal day 1 to other epileptic parents they are more likely to develop epilepsy than if they are fostered to nonepileptic parents (Kaplan, 1981). Environmental conditions also affect seizure susceptibility of mature gerbils. Adult gerbils are less likely to have seizures if they are housed with other gerbils (Pettijohn, 1978), and months of limited environmental enrichment reduces seizure incidence in adult male, but not female, gerbils (Cheal, 1987a). Gerbils, therefore, provide a model for studying the mechanisms of environmental effects on seizure probability, which could have clinical relevance for patients.
SEIZURES IN GERBILS Seizure Incidence The incidence of epilepsy in gerbils from randomly bred colonies and commercial vendors varies between 3% and 98% (Theissen et al., 1968; Harriman, 1988; Cutler and MacKintosh, 1989; Bertorelli et al., 1995). This wide range in seizure incidence may be attributable to genetic differences between colonies or to differences in seizure testing methods. For example, with repeated testing over months, 100% of a large, outbred group of gerbils displayed at least one seizure (Scotti et al., 1998). Some individuals displayed their first seizure only after weeks of testing. Therefore, less extensive testing would have resulted in lower seizure incidence values. In selectively bred gerbil lines, epilepsy occurs in virtually 100% of the population, and more than 85% will have a seizure on any given test (Loskota et al., 1974; Bartoszyk and Hamer, 1987). In selectively bred colonies epileptic gerbils tend to have a lower seizure threshold than randomly bred gerbils. For example, Löscher and Frey (1984) reported that their non-selectively bred gerbils rarely have seizures when placed in a novel environment, and less than 10% have seizures when handled. However, when challenged with a more intense stimulus (air blowing) almost 100% have seizures. The high incidence of seizures in randomly bred gerbil colonies raises doubts about the possibility of assembling adequate control groups for experiments. In many experiments gerbils are tested weekly for seizure susceptibility over a period of 3 to 5 weeks to identify a very epileptic group and a control group that never displays a seizure. However, longer and more extensive testing might reveal that “control ” gerbils actually are mildly epileptic. The low penetrance of the genetic defect(s) underlying epilepsy in gerbils makes genotypically pure seizure-resistant gerbils difficult to isolate (Scotti et al., 1998). Even in control strains, which have been developed by selective inbreeding of seizure-resistant gerbils for more than 20 generations,
epilepsy occasionally appears (Seto-Ohshima et al., 2003). Comparing very epileptic gerbils to mildly epileptic gerbils is valid for some experiments, but others (for example, genetic studies) require animals that are completely free of epilepsy. In 1995 wild gerbils were captured during a joint expedition to Central Mongolia by the Leibniz Institute of Neurobiology in Magdeburg, Germany and the State University of Mongolia (Neumann et al., 2001). Seizures were absent in gerbils trapped in the wild and subsequently housed in Germany; however, in the laboratory environment seizures occur rarely in some of the offspring of a small percentage of wild breeding pairs (Stuermer et al., 2003). This observation suggests that the epilepsy trait may be present at low levels in the wild population. Nevertheless, nonepileptic, nondomesticated gerbils and their descendants are another alternative to be considered for use as a control group, although they display anatomic differences from domestic gerbils (Gleich et al., 2000), for example, larger brain size (Stuermer et al., 2003).
Seizure Triggers One of the unusual features of epileptic gerbils is that their seizures can be triggered. Seizures are more likely to occur at night, which is the gerbil’s most active time (Thiessen et al., 1968; Schonfeld and Glick, 1980). Spontaneous seizures occur occasionally (Loskota et al., 1974; Buckmaster, 2004), but most seizures observed by investigators are triggered by external stimuli. Latency from the precipitating stimulus to seizure onset is short, typically less than 1 min (Thiessen et al., 1968; Loskota et al., 1974; Ludvig et al., 1991; Buckmaster and Wong, 2002). The ability to trigger seizures is a useful experimental feature, allowing investigators to control the timing of seizure onset. Numerous stimuli trigger seizures (Thiessen et al., 1968), and many seizure-provoking protocols have been reported. Some are simple, for example placing the gerbil in the palm of the investigator’s hand (Seregi et al., 1984), and others, like the “triple handling technique,” are elaborate. The latter involves removing the gerbil from the home cage, weighing it and placing it in a novel environment (top of a laboratory cart) for 2.5 min, handling it for 20 s, replacing it in a novel environment for another 2.5 min, suspending it by the tail above the top of the cart for 2.5 min, and finally replacing it in a novel environment (Cox and Lomax, 1976). The “S method” consists of suspending the gerbil by the tail and applying pressure to its back for 5 s, as if checking for pregnancy (Seto-Ohshima et al., 2003). The “H method” consists of a researcher moving his/her palm back and forth over the gerbil’s head at a frequency equal to or greater than 1 movement/s (Seto-Ohshima et al., 2001). Other seizure-evoking protocols include:
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• placing the gerbil in an empty plastic cage and blowing on it (10–15 cm from its back) with compressed air (five bars at outlet) for 15 seconds (Frey, 1987b) • lifting the gerbil by its tail, swinging it left and right 10 times, placing it in a cage, and then throwing it up in the air about 10 cm above the cage 30 times (Asano and Mizutani, 1980) • transferring the gerbil from the home cage to a red plastic shopping basket (Buckmaster et al., 1996) • placing the gerbil on a metal grid spanned over a sink, bouncing the grid up and down and side-to-side or patting the gerbil repeatedly while rattling a pencil over the grid (Scotti et al., 1998) • placing the gerbil on an automatic shaker (2 Hz) for 30 seconds (Bartoszyk and Hamer, 1987). • transferring the gerbil to a new cage and clapping a sheaf of papers over it without physical contact for 30 seconds (Revilla et al., 1999). Novel environment exposure and air blowing are simple, humane, and effective treatments for triggering seizures in gerbils. Novel environment exposure is one of the most salient stimuli for triggering seizures (Thiessen et al., 1968; Kaplan, 1975; Fueta et al., 1983; Spangler et al., 1997). Ludvig et al. (1991) evaluated various seizure-evoking stimuli by placing gerbils in a novel environment for 3 min or subjecting them to stimulation of a specific sensory modality (or air blowing) for 1 min. Placement in a dishdrying rack was the most effective stimulus; and next most effective were other novel environments, including a Ymaze, the surface of a laboratory cart, a sink, a Styrofoam box, and a glass jar. None of the specific sensory stimuli triggered seizures, including pain stimulation by pinching the tail, odor stimulation with ammonia, tactile stimulation by poking with a brush, acoustic stimulation with 95 dB ringing, and visual stimulation with a strobe light. These findings suggest that the gerbil’s perception of the novel environment, or reaction to it, contributes to the seizure triggering mechanism, whereas simple, monomodal stimuli are insufficient. However, colonies appear to differ in their sensitivity to stimuli. For example, air blowing evoked seizures in 98% of the gerbils in one colony (Löscher et al., 1983) but only 23% in another (Ludvig et al., 1991). Understanding why a patient has a seizure at any given point in time is a fundamental problem in epilepsy research (Schwartzkroin, 1997). The ability to accurately predict seizure onset would be a tremendous advance to help patients prepare for that event. The epileptic gerbil is a model that may help investigators discover the mechanisms underlying seizure onset. Many of the aforementioned seizure-evoking stimuli are likely to be stressful, suggesting that the stress of the moment might trigger seizures in gerbils (Kaplan and Miezejeski, 1972). However, placing a smaller gerbil in the cage of an aggressive, larger gerbil does not trigger seizures
or produce remarkable changes in the EEG (Seto-Ohshima et al., 1992). Specific sensory stimuli such as tail pinching and 95 dB ringing are likely to be stressful but do not induce seizures (Ludvig et al., 1991). And there is no positive correlation between cortisol levels and the seizure-evoking efficacy of stimuli (Revilla et al., 1999). Stress, therefore, does not appear to be a critical factor in triggering seizures in gerbils.
Seizure Behavior Behaviorally, seizures in gerbils range from brief immobility to severe, generalized tonic-clonic convulsions followed by wild running fits. Seizures begin as mild events and usually become more severe with development (Loskota et al., 1974; Löscher and Frey, 1984; Seto-Ohshima et al., 1992; Buckmaster and Wong, 2002), although some gerbils only have mild seizures throughout their adult life (Cutler and MacKintosh, 1989). Mild and severe seizures begin similarly with motor arrest and then clonus of the face and ears. There are five cumulative stages of seizure severity: (1) motor arrest, (2) clonus of discrete body areas (usually the pinnae and head), (3) tonic extension, (4) abnormal activity (automatisms or wild running), and (5) postictal depression. Kaplan and Miezejeski (1972) provide one of the earliest complete descriptions of behavioral seizures in gerbils: A seizure typically begins with cessation of ongoing activity and vibrissae twitching, eye blinking, ears flattened against the head, and small muscle twitches. Then there are contractions of the anterior part of the body, crouching, often with front paws pushing against the substrate, and later immobility, sometimes in unusual postures such as with limbs spread out laterally or the tail curved up over the body. Subsequently, there may be rolling over onto one side often with tonic-clonic spasms resulting in such random movements as pawing the air, slow head turns, and jerky movements of the head, limbs, and torso. Finally, there may be large muscular movements resulting in nonrandom behavioral sequences such as grooming, chewing, walking, circling, running, and jumping that were distinguishable from normal behavior in that the movements were jerky, violent, or abortive. These were often interspersed with periods of immobility.
At the seizure onset electrographic spikes precede the start of clonic movements by an average of 5 seconds (Buckmaster et al., 2000), and the duration of electrographic seizure activity that is contemporary with the behavioral seizure varies from 4 to 90 seconds (Buckmaster and Wong, 2002). Although the convulsive phase of the seizure typically ends within 2 min, several more minutes may elapse before normal behavior resumes (Thiessen et al., 1968; Loskota et al., 1974; Cutler and MacKintosh, 1989). Following the convulsive phase of the seizure a period of immobility and postictal depression may occur. Postictal depression is rare in gerbils under 2 months old, but common in adults. When it occurs, postictal depression lasts
Seizures in Gerbils
an average of 3 min and ends abruptly (Buckmaster and Wong, 2002). Following the seizure and the period of postictal depression, seizing gerbils are more active than nonseizing gerbils (Cheal, 1987a; Laming et al., 1989a) and experience a seizure-refractory period. When adult gerbils are tested at weekly intervals the proportion of animals having seizures remains constant, but when they are tested daily or even more frequently, the proportion decreases to zero within a few days (Theissen et al., 1968; Revilla et al., 1999). The seizure-induced release of endogenous opioids has been proposed as the underlying mechanism of the refractory period in gerbils, and the opioid antagonists naloxone and naltrexone block the seizure-refractory effect (Lee et al., 1983; Lee and Lomax, 1985). However, the high doses of antagonists used in those experiments might have produced nonspecific effects (Frey, 1987a). The activity of glutamic acid decarboxylase (GAD), the enzyme that synthesizes g-aminobutyric acid (GABA), increases after seizures and could have an inhibitory effect after a seizure. However, GAD levels return to baseline long before the seizure-refractory period ends (Löscher and Frey, 1987b). Hence, the mechanisms of seizure refractoriness in gerbils remain unknown but are of investigational interest because once better defined they could provide targets for anticonvulsant therapies.
EEG Recordings It can be difficult to localize the site of seizure onset with EEG recording in rodents, and the seizure initiation site in gerbils is unclear. Gerbils have thin skulls that are advantageous for transcranial recording (Guedes et al., 1987), but most studies have used electrodes that record from the surface of the neocortex and from the deeper hippocampal formation. Some evidence suggests that the hippocampus initiates seizures in gerbils, but most identifies the neocortex (Loskota and Lomax, 1975; Suzuki and Nakamoto, 1978). In 94% of 73 seizures recorded with electrodes in the anterior neocortex and hippocampus, the neocortical recording most clearly displayed the seizure onset, but in at least 25% of cases the hippocampal recording simultaneously displayed the seizure onset (Buckmaster and Wong, 2002). Electrographic seizures in gerbils do not resemble spikeand-wave discharges of absence epilepsy. EEG spikes or sharp waves have been recorded at seizure onset in the parietal cortex, and electrically induced neocortical seizures resemble environmentally precipitated seizures more than induced hippocampal seizures (Majkowski and Donadio, 1984). The parietal cortex, therefore, is a likely site of seizure onset, however many other regions remain to be evaluated, and many questions persist about the onset and spread of electrographic seizures in gerbils. Gerbils offer experimental advantages for ictal EEG studies, because investigators can control seizure onset.
277
Since seizures can be triggered, experiments on the dynamic effects of seizures are more feasible. For example, seizures have been evaluated by monitoring the changes of evoked responses obtained before, during, and after seizures. Dentate gyrus field potential responses to stimulation of its major afferent, the perforant path, have been analyzed during environmentally precipitated seizures in awake, unrestrained gerbils (Buckmaster et al., 2000; Buckmaster and Wong, 2002). Synchronously stimulating many axons of the perforant path is not a physiologically natural method of dentate excitation, and field potential recording is a limited technique. Despite these limitations, evoked field potentials reflect the activity of a population of neurons (seizures are a population phenomenon) and can be obtained in vivo. In gerbils experiencing environmentally induced seizures, evoked responses remain at baseline values until after seizure onset. Then, during the first part of the seizure, the responsiveness or excitability of the dentate gyrus increases dramatically and the field excitatory postsynaptic potential (fEPSP) slope and the number and amplitude of population spikes peak. These measures of excitability increase more in older gerbils that exhibit seizures that are more severe behaviorally. For example, older gerbils display multiple population spikes (an average of four per stimulus) that begin 14 seconds after seizure onset and last over a period of 13 s, whereas gerbils under 2 months old display only single population spikes. The period of hyperexcitability at the beginning of the seizure is followed by a dramatic reduction in responsiveness to perforant path stimulation in older but not younger gerbils. In many cases during this phase of reduced excitability, responses are completely abolished beginning about 30 seconds after seizure onset and lasting an average of 37 s. These findings suggest that seizures activate endogenous homeostatic mechanisms that dampen tissue excitability and help to terminate the seizure. After seizure activity ends fEPSP slope and population spike amplitude gradually recover while the gerbil is immobile and in a state of postictal depression. The fEPSP slope recovers smoothly toward its baseline value, but the population spike amplitude stalls at approximately 60% of its baseline value and remains there until the end of the postictal period. The dissociation of fEPSP slope and population spike amplitude might be attributable to selective activation of inhibitory input to specific parts of the granule cell. In gerbils as in other species different subpopulations of interneurons in the dentate gyrus selectively inhibit granule cell dendrites versus soma (Buckmaster et al., 2002). Inhibition at the level of the granule cell body reduces population spike amplitude, whereas inhibition at the dendritic level reduces fEPSP slope (Moser, 1996). In CA1 pyramidal cells it has been shown that recurrent inhibitory synaptic input to the soma can be dissociated from recurrent inhibitory input to the apical dendrites by stimulus frequency (Pouille and Scanziani, 2004). These findings
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suggest that changes in evoked responses during the period of postictal depression might reflect the independent recovery of dendritic and somatic inhibitory circuits. The switch from postictal depression to normal exploratory behavior is abrupt and coincident with a jump in population spike amplitude from approximately 60% to 100% of the baseline value. This suggests that reduced population spike amplitude and behavioral depression might share a common neurophysiologic mechanism. Population spike amplitude ratio (2nd/1st response) is a measure of inhibition that can be obtained with field potential recordings in awake, behaving animals. The amplitude ratio begins to increase (less inhibition) shortly after seizure onset, especially in older gerbils with more severe seizures. The amplitude ratio peaks approximately 1 min after seizure onset and returns to baseline approximately 11 min later. This finding suggests that paired-pulse inhibition of the major afferent to the dentate gyrus is reduced by seizure activity but recovers within minutes. In summary, these examples of changes in evoked responses correlated with different phases of
seizures illustrate opportunities to investigate the mechanisms of seizure initiation and termination afforded by a model whose seizures can be controlled.
Effects of Seizures One reason why epileptic gerbils are advantageous for studying the effects of seizures is that there are no potentially confounding treatments produced by chemical convulsants, electrical stimulation, or lesions. Furthermore, control of seizure timing facilitates time-series analyses. Many parameters have been evaluated by comparing their expression before and after seizures in epileptic gerbils. As shown in other models of epilepsy, results from gerbils indicate that seizures have myriad widespread effects on the central nervous system. Changes in neurotransmitters, their receptors, their synthesizing/degrading enzymes, their transporters, second messengers, calcium-binding proteins, ion transporters, and others have been reported after seizures in gerbils (Table 1).
TABLE 1 Effects of Seizures in Epileptic Gerbils Neurotransmitter/ neuromodulator
Effect
Aspartate Corticotropin releasing factor (CRF)
No change in cortex, hippocampus, and striation Increase in interneurons of hippocampal formation
Time 30 sec 0.5–12 h
Reference Wolf-Dieter et al., 1989 Park et al., 2003
CRF-binding protein
Increase in entorhinal cortex
0.5–12 h
Park et al., 2003
Dynorphin
Increase in hippocampus
5 min–72 h
McGinty et al., 1986
GABA, glutamate, and glycine
No change in cortex, hippocampus, and striatum
30 sec
Wolf-Dieter et al., 1989
Met-enkephalin level
Increase in hippocampus Increase in hippocampus, not in other brain regions
5 min–72 h 5 min
McGinty et al., 1986 Lee et al., 1987
Neuropeptide Y (NPY)
No change in cortex, hippocampus, and striatum Increase in dentate gyrus and subiculum; no change in entorhinal cortex
2h 0.5–12 h
Wolf-Dieter et al., 1989 Kang et al., 2000b
Prostaglandin F2a
Increase after severe seizure; largest increase in hippocampus and neocortex; decrease after mild seizure
Peak at 6 min
Simmet et al., 1987, 1988; Seregi et al., 1985
Prostaglandin D2
Largest increase in hippocampus and neocortex
3–15 min
Seregi et al., 1985
Leukotriene C4
Increase after severe seizure; decrease after mild seizure
Peak at 6 min
Simmet et al., 1987, 1988; Simmet and Tippler, 1991
Leukotriene D4
Increase after severe seizure
5 min
Simmet et al., 1987; Simmet and Tippler, 1991
Secretoneurin
Increase neuron expression after kainate-induced seizure
6–48 h
Marti et al., 2002
Somatostatin
Increase in cortex; no change in hippocampus and striatum Decrease in the dentate gyrus and subiculum Increase in the entorhinal cortex
2 h and 1 wk Up to 12 h 30 min
Wolf-Dieter et al., 1989 Kang et al., 2000a Kang et al., 2000a
Taurine
No change in cortex, hippocampus, and striatum
30 sec
Wolf-Dieter et al., 1989
Increase binding affinity in all brain regions, largest in striatum and brainstem.
Peaks at 10 min, returns to baseline by 20 min
Asano and Mizutani, 1980
Decrease in hippocampal formation
0.5–3 h
An et al., 2003a
Neurotransmitter receptors Benzodiazepine
Corticotropin-releasing factor
(continues)
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Seizures in Gerbils
TABLE 1 (continued) Neurotransmitter/ neuromodulator
Effect
Time
Reference
Opioid
Decreased binding in the interpeduncular nucleus and accessory optic nucleus
5 min
Lee et al., 1984b
NMDA receptor (NR) 1
Increase in superior blade of dentate gyrus Decrease in inferior blade of dentate gyrus and in subiculum
30 min 0.5–12 h
Suh et al., 2001 Suh et al., 2001
NR 2A/B
Increase in subiculum; decrease in dentate gyrus
12 h
Suh et al., 2001
15 min
Löscher and Frey, 1987b
Neurotransmitter synthesizing/degrading enzymes/cofactors Glutamic acid decarboxylase Activity decreases in corpus striatum, thalamus, hypothalamus; (GAD) no change in other brain regions Immunoreactivity increases in hippocampus GABA-transaminase Pyridoxine-5¢-phosphate oxidase
Increase followed by decrease in dentate gyrus Increase in hippocampus
At least 12 h
Kang et al., 2001a
0.5–12 h 12–24 h
Kang et al., 2001c Kang et al., 2002a
Pyridoxal kinase
Decrease in hippocampus
0.5–3 h
Kang et al., 2002e
Succinic semialdehyde reductase
Increase immunoreactivity but not activity in hippocampus and entorhinal cortex
3h
Kang et al., 2003b
Succinic semialdehyde dehydrogenase
Increase immunoreactivity but not activity in hippocampus and entorhinal cortex
3h
Kang et al., 2003b
Neurotransmitter transporters GABA transporter (GAT)-1
Decrease in hippocampus
30 min
Kang et al., 2001b
GAT-3
No difference in hippocampus
0.5–12 h
Kang et al., 2001b
Vesicular GAT
Increase in hippocampus
3–12 h
Kang et al., 2003e
Increase in cortex and cerebellum Decrease in cortex Increase in cerebellum
1 sec–5 min 1–5 min 0.5–15 min
Wolf-Dieter et al., 1989 Wolf-Dieter et al., 1989 Wolf-Dieter et al., 1989
Increase in dentate gyrus Increase in CA1, CA2, and subiculum
0.5–12 h 12 h
Hwang et al., 2004a Hwang et al., 2004a
Decrease in CA1, less so in CA2 and CA3
1h
Scotti et al., 1997b
Decrease then increase in most regions in hippocampal formation
0.5 h then 3–24 h Kang et al., 2002b
Second messengers cAMP cGMP
Calcium binding proteins Calbindin Parvalbumin Ion transporters Na+-K+-Cl+ cotransporter Na+-K+ ATPase
Increase in hippocampus
3 and 12 h
Kang et al., 2004a
Na+/H+ exchanger1
Increase in hippocampus
0.5–3 h
Kang et al., 2002d
Na /HCO3 cotransporter
Increase in hippocampus
0.5–3 h
Kang et al., 2002d
Na+/Ca2+ exchanger
No change in hippocampus
0.5–3 h
Kang et al., 2002d
Increase in glia of entorhinal cortex and amygdala after kainateinduced seizure Increase in neurons in thalamus after kainate-induced seizure
3–24 h
Ferrer et al., 2000
48 h
Ferrer et al., 2000
+
-
Other Caspases
Fos
Increase in many brain areas
1.5 h
Mirzaeian and Ribak, 2000
Microtubule associated protein (MAP) 1A
Increase in some dentate gyrus neurons; decrease in hilar neurons and in CA1 and CA3
0.5–3 h
An et al., 2003c An et al., 2003c
MAP 2
First decrease then increase in hilar neurons
0.5–3 h
Passive avoidance test learning
Decrease
Immediate
Schonfeld and Glick, 1978
Extent of neuropathology produced by stroke
No effect
2–24 h
Herrmann et al., 2004
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ANTICONVULSANT DRUG SCREENING IN GERBILS Gerbils initially appeared to be an ideal model for screening anticonvulsant drugs. They are easy to handle, and propagate rapidly, and have naturally occurring epilepsy with seizures that can be triggered by the investigator. Seizure severity ranges from mild to severe in different individuals. Table 2
Mild and severe seizures in gerbils are blocked most effectively by different anticonvulsants, suggesting the possibility to differentiate between drugs that are clinically useful against absences and myoclonic seizures and drugs effective against generalized tonic-clonic seizures (Löscher, 1985a; Löscher and Frey, 1984). Gerbils were used extensively during the 1980s to test the anticonvulsant effects of many drugs (Tables 2 and 3).
Drug/Treatment Effects on Seizure Behavior in Epileptic Gerbils
Drug/treatment
Dose
Time
Effect
Reference
Adrenalectomy
—
—
Proconvulsant
Adrenocorticotropin hormone (ACTH)
1 mg i.c.v.
0
No effect
Lee et al., 2003 Bajorek et al., 1984
N-allylnormetazocine
0.1–1.0 mg/kg s.c.
20 min
Anticonvulsant
Lee et al., 1984a
Aminoadamantane
2 mg/kg i.p.
2h
No effect
Rausch et al., 1988
2-amino-5–phosphonopentanoate (AP5)
200 mg/kg i.p.
30 min
Mildly anticonvulsant
Löscher et al., 1988
2-amino-7–phosphonopentanoate (AP7)
50 mg/kg i.p. 100–200 mg/kg i.p.
30 min 30 min
No effect Anticonvulsant
Löscher et al., 1988 Löscher et al., 1988
Amphetamine
1–2 and 8 mg/kg i.p. 4 mg/kg i.p.
30 min 30 min
No effect Mildly anticonvulsant
Schonfeld and Glick, 1980 Schonfeld and Glick, 1980
Apomorphine
0.1–5.0 mg/kg s.c. 0.2 mg/kg i.p. 1–5 mg/kg i.p. 2–8 mg/kg i.p. 2–10 mg/kg i.p. 16 mg/kg i.p.
20 min 20 min 30 min 30 min 30 min 30 min
Anticonvulsant No effect Anticonvulsant No effect Anticonvulsant Anticonvulsant
Lee and Lomax, 1983a Cox and Lomax, 1976 Cox and Lomax, 1976 Schonfeld and Glick, 1980 Löscher and Czuczwar, 1986 Schonfeld and Glick, 1980
Arginine vasopressin
10–100 mg/kg s.c.
20 min
Anticonvulsant
Lee and Lomax, 1983b; Bajorek et al., 1984
BRL 43694 (5-HT3 receptor agonist)
1.5 mg–1 mg/kg/day orally
—
No effect
Cutler and Piper, 1990
Bromocryptine
1–10 mg/kg s.c.
30 min
Anticonvulsant
Lee and Lomax, 1983a
Carbamazepine
2–10 mg/kg i.p. 25 mg/kg orally
30 min 1h
No effect Anticonvulsant
Araki et al., 2002 Frey, 1987a
3-(2-carboxypiperaxin-4-yl)propyl-1-phosphonic acid (CPP)
5–10 mg/kg i.p.
30 min
No effect
Löscher et al., 1988
p-chloroamphetamine
3.5 mg/kg i.p.
48 h
No effect
Cox and Lomax, 1976
Clonazepam
0.5–1.0 mg/kg i.p. 2 mg/kg i.p.
30 min 30 min
No effect Anticonvulsant
Araki et al., 2002 Araki et al., 2002
Dexamethasone
2 mg/kg i.p.
2h
Anticonvulsant
Rausch et al., 1988
Diazepam
0.5 mg/kg orally 0.5–2.0 mg/kg i.p. 1 mg/kg i.p. 2 mg/kg/day orally
45 min 30 min 2h After 1 wk of treatment
Anticonvulsant Anticonvulsant Anticonvulsant Anticonvulsant
Frey, 1987b Araki et al., 2002 Rausch et al., 1988 Kavvadias et al., 2004
Diethyldithiocarbamate
500 mg/kg i.p.
4h
Anticonvulsant
Cox and Lomax, 1976
Diphenyl hydantoin sodium (dilantin)
2–20 mg/kg i.p. 7.5 mg/gerbil i.p. 25 mg/kg i.p.
30 min 1h 1h
No effect Anticonvulsant Anticonvulsant
25 mg/kg i.p. 30 mg/kg i.p. 75 mg/kg i.p. 150 mg/kg i.p.
1h 1h 1h 1h
No effect Mildly anticonvulsant No effect Anticonvulsant
Araki et al., 2002 Thiessen et al., 1968 Loskota and Lomax, 1974; Cox and Lomax, 1976 Schonfeld and Glick, 1980 Frey, 1987b Schonfeld and Glick, 1980 Schonfeld and Glick, 1980
Domperidone
2 mg/kg i.p.
2h
No effect
Rausch et al., 1988
L-DOPA/carbidopa
10/1 mg/kg i.p 25/2.5–100/10 mg/kg i.p
30 min 30 min
No effect Anticonvulsant
Löscher and Czuczwar, 1986 Löscher and Czuczwar, 1986 (continues)
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Anticonvulsant Drug Screening in Gerbils
TABLE 2 (continued) Drug/treatment
Dose
Time
Effect
Reference
b-endorphin
0.1–3.0 mg i.c.v.
0
Anticonvulsant
Met-enkephalin
100 mg i.c.v.
0
No effect
Bajorek and Lomax, 1982 Bajorek et al., 1984
Ethosuximide
50 mg/kg i.p. 100–200 mg/kg i.p. 125–250 mg/kg i.p. 500 mg/kg i.p.
30 min 30 min 1h 1h
No effect Anticonvulsant No effect Anticonvulsant
Araki et al., 2002 Araki et al., 2002 Schonfeld and Glick, 1980 Schonfeld and Glick, 1980
Gastropodin
60 mg/kg/day orally
After 1 wk of treatment
Anticonvulsant
An et al., 2003b
Haloperidol
0.5 and 2–4 mg/kg i.p. 1 mg/kg i.p.
30 min 30 min
No effect Mildly proconvulsant
Schonfeld and Glick, 1980 Schonfeld and Glick, 1980
Hispidulin (4¢,5¢7-trihydroxy-6-methoxyflavone)
10 mg/kg/day orally
After 1 wk of treatment
Anticonvulsant
Kavvadias et al., 2004
8-hydroxy-2-(di-n-propylamino)tetralin
0.5–1.0 mg/kg s.c.
30 min
No effect
Löscher and Czuczwar, 1985
5-hydroxytryptamine
50–100 mg/kg i.p.
30 min
No effect
Löscher and Czuczwar, 1985
Ketocyclazocine
0.15–0.50 mg/kg s.c.
20 min
Anticonvulsant
Lee et al., 1984a
Lidoflazine
1 mg/kg i.p.
2h
Anticonvulsant
Rausch et al., 1988
Lisuride
0.05–0.20 mg/kg i.p. 0.5 mg/kg i.p.
30 min 30 min
No effect Anticonvulsant
Löscher and Czuczwar, 1986 Löscher and Czuczwar, 1986
Melatonin
0.1–1.0 mg/kg i.p. 25 mg s.c./day
30 min After 13 wks of treatment
No effect Anticonvulsant
Champney et al., 1996 Champney and Champney, 1996; Champney et al., 1996
a-methylmetatyrosine
250 mg/kg i.p.
16 h
Anticonvulsant
Cox and Lomax, 1976
a-methyl-para-tyrosine
75–300 mg/kg
75 min
No effect
Schonfeld and Glick, 1980
Morphine
1–10 mg/kg s.c.
20 min
Anticonvulsant
Lee et al., 1984a
Muscimol
12.5–25.0 ng into thalamus
15 min
Anticonvulsant
Lee et al., 1989
Naloxone
1 mg/kg 2 mg/kg i.p. 2–10 mg/kg
20 min 2h 20 min
No effect Anticonvulsant Mildly proconvulsant
Wong and Gray-Allan, 1991 Rausch et al., 1988 Wong and Gray-Allan, 1991
Phenobarbital
7 mg/kg orally 10–80 mg/kg i.p. 20 mg/kg i.p.
2h 1h 1h
Anticonvulsant Anticonvulsant Anticonvulsant
Frey, 1987a Schonfeld and Glick, 1980 Loskota and Lomax, 1974; Cox and Lomax, 1976
Pentobarbital
20–40 mg/kg i.p.
1h
Anticonvulsant
Schonfeld and Glick, 1980
Pimozide
0.05–0.50 mg/kg
3h
No effect
Cox and Lomax, 1976
Pinealectomy
—
0.5–1 h
Proconvulsant
Philo and Reiter, 1978, 1981
(+)-4-propyl-9-hydroxynaphthoxazine (PHNO)
0.05–0.50 mg/kg i.p
30 min
Anticonvulsant
Löscher and Czuczwar, 1986
Reserpine
0.25 mg/gerbil i.p.
6h
Anticonvulsant
Thiessen et al., 1968
SKF 38393-A
2–20 mg/kg i.p.
30 min
No effect
Löscher and Czuczwar, 1986
D -tetrahydrocannabinol
20 mg/kg i.p. 50 mg/kg i.p.
2–3 h 2–3 h
No effect Anticonvulsant
ten Ham et al., 1975 ten Ham et al., 1975
DL-threodihydroxyphenylserine
50 mg/kg i.p.
1h
Anticonvulsant
Cox and Lomax, 1976
Thyrotopin-releasing hormone (TRH)
10 mg i.c.v
0
Proconvulsant
Bajorek et al., 1984
Trimethadione
150–600 mg/kg i.p.
1h
No effect
Schonfeld and Glick, 1980
Valproate
1 mg/kg i.p. 50–100 mg/kg i.p. 200 mg/kg i.p. 82–111 mg/kg orally
2h 30 min 30 min After 1 yr of treatment
Anticonvulsant No effect Anticonvulsant Proconvulsant
Rausch et al., 1988 Araki et al., 2002 Araki et al., 2002 Cutler and Horton, 1988
Vigabatrin
60 mg/kg/day orally
After 1 week of treatment
Anticonvulsant
Kang et al., 2003a
Zonisamide
12.5–50.0 mg/kg i.p.
1h
No effect
Araki et al., 2002
9
Table 3
ED50s for Complete Suppression of Environmentally Provoked Seizures in Epileptic Gerbils
Drug
ED50
Time
Abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-b-carboxylate) (ZK 112119) g-acetylenic GABA AHR-11784 Aminooxyacetic acid (±)-2-amino-7-phophonoheptanoic acid (APH) (±)-2-amino-5-phosphonopentanoci acid (APP) Apomorphine Atropine 5-benzyloxy-4-methoxymethyl-b-carboline-3-carboxylic acid ethyl ester (ZK 91 296) 6-benzyloxy-4-methoxymethyl-b-carboline-3-carboxylic acid ethyl ester (ZK 93 423) Biperiden Carbamazepine Cetyl g-aminobutryic acid
0.075 mg/kg i.p. 2.1 mg/kg i.p. 55.6 mg/kg orally 0.9 mg/kg i.p. 120 mg/kg i.p. >200 mg/kg i.p. ~5 mg/kg i.p. >10 mg/kg i.p. 0.45 mg/kg i.p. 0.14 mg/kg i.p. 12 mg/kg i.p. 11.8 mg/kg orally 4.5 mg/kg i.p.
0.5 h 4h 1h 6h 0.5 h 0.5 h 0.5 h 0.5 h 0.5 h 0.5 h 0.5 h 1h 1h
4-{[(4-chlorophenyl)(5-fluoro-2-hydroxyphenyl)methylene]amino}butyric acid (SL 75 102) 2-(2-chloro-5-trifluoromethylphenylimino)imida zolidine (St 587) (±)-cis-4-hyoxynipecotic acid methyl ester Clonazepam Clonidine
45 mg/kg i.p.
0.5 h
3.7 mg/kg i.p. 164 mg/kg i.p. 0.023 mg/kg i.p. 0.38 mg/kg i.p.
0.5 h 0.5 h 0.5 h 0.5 h
Diazepam N-[4,4-diphenyl-3-butenyl]-nipecotic acid L-DOPA plus carbidopa Ethanolamine-O-sulfate (EOS) Felbamate Fenfluramine Gabapentin Hydroxamic acid of GABA L-5-hydroxytryptophan (HTP) L-5-HTP plus carbidopa 5-isopropoxy-4-methyl-b-carboline-3-carboxylic acid ethyl ester (ZK 93 426) Ketanserin Lisuride Meprobamate Milacemide Muscimol (-)-nipecotic acid ethyl ester Phenobarbital Phenytoin Primidone Progabide
0.22 mg/kg i.p. 4.1 mg/kg i.p. 34 mg/kg i.p. 1 g/kg i.p. 63 mg/kg orally >10 mg/kg i.p. 15.1 mg/kg orally >100 mg/kg i.p. >200 mg/kg i.p. >100 mg/kg i.p. no effect >0.1 mg/kg >5 mg/kg s.c. 34 mg/kg orally >300 mg/kg i.p. 0.66 mg/kg i.p. 21 mg/kg i.p. 9.1 mg/kg i.p. 7.1 mg/kg orally 29.4 mg/kg orally 10–26 mg/kg orally 50 mg/kg i.p.
0.5 h 0.5 h 0.5 h 0.5 h 1h 0.5–2 h 1.5 h 0.25–0.50 h 0.5 h 0.5 h 0.5 h 0.5 h 0.5 h 1h 0.5–3.0 h 0.5 h 0.5 h 2h 2h 2h 0.5–18.0 h 0.5 h
2-propyl-2-pentenoic acid (2-en-VPA) Ralitoline threo-DOPS (24 h after 50 mg/kg of iproniazid) 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP)
90 mg/kg i.p. 10.2 mg/kg orally >200 mg/kg i.p. 1.34 mg/kg i.p.
0.5 h 0.25 h 1h 0.5 h
Valproic acid
73 mg/kg i.p.
0.5 h
g-vinyl GABA (vigabatrin) Xylazine Zonisamide
231 mg/kg orally 50 mg/kg i.p. ~10 mg/kg i.p. 112 mg/kg orally
0.5 h 6h 0.5 h 2h
Reference Turski et al., 1990 Löscher et al., 1983 Bartoszyk and Hamer, 1987 Löscher et al., 1983 Löscher, 1985b Löscher, 1985b Löscher, 1985b Löscher, 1985b Löscher et al., 1985 Löscher et al., 1985 Löscher, 1985b Bartoszyk and Hamer, 1987 Löscher et al., 1983; Löscher, 1985c Löscher, 1985b Löscher and Czuczwar, 1987 Löscher et al., 1983 Löscher, 1985c Löscher, 1985b; Löscher and Czuczwar, 1987 Löscher, 1985c Löscher, 1985c Löscher, 1985b Löscher, 1985b Frey and Bartels, 1997 Löscher, 1985b Bartoszyk and Hamer, 1987 Löscher and Frey, 1984 Löscher, 1985b Löscher, 1985b Löscher et al., 1985 Löscher, 1985b Löscher, 1985b Frey and Bartels, 1997 Löscher, 1985b Löscher, 1985b Löscher et al., 1983 Löscher, 1985c Bartoszyk and Hamer, 1987 Bartoszyk and Hamer, 1987 Frey et al., 1984 Löscher et al., 1983; Löscher, 1985c Löscher et al., 1984 Bartoszyk and Hamer, 1987 Löscher, 1985b Löscher et al., 1983; Löscher, 1985c Löscher et al., 1983; Löscher et al., 1984 Bartoszyk and Hamer, 1987 Löscher and Frey, 1987a Löscher, 1985b Bartoszyk and Hamer, 1987
What Causes Epilepsy in Gerbils?
However, increased experimental use revealed peculiarities and drawbacks of the model, and investigators began to question the usefulness of gerbils for anticonvulsant screening (Schonfeld and Glick, 1980; Majkowski and Kaplan, 1983; Bajorek et al., 1984; Bertorelli et al., 1995). Compared to other rodent models, gerbils are more sensitive to the anticonvulsant effects of drugs that enhance GABA and dopamine, and they are less sensitive to drugs that block excitatory amino acid neurotransmission and increase serotonin levels (Löscher, 1985b). These findings suggest that the anticonvulsant drug sensitivities of gerbils may not represent those of patients. In addition, experiments with gerbils are time consuming, because their seizure refractory period only permits testing once a week (Löscher and Frey, 1984). The half-lives of most anticonvulsants are very short in gerbils, and their rapid elimination only allows for the evaluation of the acute potency (Löscher and Frey, 1984). In some cases when it has been possible to administer drugs chronically other problems arose. Adult gerbils develop tolerance to chronically administered anticonvulsants (Löscher, 1986; Löscher and Frey, 1987a), and chronic treatment in young gerbils can permanently modify seizure threshold. For example, treatment with phenobarbital starting at 1 month old and continuing for 4 months paradoxically intensifies seizure activity even during a 7-month period following treatment (Watanabe et al., 1978; Schain and Watanabe, 1982). Drug administration is a major problem when working with gerbils, because handling can trigger seizures, making them refractory at the time of testing. Consequently, even vehicle treatments inhibit seizure activity when compared to pretreatment baselines (Schonfeld and Glick, 1980). This problem has been dealt with by using outbred gerbils with a higher threshold for handling-induced seizures (Löscher and Frey, 1984), by adapting animals to the stress of oral drug application by repeated exposure (Bartoszyk and Hamer, 1987), and by adding drugs to drinking water (Cutler and Horton, 1988; Cutler and Piper, 1990) or using surgically implanted osmotic minipumps (Löscher, 1986). However, not all drugs are stable at body temperature or soluble in aqueous solutions at high concentration. Some studies used nitrous oxide or halothane to anesthetize gerbils in their cage and then quickly remove them for weighing and drug administration (Loskota and Lomax, 1974; Wong and Gray-Allan, 1991). However, even brief anesthesia might affect seizure threshold. Most importantly, the underlying causes of epilepsy and the seizure-triggering mechanisms are unknown, which makes the relationship of gerbil epilepsy to human epilepsy unclear.
WHAT CAUSES EPILEPSY IN GERBILS? The value of gerbils as a model of human epilepsy would be clarified if the mechanisms of their seizure disorder were
283
better understood. Several strategies have been used to investigate the etiology of epilepsy in gerbils. Some studies have focused on species-specific characteristics of Meriones unguiculatus that might explain the propensity for developing epilepsy. Others have used genetic analyses to compare epileptic and control gerbils. Another approach has been to compare the brains of seizure-sensitive and seizure-resistant gerbils to identify factors that correlate with seizure susceptibility. And lesion studies suggest that an epileptogenic focus probably does not exist in the corpus striatum (Schonfeld and Glick, 1981), substantia nigra pars compacta (Van Ness et al., 1989), or CA1 field (Winkler et al., 2001). As reviewed below, many hypotheses have been proposed, but none yet satisfactorily and comprehensively accounts for epileptogenesis in gerbils. Species-specific characteristics. There might be speciesspecific characteristics that predispose gerbils to epilepsy. It has been suggested that a nutritional deficiency (Robinson, 1968) or an irregularity in the metabolism of magnesium causes seizures (Harriman, 1974, 1978, 1980, 1988). But feeding supplemented rodent food does not reduce seizure susceptibility (Zeman, 1967). Gerbils are unique among small animals in that approximately one-third develop cerebral ischemic lesions following only unilateral carotid ligation (Levine and Payan, 1966; Berry et al., 1975). This makes them useful for stroke research and offers opportunities for certain types of epilepsy experiments (Payan, 1970). It has been proposed that the vascular anomaly that distinguishes stroke-sensitive from stroke-resistant gerbils also distinguishes seizure-sensitive from seizure-resistant gerbils (Schonfeld and Glick, 1979). However, neither the vascular pattern nor the frequency of occurrence of stroke following unilateral carotid ligation is related to seizure propensity (Donadio et al., 1982). Immunocytochemical evaluations of the gerbil hippocampus have revealed species-specific characteristics that might be related to seizure sensitivity. It has been suggested that the expression of the calcium-binding protein calretinin makes mossy cells in the hilus of the dentate gyrus resistant to excitotoxicity by kainic acid (Kotti et al., 1996). However, calretinin’s neuroprotective role is questionable, because in addition to mossy cells many other types of neurons in limbic structures are more resistant to kainate-induced excitotoxicity in gerbils compared to rats (Ferrer et al., 2000; Marti et al., 2002). The neuroprotective mechanism instead may be related to species-specific differences in kainate receptors (Kataoka et al., 1993) or protective effects of prior seizures (Kelly and McIntyre, 1994; Zhang et al., 2002) in epileptic gerbils. Another immunocytochemical oddity of gerbils is the expression of parvalbumin in perforant path fibers, which project from layer II of the entorhinal cortex to the molecular layer of the dentate gyrus. Unlike other species that have been examined, layer II neurons in the entorhinal cortex of gerbils are parvalbumin-immunoreactive (Scotti et
284
Chapter 21/Inherited Epilepsy in Mongolian Gerbils
al., 1997a; Bender et al., 2000). The axons of layer II entorhinal cortical neurons concentrate in the molecular layer where they form a parvalbumin-positive band, which is especially evident in the middle third of the molecular layer. The presence of parvalbumin in these glutamatergic axons in gerbils has been used to investigate its role in neurotransmitter release (Scotti et al., 1993). It has been suggested that expression of the calcium-binding protein affects the physiology of this major afferent projection to the dentate area and could be responsible for increased excitability and seizures (Scotti and Nitsch, 1991). However, parvalbumin staining in the molecular layer is similar in seizure-sensitive and seizure-resistant gerbils (SetoOhshima et al., 1994; Buckmaster et al., 1996). In addition to perforant path fibers in the molecular layer, a class of interneurons is parvalbumin-positive in the hippocampal formation. Seizure activity in gerbils reduces the expression of parvalbumin-immunoreactivity in the somata and dendrites (but not axons) of interneurons in CA1, to a smaller extent in CA3, but not in the dentate gyrus (Scotti et al., 1997b). The maximal effect occurs 1 hour postseizure, and GAD staining shows that the loss of parvalbumin staining is not attributable to cell death, because the total number of interneurons is stable (Scotti et al., 1997b). Lesioning by ischemia reveals that the presence of CA1 pyramidal cells is necessary for seizure-induced reduction of parvalbumin-immunoreactivity in interneurons (Winkler et al., 2001). A similar seizure-induced reduction of parvalbumin-immunoreactivity may occur in the dentate gyrus and proximal CA3 region in patients with temporal lobe epilepsy (Sloviter et al., 1991; Wittner et al., 2001; Zhu et al., 1997). It has been suggested that reduced parvalbumin expression in interneurons may affect their spiking characteristics and reduce their inhibitory control of pyramidal cells (Scotti et al., 1997b), but this prediction remains to be tested. Gerbils also show evidence of species-specific degenerative changes in the brain. As gerbils age, lipofuscin granules accumulate in neurons, but their presence does not correlate with seizure-sensitivity (Seto-Ohshima et al., 1999). Older gerbils develop spongiform degeneration of the cochlear nucleus (Faddis and McGinn, 1993), but it also occurs in the nonepileptic species Meriones libycus (Ostapoff and Morest, 1989). Purkinje cells develop dystrophic axon terminals with age, but this occurs in both seizure-sensitive and seizure-resistant gerbils (Takeuchi et al., 1997). It has been reported that Purkinje cells are vulnerable to seizure activity in gerbils (Dam et al., 1984). However, the differences in Purkinje cell densities may be attributable to age differences between the epileptic and nonepileptic groups used in the study and the age-dependent development of the aforementioned progressive degenerative disorder involving dystrophic axons. Chronic treatment with the anticonvulsant phenytoin has been proposed to kill Purkinje cells. Consistent with this hypothesis,
phenytoin is concentrated in the cerebellum of gerbils after systemic administration (Navarro-Ruiz et al., 1982). Genetic studies. The genetic diversity of laboratory gerbils is below that observed in inbred mouse or rat strains (Neumann et al., 2001; Razzoli et al., 2003), which is not surprising considering the severe population bottleneck that occurred when they were domesticated. Low genetic diversity hampers identification of disease-associated genes, and the gerbil genome is poorly characterized compared to that of the mouse and rat. Nevertheless, several genetic studies have been conducted on epilepsy in gerbils. Familial transmission of epilepsy in domestic gerbils has been clear from the start (Thiessen et al., 1968; Kaplan, 1981), and seizure sensitivity is inherited equally by both sexes (Loskota et al., 1974; Harriman, 1978; Cheal, 1987a; Cutler and MacKintosh, 1989; Seto-Ohshima et al., 1992). Seizure susceptibility correlates with coat color, of which there are three types: wild-type agouti, black, and sandy (“albino”). Epilepsy is less common and less severe in sandy gerbils (Robbins, 1976) that are homozygous-recessive at the pink-eyed dilution locus (Gray-Allan and Wong, 1990; Fujisawa et al., 2003). It is unclear whether the relationship between seizure sensitivity and the pink-eyed dilution gene is direct (the gene that determines coat color also regulates seizure activity) or indirect (the pink-eyed dilution gene is associated or linked with a gene controlling seizures). Epileptic and control gerbils have been crossed to evaluate the heritability of seizure sensitivity. Gerbils derived from the seizure-sensitive WJL/UC strain were crossed with descendents of nonepileptic, nondomestic gerbils (kindly provided by Drs. Weinandy and Gatterman, Martin Luther University, Halle, Germany) (Buckmaster, 2004). Six pairs of seizure-sensitive X control parents produced a total of 210 F1 offspring that were tested with 4 weekly novel environment exposures starting at 2 months old, and only 10% (5 males and 16 females) displayed seizure activity. Three pairs of nonepileptic F1 gerbils were crossed to produce F2 offspring, of which only 1 of 59 displayed a seizure. In another study 91% of the F1 offspring of the seizure-sensitive MGS/Idr X seizure-resistant MGR strains displayed seizures, but the seizures were milder and slightly delayed in age at onset (Seto-Ohshima et al., 2003). It is unclear what accounts for the large difference in the proportion of F1 gerbils that displayed seizures in these two studies, but duration and type of seizure testing and genetic contamination of the seizure-resistant strain are possibilities. Nevertheless, the results indicate that seizure-susceptibility in gerbils is not inherited as a simple, Mendelian single-locus trait. Comparisons of seizure-sensitive and seizure-resistant gerbils. Gerbils provide an opportunity for studying mechanisms of epileptogenesis, because seizure-sensitive and seizure-resistant animals can be compared to detect differences that correlate with seizure behavior. Many differences
What Causes Epilepsy in Gerbils?
have been reported between seizure-sensitive and seizureresistant gerbils, and they include changes in neurotransmitters, metabolism, anatomy, electrophysiology, and behavior (Table 4). In most studies that have compared seizure-sensitive with seizure-resistant gerbils, however, it is unclear whether the differences are a cause or an effect of seizures. This problem of comparing epileptic with control groups is encountered frequently in epilepsy research and is not unique to studies that have used the gerbil model. However, gerbils offer a solution, because seizures do not develop until they are more than one month old, so they can be evaluated before seizures begin. Studies that have identified abnormal parameters in pre-epileptic gerbils have generated the most compelling hypotheses. All of the abnormalities reported for pre-epileptic gerbils involve GABAergic synaptic transmission, which is consistent with the unusually high anticonvulsant potency of GABAmimetic drugs in gerbils (Löscher et al., 1983; Löscher and Frey, 1984). One hypothesis holds that a defect in neocortical inhibition underlies epilepsy in gerbils. In epileptic gerbils GAD activity is reduced in the frontal cortex but not in other brain regions (Löscher, 1987). In epileptic (Kato et al., 2000) and pre-epileptic gerbils (SetoOhshima et al., 2003), evoked responses in the barrel field of the somatosensory cortex appear to lack a component of the waveform that normally may be generated by inhibitory postsynaptic potentials. Electrical stimulation or focal application of bicuculline to the same neocortical area elicits behavioral and EEG responses like those that occur at the onset of environmentally induced seizures (Seto-Ohshima et al., 2001). Comparing seizure-sensitive with seizureresistant gerbils using two-dimensional gel electrophoresis of proteins from the cerebral cortex reveals differences in mitofilin, a mitochondrial inner membrane protein, caused at least in part by a base amino acid substitution near a putative transmembrane domain (Omori et al., 2002). Together these findings support the hypothesis that a mutation in mitofilin impairs GABAergic inhibition and reduces seizure threshold in the somatosensory cortex of epileptic gerbils. But many aspects of the hypothesis, for example how mitofilin affects cortical evoked potentials, remain to be clarified. Another hypothesis proposes a deficit of GABAergic transmission in the substantia nigra pars reticularis, a region that normally plays a role in controlling seizure activity (Iadorala and Gale, 1982; McNamara et al., 1984). GABAA receptors in gerbils are similar to those in rat and human brain (Asano and Mizutani, 1980), but compared to controls, epileptic and pre-epileptic gerbils have fewer GABAA receptor binding sites in the substantia nigra pars reticularis (Olsen et al., 1984, 1985, 1986). The deficit probably is attributable to fewer receptors expressed per neuron, since neuron numbers in this region appear to be similar in control and epileptic gerbils (Peterson and Ribak, 1987). However,
285
GABAergic transmission has not been evaluated in the substantia nigra pars reticularis of gerbils, and the mechanisms underlying reduced receptor expression are unclear. The “disinhibition” hypothesis contends that seizures initiate in the hippocampus. Temporal lobe epilepsy is the most common type of epilepsy associated with the hippocampus. However, epileptic gerbils do not display CA1 pyramidal cell loss (Mouritzen Dam et al., 1981), hilar neuron loss (Buckmaster et al., 1996), or granule cell axon sprouting (Peterson and Ribak, 1987; Ribak and Peterson, 1991) like patients with temporal lobe epilepsy. Gerbils with the most severe seizure susceptibility have fewer CA3 pyramidal cells (Mouritzen Dam et al., 1981) with fewer dendritic spines (Paul et al., 1981). And granule cell axon terminals in epileptic gerbils display evidence of accelerated synaptic vesicle recycling (Peterson et al., 1985), which can be rescued by transecting the perforant path (Farias et al., 1992). However, all of these changes could be effects of seizures rather than causes. Electrographic seizures are recorded in the hippocampus (Loskota and Lomax, 1975; Majkowski and Donadio, 1984; Buckmaster and Wong, 1992), and Fos-immunoreactivity increases after seizures (Mirzaeian and Ribak, 2000), but these findings do not prove that the hippocampus is the seizure initiation site. The hippocampus plays an important role in spatial navigation, and the seizure-triggering effectiveness of novel environment exposure is intriguing in this regard (Ludvig et al., 1991). Lesioning CA1 pyramidal cells reduces seizure severity in some gerbils (Winkler et al., 2001), and lesioning the perforant path eliminates seizures (Ribak and Kahn, 1987), but extrahippocampal structures also were damaged in those studies. Nevertheless, on the whole there is support for the hypothesis that seizures begin in the hippocampus, and the disinhibition hypothesis proposes a mechanism. Within the hippocampal dentate gyrus of gerbils interneurons have far-reaching and dense axon arbors that could affect the activity of many neighboring neurons (Buckmaster and Schwartzkroin, 1995; Buckmaster et al., 2002). The disinhibition hypothesis proposes that defects in the number and connectivity of interneurons in the dentate gyrus are a critical epileptogenic factor. The hypothesis was prompted by the paradoxical finding of more GAD-immunoreactive interneurons in the dentate gyrus and CA3 field of seizuresensitive gerbils compared with controls (Peterson et al., 1985). The differences were most significant in the septal or dorsal hippocampus, and no differences were found in other brain regions, suggesting a specific defect in the septal hippocampus (Peterson and Ribak, 1987). Alone, an excess of interneurons might be expected to increase the level of inhibition. However, seizure-sensitive gerbils also displayed more GAD-immunoreactive axon terminals that appeared to selectively target interneurons (Farias et al., 1992), forming a circuit in which inhibitory interneurons inhibit other
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Chapter 21/Inherited Epilepsy in Mongolian Gerbils
Table 4
Comparisons of Seizure-Sensitive and Seizure-Resistant Adult Gerbils (No Immediately Preceding Seizure Specified)
Parameter Neurotransmitter/neuromodulator
Effect
Reference
Aspartate, glutamate, glutamine, glycine, citrulline, arginine, taurine, and cysteine
No difference in neocortex or in brainstem
Gardiner et al., 1993
GABA
Reduced in right neocortex; no difference in left or in brainstem
Gardiner et al., 1993
Dynorphin
Increased in hippocampus, not in other brain regions
Lee et al., 1987
Neuropeptide Y (NPY)
Decreased in dentate gyrus, subiculum, and entorhinal cortex
Kang et al., 2000b
Prostanoids
Decreased total amount of cycylo-oxygenase products, and significantly less PGD2, PGE2, and 6-keto-PGF1a
Seregi et al, 1984
No difference in hippocampus
An et al., 2003a
Neurotransmitter receptors Corticotropin-releasing factor (CRF) GABAA a1 and a2 subunits
Decreased in Ammon’s horn; increase in dentate gyrus
Hwang et al., 2004b
GABAA a5 subunit
Increased in Ammon’s horn; decrease in dentate gyrus
Hwang et al., 2004b
GABAA g2 subunit
Decreased in Ammon’s horn; no difference in dentate gyrus
Hwang et al., 2004b
GABAA a3, a4, pan b, and d subunits
No differences in hippocampus
Hwang et al., 2004b
GABAB
No difference in hippocampus
Park et al. 2004; Hwang et al., 2004b
Opioid binding
Increased
Lee et al., 1984b, 1986
P2X2 and P2X4
Decreased in hippocampus
Kang et al., 2003c
P2X7
No difference in hippocampus
Kang et al., 2004b
Somatostatin (SST) 2A
Decreased in hippocampus
Kang et al., 2003d
SST 2B, 3, 4, and 5
No difference in hippocampus
Kang et al., 2003d
Neurotransmitter synthesizing/degrading enzyme/cofactors GABA-transaminase No difference in hippocampus
Kang et al., 2001c
Glutamine synthetase activity
Increased in right cerebral hemisphere
Laming et al., 1989b
Pyridoxal kinase
Increased in hippocampus
Kang et al., 2002e
Pyridoxine-5¢-phosphate oxidase
Increased in hippocampus
Kang et al., 2002a
Succinic semialdehyde dehydrogenase
Immunoreactivity but not activity increased in hippocampus; decreased in entorhinal cortex Immunoreactivity but not activity increased in hippocampus; decreased in entorhinal cortex
Kang et al., 2003b
Succinic semialdehyde reductase
Kang et al., 2003b
Neurotransmitter transporter GABA transporter (GAT)-1
Increased in hippocampus
Kang et al., 2001b
vesicular GAT
Decreased in hippocampus
Kang et al., 2003e
Liver microsomal P-450 enzymes Debrisoquine 4 monooxygenase
Increased
Iwahashi et al., 1994
No difference
Iwahashi et al., 1994
Decreased in Purkinje cells Decreased in hippocampus
Kang et al., 2002c Hwang et al., 2004a
Decreased in Purkinje cells
Kang et al., 2002c
Decreased in hippocampus Increased in CA2-3 field, subiculum, and entorhinal cortex
Kang et al., 2004a Kang et al., 2002b
p-nitroanisole, O-demethylase, aminopyrine demethylase, and benzo[a]pyrene-3monooxygenase Calcium binding proteins Calbindin Parvalbumin Ion transporters Na+-K+ ATPase Na+-K+-Cl- cotransporter
(continues)
287
What Causes Epilepsy in Gerbils?
TABLE 4 (continued) Parameter Neurotransmitter/neuromodulator
Effect
Reference
Electrophysiology Cortical slow potential shifts
Larger amplitude
Roughan and Laming, 1998a,b
Cortical somatosensory evoked potentials
Different waveform in barrel cortex
Kato et al., 2000
Cortical visual evoked potentials
Increased amplitude and shorter latency
Roughan and Laming, 1998a
Effect of 0 magnesium and hypoglycemia on excitability of CA1 in hippocampal slices
Decreased compared to rats
Stittsworth and Lanthorn, 1994
Excitability of CA1 in hippocampal slices
Increased compared to rats
Stittsworth and Lanthorn, 1994
Paired-pulse (PP) responses in the dentate gyrus
Increased PP depression at 30 msec interval; increased PP facilitation at 70 ms interval
Buckmaster and Schwartzkroin, 1994; Buckmaster et al., 1996
Stimulus threshold for maximal dentate activation
No significant difference
Buckmaster et al., 1996
Behavior Behavior in a resident-intruder paradigm
Less social and sexual investigation; no other differences
Cutler and MacKintosh, 1989
Level of arousal
Increased
Laming et al., 1989a
Other Microtubule associated protein (MAP) 1A
Increased in dentate gyrus, CA1, and CA3
An et al., 2003a
MAP 2
Increased in dentate gyrus and CA1
An et al., 2003a
Neurofilaments (NF150, NF200, RT97)
Decreased in Purkinje cells
Kang et al., 2002c
interneurons thereby disinhibiting (exciting) granule cells (Peterson and Ribak, 1989). The disinhibitory mechanism proposed for epileptic gerbils might also contribute to seizure genesis in temporal lobe epilepsy, and gerbils have been proposed as a model of limbic epilepsy (Scotti et al., 1997b). In the hippocampus of patients with temporal lobe epilepsy many excitatory neurons are missing, but GABAergic interneurons have been reported to be relatively spared, and they appear to sprout axon collaterals and form new inhibitory synapses (Babb et al., 1989). Despite the apparent sparing of interneurons, granule cells are less inhibited in patients with hippocampal neuron loss compared with controls (Williamson et al., 1999). Together these findings suggest that granule cells may be disinhibited because GABAergic interneurons are hyperinnervated by other GABAergic interneurons. However, there is increasing evidence of a significant loss of GABAergic interneurons in the hippocampus of patients (de Lanerolle et al., 1989; Sloviter et al., 1991; Mathern et al., 1995; Zhu et al., 1997; Maglóczky et al., 2000; Wittner et al., 2001), and compelling evidence of GABAergic axon sprouting and selective synaptogenesis with interneuron targets is lacking. The differences between seizure-sensitive and seizureresistant gerbils in GAD-immunoreactive interneuron numbers were thought not to be attributable to seizureinduced increased expression of GAD, because pre-epileptic gerbils also had more GAD-immunoreactive neurons in
the septal part of the dentate gyrus than controls (Peterson et al., 1985). However, the difference between pre-epileptic gerbils and controls was small, there was no difference in the total number of GAD-immunoreactive cells in the dentate gyrus, and no difference in CA3 (Peterson and Ribak, 1987). Furthermore, in epileptic gerbils the number of GAD-positive neurons was proportional to seizure severity (Peterson and Ribak, 1985), suggesting that seizure activity might have increased GAD expression. Previous studies have shown that seizure activity increases the expression of GAD (Feldblum et al., 1990; Schwarzer and Sperk, 1995; Esclapez and Houser, 1999), including in the hippocampus of epileptic gerbils (Kang et al., 2001a). Therefore, seizure activity might bring borderline GAD-positive cells beyond the threshold for immunocytochemical detection in epileptic but not control gerbils. Other studies compared seizuresensitive, seizure-resistant, and pre-epileptic gerbils using a variety of interneuron markers. Those studies found no seizure-related differences in numbers of GAD- (Scotti et al., 1997a), parvalbumin- (Scotti et al., 1997b), somatostatin- (Buckmaster et al., 1996), or GABA-immunoreactive neurons or in numbers of neurons labeled with in situ hybridization for GAD (Buckmaster et al., 2000). Differences in results might be attributable to the use of more sensitive interneuron labeling methods (Szabat et al., 1992; Obenaus et al., 1993; Houser and Esclapez, 1994) and modern stereological techniques (West et al., 1991).
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Hence, most studies suggest that epileptic and control gerbils have similar numbers of GABAergic interneurons in the hippocampus. However, that does not exclude the possibility that epileptic gerbils might have an abnormally exaggerated disinhibitory circuit. Inhibitory circuits in the dentate gyrus of epileptic gerbils have been evaluated electrophysiologically. Compared to controls, chronically epileptic adult gerbils and juvenile gerbils that have just begun having seizures both exhibit increased paired-pulse facilitation of dentate gyrus field potential responses to perforant path stimulation at a 70 ms interstimulus interval (Buckmaster and Schwartzkroin, 1994), which is consistent with a disinhibitory mechanism. However, the most dramatic difference is increased paired-pulse inhibition at shorter interstimulus intervals in epileptic gerbils (Buckmaster et al., 1996), which is difficult to reconcile with a simple disinhibitory mechanism. Other models of epilepsy also display increased paired-pulse inhibition in the dentate gyrus (Tuff et al., 1983; Milgram et al., 1991; Buckmaster and Dudek, 1997), suggesting this might be a side effect of seizures. Intracellular recording provides a more direct measure of granule cell inhibition. The conductances of fast (GABAA receptor mediated) or slow (GABAB receptor mediated) evoked inhibitory postsynaptic potentials in granule cells recorded in vivo with dentate gyrus circuitry intact are similar in seizure-sensitive and seizure-resistant gerbils (Buckmaster et al., 2000). However, inhibition is dynamic and an imbalance might appear only in the moments preceding a seizure. The hypothesis contends that seizures begin when granule cells in the septal dentate gyrus are disinhibited. However, in this region paired-pulse inhibition of granule cells and other measures of excitability (fEPSP slope and population spike amplitude) are maintained at baseline values through the onset of seizures. Only after a seizure continues for more than 5 seconds do signs of reduced inhibition begin to appear (Buckmaster et al., 2000, Buckmaster and Wong, 2002). These findings suggest that granule cell inhibition is not reduced either between seizures or at seizure onset. Hence, the epileptogenic role of disinhibition in the dentate gyrus of epileptic gerbils is questionable.
WHAT TYPE OF HUMAN EPILEPSY DO EPILEPTIC GERBILS MODEL? Seizures and epilepsy in gerbils do not easily fall within the diagnostic scheme of the International League Against Epilepsy (Engel, 2001). In gerbils the onset of motor seizures appears to be focal, because discrete body parts are involved. Seizures sometimes begin asymmetrically, for example, with unilateral clonus of an ear. However, most seizures begin symmetrically, suggesting a generalized onset. Epilepsy in gerbils could be classified as a reflex
epilepsy, but not if spontaneous seizures occur. Spontaneous seizures have been observed occasionally (Loskota et al., 1974; Buckmaster, 2004), but in those cases undetected precipitating sensory stimuli cannot be excluded with certainty. Epilepsy in gerbils does not appear to be a simple reflex epilepsy (Binnie, 1997), because the precipitating stimuli are more complex than flashing light, visual patterns, touch, pain, sound, or startle (Ludvig et al., 1991). Seizures in complex reflex epilepsies are triggered by more elaborate stimuli that involve integrative, higher, cortical function (Zifkin and Andermann, 1997). In gerbils novel environment exposure is an effective trigger, and it could evoke cognition of spatial relationships within the environment, an activity involving the hippocampal formation. However, air blowing is a very different type of stimulus, yet it also is effective at triggering seizures in gerbils. To the author’s knowledge there is no human reflex epilepsy with seizures precipitated by novel environment exposure or air blowing. Hence, it is unclear whether gerbils have complex reflex epilepsy. If not, then perhaps they have an idiopathic epilepsy syndrome. Inheritance, age dependence, and lack of structural brain lesions or other neurologic signs or symptoms are all features that match human idiopathic epilepsy. Although the direct relevance of epilepsy in gerbils to human epileptic syndromes is unclear, epileptic gerbils offer many opportunities to increase understanding of human epilepsy. Revealing the mechanisms that control the agedependent expression of epilepsy in gerbils may help to explain how some inherited human epilepsies appear and fade with development. Unraveling the seizure-triggering mechanisms in gerbils may provide insight into the neurophysiologic events that initiate seizures in patients. And identifying the underlying mutation(s) of epilepsy in gerbils may lead to the discovery of novel epilepsy-causing mutations in humans. Hence, Mongolian gerbils are likely to continue to be a useful model for epilepsy research in the future.
Acknowledgments The author’s work is supported by NIH/NINDS. The author is grateful to Jennifer Chung for assistance with data analysis.
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niques. Depending on the experimental protocol, it induces a three- or four-layered cortex (microgyrus), focal heterotopia in layer I, or a cortical cleft (schizencephaly). This model has been regularly studied in the rat and to some extent in the mouse. In vitro electrophysiologic studies have demonstrated that the epileptogenic region does not correlate with the structural lesion and that a small region surrounding the microgyrus generates the epileptiform activity (Jacobs et al., 1996; Luhmann and Raabe, 1996; Luhmann et al., 1998b; Jacobs et al., 1999a; Scantlebury et al., 2004). Epileptiform activity originating from this hyperexcitable region propagates over large distances into neighboring cortical areas with a normal histologic appearance but significant alterations in glutamatergic and GABAergic receptors (Zilles et al., 1998; Redecker et al., 2000). The lack of a correlation between the structural and functional lesion and the widespread pathophysiologic alterations in cortical regions surrounding the lesion replicate the clinical features of cortical dysplasia associated with neuronal migration disorders (Palmini et al., 1991a; Palmini et al., 1995).
GENERAL DESCRIPTION OF THE MODEL The neocortical freeze lesion model in the newborn rodent as initially described by Dvorak and Feit (Dvorak and Feit, 1977) and Dvorak et al. (Dvorak et al., 1978) reproduces the pathology of certain types of human neuronal migration disorders (polymicrogyria, focal heterotopia, cortical dysplasia, schizencephaly). Neuronal migration disorders are structural malformations in the cerebellum, hippocampus, or cerebral cortex, which result from early developmental disturbances or genetic defects in the migration of newly generated neurons to their target region (Aicardi, 1994; Kuzniecky and Barkovich, 1996; Feng and Walsh, 2001; Leventer et al., 2001; Chan et al., 2002; Guerrini et al., 2003; Palmini et al., 1994a). In the cerebral cortex, neuronal migration disorders are often associated with pharmaco-resistant epilepsy (Palmini et al., 1991b; Guerrini et al., 2003) and a therapeutical success might be only obtained after surgical removal of the structural abnormality (Mathern et al., 1999; Wyllie et al., 1998; Kral et al., 2003; Cohen-Gadol et al., 2004). With the recent improvement of imaging techniques it became clear that neuronal migration disorders are more common than previously estimated (Kuzniecky, 1994; Janszky et al., 2003; Bernasconi, 2003) and that a large population of patients suffering from pharmaco-resistant epilepsy show obvious cortical neuronal migration disorders (Bast et al., 2004; Guerrini et al., 2003). The cortical freeze lesion model can be easily developed, generates reproducible pathologic results and has been well studied over the last decade by various groups using in vitro electrophysiologic, immunohistochemical, receptor autoradiographic, molecularbiologic and neuroanatomic tech-
Models of Seizures and Epilepsy
WHAT DOES IT MODEL? So far spontaneous epileptiform activity in vivo and pathophysiologic electrographic features in the EEG have not been described in the cortical freeze lesion model. This model has been studied in vivo only in combination with hyperthermia-induced seizures (Scantlebury et al., 2004). Therefore, a classification of this model on the basis of epileptic seizure types or epileptic syndromes is currently not possible. The freeze lesion induced pathologic alterations in the cerebral cortex replicate “malformations due to
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abnormal cortical developments” (see Table 6 in Engel, Jr., 2001) and resemble the pathology of polymicrogyria, focal heterotopia, focal or multifocal cortical dysplasia, MillerDieker syndrome, or schizencephalies described in humans.
METHODS OF GENERATION Animal Issues The neocortical freeze lesion model has been used predominantly in newborn rats of various strains (see References). Comparable results could be also obtained with newborn mice (Supèr et al., 1997; Rosen et al., 1995). Other small mammals (e.g., the hamster) are probably also suited; however, prominent differences in cortical development during the early postnatal period have to be considered (e.g., hamsters are born more immature compared to rats and mice). Guinea pigs are inappropriate, since they are born relatively mature. The age of the animal is very critical. Using the identical lesion protocol, very different cortical pathologies can be obtained. In adult mice and rats, the cortical freeze lesion model has been used as a model for traumatic brain injury or brain edema (Stoffel et al., 2001; Murakami et al., 1999; Chan et al., 1987; Pedley et al., 1976). Cortical migration disorders with a pathology described in the following text (cortical dysplasia, three- or four-layered cerebral cortex, microgyria, heterotopia in layer I) can only be obtained when the lesion is induced during the period of neuronal migration in the neocortex (in rats and mice approximately up to postnatal day 4). The best reproducible results can be obtained when the lesion is induced during the first 24 hours after birth. However, other factors (e.g., number of newborns per litter, variability in developmental status at birth) may have some influence on the outcome. Sex differences in this model have been reported in rats by Glenn Morris and co-workers. Neonatal cortical freeze lesions caused a defect in fast auditory processing (Herman et al., 1997) and a shift toward greater numbers of small neurons in the medial geniculate nucleus (Rosen et al., 1999) in adult male, but not female, rats. For that reason and to avoid cyclic hormonal influences in adult females, newborn male animals should be preferred.
Procedures The methods described below are based on a model initially described by Dvorak and Feit (1977) and are most appropriate to induce a focal microgyrus in newborn (<24 hours after birth) rats (Figure 1). For other species, experimental procedures have to be modified. (1) Anaesthesia by hypothermia: place newborn rats for 5–10 min in normal ice.
(2) Remove animals from ice box and carefully cut the skin overlying the cortical area of interest with a small scalpel in rostro-caudal direction over a length of 5–8 mm (operation microscope is helpful, but not absolutely necessary). (3) To induce a cortical freeze lesion under visual control, a custom-built small plastic cylinder attached to a simple 3D micromanipulator is needed (Figure 1B). The dimensions (in cm) of the plastic container which is completely filled with liquid nitrogen and to which a copper rod with a tip diameter of 0.5, 1, or 2 mm can be attached, are shown in Figure 1C. (4) To induce a microgyrus as shown in Figure 2, use the copper cylinder with 1 mm tip diameter and place the tip of the liquid nitrogen-cooled probe for 8 seconds on the exposed calvarium above the cortical area of interest (in Figure 2 the frontoparietal cortex in the left hemisphere). Induce an identical second and third freeze lesion at a distance of 1.5 mm from the first lesion in rostral and caudal direction, respectively. These three lesions generate a 4–6 mm long microsulcus in rostrocaudal direction, as shown in Figure 2. (5) Close wound with histoacryl tissue glue (e.g., from Braun-Dexon, Melsungen, Germany). (6) Place animals below infrared light (avoid overheating!). When pups start to move again, place them back in the cage with their mother. Always leave a few rat pups with the mother during the operation. Pups can be labelled with a water-resistant marker, but the label is not permanent due to cleaning of the pups by the mother, and usually the label has to be renewed every day. If the ethical committee agrees, pups can be also marked by cutting off the tip of the tail. It is important that sham-operated control animals are exposed to the same degree of hypothermia as the lesioned animals (see Kolb and Cioe, 2001). Sham-operated animals are treated the same way as freeze-lesioned animals with the exception that the copper cylinder is not cooled.
Monitoring The cortical freeze lesion model in newborn rats (and mice) is very easy to establish and does not require costintensive materials. The type and size of the cortical lesion depends on the age of the animal, the type of the copper cooling probe (Figure 1C), and the duration of the freezing procedure (see also Rosen and Galaburda, 2000). With the methods described above, a cortical microgyrus as shown in Figure 2 can be reliably induced. Using a larger copper probe (e.g., a 2-mm tip diameter) and/or longer exposure times (>20 seconds instead of 8–10 seconds), other cortical malformations can be obtained (Rosen and Galaburda, 2000).
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Characteristics/Defining Features
FIGURE 1 Materials to induce a cortical freeze lesion in the newborn rodent. A: Schematic illustration of the experimental protocol. After anaesthesia by hypothermia and cutting the skin overlying the cortical area of interest, a liquid nitrogen-cooled copper rod with a tip diameter of 1 mm (see Figure 1C for exact dimensions of copper rod) is positioned by the use of a 3D-micromanipulator (Figure 1B) for 8 seconds on the exposed calvarium.
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Spontaneous seizures have not been described so far in the cortical freeze lesion model. Holmes and coworkers studied the consequences of cortical dysplasia on seizure susceptibility in adult rats following amygdala kindling and did not detect any significant differences in seizure susceptibility (Holmes et al., 1999). However, hyperthermiainduced seizures recorded in young freeze-lesioned rats reveal a significantly lower threshold temperature and latency and a longer duration as compared to control animals, indicating a pathophysiological link between focal cortical dysplasia and atypical febrile seizures (Scantlebury et al., 2004). Similar results have been obtained in the MAM model of cortical neuronal migration disorders (Germano et al., 1996) (see also Chapter 23 on MAM model).
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FIGURE 2 Typical structure of a cortical microgyrus in the adult rat brain resulting from a longitudinal freeze lesion at the day of birth. A: Dorsal view of the adult rat brain with a freeze lesion in the fronto-parietal cortex of the left hemisphere (marked by arrowheads). B, C: Photomicrographs of Nissl-stained frontal sections in the region of the primary somatosensory cortex at different magnifications. The microgyrus shows the characteristic four-layered structure (i to iv) and is surrounded by a normal six-layered cortex (I to VI). The white matter is labelled by WM. Modified from (Schwarz et al., 2000) and (Luhmann and Raabe, 1996).
CHARACTERISTICS/DEFINING FEATURES Behavioral Features Behavioral analyses on the cortical freeze lesion model in the newborn mouse or rat have been performed by Glenn Rosen and coworkers (Herman et al., 1997; Rosen et al.,
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1995; Fitch et al., 1994). Freeze-lesioned mice performed poorly when compared to sham-operated animals in discrimination learning, in a spatial Morris Maze Match-toSample task, and in a Lashley Type III maze (Rosen et al., 1995). Male rats with a cortical freeze lesion also show a defect in fast auditory processing (Herman et al., 1997; Fitch et al., 1994). Behavioral analyses in somatosensory function have been recently performed in mice, which received a local freeze lesion in the barrel cortex when they were adult (Luhmann et al., 2005). These mice showed significant deficits in thigmotactic scanning.
Electrographic/EEG Features and Electrophysiology Scantlebury et al. reported that hyperthermia-induced seizures recorded in freeze-lesioned rats begin with jaw myoclonus, followed by hindlimb clonus and generalized convulsions, and are terminated by a period of posthyperthermia depression (Scantlebury et al., 2004). Recurrent seizures and status epilepticus have not been described so far in the cortical freeze lesion model. Functional alterations in the cortical freeze lesion model have been analyzed in more detail by various groups using in vitro brain slice techniques. Spontaneous epileptiform activity using extra- and intracellular recordings in physiologic extracellular bathing solutions have not been observed in this model. However, epileptiform synaptic responses upon electrical stimulation of various afferent inputs have been reliably demonstrated by various groups (Jacobs et al., 1996; Luhmann and Raabe, 1996; Defazio and Hablitz, 2000; Kraemer et al., 2001). This epileptiform activity originates from a focal region of hyperexcitability around the lesion and not from the microgyrus itself (Jacobs et al., 1996; Luhmann et al., 1998b; Jacobs et al., 1999a). This hyperexcitable belt region around the microgyrus shows a relatively normal architecture, but a significant imbalance between excitatory and inhibitory receptors (Zilles et al., 1998) and prominent alterations in intracellularly recorded IPSPs and EPSPs (Jacobs et al., 1996; Luhmann et al., 1998a; Jacobs et al., 1999a). Functional modifications in NMDA receptor subunit composition have been described in the rat cortical freeze lesion model (Defazio and Hablitz, 2000) and in human cortical dysplastic tissue (André et al., 2004). Stimulus-induced epileptiform responses propagate from this hyperexcitable belt region at approximately 0.06 m/second over several millimetres across the neocortex (Jacobs et al., 1996; Luhmann and Raabe, 1996; Luhmann et al., 1998b) into cortical areas with normal morphologic appearance, but with significant disturbances in excitatory and inhibitory receptors or receptor subunits (Zilles et al., 1998; Redecker et al., 2000).
Neuropathology The most consistent result of a focal cortical freeze in the newborn rodent is the formation of a four-layered microgyrus (Dvorak and Feit, 1977; Dvorak et al., 1978) (Figure 2). When the lesion is located in the face representation of the somatosensory cortex (so-called barrel cortex), a widespread disruption of the characteristic whisker barrel field pattern can be achieved (Jacobs et al., 1999c; Rosen et al., 2001). This disorganization in cortical architecture results from disturbances in cortical barrel formation and from abnormal ingrowth of thalamocortical afferents into layer IV (Jacobs et al., 1999c). Rosen et al. (Rosen et al., 2000) reported an almost complete lack of thalamocortical or corticothalamic projections between the ventrobasal complex in the somatosensory thalamus and the cortical microgyrus. Other freeze lesion induced cortical malformations like layer I and white matter heterotopia, laminar necrosis, status verrucosus, and porencephaly have also been described (Ferrer et al., 1993). Larger freezing probes (e.g., 2 mm copper rod) and freezing times (20–30 seconds) result in the formation of a cortical cleft, resembling the pathology of schizencephaly described in humans (Figure 3). The formation of the cortical microgyrus consists of two processes: (1) destruction of neurons and glial cells by the freezing procedure and (2) regrowth of damaged radial glial fibers and migration of neurons of different origin that repair the damaged cortical region within the first postnatal week (Figure 4). The freezing probe induces a focal necrotic
A
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FIGURE 3 Formation of a cortical cleft (open arrow in A) resembling human schizencephaly in the cerebral cortex of an adult rat. This animal received a focal freeze lesion with the 2 mm copper probe and freezing time was 30 seconds. Photographs show Nissl-stained frontal sections from the same tissue section at different magnifications. Arrows in B mark cellsparse layer near cortical cleft and in surrounding normal cortex. Scale bar in A to C corresponds to 1 mm.
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Characteristics/Defining Features
migrate into the lesioned area (Kharazia et al., 2003). It has been postulated that neurogenesis in response to early cortical freeze lesioning may take place during the postnatal period (Rosen et al., 1996). The damaged cortex begins to assume its adult-like microgyric appearance from P5 to P10. The afferent, intracortical, and efferent axonal connectivity of the freeze-lesioned cortex is profoundly altered. Disorganized thalamocortical and corticothalamic projections (Jacobs et al., 1999c; Rosen et al., 2000) and aberrant callosal connections (Giannetti et al., 1999) have been reported.
Imaging and Metabolic Changes
FIGURE 4 Formation of three- or four-layered microgyrus by focal cortical freeze lesion in the newborn rodent. The freeze lesion induces death of immature cortical neurons and a necrotic center. During the first postnatal days, newly migrating neurons destined for layer II/III and surviving Cajal-Retzius neurons in layer I invade the lesion. Modified from Redecker et al., 2000.
Modifications in cortical metabolism following cortical freeze lesioning have been studied in the adult rat by Kraemer et al. (Kraemer et al., 2001). The authors performed autoradiographic measurements of basic cortical [14C]deoxyglucose metabolism and demonstrated a significant reduction up to 1 mm from the microgyrus, whereas basic metabolism was unchanged in remote cortical regions. Using functional magnetic resonance imaging (fMRI), Schwindt et al. (Schwindt et al., 2004) recently reported in this model that fMRI activation in the somatosensory cortex of adult rats was significantly weaker in the lesioned hemisphere as compared to the untreated hemisphere. In a few animals with a neonatal cortical freeze lesion, cortical areas outside the primary sensory cortex were activated, indicating a functional reorganization following the early cortical lesion.
Molecular Changes lesion on the cortical layers which are already present at birth (layers IV to VI and marginal zone/layer I). Reactive astrocytes and macrophages arrive at the site of the cortical lesion within 24 hours following the injury (Rosen et al., 1992). Reactive gliosis accompanied by a loss of inwardly rectifying K+ channels (Kir) and reduced gap junction coupling have been described in astrocytes in freeze-lesioned dysplastic cortex (Bordey et al., 2001). A marked increase in GFAP immunoreactivity along the entire length of the microgyrus and clusters of proliferative BrdU-positive astrocytes were observed near the depth of the lesion (Bordey et al., 2001). During the first postnatal days, migrating neurons destined to form layer II/III invade the necrotic core. Interestingly, radial glial cells are preserved in freezelesioned cortex of the adult rat (Rosen et al., 1994). These glial cells might serve as mechanical guiding structures for the migrating neurons. In addition, Cajal-Retzius neurons in the marginal zone/layer I survive for longer developmental periods following cortical injury and probably migrate tangentially from the cortical areas surrounding the lesioned zone (Supèr et al., 1997). Subplate neurons might also
Molecular changes in the cortical freeze lesion model have been demonstrated with different techniques. With quantitative in vitro receptor autoradiography, Zilles et al. (Zilles et al., 1998) demonstrated that the binding to NMDA, AMPA, and kainate receptors is significantly increased in the dysplastic cortex, whereas the binding to GABA-A and GABA-B receptors is reduced. Significant changes could be also observed in remote regions and even span the whole cortical hemisphere. These remote areas of the lesioned hemisphere showed an imbalance between excitatory and inhibitory receptor binding with an up-regulation of the binding to AMPA and kainate, and a down-regulation to GABA-A receptors. The binding to GABA-B and NMDA receptors was not significantly changed in these remote cortical regions (Zilles et al., 1998). These observations are in good agreement with in vitro electrophysiologic data demonstrating widespread propagating epileptiform activity which could be blocked by AMPA but not NMDA receptor antagonists (Luhmann et al., 1998b). However, epileptiform activity recorded in the vicinity of the microgyrus, where NMDA receptors are up-regulated, could be blocked by
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NMDA antagonists (Luhmann et al., 1998b; Jacobs et al., 1999a; Luhmann et al., 1998a). This observation could be confirmed by Defazio and Hablitz, who demonstrated with electrophysiologic and neuropharmacologic studies that NMDA receptors containing the 2B-subunit are functionally enhanced in freeze-lesioned cortex (Defazio and Hablitz, 2000). Immunohistochemical studies on the regional expression of four NMDA receptor subunits (NR1, NR2A, NR2B, NR2D) and two major AMPA- (GluR2,3) and kainatereceptor (GluR5,6,7) complexes have been performed by Hagemann et al. (Hagemann et al., 2003) in adult rats after neonatal freeze lesions. Surprisingly, no local or widespread changes of glutamate-receptor subunit distribution could be detected, suggesting that other or additional mechanisms (e.g., modifications in receptor affinity) may contribute to the increased excitatory neurotransmitter binding and excitability in cortical malformations (Hagemann et al., 2003). The distribution pattern of GABA receptors and GABAA receptor subunits in the adult rat with a neonatal cortical freeze lesion has also been investigated in detail. The pronounced down-regulation of GABA-A receptors in the dysplastic cortex and in remote cortical regions of the ipsilateral hemisphere as described by Zilles et al. (Zilles et al., 1998) could be reproduced by Redecker et al. (Redecker et al., 2000) in their immunohistochemical analyses of GABA-A receptor subunits. A widespread regionally differential reduction of the GABA-A receptor subunits a1, a2, a3, a5, and g2 was observed. Furthermore, the down-regulation of GABA-A receptor subunits also involved the ipsilateral hippocampal formation, as well as restricted contralateral neocortical areas, indicating widespread disturbances in the inhibitory neocortical and hippocampal network (Redecker et al., 2000). These molecular data are in good agreement with electrophysiologic studies demonstrating a functional down-regulation of GABAergic inhibition (Luhmann et al., 1998a) and modifications in GABA-A receptor subunit composition (Defazio and Hablitz, 1999). Inhibitory interneurons immunoreactive for GABA, calbindin, or parvalbumin and GluR1/calbindin-containing interneurons are not significantly reduced in density near to or remote from the lesion (Schwarz et al., 2000; Kharazia et al., 2003). In contrast, Rosen et al. (Rosen et al., 1998) demonstrated a transient (up to postnatal day 21) decrease in the expression of parvalbumin immunoreactivity in supragranular neurons, both within the malformation itself and up to 2 mm adjacent to it. In infragranular layers, parvalbumin immunoreactivity neurons were permanently reduced in their density both within and immediately adjacent to the microgyrus (Rosen et al., 1998). It has also been suggested that GABAergic inhibition is enhanced in the dysplastic cortex due to an increased excitatory drive onto inhibitory interneurons (for review, see Prince and Jacobs, 1998; Prince et al., 1997; Jacobs et al., 1999b).
In a recent study using conventional protocols to induce long-term potentiation (LTP) in neocortical slices, Peters et al. (Peters et al., 2004) demonstrated a layer-specific modification in synaptic plasticity at a distance of 2–3 mm lateral to the microgyrus. Whereas field potentials recorded in layer II/III were strongly enhanced following theta-burst stimulation in layer VI, layer IV revealed an impaired plasticity. Cytochrome-oxidase staining revealed that LTP in layer IV of the freeze-lesioned cortex could only be elicited, when stimulation was applied within a preserved barrel cortex (Peters et al., 2004).
Response to antiepileptic drugs and usefulness in screening drugs In vivo studies on responsiveness to antiepileptic drugs have so far not been performed. Different antagonists or modulators acting on GABA and glutamate receptors have been studied in vitro using neocortical slices (Luhmann et al., 1998a; Luhmann et al., 1998b; Defazio and Hablitz, 1999; Defazio and Hablitz, 2000; Hablitz and Defazio, 2000). The cortical freeze lesion model might become a practical drug screening in vitro model using acute neocortical slices and electrical stimulation protocols to elicit propagating epileptiform responses (Jacobs et al., 1996; Luhmann and Raabe, 1996; Luhmann et al., 1998b) or with modified extracellular bathing solutions to induce spontaneous epileptiform activity.
LIMITATIONS The cortical freeze lesion model is easy to develop and produces a focal cortical pathology which resembles human migration disorders in the cerebral cortex (Andermann, 2000; Rosen and Galaburda, 2000). The model gives very reproducible results when the same lesion protocol is used. The freeze-lesion induced cortical malformations (focal microgyrus) published by various groups (Dvorak et al., 1978; Rosen et al., 1992; Ferrer et al., 1993; Jacobs et al., 1996; Luhmann and Raabe, 1996; Defazio and Hablitz, 1999; Scantlebury et al., 2004) confirm this observation. The type of lesion is strongly dependent on the age of the animal, and reproducible results can be only obtained when the identical age group is used. The mortality of this model is low (<5%). Problems may arise when the mother does not accept the lesioned rat pups. If this problem arises, only litters with an “experienced” mother should be selected. The electrographic/EEG features of this model need to be analyzed in more detail. It is currently unclear at what age experimental animals with a cortical freeze lesion might reveal spontaneous seizures, whether seizures can be provoked by systemic application of drugs and what type of
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spontaneous seizures can be provoked by lowering the seizure threshold (Roper et al., 1995). The question of whether intracortical GABAergic inhibition is reduced or enhanced in the cortical freeze lesion model is not completely solved (for a discussion of this issue see, for example, Prince et al., 1997). The excitatory drive onto certain types of inhibitory interneurons may be enhanced or reduced, the intracellular Cl+ homeostasis might be modified, and the functional consequences of changes in GABA-A receptor subunit composition are also not completely understood.
INSIGHTS INTO HUMAN DISORDERS Molecular and electrophysiologic alterations in the function of GABA-A receptor-mediated inhibition and glutamateric excitation mediated by ionotropic glutamate receptors have been consistently demonstrated in this model (see the preceding and the References) and represent the cause of the intracortical imbalance between synaptic excitation and inhibition. Interestingly, these alterations are not restricted to the site of the structural lesion but also involve macroscopically normal cortical regions surrounding the microgyrus. This picture corresponds to clinical data reporting that focal neuronal migration disorders as the cause of intractable partial epilepsy (Kuzniecky, 1994; Palmini et al., 1991b) and that the lesion is microscopically more extensive, as revealed by MRI (Wyllie et al., 1998; Palmini et al., 1995; Palmini et al., 1994b; Palmini et al., 1991a). As in the rodent cortical freeze lesion model, the site of the structural lesion in patients with neuronal migration disorders and intractable partial epilepsy does not necessarily overlap with the site of the epileptogenic tissue. In some studies, the surgical outcome correlated with the amount of tissue removed (Wyllie et al., 1998; Palmini et al., 1994b; Palmini et al., 1991b; Palmini et al., 1991a), supporting the notion that focal neuronal migration disorders cause widespread alterations in cortical function. The mechanisms underlying this pathophysiologic enlargement of the cortical lesion are currently unknown. It may well be that during early development some molecular or electrophysiologic signals spread from the site of the lesion to neighbouring intact areas, generating secondary alterations in cortical function. Interestingly, early neurosurgical interventions in children with intractable epilepsy often produce the best result (Holmes, 1996), maybe because removal of the lesion during early development prevents the enlargement of the cortical lesion. The cortical freeze lesion model in the newborn rodent serves as a valuable model to study the consequences of a cortical lesion due to focal neuronal migration disorders. It replicates many of the pathologic malformations described in humans. The model is easy to establish and generates reproducible results concerning the pathology of the lesion.
For drug screening purposes, in vitro electrophysiologic studies are suitable. In vivo screening studies are less favorable, since the cortical freeze lesion model probably does not generate spontaneous epileptiform activity in vivo.
Acknowledgments This work was supported by DFG grants SFB194, Lu375/3 and Lu375/4.
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23 MAM and Other “Lesion” Models of Developmental Epilepsy GIORGIO BATTAGLIA AND STEFANIA BASSANINI
The present chapter reviews different acquired “lesion” models, i.e., experimental animals in which developmental brain malformations are produced by the action of toxic compounds during the gestational period. Several compounds interfering with DNA synthesis have been used to generate animal models with cerebral malformations, but the only widely used model is based on the administration of methylazoxymethanol-acetate, or MAM. In comparison to other teratogenic compounds, MAM neither affects gestational parameters nor displays teratogenic effects outside the central nervous system (CNS); by contrast, it is able to kill actively dividing neuroepithelial cells in a narrow time window, thus selectively affecting the proliferation of specific neuronal cell populations. Single or double MAM administrations on gestational day 15 are able to induce cerebral hypoplasia with a clear rostro-caudal and mediolateral gradient; in addition, particularly after double MAM administrations, cerebral heterotopia are generated, closely mimicking those observed in human periventricular nodular heterotopia. Heterotopic neurons are hyperexcitable, and different cellular mechanisms, including intrinsic membrane abnormalities and receptor dysfunctions, may have a role in their abnormal firing behavior. In addition, even if spontaneous seizures have not been reported, MAM-treated rats display decreased seizure threshold in vivo to a variety of proconvulsant agents both in adulthood and during postnatal development. This lower seizure threshold may be explained not only by the hyperexcitability of the heterotopic neurons but also by the abnormal corticosubcortical circuitry connecting the heterotopia with both hippocampus and neocortex. All these features make the MAM model a good model for studying epileptogenesis in the malformed brain.
Models of Seizures and Epilepsy
INTRODUCTION Epilepsy is a group of heterogeneous syndromes affecting more than 50 million people worldwide, and about 30% of epileptic patients are intractable to antiepileptic drugs. The goal of identifying the molecular mechanisms of epileptogenesis in the different epileptic syndromes is the actual challenging task for both clinical epileptologists and basic scientists and possibly the most rational approach for achieving better therapeutic results. To achieve these goals, a useful approach is to produce appropriate models in experimental animals. Theoretically, an appropriate animal model should meet specific criteria, such as: (1) it should recapitulate the pathophysiology of the human disease and display behavioral abnormalities similar to those observed in affected humans; (2) it should be suitable for testing pharmacologically active compounds, exerting the same effects on both the animal model and affected humans; (3) it should help clarify the neurobiological mechanisms underlying the human disorder. In the specific case of animal models mimicking epileptic syndromes associated with acquired or genetically determined malformations of cortical development (MCDs), the model should be characterized not only by hyperexcitable brain regions, but also by structural cerebral abnormalities both macroscopically and microscopically similar to those observed in human patients. Models with these features could be of great value for clarifying the ontogenesis of cerebral malformations as a substrate to understanding why those malformations eventually become hyperexcitable. The need for such animal models is now made more critical by two pieces of clinical evidence. First, the large-scale use of magnetic resonance imaging (MRI) since the mid-80s
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has clearly demonstrated that developmental brain abnormalities are an important cause of developmental disabilities and particularly of focal epilepsy. Second, a relevant fraction of patients with intractable epilepsy harbors developmental cerebral malformations, as demonstrated by the high percentage (ranging from 20 to 50%) of neuropathologically proven MCDs in patients undergoing epilepsy surgery for the relief of intractable seizures (Guerrini et al., 2003). Different animal models for both acquired and genetically determined human MCDs have been developed and studied in the last decades, and many of these models are described in detail in other chapters of this book. The present chapter reviews the different acquired “lesion” models, i.e., experimental animals in which structural brain abnormalities are produced by the action of toxic compounds during the gestational period. We will briefly illustrate the different antimitotic compounds used for this scope, and we will then focus on the model more frequently—and successfully— used, i.e., the rat model based on the administration of methylazoxymethanol-acetate, or MAM.
ANTIPROLIFERATIVE AGENTS Several compounds inducing inhibition of DNA synthesis and therefore displaying antiproliferative activity have been used to generate cerebral malformations during embryonic development: 5-azacytidine (Taylor and Jones, 1979), methyl-mercury (Rodier, 1990), nitrosoureas (Hallas and Das, 1978), and carmustine or BCNU (Benardete and Kriegstein, 2002). All these agents are able to induce neuronal cellular ablations when given at different times during embryogenesis or in the early postnatal phase (Rodier et al., 1986), and they are all used by developmental neurotoxicologists to create experimental animals with functional neurological abnormalities (Rodier, 1990). However, as briefly outlined below, data demonstrating the presence of an epileptic phenotype in animals treated with all these compounds are quite limited.
5-Azacytidine, Methyl-Mercury, Nitrosoureas, and Carmustine 5-Azacytidine, synthesized in 1964 (Sorm et al., 1964), is a cytidine analogue possessing a potent inhibiting action against the cellular synthesis of RNA and proteins and used as an antiblastic agent in the chemotherapy of acute myeloid leukemia and myelodysplastic syndrome (Pinto and Zagonel, 1993). It is also a teratogen agent able to cross the placental barrier (Gutova et al., 1971) and to accumulate preferentially in the developing neural tissue (Seifertova et al., 1968, 1974), and therefore is used in experimental teratology of the CNS. When administered to pregnant mice, it
interferes with the mitotic activity of neuroepithelial cells, selectively killing proliferating cells and sparing mature cells. Repeated small doses of 5-azacytidine during prenatal life cause severe microcephaly at birth (Rodier and Reynolds, 1977). In mice treated on embryonic day 15 (E15) the neocortical thickness is reduced, whereas mice treated on E16 or E18 are characterized by hippocampal hypoplasia and cytologically abnormal neurons; these latter mice display behavioral features characteristic of hippocampal damage (Rodier and Reynolds, 1977). However, 5-azacytidine is not specific for the nervous system, and treated mice are smaller than controls at birth, the size difference persisting into adulthood. In addition, no data have so far demonstrated that 5-azacytidine is a compound useful for creating an animal model with epileptic phenotype. Methyl-mercury is neurotoxic in adults, but it is even more deleterious to the developing brain, as demonstrated by the effects of intrauterine methyl-mercury poisoning in human patients affected by Minamata disease in Japan (Matsumoto et al., 1975). In human subjects exposed to methylmercury in utero the presence of heterotopic neurons was also reported. This compound is also effective in experimental animals, since a single dose of methyl-mercury in mice is able to induce rapid changes of the mitotic rate in neonatal cerebellum (Rodier, 1983). However, attempts to create migrational defects in experimental animals were unsuccessful: for instance, very high doses of methylmercury during early guinea pig cortical development produced some cytologic abnormalities of neocortical neurons but no evidence of well-defined heterotopia (Inouye and Kajiwara, 1988). N-Ethyl-N-nitrosourea, or ENU, is able to induce brain tumors if administered to pregnant rats (Druckrey et al., 1966). In addition, it is also able to function as a teratogen and to produce cortical hypoplasia associated to cytologic abnormalities of the neocortical neurons. Rats receiving ENU during early stages of embryogenesis, such as E14 and E15, exhibit a more pronounced reduction in cortical size than those receiving ENU later during embyogenesis. Hallas and co-workers carefully investigated the effects of ENU injections at different embryonic stages on the development of the rat neocortex (Hallas and Das, 1978). Reduction in cortical thickness was equally observed in all neocortical layers, and pyramidal neurons throughout the cortex were characterized by small perikarya and fewer and less extensively branched apical and basal dendrites (Hallas and Das, 1978). The electrophysiology of these animals, however, was not investigated. Carmustine, or 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU), is a DNA-alkylating and DNA-cross-linking agent, widely used as a cytostatic drug in the therapy of brain tumors, lymphomas, leukemia, malignant melanoma, and other neoplasms. The mechanism of action of carmustine is most likely the formation of DNA interstrand cross-links
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(Gombar et al., 1980). As for the other teratogenic compounds, BCNU has high lipophilicity and therefore high CNS penetration. It has been recently reported that the neocortex of rats treated with BCNU at E15 displays cytologic abnormalities suggestive of cortical dysplasia (Benardete and Kriegstein, 2002). Neurons from the malformed cortex are hyperexcitable and display reduced neuronal sensitivity to GABA inhibition (Benardete and Kriegstein, 2002). In general, the BCNU-treated rats have anatomic features similar to those observed in rats treated with MAM (see below). However, the experience with BCNU is quite limited, if compared to the bulk of data obtained with the MAM model; in addition, BCNU activity is probably less selective for the developing CNS, given the reduced litter size and body weight at birth after BCNU treatment (Benardete and Kriegstein, 2002).
THE MAM MODEL OF EPILEPSY The antimitotic agent methylazoxymethanol-acetate (MAM) has been long used to induce developmental brain dysfunction in rodents (Cattabeni and Di Luca, 1997). The main advantage of this compound over the other teratogenic agents is the higher selectivity on neuronal precursors undergoing their final mitotic division at the time of administration. Indeed, MAM treatment has been shown to be more toxic in vitro on neuroepithelial cells rather than on astrocytes or vascular smooth muscle cells (Cattaneo et al., 1995). In keeping with this experimental observation, MAM does not affect gestational parameters of the dams and it has no teratogenic effects on other organs of the offspring (Cattabeni and Di Luca, 1997). Therefore, MAM can be considered an ideal tool to induce selective cellular ablations of neuronal precursors at specific and well-defined neurogenetic times in experimental animals.
Mechanism of Action of MAM MAM is an alkylating agent that occurs naturally in palmlike plants belonging to the Cycas genus of the Cycads, commonly found in tropical and subtropical regions all over the world (Matsumoto and Strong, 1963). Both the glucoside cycasin (b-D-Glucosyloxy-azoxymethane) and its aglycone MAM are extractable from the seeds, roots, and leaves of the Cycas plants. When administered intraperitoneally to pregnant rats, MAM is rapidly converted to methyl-diazonium, which passes the placental and emato-encephalic barriers, and damages DNA by methylating the O6 or N7 position of guanine nucleic acids (Matsumoto et al., 1972). Actively dividing neuroepithelial cells during the S-phase are affected, whereas postmitotic neurons or neuroblasts in the G0 phase are spared (Johnston and Coyle, 1979). Another interesting feature of the biological activity of
MAM is the narrow time window of action: the cell killing effect of MAM is confined to a period of 2–24 hours, with maximal activity at 12 hours after administration (Matsumoto et al., 1972). Therefore, since neurogenesis follows a relatively rigid timetable (Rodier et al., 1977; Bayer and Altman 1993), the administration of MAM allows one to affect rather selectively the proliferation of specific neuronal cell populations.
Toxic Effect of MAM in Humans Since the Cycad plants are able to withstand drought and even typhoons, their seeds have been used as a source of emergency food by traditional populations, such as the Chamorro people. Toxic properties of cycad seeds were, however, well known to the consumers long before the chemical identification of the active compounds was established. Accordingly, consumers used to wash repeatedly and sun-dry the seeds for detoxification before grinding them into flour. Consumption of insufficiently washed seeds has led to sickness and death (Whiting, 1963). For instance, the Guamanians relied on Cycas to a considerable degree as source of food during World War II. As a consequence of that, amyotrophic lateral sclerosis, Parkinsonism-dementia complex (Kisby et al., 1992), and also Alzheimer-like dementia were found at extraordinary high rates among the Chamorro people of Guam and Rota before the flour from Cycad plants was suggested as responsible for the conditions (Whiting, 1963).
MAM Administration: Anatomic Effects Single MAM Administration Early reports on MAM effects demonstrated that a single MAM administration during the gestational period was able to produce microencephaly in rodents. It was also apparent from early studies that the degree and type of cerebral malformation were dependent on the embryonic day at which MAM was injected (Johnston and Coyle 1982). According to the timetable of neurogenesis, MAM treatment on E14 resulted in thickness reduction of all cortical layers, whereas MAM treatments on E15 or E16 resulted in the selective ablation of layers II–IV and sparing of layers V–VI (Ashwell, 1987). Another interesting feature revealed by early studies was the dose-dependent effect of MAM. The smallest effective dose was shown to be 10 mg/kg of body weight in rats (Tamaru et al., 1988), whereas the largest dose compatible with offspring survival was 30 mg/kg (Haddad et al., 1972). The use of this highest dose was, however, hampered by the fact that the effect was less specific; indeed, in addition to the cerebral cortex, other brain areas, such as the cerebellum, were affected by the treatment (Vorhees et al., 1984).
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Most early studies were aimed at evaluating the effect of the neurotoxin on cognitive functions and therefore focused on the effects of MAM exposure at E15. In fact, telencephalic areas of crucial importance for cognitive functions, such as neocortex and hippocampus, were most selectively affected by treatment within this time window, corresponding in rodents to the neurogenetic peak of the neocortex. Single MAM exposures (25 mg/kg of maternal body weight) on E15 resulted in cerebral abnormalities primarily involving telencephalic neocortical and hippocampal areas, with clear reduction of the neocortical thickness (Johnston and Coyle, 1979). In addition, such injections produced subcortical neuronal heterotopia primarily located in the CA1 and CA2 regions (Singh et al., 1977; Dambska et al., 1982; Collier and Ashwell, 1993). Double MAM Administration Capitalizing on the experience of previous investigators, our group attempted to increase the effect of MAM during the neurogenetic peak of the neocortex in order to create a model for epileptogenic cortical dysgeneses. Toward this goal, we devised a modified schedule of MAM administration on E15 (Sancini et al., 1998; Colacitti et al., 1999). Pregnant Sprague-Dawley rats received two MAM acetate intraperitoneal doses (15 mg/kg maternal body weight, in sterile saline) on E15, the first injection at 12.00 a.m. and the second at 12.00 p.m. (the day after conception, as determined by vaginal smear, was designated as E1). As expected, even after this double MAM treatment, no differences in gestational parameters, pup weight, and litter size were observed. By contrast, the double MAM treatment consistently produced severe cortical hypoplasia and layering abnormalities, with a clear rostro-caudal and mediolateral gradient of the abnormalities (i.e., the rostral and lateral cortical areas being less severely impaired; Figure 1A). In addition, conspicuous cerebral heterotopia were generated: subpial bands of heterotopic neurons in layer I and intracortical, periventricular, and intrahippocampal nodules of heterotopic neurons (Figures 1B–D) (Colacitti et al., 1999). The tract-tracing analysis of MAM-treated brains in vivo revealed that MAM-exposed rats were characterized by abnormal corticocortical and corticohippocampal connections, since subcortical and even intrahippocampal heterotopia were reciprocally connected with ipsilateral and contralateral cortical areas (Colacitti et al., 1998). In addition, the staining pattern of hippocampal heterotopia was strictly similar to that obtained for neocortical, and not hippocampal, structures (Colacitti et al., 1998, 1999). Similar results were independently obtained in rats single-treated with MAM on the same gestational day (Chevassus-AuLouis et al., 1998a,b; Castro et al., 2001), thus supporting the idea that heterotopia were composed by neurons originally destined to the neocortex and inserted in an abnormal
FIGURE 1 Cerebral cytoarchitectural features of adult rats treated with a double MAM injection on E15. Thionine stain. A: The rostral cortical areas are largely unaffected by the MAM injections. B: Approximately at the rostro-caudal level of the anterior commissure, the sensorimotor cortex is characterized by reduced cortical thickness (arrow), layering abnormalities, and intracortical heterotopia (arrowheads). C–D: Proceding more caudally, periventricular (PV) heterotopia (C), and intrahippocampal (IH) heterotopia (D) become evident. Note the hypoplasia of both neocortex and hippocampus. Calibration bars: 250 mm.
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corticosubcortical circuitry involving both hippocampus and neocortex. The macroscopic and microscopic analysis of the doubletreated MAM rats revealed anatomic features also found in human malformations of cortical development: for instance, abnormally located vessels associated with intracortical heterotopia, fusiform neurons marginally located at the borders of the heterotopic nodules, and the dense GABA immunoreactive neuropil within the subcortical nodules. In addition (see below), a substantial fraction of heterotopic neurons were prone to abnormal burst firing (Sancini et al., 1998; Chevassus-Au-Louis et al., 1998a; Colacitti et al., 1999). For all these features, the MAM-treated rats can be considered a useful model for human brain dysgeneses, particularly for human periventricular nodular heterotopia (PNH), a cerebral dysgenesis characterized by nodular masses of gray matter located in close apposition to the periventricular germinative neuroepithelium (Battaglia et al., 1996; Battaglia et al., 1997).
Genesis of Cerebral Heterotopia in MAM Model The genesis of the heterotopia was also studied by means of morphologic analysis and birth-dating experiments with bromodeoxyuridine (BrdU) immunocytochemistry in both developing and adult MAM-treated brains (Chevassus-AuLouis et al., 1998b; Battaglia et al., 2003a). All types of cerebral heterotopia are still developing and increasing in size during the early postnatal period, and they are always associated with white matter bands of developing neurons serving as reservoirs of cells already committed to form the
heterotopia (Figure 2). BrdU experiments have demonstrated that the time of generation of neurons within the heterotopia is extended in comparison to the corticogenesis in normal rat brains, and that heterotopia are formed through rather precise outside-in (for cortical and periventricular heterotopia) and dorso-ventral (for intra-hippocampal heterotopia) neurogenetic patterns. The genesis of the heterotopia can be explained as follows (Figure 3): (1) first, the MAM-induced ablation of an early wave of neurons committed to the neocortex impairs the correct migration of a following wave of neurons, which therefore settle as heterotopic neurons at different points of the migration pathway; (2) second, the heterotopic neurons influence the migration of the subsequent waves of young neurons, which first sojourn in the white matter neuronal bands, and then migrate within the heterotopia through the abovementioned neurogenetic gradient; (3) finally, heterotopic neurons send their axons to (and receive axons from) neocortical and archicortical areas, thus establishing aberrant connections between the neocortex and the hippocampus (Battaglia et al., 2003b). The neurogenesis of the MAM-induced heterotopia is also a useful neurogenetic scheme to explain the origin of the heterotopic nodules in human periventricular nodular heterotopia, and the propagation and maintenance of epileptic discharges through the abnormal corticosubcortical connections (Battaglia et al., 2003b).
MAM Administration: Neurophysiologic Effects As mentioned above, the majority of early studies on MAM-treated rats investigated the effects of MAM ex-
FIGURE 2 Cytoarchitectural features of cerebral heterotopia during early postnatal development (postnatal days 3 and 5) of MAM-treated rats. Thionine stain. A–B: Cortical heterotopia. Note the bands of densely packed neurons in the white matter close to the heterotopia (arrow in A), and the reduction of cellular density in the cortical layers overlying the heterotopia (asterisks). C: Periventricular heterotopia. Note the close anatomic contiguity with the cellular bands in the overlying white matter (arrows). Calibration bars: 100 mm in A–B, 50 mm in C.
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FIGURE 3 Summary diagram illustrating the mode of neurogenesis of cerebral heterotopia suggested by the present experiments. The dotted lines represent the normal pathways of migration; arrows indicate the outside-in and dorso-ventral neurogenetic patterns by which heterotopia are formed; the different heterotopia are represented in close proximity with the white matter bands of neurons (solid black bars); finally the dashed lines represent the reciprocal connections between the neocortex and the different heterotopia. See text for more specifications on the different neurogenetic steps (from 1 to 3).
posure at E15 on cognitive functions and demonstrated impairments of learning and memory functions (Haddad et al., 1969; Vorhees et al., 1984) and long-term potentiation (Ramakers et al., 1993). More recently, given the consistent report of intrahippocampal heterotopia disrupting the normal layering of the CA1-CA2 pyramidal layer, the rats treated with MAM injections at E15 have been the subject of a growing number of studies specifically investigating the existence of epileptic phenomena and/or hyperexcitable firing patterns. These studies have all convincingly demonstrated the relevance of this model to study epileptic phenomena. Many reports have revealed that MAM-treated rats possess decreased seizure threshold in vivo. They are indeed more sensible to different proconvulsant agents mimicking different seizure types, such as generalized seizures (fluorothyl: Baraban and Schwartzkroin, 1996; pentylentetrazole: Chevassous-Au-Louis et al., 1998a), temporal or limbic seizures (kainate: Germano and Sperber, 1997), and focal seizures with secondary generalization (hippocampal kindling: Chevassous-Au-Louis et al., 1998a). Decreased seizure threshold has also been reported during postnatal development, since MAM rats at postnatal day 15 (P15) are more susceptible to bicuculline- and kainate-induced seizures (De Feo et al., 1995), and P14 rats more easily develop behavioral seizures if exposed to hyperthermia (Germano et al., 1997).
The heterotopic neurons of MAM-treated rats have been also been shown to possess increased seizure susceptibility or neuronal hyperexcitability in vitro. Indeed, hippocampal heterotopic neurons display bursts of action potentials in response to suprathreshold current injections, and they produce epileptiform discharges if exposed to increased potassium concentrations (Baraban and Schwartzkroin, 1995). They are also able to generate independent epileptiform activities if perfused with either bicucullin or 4aminopiridine (Baraban et al., 2000). Our group has extended the electrophysiologic analysis to heterotopic neurons outside the hippocampus in the MAM model with double treatment at E15. Intracellular recordings have demonstrated the existence, in both the subcortical and the intracortical heterotopia, of neurons showing hyperexcitability phenomena leading to aberrant firing patterns (Sancini et al., 1998; Colacitti et al., 1999). These neurons, dubbed excessively bursting neurons (Figure 4), fire progressively with long repetitive bursts of action potentials in response to low-amplitude depolarizing current pulses, until reaching long-lasting trains of highfrequency action potentials outlasting the duration of the intracellular pulses (Sancini et al., 1998; Colacitti et al., 1999). Progress has also been made in investigating the molecular and cellular mechanisms underlying MAM-induced hyperexcitability. Most likely, diverse mechanisms contribute to generate the abnormal firing patterns in heterotopic neurons. Sancini and co-workers have postulated that abnormalities of intrinsic membrane excitability, namely a decline in the Ca2+-activated K + current, responsible for hyperpolarizing the membrane after action potentials, account for the observed abnormal firing behavior in the excessively bursting neurons (Sancini et al., 1998). In line with the hypothesis of intrinsic membrane alterations in MAM rats, the reduction of A-type Kv4.2 potassium channels has been reported as a possible cause of the decreased seizure threshold observed in single-treated MAM rats (Castro et al., 2001). More recently, our group has reported, in the heterotopic neocortical pyramidal neurons of double-treated MAM rats, a selective impairment of the targeting to the postsynaptic membrane of the regulatory subunits NR2A/B of the NMDA receptor complex, and abnormalities of the NR2A/B phosphorylation mediated by the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (Gardoni et al., 2003). Interestingly, data suggesting a reduction of NR2A/B subunits and associated alphaCaMKII have been also reported in patients affected by periventricular nodular heterotopia and drug-resistant focal epilepsy (Battaglia et al., 2002; Finardi et al., in preparation). These data demonstrate that the MAM model can also be useful in investigating molecular mechanisms of epileptogenesis also relevant in human malformations of cortical development.
MAM Model: Limitations and Cautions
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FIGURE 4 Intracellular recordings of an excessively bursting neuron located within an intracortical heterotopia. Note the progressive increase in burst duration during a sequence of suprathreshold depolarizing current pulses, gradually leading to a sustained tonic firing outlasting the stimulus duration. (Courtesy of Dr. Giulio Sancini.)
MAM MODEL: LIMITATIONS AND CAUTIONS Despite the bulk of studies exploring and demonstrating the hyperexcitability phenomena of MAM-treated rats, spontaneous seizures were never convincingly reported in any of the rats treated with the different MAM administration. This peculiar feature, which is common to all models of experimentally induced cerebral developmental malformations, such as the freeze lesion model of human microgyria (Dvorak et al., 1978), or the X-irradiation model (Hicks et al., 1959; Roper et al., 1997), should not, however, be taken as the definitive proof that MAM-treated rats do not develop spontaneous seizures, given that continuous video-EEG monitoring of electrophysiologic and behavioral activities (the only way of investigating the presence of spontaneous seizures) has never been reported or investigated by the different groups working with this model (Chevassus-Au-Louis et al., 1999). The apparently low aptitude for spontaneous seizures in MAM rats could be related to the limited anatomic extent of the affected cortex, or to the presence of enhanced inhibitory systems in the MAM-induced heterotopia, as
recently postulated (Calcagnotto et al., 2002). It should also be underlined that even in human patients cortical malformations are not necessarily associated with a spontaneous epileptic phenotype. In dealing with the prenatal administration of MAM, care should be paid in using a uniform dating of the beginning of pregnancy and birth. Some groups, including ours, designate the first day of pregnancy as E1 and the day of birth as P1, whereas others designate them as E0 and P0, respectively. Given the rigid timetable of neurogenesis in rodents, and the potential relevance of even minor differences in the time of administration, attention should be paid not only to choosing and indicating the precise time of MAM administration but also to the nomenclature used for indicating the time of pregnancy and birth. These details should be of value for a correct comparison of results obtained in different laboratories. In this context, caution should also be paid in comparing data obtained with the single or double MAM treatment on E15: even if many results from the different labs indicate than they are similar, they are obtained by treatments differently affecting neuroepithelial cell proliferation, and they should therefore by no means be taken as identical models.
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CONCLUSION The MAM model, despite the limitations mentioned above, has many practical advantages. First, MAM is inexpensive, easy to handle, more selective on neuroepithelial cells than any other teratogenic compound, and it does not affect gestational parameters and litter size. Second, it has been widely used, and much experience has been gathered by different labs in demonstrating its relevance in investigating different aspects of epileptogenesis. Third, the effects are reproducible, and, particularly in the case of the double MAM exposure, the similarities between treated rats and at least a particular type of human malformation of cortical development, i.e., periventricular nodular heterotopia or PNH, are striking. In the opinion of the authors, the lack of spontaneous seizures may be overcome by the combined use of MAM and agents known to induce chronic seizures— pilocarpine or kainate may be good candidates.
Acknowledgments The authors wish to thank Drs. Veronica Setola, Adele Finardi, Giulio Sancini, and Giovanni Carriero for their help in the preparation of the manuscript. Work from our group reported in this review have been partially supported by grants ICS 030.3/RF98.36 and RF162/02 from the Italian Ministry of Health.
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24 In Utero Irradiation as a Model of Cortical Dysplasia DEAN D. LIN AND STEVEN N. ROPER
and GABA receptors (Crino et al., 2001; Ying et al., 1999). Single-cell recordings from dysplastic neurons have shown enhanced Ca2+ currents and reduced Mg2+ sensitivity in NMDA receptors (Andre et al., 2004; Cepeda et al., 2003). But human studies, by definition, are limited by lack of appropriate control tissue and inability to study the disease in its early stages. The in utero irradiation model was adopted to study certain aspects of CD in an animal setting that would allow quantitative comparisons between affected animals and controls with regard to cortical structure and physiology at multiple time points throughout development and epileptogenesis.
INTRODUCTION Abnormalities of cortical development are recognized as a major cause of intractable epilepsy in children and adults, but a clear understanding of the relationship between the structural abnormalities and the seizures themselves is still lacking. Cortical dysplasia (CD) represents a broad spectrum of pathologies (Barth, 1987) ranging from lissencephaly to focal cortical dysplasia (FCD). Individuals born with the more severe forms of CD often possess significant cognitive deficits in addition to epilepsy (Barkovich and Kjos, 1992a,b; Leventer et al., 1999). At a microscopic level, CD can range from subtle alterations in lamination and spatial orientation of the neurons to major abnormalities of neuronal morphology and cellular commitment (Mischel et al., 1995). Several classification systems have been devised in an attempt to correlate histologic changes with clinical presentation and developmental pathology (Barkovich et al., 2001; Palmini et al., 1994b). Mild forms of CD are characterized by loss of lamination and spatial disorientation of otherwise normal-appearing neurons. More severe forms show major migrational abnormalities, such as neuronal heterotopia; i.e., islands of gray matter in the subcortical white matter or periventricular region. The most severe forms show large, dysmorphic neurons and balloon cells. Numerous important findings regarding the epileptogenic nature of CD have been achieved by studying human tissue directly. These include ECoG recordings of sustained ictal activity from regions of FCD (Palmini et al., 1995) and pronounced bursting in in vitro slices from areas of human CD (Avoli et al., 1999; Mattia et al., 1995). Other studies have demonstrated alterations in subunit composition of NMDA
Models of Seizures and Epilepsy
GENERAL DESCRIPTION OF MODEL Some models of CD have been classified as injury-based because their ultimate goal is to disrupt normal cortical development at specific time points in order to evaluate the physiologic and histologic sequelae. One model, perinatal freeze-lesioning of cortex, generates a focal lesion, as opposed to treating animals with methylazoxymethanolacetate (MAM) or with in utero irradiation, which generates more diffuse cortical lesions. The remainder of this discussion will focus on the in utero irradiation model of cortical dysplasia. Irradiation in utero was initially used as a means to study normal cortical development by disrupting the process at various stages and to better understand the deleterious effects of radiation exposure (Hicks et al., 1959; Riggs et al., 1956). More recently, the model has undergone a reassessment in an attempt to relate physiologic changes in the dysplastic cortex with CD-associated epilepsy (Roper,
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1998). Irradiated animals typically exhibit behavioral deficits that vary depending on the time of exposure. These include increased reactivity to novel stimuli, impaired maze learning, and abnormalities of locomotion (for review, see Hicks and D’Amato, 1978). The basic histologic features (Figure 1A) of rats irradiated on embryonic day 17 (E17) are microcephaly, diffuse CD, heterotopic neurons in the hippocampus, and agenesis or hypoplasia of the corpus callosum (Cowan and Geller, 1960). While the overall decreased number of neurons and neuronal fibers is believed to be caused by the initial, toxic effects of radiation on brain cells (Altman et al., 1968; Bayer and Altman, 1990; Roper et al., 1997a), the abnormal corticogenesis is thought to be
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a secondary effect of continued cortical development in an abnormal cellular environment (Marin-Padilla et al., 2003).
WHAT DOES IT MODEL? The in utero irradiation model is an attempt to model human cortical dysplasia; but it clearly does not replicate all features of the condition in humans. It does not produce balloon cells that are characteristic of tuberous sclerosis and Taylor’s type CD (Taylor et al., 1971). Although dysmorphic neurons are present, the giant neurons reported in some human cases are not a regular feature of the in utero irradiation model. Irradiation in utero produces a diffuse injury in the developing cortex, therefore it is not a specific model of focal CD; although the changes seen in irradiated cortex could certainly be present in a focal distribution in some types of CD. Marin-Padilla (1999) has documented “acquired CD” in children who suffered known in utero and perinatal insults and died later in childhood. Many of the changes that he described are also seen in the irradiated rat cortex. Given that in utero irradiation is an injury-based model, it most closely replicates the features of this “acquired CD” in humans. Of note, most of the human cases reported by Marin-Padilla had defined encephaloclastic lesions (e.g., porencephalic cysts). Most cases of human CD do not. It is currently unknown how often in utero injuries at a certain stage of development could disrupt normal cortical development without producing identifiable encephaloclastic lesions because of the generative capacity of the remaining progenitors and immature neurons at the time of the injury.
Dysplastic Cortex METHODS OF GENERATION
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FIGURE 1 Irradiated rat model of cortical dysgenesis: A. A cresyl violetstained coronal section of the brain of a rat that was irradiated on E17 shows cortical dysplasia with dense accumulations of neurons extending up to the pial surface (filled arrowhead), periventricular heterotopia (filled arrow), subcortical heterotopia (empty arrowheads), and heterotopia in the hippocampus (empty arrow). The corpus callosum is also absent. V = lateral ventricle. B. Whole-cell patch clamp recordings of miniature inhibitory posy-synaptic currents from pyramidal cells in control (left) and dysplastic (right) cortex show that the frequency of the currents is reduced in dysplastic cortex; representing an impairment of inhibition. (Reprinted from Zhu and Roper, J Neurosci, 2000, with permission of the Society for Neuroscience, copyright 2000, and Roper and Yachnis, The Neuroscientist, 2002, with permission of Sage Publications, Inc., copyright 2002.)
In order to generate irradiated animals with diffuse cortical dysplasia, pregnant rats with known insemination dates are obtained commercially. Multiple different radiation sources have been used with similar results. In this author’s lab, a linear accelerator is used to apply 225 cGy of gradiation to pregnant animals on E17 as described by Roper at al. (1997b). The pups are born normally on E21. Litter sizes range from 7–10 pups and there are rarely any problems with animals feeding or surviving into adulthood. Some authors have suggested that the dose of radiation has an impact on the structural phenotype and seizure susceptibility. Kellinghaus et al. (2004) irradiated rats on E17 with 100 cGy, 145 cGy, and 175 cGy. They reported progressively more severe CD with increasing radiation dosage. They also reported spontaneous seizures only in the 145 cGy group.
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Although the model has been used primarily in rats, mice have also been studied in some laboratories. Mice exposed to 150 cGy on E13 develop ectopic gray matter (Sun et al., 1996). Mice exposed to 200 or 300 cGy on E16 show absence of the corpus callosum and hypotrophic cortex with some cortical disorganization that is most pronounced in the 300 cGy group (Lent and Schmidt, 1986). However, dysplasia of the normotopic cortex has not been extensively described or studied in the mouse model. The histologic result of the irradiation is very sensitive to the timing of the irradiation. For example, irradiation early in cortical development (E12) produces animals with almost normal lamination but a thin cortex. Irradiation at E16, alternatively, generates animals with the most severe dysplasia, including a markedly thinned cortical mantle and heterotopic gray matter. By E17, the neurons in the most superficial (I) and deep layers (V, VIa, VIb) have already reached the cortical plate. Therefore, neurons destined for the intermediate layers (II, III, and IV) and the interneurons are the most severely affected by radiation. There have been no reported sex differences and evolution of the histological changes over time has not been thoroughly studied.
Monitoring Continuous in vivo EEG monitoring has demonstrated that animals exposed to in utero irradiation display interictal spikes and spontaneous epileptic activity. Two studies from Najm, Luders, and colleagues describe long-term EEG monitoring of irradiated rats (Kellinghaus et al., 2004, Kondo et al., 2001). In these studies, bifrontal as well as bilateral hippocampal depth electrodes were placed stereotactically under pentobarbital anesthesia at P45. For the bifrontal electrodes, stainless steel screw electrodes were placed in the dura mater overlying bilateral frontal cortices. Bipolar twisted wire electrodes were used to monitor electrical activity in bilateral hippocampi. A reference electrode was also placed in the frontal sinus. One of the studies included continuous video monitoring as well (Kellinghaus et al., 2004).
CHARACTERISTICS/DEFINING FEATURES Behavioral/Clinical Features Although learning deficits and hyperactivity have been reported in irradiated rats (Hick and D’Amato, 1978), one may be more impressed by how normal they appear on visual examination. Irradiated animals feed normally and live long lifespans. Although seizures have been reported (Kellinghaus et al., 2004, Kondo et al., 2001), they do not die of status epilepticus as in some genetic models of CD.
Incidence of seizures in irradiated rats is not clear due to a relatively small number of animals that have undergone long-term monitoring. One EEG-only study (i.e., no behavioral monitoring) showed ictal activity in 4 of 7 animals that were treated with 145 cGy on E17 (Kondo et al., 2001). A second study that included video monitoring, documented seizures in 3 of 5 rats exposed to 145 cGy on E17; but no rats given 100 cGy (n = 4) or 175 cGy (n = 8) demonstrated seizures (Kellinghaus et al. 2004). This suggests that there is a narrow treatment window for producing seizures in this model and that the more severe histologic effects do not necessarily correlate with the animals having seizures. The seizures lasted 30 seconds to 2 minutes. Seizure semiology included staring, behavioral arrest, facial twitching, wet dog shakes, loss of postural reflexes, and asymmetrical fore- and hindlimb clonus. As both of these studies were performed on adult animals, there is no information on seizures in immature animals in this model.
EEG Features Kondo et al. (2001) characterized ictal and interictal epileptiform discharges from irradiated animals. Using the monitoring system described above, Kondo demonstrated that all irradiated animals demonstrated interictal epileptiform discharges (IEDs) in the initial 6 days following electrode implantation that localized to both cortical and hippocampal regions. The control animals, in comparison, only rarely demonstrated IEDs. Furthermore, four of seven irradiated animals demonstrated spontaneous ictal epileptiform EEG activity. These seizures could arise from the frontal cortex or the hippocampus and they subsequently secondarily generalized. The seizures were characterized by either fast beta activity or a spiking theta pattern. In this study, after the initial 6 days of monitoring, the frequency of IEDs diminished significantly and no seizures were recorded. This raised the concern that the seizures were simply a result of irritation from the electrode placement. However, in their follow-up study, Kellinghaus et al. (2004) documented seizures 7–14 days after electrode implantation (see below). A follow-up study by Kellinghaus et al. (2004) included video monitoring in addition to EEG. Kellinghaus demonstrated that the frequency of the seizures was dosedependent in nature. Animals exposed to all doses of radiation developed IEDs. The frequency of IEDs, however, was highest in the low- (100 cGy) and medium- (145 cGy) dose groups (0.89 and 0.83 spikes/hour, respectively) whereas the frequency in the high-dose (175 cGy) group was only 0.29 spikes/hour. Additionally, only animals in the medium-dose group demonstrated spontaneous seizure activity (three of five animals) that correlated with EEG activity. Seizure onset was localized to the cortical or hippocampal electrodes, with secondary generalization. No
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cases of status epilepticus have been documented in this model of CD. Several electrophysiologic studies have emerged characterizing this model of cortical dysplasia using in vivo brain electrodes as well as in vitro extracellular field potential and individual whole-cell patch-clamp recordings. Initial studies using in vivo EEG recording electrodes demonstrated an increased propensity for epileptiform activity in the presence of the anesthetic agents, acepromazine (1–2 mg/kg, I.P.) and xylazine (8–14 mg/kg, I.P.), in irradiated but not control animals (Roper et al., 1995). Subsequent studies focused on in vitro brain slice physiology. Roper and colleagues (1997b) used extracellular field potential recordings to demonstrate that neocortical slices obtained from irradiated animals exhibited a heightened intrinsic excitability. In this study, bicuculline methiodide (10 mM) was applied to slices obtained from both control and irradiated animals at a concentration sufficient to block all inhibitory GABAa receptor activity. Both spontaneous and evoked field potentials recordings were then performed in neocortex. Under both evoked and spontaneous conditions, more robust epileptiform bursting was observed in slices from irradiated animals as compared to slices from control animals, thereby revealing a heightened excitability in irradiated neocortex when inhibitory receptors were blocked. Whole-cell patch clamp recordings obtained from pyramidal cells in dysplastic neocortex of irradiated animals demonstrated an impairment of inhibitory synaptic transmission compared to pyramidal cells from control neocortical tissue (Zhu and Roper, 2000). Under conditions when glutamatergic activity was blocked, the frequency and amplitude of spontaneous inhibitory post-synaptic currents (sIPSCs) was significantly decreased, by 70% and 35%, respectively, in dysplastic neocortical pyramidal neurons compared to control pyramidal neurons. Furthermore, a 66% decrease in the frequency of miniature IPSCs (mIPSCs) was observed in dysplastic cortex without any change in mIPSC amplitude (Figure 1B). This reduction in mIPSC frequency suggests a presynaptic locus for the impaired inhibition. When excitatory postsynaptic currents (EPSCs) were evaluated, a significant increase in amplitude (42%) and frequency (77%) of spontaneous EPSCs was noted in dysplastic cortex. But when mEPSCs were analyzed, there was no significant difference in the amplitude or frequency of these currents between control and dysplastic pyramidal cells. When stimulation was applied, derangement in inhibitory transmission was also noted, as paired-pulse depression of EPSCs and monosynaptic evoked IPSCs were reduced in irradiated animals. This study identified a selective reduction in inhibitory synaptic transmission as a primary abnormality in the neocortical circuitry in this model. A follow-up study confirmed these findings in areas of subcortical heterotopic gray matter with a significant
reduction in spontaneous and miniature IPSCs in heterotopic pyramidal cells compared to neocortical pyramidal cells from control animals (Chen and Roper, 2003).
Neuropathology Brains from irradiated rats are microcephalic (Cowan and Geller, 1960; Riggs, 1956). The thickness of the affected cortex is reduced to about one-half that of control neocortex (Marin-Padilla et al., 2003). Although the effect of the radiation is diffuse, it is not uniform throughout the entire neocortex. The dorsomedial cortex is most severely affected, with relative preservation of normal lamination of the lateral neocortex near the rhinal sulcus. This is probably due to a developmental gradient with the lateral neocortex maturing earlier than the medial cortex (Bayer and Altman, 1990). Severely affected neocortex shows loss of any recognizable lamination (Roper et al., 1995). Small and large neurons appear to be scattered randomly throughout the thickness of the cortical mantle. Clusters of neurons sometimes extend through the (normally) cell-sparse layer I up to the pial surface. In milder examples (Figure 2), a putative layer V can be appreciated (large pyramidal cells lined side-by-side) but this structure is displaced superficially (Roper et al., 1998). Presumably, this is due to the fact that irradiation on E17 most severely affects the layer II/III neurons that are still migrating at this time so that they never assume their proper place above the layer V pyramids. Pyramidal cells lack their normal vertical orientation (apical dendrite toward the pia) and may be inverted or laterally oriented. Abnormalities of neuronal morphology are abundant with bizarre dendritic profiles (Figure 3) that extend laterally well beyond the columnar arrangement seen in normal neocortex (Marin-Padilla et al., 2003). In addition, abnormally large pyramidal cells and basket cells can be found in dysplastic cortex (Marin-Padilla et al., 2003). Irradiated and control animals were studied as adults with immunohistochemical markers of two types of cortical interneurons, parvalbumin and calbindin (Roper et al., 1999). The density of these cells was reduced by about 50% in dysplastic cortex compared to control neocortex, but the overall neuronal density was not different between irradiated animals and controls. This showed a selective reduction in interneurons in dysplastic cortex in this model. These findings complemented the physiologic data that showed a selective reduction in mIPSCs in dysplastic pyramidal cells (see preceding text). Recent studies have focused on cortical interneurons in the perinatal period (Deukmedjian et al., in press). Stereologic methods were used to obtain absolute numbers of total neurons and GABAergic neurons in the cortex at E21 and postnatal day 6 (P6), using rats irradiated on E17 and controls. As expected, the number of total neurons was reduced
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by >50% at both time points in irradiated animals. However, the number of total neurons in the cortex doubled from E21 to P6, even in the irradiated group. In controls, GABAergic neurons in the cortex demonstrated a 10-fold increase from E21 to P6. In sharp contrast, there was no increase in the number of cortical GABAergic neurons during this period in the irradiated animals. The net result of these changes was that, by P6, 18% of cortical neurons were GABAergic in control neocortex but only 9% of cortical neurons were GABAergic in the irradiated animals. These data suggest that the GABAergic system has a reduced capacity to recover after in utero radiation injury (Deukmedjian et al., in press), possibly secondary to the increased distance and duration of the migratory journey for immature interneurons going from the ganglionic eminence to the cortical plate. To date, there are no electrophysiologic studies in P6 rats.
LIMITATIONS
FIGURE 2 Camera lucida drawings of the primary somatosensory cortex of irradiated (left) and control (right), 7-week-old rats, comparing their overall cytoarchitectural organization, thickness, and lamination and the relative size and number of neurons. The irradiated cortex is characterized by altered corticogenesis, generalized thickness reduction, reduction in the number of gray matter neurons and fibers, lack of distinct laminar demarcations, formation of neuronal aggregates (nodules) throughout the upper layers, and reduction of white matter tissue. The upper layers (II, III, IV, and V) seem to be more affected than the lower (VIa, VIb) ones, reflecting the timing of irradiation. The size of some neurons is larger in the irradiated than in the untreated cortex. The interstitial neurons of the white matter are more prominent in the irradiated than in the untreated cortex and their number does not seem to be reduced. Layer I has scattered, displaced neurons and focal subpial gliosis. Key: P = pia, V = ventricle, WM and W = white matter, I, II, III, IV, V, VIa, VIb = cortical layers, and V = blood vessels. Scale = 100 mm per division. (Reprinted from Marin-Padilla et al. J Neuropathol Exp Neurol, 2003, with permission of the American Association of Neuropathologists, copyright 2003.)
There are very few technical drawbacks to the in utero irradiation model. It requires a reliable source of radiation delivery and that requires the hardware and special training on the part of the laboratory personnel. This could be a constraint at some institutions. The animals are hardy and easy to handle. Rats offer the advantage of an extensive body of knowledge on normal physiology and comparison with numerous alternate epilepsy models. Differences in genetic strains are not a major problem in rats. The primary limitation of this model lies in how to apply it to human disease. Without balloon cells and giant/monstrous neurons, it is clearly not a model of Taylor’s type or type III CD. Histologically, it most closely resembles mild-to-moderate sporadic (i.e., nonfamilial) CD. As it arises from a known injury, it most closely relates to the acquired CD described by Marin-Padilla (1999). However, most of these children had perinatal insults with obvious encephaloclastic lesions. Most children with CD-associated epilepsy do not. This raises the possibility that more focal injuries may occur earlier in development such that the destructive aspect of the injury is largely masked by generative processes that continue after the injury. This would result in a condition where the initial, toxic effect of the process is minimized and the subsequent altered corticogenesis is maximized in the ultimate structural abnormality. The fact that potentially harmful prenatal events occur more commonly in CD-associated epilepsy than non-CD epileptic controls lends some credence to this hypothesis (Palmini et al., 1994a). However, the existence of such a condition is still speculative at this point, and defining it will be very difficult in humans since we have no way of detecting these events until well after the fact.
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FIGURE 3 Composite figure showing (A, B) photomicrographs and (C, D) corresponding camera lucida drawings of the morphologic features of large excitatory pyramidal neurons with altered dendritic profiles of the visual (A, C) and sensorimotor (B, D) cortex, from Golgi-Cox preparations of irradiated, 7- and 6-week-old rats. The altered pyramidal neurons are characterized by their large size (hypertrophy), the predominant horizontal/oblique distribution of their long spiny dendrites, and the wanting of distinct apical dendrites. However, some dendritic terminals reach into layer I. The predominant horizontal distribution of these altered neurons’ dendrites contrasts sharply with the vertical (columnar) dendritic profiles of normal pyramidal cells. Most dendrites are distributed within a rectangular (horizontal) functional territory, located between 100 and 300 mm from the pial surface, that represent a concentration of excitatory (axo-spinodendritic) synaptic contacts at that location. One relatively normal (unaffected) pyramidal neuron with a distinct apical dendrite and essentially vertical (columnar) dendritic profile is recognized in B and D (arrow). Altered pyramidal neurons are invariably associated with stellate neurons (C) characterized by long ascending, descending, and horizontal spineless dendrites with fusiform dilatations. Some of them are morphologically compatible with large (hypertrophic) inhibitory basket cells. The camera lucida drawings of the stellate neurons depicted in (C) are completed showing their essential morphologic features. The drawings of the stellate cells illustrated in (D) are only outlined (unfinished) to simply indicate size, number, and location. The pial surface is at the figures’ top. Scale = 100 mm per division. (Reprinted from Marin-Padilla et al. J Neuropathol Exp Neurol, 2003, with permission of the American Association of Neuropathologists, copyright 2003.)
INSIGHTS INTO HUMAN DISORDERS The primary finding from this model has been a selective loss of cortical interneurons after in utero irradiation. This has been confirmed in two immunohistochemical studies (Deukmedjian et al., in press; Roper et al., 1999) and two
physiologic studies (Chen and Roper 2003; Zhu and Roper, 2000). The exact nature of this increased susceptibility is unknown, but it may relate to a protracted period of migration for cortical interneurons as opposed to pyramidal cells. Because they must migrate from the ganglionic eminence rather than the telencephalic ventricular zone, a greater
References
number of immature interneuorns are “in transit” at the time of the irradiation on E17. Previous work has suggested that migrating cells may be more susceptible to radiation injury than nonmigrating cells (Altman et al., 1968). However, the specific mechanisms by which in utero irradiation results in a relative deficit of cortical interneurons await further study. Two other animal models have demonstrated the importance of interneuron development in epilepsy. Flathead is a spontaneous mutant rat with a defect in the citron K gene which regulates cytokinesis (Sarkisian et al., 2002). This results in a failure of interneuron progenitors and a reduction in the percentage of GABAergic cells in somatosensory cortex and entorhinal cortex (Sarkisian et al., 2001). These animals demonstrate a markedly reduced brain size, ataxia, spontaneous seizures, and premature death at about 4 weeks postnatal (Roberts et al., 2000). The uPAR-/- mouse is transgenic with a defect in the urokinase plasminogen activator receptor, a protein that affects migration of interneurons via the hepatocyte growth factor/scatter factor pathway (Powell et al., 2003). These animals demonstrate a 50% reduction of interneurons in the anterior cingulated and parietal cortices. They also have spontaneous seizures that are sometime lethal. The fact that interference of interneuronal development by three very different mechanisms results in a common endpoint, epilepsy, suggests that loss of interneurons may be important in human epilepsy as well. But it does not prove that this is the cause of epilepsy in most or even any human epileptic disorders. Some human studies have reported reduced numbers of interneurons in surgical specimens from people with epilepsy due to focal cortical dysplasia (Ferrer et al., 1994; Spreafico et al., 2000). But confirmation of these findings will require quantitative, controlled pathologic studies. Further studies are needed, both in animals and human tissue, to determine if this phenomenon is limited to in utero irradiation or if other insults, such as hypoxia/ischemia, might have the same effect. If so, then vulnerability of immature interneurons and restoration of cortical interneurons may represent a major avenue of therapeutic intervention in diseases associated with prenatal injury.
References Altman, J., Anderson, W.J., and Wright, K.A. 1968. Differential radiosensitivity of stationary and migratory primitive cells in the brains of infant rats. Exp Neurol 22: 52–74. Avoli, M., Bernasconi, A., Mattia, D., Olivier, A., and Hwa, G.G.C. 1999. Epileptiform discharges in the human dysplastic neocortex: in vitro physiology and pharmacology. Ann Neurol 46: 816–826. Barkovich, A.J., and Kjos, B.O. 1992a. Gray matter heterotopias: MR characteristics and correlation with developmental and neurologic manifestations. Radiology 182: 493–499. Barkovich, A.J., and Kjos, B.O. 1992b. Nonlissencephalic cortical dysplasias: Correlation of imaging findings with clinical deficits. AJNAJNR 13: 95–103.
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Barkovich, A.J., Kuzniecky, R.I., Dobyns, W.B., Jackson, G.D., Becker, L.E., and Evrard, P. 1996. A classification scheme for malformations of cortical development. Neuropediatrics 27: 59–63. Barkovich, A.J., Kuzniecky, R.I., Jackson, G.D., Guerrini, R., and Dobyns, W.B. 2001. Classification system for malformations of cortical development: update 2001. Neurology 57: 2168–2178. Barth, P.G. 1987. Disorders of neuronal migration. Can J Neurol Sci 14: 1–16. Bayer, S.A., and Altman, J. 1991. Neocortical Development. New York: Raven Press. Chen, H.X., and Roper, S.N. 2003. Reduction of spontaneous inhibitory synaptic activity in experimental heterotopic gray matter. J. Neurophysiol 89: 150–158. Cowan, D., and Geller, L.M. 1960. Long-term pathological effects of prenatal X-irradiation on the central nervous system of the rat. J Neuropathol Exp Neurol 19: 488–527. Crino, P.B., Duhaime, A.C., Baltuch, G., and White, R. 2001. Differential expression of glutamate and GABA-A receptor subunit mRNA in cortical dysplasia. Neurology 56: 906–913. Deukmedjian, A.J., King, M.A., Cuda, C., and Roper, S.N. (in press). The GABAergic system of the developing neocortex has a reduced capacity to recover from in utero injury in experimental cortical dysplasia. J Neuropathol Exp Neurol Ferrer, I., Oliver, B., Russi, A., Casas, R., and Rivera, R. 1994. Parvalbumin and calbindin-D28k immunocytochemistry in human neocortical epileptic foci. J Neurol Sci 23: 18–25. Hicks, S.P., D’Amato, C.J., and Lowe, M.J. 1959. The development of the mammalian nervous system. I. Malformations of the brain, especially the cerebral cortex, induced in rats by radiation; II. Some mechanisms of the malformations of the cortex. J Comp Neurol 113: 435–469. Hicks, S.P., and D’Amato, C.J. 1978. Effects of ionizing radiation on developing brain and behavior. In Studies on the Development of Behavior and the Nervous System. Ed. G. Gottleib. pp. 35–72. New York: Academic Press. Kellinghaus, C., Kunieda, T., Ying, Z., Pan, A., Luders, H.O., and Najm, I.M. 2004. Severity of histopathologic abnormalities and in vivo epileptogenicity in the in utero radiation model of rats is dose dependent. Epilepsia 45: 583–591. Kondo, S., Najm, I., Kunieda, T., Perryman, S., Yacubova, K., and Luders, H.O. 2001. Electroencephalographic characterization of an adult rat model of radiation-induced cortical dysplasia. Epilepsia 42: 1221–1227. Lent, R., and Schmidt, S.L. 1986. Dose-dependent occurrence of the aberrant longitudinal bundle in the brains of mice born acallosal after prenatal gamma irradiation. Dev Brain Res 25: 127–132. Leventer, R.J., Phelan, E.M., Coleman, L.T., Kean, M.J., Jackson, G.D., and Harvey, A.S. 1999. Clinical and imaging features of cortical malformations in childhood. Neurology 53: 715–722. Marín-Padilla, M. 1999. Developmental neuropathology and impact of perinatal brain damage. III. Gray matter lesions of the neocortex. J Neuropathol Exp Neurol 58: 407–429. Marin-Padilla, M., Tsai, R., King, M.A., and Roper, S.N. 2003. Altered corticogenesis and neuronal morphology in irradiation-induced cortical dysplasia. A Golgi-Cox study. J Neuropathol Exp Neurol 62: 1129–1143. Mattia, D., Olivier, A., and Avoli, M. 1995. Seizure-like discharges recorded in human dysplastic neocortex maintained in vitro. Neurology 45: 1391–1395. Mischel, P.S., Nguyen, L.P., and Vinters, H.V. 1995. Cerebral cortical dysplasia associated with pediatric epilepsy. Review of neuropathologic features and proposal for a grading system. J Neuropathol Exp Neurol 54: 137–153. Palmini, A., Andermann, E., and Andermann, F. 1994a. Prenatal events and genetic factors in epileptic patients with neuronal migration disorders. Epilepsia 35: 965–973.
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Palmini, A., Gambardella, A., Andermann, F., Dubeau, F., Cost da Costa, J., Olivier, A. et al.1994b. Operative strategies for patients with cortical dysplastic lesions and intractable epilepsy. Epilepsia 35(Suppl 6): S57–S71. Palmini, A., Gambardella, A., Andermann, F., Dubeau, F., Cost da Costa, J., Olivier, A. et al.1995. Intrinsic epileptogenicity of human dysplastic cortex as suggested by corticography and surgical results. Ann Neurol 37: 476–487. Powell, E.M., Campbell, D.B., Stanwood, G.D., Davis, C., Noebels, J.L., and Levitt, P. 2003. Genetic disruption of cortical interneuron development causes region- and GABA cell type-specific deficits, epilepsy, and behavioral dysfunction. J Neuro Sci 23: 622–631. Riggs, H.E., McGrath, J.J., Schwartz, H.P. 1956. Malformation of the adult brain (albino rat) resulting from prenatal irradiation. J Neuropathol Exp Neurol 15: 432–447. Roberts, M.R., Bittman, K., Li, W.-W., French, R., Mitchell, B., LoTurco, J.J., and D’Mello, S.R. 2000. The flathead mutation causes CNSspecific developmental abnormalities and apoptosis. J Neurosci 20: 2295–2306. Roper, S.N., Gilmore, R.L., and Houser, C.R. 1995. Experimentallyinduced disorders of neuronal migration produce an increased propensity for electrographic seizures in rats. Epilepsy Res 21: 205–219. Roper, S.N., Abraham, L.A., and Streit, W.J. 1997a. Exposure to in utero irradiation produces disruption of radial glia in rats. Dev Neuro Sci 19: 521–528. Roper, S.N., King, M.A., Abraham, L.A., and Boillot, M.A. 1997b. Disinhibited in vitro neocortical slices containing experimentally induced
cortical dysplasia demonstrate hyperexcitability. Epilepsy Res 26: 443–449. Roper, S.N. 1998. in utero irradiation of rats as a model of human cerebrocortical dysgenesis: a review. Epilepsy Res 32: 63–74. Sarkisian, M.R., Frenkel, M., Li, W., Oborski, J.A., and LoTurco, J.J. 2001. Altered interneuron development in the cerebral cortex of the flathead mutant. Cereb Cortex 11: 734–743. Sarkisian, M.R., Li, W., Di Cunto, F., D’Mello, S.R., and LoTurco, J.J. 2002. Citron-kinase, a protein essential to cytokinesis in neuronal progenitors, is deleted in the flathead mutant rat. J Neuro Sci 22: RC217. Spreafico, R., Tassi, L., Colombo, N., Bramerio, M., Galli, C., Garbelli, R. et al. 2000. Inhibitory circuits in human dysplastic tissue. Epilepsia 41(Suppl 6): S168–S173. Sun, X-Z., Inouye, M., Takagishi, Y., Hayasaka, S., and Yamamura, H. 1996. Follow-up study on histogenesis of microcephaly associated with ectopic gray matter induced by prenatal g-irradiation in the mouse. J Neuropathol Exp Neurol 55: 357–365. Taylor, D.C., Falconer, M.A., Bruton, C.J., and Corsellis, J.A.N. 1971. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Pyschiatry 34: 369–387. Ying, Z., Babb, T.L., Mikuni, N., Najm, I., Drazba, J., and Bingaman, W. 1999. Selective coexpression of NMDAR2A/B and NMDAR1 subunits proteins in dysplastic neurons of human epileptic cortex. Exp Neurol 159: 409–418. Zhu, W.J., and Roper, S.N. 2000. Reduced inhibition in an animal model of cortical dysplasia. J Neuro Sci 20: 8925–8931.
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25 Modeling Hypoxia-Induced Seizures and Hypoxic Encephalopathy in the Neonatal Period RUSSELL M. SANCHEZ AND FRANCES E. JENSEN
hypoxia with focal ischemia. These models are discussed in the final portion of this chapter.
This work was supported by National Institutes of Health RO1 NS31718 (F.E.J.) and RO1 NS047385 (R.M.S.) from the National Institute on Neurological Disorders and Stroke and by a Mental Retardation Research Center Grant P30 HD18655 from the National Institute of Child Health and Human Development.
METHODS OF GENERATION We previously developed a rodent model of perinatal hypoxia-induced seizures (Jensen, 1991). The model replicates the (1) unique age-dependent susceptibility to hypoxia, (2) refractory nature of the seizures to conventional AEDs, and (3) long-term increases in seizure susceptibility.
INTRODUCTION The neonatal period represents a stage of life characterized by a high incidence of seizures. The incidence of seizures is highest in the neonatal period, with recent reports ranging from 1–3.5/1000 live births (Aicardi and Chevrie, 1970; Saliba et al., 1999). The most common cause of neonatal seizures is hypoxic encephalopathy (Aicardi and Chevrie, 1970; Hauser et al., 1993; Volpe, 2001). Seizures occurring in this setting can be prolonged and refractory to conventional antiepileptic drug (AED) therapy. Sequelae of hypoxic encephalopathy include neuromotor and neurocognitive deficits, and, in some cases, epilepsy. Models of neonatal seizures are required to elucidate factors contributing to the high seizure susceptibility of the immature brain and age-specific mechanisms of epileptogenesis. In this chapter, we describe a rodent model of perinatal seizures induced by global hypoxia that can be used to address these questions. Similarly to the human, the neonatal rat exhibits a narrow age window of susceptibility to seizures induced by hypoxia, and these seizures result in a long-term increase in subsequent susceptibility to seizures and seizure-induced neuronal death. The model described here in detail is one of global hypoxia (lower inhaled oxygen concentration), while other models in the literature combine
Models of Seizures and Epilepsy
Animal Issues The model has been best developed in the Long-Evans rat. There is a clear age-dependence in susceptibility to seizure activity induced by hypoxic conditions. For LongEvans rats, postnatal days (P) 9–12 are recommended, with most seizure activity being observed at P10 in pups weighing between 18 and 22 g. Other rat strains have been used, and the Sprague-Dawley rat appears to be somewhat more mature for a given age, exhibiting peak seizures at P8–9 (Owens, 1997). Unpublished studies in mice (C57/BL6) reveal a window of susceptibility at P8–9. For both rat and mouse, animals at ages younger or older than these peak ages do not exhibit seizure activity.
Procedures Hypoxia The model uses a brief (15 minute) exposure to graded global hypoxia (7–4% O2) in an airtight chamber. Animals
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are placed in an airtight chamber (1 ft3) with light restraint of each limb. Body temperature is maintained at the ageappropriate basal temperature (32–33° C for the P10 LongEvans rat) by the use of a heating pad placed under the animal. An oxygen meter in the chamber is used to monitor % O2, and a fan in the ceiling of the chamber is activated to circulate the air. Nitrogen is infused into the chamber to create a graded hypoxia over 15 minutes: for the first 7 minutes to 7%, for the next 4 minutes to 6%, the next 3 minutes to 5% and the final minute to 4%. Following hypoxia, temperature is re-measured to assure that the pup is not hypothermic or hyperthermic, and then the pup is returned to its dam. Pups with end-hypoxia temperatures outside of 31–34° C are disqualified from further study (Jensen et al., 1991; Jensen et al., 1992; Jensen et al., 1993a; Jensen et al., 1995; Jensen et al., 1998).
Monitoring ECG Subcutaneous electrodes are placed on the dorsal trunk to measure heart rate. The heart rate typically declines by 20% over the course of the 15 minutes of hypoxia. Temperature Rectal temperature does not change significantly over the 15 minutes of hypoxia, due to the use of the heating pad. Blood Gas Experiments were designed to measure blood gases by a terminal left atrial puncture, and showed a decrease in O2 saturation to 62.3 ± 11.9% at the end of the hypoxic period. Behavioral Monitoring Animals are observed during and after the period of hypoxia for convulsive seizures. The number, time of onset (relative to hypoxia), and duration of tonic-clonic episodes are recorded. EEG Under ether anesthesia, epidural electrodes or depth electrodes are implanted 2–4 hours prior to hypoxia. Following a midline scalp incision, the scalp is retracted and burr holes are made over parietal cortices so that a pair of electrode plugs can be placed epidurally. Electrodes are fixed to the skull with dental cement and fast-acting glue, and the scalp wound is sutured closed. The procedure takes 5–7 minutes total per animal, and the animals are fully alert and awake within 15 minutes after the procedure.
For depth electrode placement, the animals are anesthetized 24 hours prior to hypoxia using pentobarbital. The pups are placed in a modified stereotaxic apparatus, and, following scalp incision and retraction, burr holes are placed bilaterally. Epidural electrodes are then lowered into the desired position using the stereotaxic apparatus. The electrodes are fixed to the skull with rapid drying glue and dental cement. Following scalp resuturing, the pups are permitted to recover for 24 hours.
Characteristics/Defining Features Behavior Tonic-clonic seizures are observed only during a narrow age window in the developing brain (day P10–12 for LongEvans rat) (Jensen et al., 1991) (see also Veliskova in this volume). The onset of tonic-clonic head and trunk movement is observed at 3–7 minutes into the hypoxic period. These episodes may be brief initially, between 5–15 seconds, and then, with time, they become increasingly prolonged. The number of episodes typically ranges from 3–15 per animal. Between the episodes, the animals breathe normally and move spontaneously. At the end of hypoxia, the animals exhibit lowered heart rate and breathing rate. In a given litter of male rat pups, approximately 80–90% of the animals exhibit tonic-clonic behavior. In the recovery period at room air, 50% of the rats will have brief episodes of recurrent tonic-clonic activity. Observation of rat pups at intervals over 4 days after hypoxia shows persistent brief events, decreasing over time. The possible occurrence of spontaneous seizures past 4 days has not been examined. EEG Features The EEG shows rapid spike activity that often starts at low amplitude and then builds over the first 5–10 seconds after onset. Animals at younger and older ages have no ictal activity, and rats over P20 have diminished EEG activity in response to hypoxia, with an isoelectric EEG developing in the adult group (Figure 1). There does not appear to be a focal onset of these seizures behaviorally, and depth electrodes reveal that seizure onset occurs almost simultaneously in neocortex and hippocampus (Jensen and Wang, 1996). Unlike pilocarpine- and kainate-induced seizures in the adult, hypoxia does not activate typical extrahippocampal limbic areas such as amygdala or pyriform cortex. Neuropathology Using routine H&E staining, there is no evident cell loss or gliosis. In situ end labeling for single-stranded DNA damage as a marker of cell death reveals no cell death when tested at 24 hour intervals up to 1 week following hypoxia-
Methods of Generation
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Metabolic Changes 31
PNMR spectroscopy reveals a rapid and reversible reduction in phosphocreatine and NTP during hypoxia at P10. This appears to occur in the absence of significant brain acidosis (increased lactate) compared to the response in older animals (Jensen et al., 1993b). Response to Antiepileptic Drugs The acute seizures and the long-term enhanced seizure susceptibility are blocked by systemic administration of the AMPAR receptor antagonist 6-nitro-7sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX), whereas there is no effect of the NMDAR receptor antagonists MK-801, GABAA receptor agonists (lorazepam and phenobarbital), or the conventional AED phenytoin (Jensen et al., 1995). Topiramate effectively suppresses the acute seizures in a dose-related manner with a calculated ED50 of 2.1 mg/kg, i.p. (Koh and Jensen, 2001b). Altered Network Excitability
FIGURE 1 EEG activity during exposure to graded hypoxia in representative animals from different age groups. A: Spike discharges are seen in isolation and in association with a myoclonic jerk (MJ) in P5 rats. B: Tracing from a rat in the P10–12 age group showing a train of spike discharges in association with tonic-clonic head and trunk activity (T/C). C: Rhythmic spike discharges seen in a rat in the P15–17 group, but no behavioral correlates or ictal EEG activity. D: Low amplitude spike activity in a rat from the P25–27 group, with no behavioral correlate. E: Adult rat (P50–60) response to hypoxia with a gradual decrement in EEG frequency and amplitude, until an isloelectric state is reached. (Reprinted with permission from Jensen et al., 1991.)
induced seizures at P10 (Sanchez et al., 2001). As a marker of neuronal populations involved in the seizures, c-fos immunocytochemistry revealed that expression was confined to deep neocortex, in layer VI and subplate neurons, and minimally in limbic structures, consistent with the depth electrode recordings (Jensen et al., 1993a).
A strength of this model is that hyperexcitability can be demonstrated in ex vivo hippocampal and cortical slices following seizures in vivo. There are immediate changes in network excitability, as hippocampal slices prepared from P10 pups euthanized at 10 minutes after recovery from hypoxia show increased amplitude and duration of longterm potentiation (LTP) and “in vitro kindling” in area CA1 (Jensen et al., 1998; Sanchez et al., 2001) (Figure 2). The immediate early gene c-fos is induced in neurons in both hippocampus and neocortex within 4 hours following seizures at P10 (Jensen et al., 1993a). Excitability remains increased within hippocampal networks in adulthood (Jensen et al., 1998). These functional changes occur in the absence of neuronal death, suggesting that they arise from mechanisms of plasticity that are intrinsic to the developing brain (Sanchez et al., 2001) Altered Gene and Protein Expression The pharmacologic sensitivity to AMPAR antagonists suggests that these receptors play a critical role in the genesis of hypoxia-induced seizures as well as their consequences (see the following). Notably, P10 falls within a developmental window during which there is an increase in calcium-permeable AMPA receptors in principal neurons of the hippocampus and neocortex compared to earlier or later in life. The permeability of AMPARs to calcium is determined by subunit composition, with the presence of the GluR2 subunit conferring impermeability to divalent cations (Burnashev et al., 1992; Hollmann and Heinemann, 1994). Hypoxia-induced seizures down-regulate GluR2 gene and
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Hypoxia-Induced Seizures and Hypoxic
+APV
Control
Encephalopathy in the Neonatal Period
Hypoxia
2nd Tetanus mV
4th Tetanus
6th Tetanus
FIGURE 2 Repeated tetanic stimulation in the presence of the NMDA receptor antagonist APV (100 PM) revealed that hippocampal slices from rat pups that experienced hypoxia-induced seizures 4-9 days earlier (at P 10) had the capacity for purely AMPA receptor-mediated epileptogenesis, whereas control slices did not. Shown are representative field recordings from a control slice and a slice from a same-age hypoxia-treated animal. Tetanus-induced afterdischarges were always observed in the presence of APV but increased with repeated stimulation only in the majority of slices (7 of 10) from hypoxia-treated animals. This type of increase was observed in only 1 of 10 control slices. (Reprinted with permission from Sanchez et al., 2001.)
B.
C
n
96 hours post-hypoxia GluR2
GluRl
GAPDH
GluR2
FIGURE 3
A: GluR2 mRNA was significantly decreased in neocortex and hippocampus by 48 hours after hypoxiainduced seizures. B: In situ hybridization showed that GluR2 mRNA was significantly decreased in the pyramidal cell layers within 48 hours after hypoxia-induced seizures at PlO. The most significant decrease was observed in CAl/CA2 pyramidal cells (arrows). C: GluR2 protein expression was significantly decreased within 96 hours after hypoxia-induced seizures. Western blots show that GluR2 protein expression was significantly decreased in hippocampus within 96 hours after hypoxia treatment. In contrast, GluRl expression did not change significantly after hypoxia-induced seizures. (See color insert.)
protein expression within 96 hours after the seizures, with a concomitant increase in the numbers of hippocampal pyramidal neurons that exhibit calcium-permeable AMPARs (Sanchez et al., 2001) (Figure 3). These receptors lacking GluR2 are functional: whole-cell recordings demonstrate inwardly rectifying AMPAR currents, and cobalt staining
indicates cation permeability in CA1 pyramidal neurons in hippocampal slices as early as 48 hours after seizures (Sanchez et al., 2001). This increase in permeability to calcium may have a number of consequences, including the activation of kinases, phosphatases, and other calciumactivated signaling cascades. Hypothetically, such pathways
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Long-Term Effects
may underlie the alterations in long-term function of neurons and synaptic circuits following hypoxia-induced seizures, in the absence of cell death. The lack of cell death in this model makes it particularly useful in investigating cellular and molecular changes related purely to the upregulation of network excitability, without the complication of those related to cell death pathways or anatomic reorganization due to cell loss. Imaging studies are not currently available.
LONG-TERM EFFECTS
and the mechanisms of possible long-term learning impairment await further study.
Seizure Susceptibility Long-term increases in susceptibility to seizures induced by chemical convulsants (pentylenetetrazol, flurothyl, and kainate) were observed in adult rats with prior seizures induced by hypoxia at P10 (Jensen et al., 1992). The most widely studied effects are those of kainate: hypoxic seizures significantly increase susceptibility to kainate-induced seizures as early as 96 hours after hypoxia (P14) (Jensen et al., 1992; Koh and Jensen, 2001b).
Neurobehavior Results of studies to assess the long-term neurobehavioral consequences of hypoxia-induced seizures have been variable. In early studies, Long-Evans rat pups that experienced hypoxia-induced seizures at P10 were shown to have no significant impairment of water maze performance compared to controls (Jensen et al., 1992). However, a recent similar study using Sprague-Dawley rats found that studies with hypoxia at P10 resulted in impairment of spatial learning at P45 (Yang et al., 2004). The basis for this variability
Enhanced Susceptibility to Later Life Status-Induced Neuronal Injury In addition to long-term increases in seizure susceptibility, animals exposed to hypoxia at P10, but not at older or younger ages, demonstrate enhanced neuronal injury when administered kainate at P21 or older. Perinatal hypoxia increases kainate-induced neuronal injury at P21 and P28/30 (Koh, 2001) (Figure 4). The areas most affected
FIGURE 4 Perinatal hypoxia increases susceptibility to later-life seizure-induced neuronal injury. DNA fragmentation as a marker of cell death in hippocampal CA1 pyramidal neurons. A–D: upper panels. Naïve rats after kainate-status at increasing ages. E–H lower panels. Rats with prior hypoxic seizures with kainate status at same ages as in upper panels. Note a lack of cell death with status at P14 in either group (A, E), but significantly more DNA fragmentation at older ages in the P21 (B, F) and P28 (C, G) rats that had prior hypoxia compared to naïve controls. At P45, cell death is extensive in both groups (D, H). (Reprinted with permission from Koh et al., 2004.)
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with enhanced injury include hippocampal areas CA3 and CA1.
Protection from Long-Term Effects with Preand Post-Treatment with Antiepileptic Drugs Pretreatment with either NBQX (20 mg/kg) or topiramate (30 mg/kg) results not only in reduction in the number of seizures during hypoxia but also prevents the long-term increases in seizure susceptibility and seizure-induced neuronal death (Koh and Jensen, 2001a). Furthermore, repeated doses of NBQX (20 mg/kg) or topiramate (30 mg/kg) given for 48 hours after hypoxia-induced seizures prevent the increase in susceptibility to KA seizure-induced hippocampal neuronal injury at P28/30 (Koh et al., 2004) (Figure 5).
Care must be taken to avoid hyperthermia by ensuring that the pups remain at their basal physiologic temperature, and the age window of susceptibility for seizures may vary depending on the rat strain or rodent species used. For rat strains other than Long-Evans, such as Sprague-Dawley, the age is approximately 2 days earlier (P8/9) (Owens et al., 1997), and the same is true for the mouse. This is likely due to strain- /species-specific differences in maturation of critical factors such as neurotransmitter receptor expression (Sanchez and Jensen, 2001).
Mortality Mortality is extremely low if hyperthermia is avoided— less than 2–5% of the animals succumb under the hypoxic conditions—and there is little to no mortality in the recovery phase.
LIMITATIONS Reproducibility Ease of Development This model utilizes the physiologic stimulus of hypoxia. The chamber is simple to construct from Plexiglas panes, and O2 concentration is easily measured during N2 infusion.
The model has been reproduced in different rat strains and in the mouse. Problems can arise if the age of the pup is miscalculated, and, hence, a general weight range typical of the susceptible age can be used to confirm age.
FIGURE 5 Posthypoxia 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f) quinoxaline-2,3-dione (NBQX) or topiramate (TPM) treatment prevents hypoxia-induced long-term susceptibility to neuronal injury. A: DNA fragmentation in the hippocampus 2 days after kainate (KA) seizures at P30. A, E: Control, KA alone on P30 without prior exposure to hypoxia on P10. B, F: An animal exposed to hypoxia on P10 and to KA on P30. C, G: An animal treated with NBQX after hypoxia on P10 and KA on P30. D, H: An animal treated with topiramate after hypoxia on P10 and KA on P30. Notice widespread KA seizure-induced DNA fragmentation throughout all subfields of the hippocampus of the animals previously exposed to hypoxia (B), whereas neuronal injury appears limited to CA1 and CA3 subfields in the control (A). NBQX (C) or TPM post-treated animal (D) is comparable to the control. Lower panels (E–H) are highermagnification views of CA3 hippocampal subfield. Bar, 50 mm. (Reprinted with permission from Koh et al., 2004.)
Need for Future Development and Existing Variants of the Model
Age-Related Features The hallmark of this model is its age-specificity. Younger animals exhibit occasional myoclonic jerks, and animals older than P12 show EEG suppression during hypoxia. The tolerance of hypoxia is clearly age-related, and ECG reveals that the heart rate response is inversely related to age, with adult animals rapidly becoming bradycardic and expiring within 4–5 minutes of hypoxia. In addition, adult animals exhibit a commensurate rapid drop in blood O2 saturation (Jensen et al., 1991).
NEED FOR FUTURE DEVELOPMENT AND EXISTING VARIANTS OF THE MODEL At present, this model reveals acute seizures followed by a chronically lowered seizure threshold and susceptibility to seizure-induced neuronal injury. Spontaneous seizures have been documented within the first week after hypoxia at P10 (Jensen et al., 1991), but a systematic study at later ages has yet to be done. Long-term video/EEG monitoring of animals after hypoxia-induced seizures should resolve this issue. Given the priming effect of hypoxia for chemoconvulsantinduced seizures later in life, an interesting direction might be to combine early hypoxia with other seizure stimuli later in life. For instance, perinatal hypoxia may precede childhood febrile seizures, and a study could be undertaken to evaluate whether the serial insults (first, hypoxia and second, hyperthermia) might lead to more pronounced molecular and cellular abnormalities or even to hippocampal sclerosis (Jensen and Sanchez, 2001). Hypoxia in the second postnatal week in the rodent could be used to screen transgenic mouse lines for seizure susceptibility. This may be particularly relevant for genetic mutations thought to underlie seizures in infancy, such as those related to infantile spasms. Recurrent hypoxia is a frequent occurrence in the clinic, and given the fact that P10 hypoxia causes immediate increases in network excitability in hippocampus, future evaluation of the effects of repeated hypoxia in the rat model may reveal new mechanisms underlying the clinical condition. Indeed, Owens et al. (1997) employed a model of repeated hypoxic insults on three successive days beginning at P8, and this also resulted in long-term decreases in seizure threshold. Further examination of recurrent hypoxia may be warranted, as the consequences of recurrent versus a single episode of hypoxia have not been extensively studied. While a major advantage of the hypoxia-induced seizure model is the lack of cell death, neonatal seizures can also occur in the setting of perinatal stroke. A number of rodent models of rodent hypoxia/ischemia exist and these tend to employ animals in between 3–14 days of age. The ischemia is most frequently accomplished by ligation of a cerebral
329
artery, usually a unilateral carotid or middle cerebral artery ligation. The lesion resulting from such experimental manipulation is that of a cerebral infarct, or stroke, and in the neonatal animal the insult is often accompanied by acute seizures followed by the development of regional neuronal necrosis. Hypoxia/ischemia can be modeled at this age (P10) using a method of unilateral carotid ligation in combination with hypoxia (Jensen et al., 1994). A high percentage of pups (70–80%) undergoing this procedure exhibit seizure activity of similar phenotype as those with hypoxia alone. Williams et al. employed a model of hypoxia/ischemia in neonatal rat that does not produce acute seizures, but results in a large percentage of animals becoming spontaneously epileptic in later life (Williams et al., 2004). In this model, P7 Sprague-Dawley rat pups (male and female) underwent unilateral carotid artery ligation, were allowed to recover for 2 hours, and then were exposed for 2 hours to 8% O2. They then were monitored visually for 1–2 hour periods for a total of 6 hours per week for 7–24 months before being euthanized for histologic analyses. Significant lesions were observed in the hippocampus ipsilateral to the ligation, and 40% of the surviving pups exhibited spontaneous motor seizures, the frequency of which was not correlated with the extent of hippocampal lesion. Timm staining revealed mossy fiber sprouting in the inner molecular layer of the ipsilateral dentate gyrus, but, interestingly, also revealed sprouting in the nonlesioned contralateral hippocampus only in the rats that had spontaneous seizures, suggesting the development of a “mirror focus” in the epileptic rats. Notably, no correlation was found between seizure rate and the extent of sprouting in either hippocampus. While this hypoxia/ischemia model does not necessarily mimic perinatal seizures, its primary advantage as a model of epileptogenesis associated with perinatal hypoxic/ischemic encephalopathy is that a large percentage of animals become epileptic as adults. Additionally, although morphologic changes in the hippocampus ipsilateral to the insult may be reactive to the lesion, the contralateral hippocampus may undergo epileptogenic changes that can be studied in the absence of lesion. Hypoxia/ischemia also has been combined with kainateinduced status epilepticus to examine the interaction between neonatal hypoxia-ischemia and seizures (Wirrell, et al., 2001; Yager et al., 2002). Several models have evaluated the effects of using chemoconvulsants in addition to hypoxia/ischemia in an attempt to determine whether seizures exacerbate the stroke-like pattern of neuronal injury. Wirrell et al. (2001) evaluated stroke size in P10 Wistar rats exposed to unilateral carotid artery ligation and hypoxia (8% O2 for either 15 or 30 minutes). Thirty minutes after hypoxia, they were given 3 mg/kg of kainate subcutaneously through a catheter inserted between the scapulae, which was then continuously infused with kainate at a rate
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of 2 mg/kg per hour for 3 hours. Histopathologic analyses at 3 and 20 days post-treatment showed that kainate-induced status epilepticus or 15 minutes of hypoxia/ischemia caused no injury at this developmental stage. However, 30 minutes of hypoxia/ischemia alone was sufficient to cause hippocampal lesions in the majority animals assessed at 3 days, and in half of the animals assessed at 20 days post-treatment. When kainate was combined with 30 minutes of hypoxia/ischemia, this injury was exacerbated. All of these animals showed significant hippocampal injury at 3 days and over 90% exhibited significant hippocampal injury at 20 days, and the mean damage score increased 6-fold at both time points. The authors concluded that neonatal status epilepticus alone does not induce neuropathologic injury in an otherwise healthy brain but can exacerbate damage when superimposed on hypoxia/ischemia. In another study, Yager et al. (2002) induced seizures using kainate injections administered following unilateral carotid ligation and 30 minutes of hypoxia (8% O2). Animals were euthanized for determination of cerebral glutamate levels at intervals after hypoxia/ischemia with and without kainate injections. Compared to the hypoxia/ischemia-alone group, the combined hypoxia/ischemia and kainate group showed a significant rise in glutamate during seizures and for the 4 hours of recovery (Yager et al. 2002). These studies suggest that prolonged neonatal seizures may exacerbate hypoxic/ischemic brain damage. However, other studies have failed to show this correlation. Towfighi et al. (1999) evaluated the effects of inducing seizures 24 hours and 6 hours prior to, versus following, unilateral carotid ligation and hypoxia in P7 and P13 rats. Seizures were induced with either kainate or fluorothyl. Animals exposed to seizures prior to hypoxia/ischemia actually showed modest protection for lesion size compared to naïve rat exposed to hypoxia/ischemia, and this protective effect was more prominent in the P13 rats. In contrast to Wirrell et al. (2001), the induction of seizures following hypoxia/ischemia did not appear to alter lesion size in either age group. These studies suggest that the interaction between seizures and excitotoxicity is complex in the developing brain and in need of further study. Each of these models was designed to address specific questions regarding the pathogenesis of injury and epilepsy consequent to common perinatal encephalopathies. Judicious use of these models is expected to continue to provide critical insights into mechanisms and treatment of perinatal seizures and hypoxic/ischemic encephalopathy in humans.
INSIGHTS INTO HUMAN DISORDERS Underlying Mechanisms AMPAR expression appears to be necessary and sufficient for the initiation of hypoxia-induced seizures in the
P10/12 rat. This is an age window characterized by a predominance of excitatory drive related to maturational differences in a number of factors that regulate neuronal excitability. Most relevant to this model is the transient overshoot in calcium-permeable glutamate receptor expression at this age. AMPARs are abundant on neocortical and hippocampal neurons, and they are relatively lacking in GluR2 (Sanchez et al., 2001; Kumar et al., 2002). In addition, GABA-mediated inhibition is lower than adult levels, as some GABAARs remain depolarizing due to the fact that the chloride transporter KCC2 is still below adult levels until P11/12 (Rivera et al., 1999). Information regarding the developmental regulation of such factors in the human will be important in translating these findings to the clinic. Thus far, examination of human postmortem tissue reveals that indeed AMPARs lacking GluR2 are prevalent in the term infant, with GluR2 subsequently increasing over the first years of life. Identifying such factors in the human will suggest rational therapeutic strategies for use in neonatal seizures associated with hypoxic encephalopathy.
Usefulness for Therapeutic Development Given that this model employs the same physiologic stimulus that occurs clinically, it may be superior to chemoconvulsant studies in the development of therapies for the human. For example, the demonstration of efficacy with NBQX, an agent that is not clinically available, led to the investigation of topiramate, as it was a clinically available modulator of AMPARs. As additional factors are revealed in the animal model, their presence can be confirmed in human studies to develop new therapeutic strategies.
References Aicardi, J., and Chevrie, J.J. 1970. Convulsive status epilepticus in infants and children. A study of 239 cases. Epilepsia 11: 187–197. Burnashev, N., Monyer, H., Seeburg, P.H., and Sakmann, B. 1992. Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron 8: 189–198. Hauser, W.A., Annegers, J.F., and Kurland, L.T. 1993. Incidence of epilepsy and unprovoked seizures in Rochester, Minnesota: 1935–1984. Epilepsia 34: 453–468. Hollmann, M., and Heinemann, S. 1994. Cloned glutamate receptors. Ann Rev Neurosci 17: 31–108. Jensen, F.E., Alvarado, S., Firkusny, I.R., and Geary, C. 1995. NBQX blocks the acute and late epileptogenic effects of perinatal hypoxia. Epilepsia 36(Suppl 10): 966–972. Jensen, F.E., Applegate, C.D., Holtzman, D., Belin, T., and Burchfield, J. 1991. Epileptogenic effect of hypoxia in the immature rodent brain. Ann Neurol 29: 629–637. Jensen, F.E., Firkusny, I., and Mower, G. 1993a. Differences in c-fos immunoreactivity due to age and mode of seizure induction. Mol Brain Res 17: 185–193. Jensen, F.E., Gardner, G., Williams, A., Gallop, P., Aizenman, E., and Rosenberg, P.A. 1994. The putative essential nutrient pyrroloquinoline quinone is neuroprotective in a rodent model of hypoxic/ischemic brain injury. Neuroscience 62: 399–406.
References Jensen, F.E., Holmes, G., Lombroso, C.T., Blume, H., and Firkusny, I. 1992. Age-dependent long term changes in seizure susceptibility and neurobehavior following hypoxia in the rat. Epilepsia 33(6): 971–980. Jensen, F.E., and Sanchez, R.M. 2001. Why does the developing brain demonstrate heightened susceptibility to febrile and other provoked seizures? (In Press). In Febrile Seizures. Ed. T.Z. Baram, and S. Shinnar. New York: Butterworth Heinneman. Jensen, F.E., Tsuji, M., Offut, M., Firkusny, I., and Holtzman, D. 1993b. Age dependent effects of hypoxia on high energy phosphate concentrations and pH in the rat brain. Dev Brain Res 73: 99–105. Jensen, F.E., and Wang, C. 1996. Hypoxia-induced hyperexcitability in vivo and in vitro in the immature hippocampus. Epilepsy Research 26(1): 131–140. Jensen, F.E., Wang, C., Stafstrom, C.E., Liu, Z., Geary, C., and Stevens, M.C. 1998. Acute and chronic increases in excitability in rat hippocampal slices after perinatal hypoxia in vivo. J Neurophysiol 79: 73–81. Koh, S., and Jensen, F.E. 2001a. Topiramate blocks perinatal hypoxiainduced seizures in rat pups. Ann Neurol 50: 366–372. Koh, S., and Jensen, F.E. 2001b. Topiramate blocks seizures in a rodent model of perinatal hypoxic encephalopathy. Ann Neurol 50: 366–372. Koh, S., Tibayan, F.D., Simpson, J., and Jensen, F.E. 2004. NBQX or topiramate treatment following perinatal hypoxia-induced seizures prevents later increases in seizure-induced neuronal injury. Epilepsia 45: 569–575. Kumar, S.S., Bacci, A., Kharazia, V., and Huguenard, J.R. 2002. A developmental switch of AMPA receptor subunits in neocortical pyramidal neurons. J Neurosci 22: 3005–3015. Owens, J., Robbins, C.A., Wenzel, J., and Schwartzkroin, P.A. 1997. Acute and chronic effects of hypoxia on the developing hippocampus. Ann Neurol 41: 187–199. Rivera, C., Voipio, J., Payne, J.A., Ruusuvuoiri, E., Lahtinen, H., Lamsa, K., Pirvola, U. et al. 1999. The K co-transporter KCC2 renders GABAhyperpolarizing during neuronal maturation. Nature 397(6716): 251–255. Saliba, R.M., Annegers, J.F., Waller, D.K., Tyson, J.E., and Mizrahi, E.M. 1999. Incidence of neonatal seizures in Harris County, Texas, 1992–1994. Am J Epidemiol 150: 763–769.
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Sanchez, R.M., and Jensen, F.E. 2001. Maturational aspects of epilepsy mechanisms and consequences for the immature brain. Epilepsia 42: 577–585. Sanchez, R.M., Koh, S., Rio, C., Wang, C., Lamperti, E.D., Sharma, D., Corfas, G. et al. 2001. Decreased glutamate receptor 2 expression and enhanced epileptogenesis in immature rat hippocampus after perinatal hypoxia-induced seizures. J Neurosci 21: 8154–8163. Skradski, S., and White, H.S. 2000. Topiramate blocks kainate-evoked cobalt influx into cultured neurons. Epilepsia 41(Suppl 1): S45–S47. Towfighi, J., Housman, C., Mauger, D., and Vannucci, R.C. 1999. Effect of seizures on cerebral hypoxic-ischemic lesions in immature rats. Brain Res Dev Mar 12; 11(31–32): 83–95. Volpe, J.J. 2001. Neurology of the Newborn. pp. 217–276. Philadelphia: Saunders. White, H.S., Brown, S.D., Woodhead, J.H., Skeen, G.A., and Wolf, H.H. 2000. Topiramate modulates GABA-evoked currents in murine cortical neurons by a nonbenzodiazepine mechanism. Epilepsia 41(Suppl 1): S17–S20. Wirrell, E.C., Armstrong, E.A., Osman, L.D., and Yager, J.Y. 2001. Prolonged seizures exacerbate perinatal hypoxic-ischemic brain damage. Pediatr Res 50(4): 445–454. Williams, P.A., Dou, P., and Dudek, F.E. 2004. Epilepsy and synaptic reorganization in a perinatal rat model of hypoxia-ischemia. Epilepsia 45: 1210–1218. Yager, J.Y., Armstrong, E.A., Miyashita, H., and Wirrell, E.C. 2002. Prolonged neonatal seizures exacerbate hypoxic-ischemic brain damage: correlation with cerebral energy metabolism and excitatory amino acid release. Dev Neurosci 24(5): 367–381. Yang, S.N., Huang, C.B., Yang, C.H., Lai, M.C., Chen, W.F., Wang, C.L., Wu, C.L. et al. 2004. Impaired SynGAP expression and long-term spatial learning and memory in hippocampal CA1 area from rats previously exposed to perinatal hypoxia-induced insults: beneficial effects of A68930. Neurosci Lett 371: 73–78. Zona, C., Ciotti, M.T., and Avoli, M. 1997. Topiramate attenuates voltagegated sodium currents in rat cerebellar granule cells. Neurosci Lett 231: 123–126.
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FIGURE 25--3 A: GluR2 mRNA was significantly decreased in neocortex and hippocampus by 48 hours after hypoxia-induced seizures. B: In situ hybridization showed that GluR2 mRNA was significantly decreased in the pyramidal cell layers within 48 hours after hypoxia-induced seizures at P10. The most significant decrease was observed in CA1/CA2 pyramidal cells (arrows). C: GluR2 protein expression was significantly decreased within 96 hours after hypoxia-induced seizures. Western blots show that GluR2 protein expression was significantly decreased in hippocampus within 96 hours after hypoxia treatment. In contrast, GluR1 expression did not change significantly after hypoxiainduced seizures.
FIGURE 3 6 - 3 Timm staining was positive in the animals killed during the latent period, 20 days after selfsustaining status epilepticus (SSSE) (A), and 6 hours after the first spontaneous seizure (B). Timm staining in the animals euthanized 2-month after SSSE, after a total of 203 (C) and 42 (D) spontaneous seizures. (Reproduced from Mazarati et al., 2002a. Copyright Blackwell Publishing, 2002.)
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26 Complex Febrile Seizures—An Experimental Model in Immature Rodents CÉLINE M. DUBÉ AND TALLIE Z. BARAM
1978) do not lead to long-term sequelae. Thus, neither epilepsy nor cognitive dysfunction are described in children with a limited number of short febrile seizures (Verity et al., 1985, 1998; Berg and Shinnar, 1996a). However, complex febrile seizures, defined as prolonged, having focal features, or that recur within a single febrile episode, are more controversial (Annegers et al., 1987; Berg and Shinnar, 1996b). Whereas there is limited epidemiologic evidence for adverse outcome, retrospective analyses strongly link a history of prolonged febrile seizures to temporal lobe epilepsy (TLE) (Cendes et al., 1993; French et al., 1993; Hamati-Haddad and Abou-Khalil, 1998; Theodore et al., 1999). The controversy over the clinical outcome of prolonged febrile seizures, and the potential that they may promote epileptogenesis, provides a strong impetus for modeling them. Animal models, unlike the human condition, allow direct investigation of the potential consequences of these seizures. Hypotheses about mechanisms by which febrile seizures might influence the developing brain can be formulated and tested directly, using diverse neuroanatomic, molecular, electrophysiologic, and imaging methods. A second impetus for developing a model of febrile seizures is to understand the mechanisms by which they are generated. Even if not causing epilepsy, febrile seizures are common, frightening, and associated with iatrogenic complications from treatment; their prevention requires an understanding of how they arise. Thirdly, febrile seizures constitute a common manifestation of hyperexcitability in the developing human brain. Because they do not occur in adults, they provide an excellent tool for studying the unique characteristics (and underlying mechanisms) of abnormal excitability during development. In addition, as a generalizable model of developmental hyperexcitability,
This chapter describes a rodent (rat and mouse) model of prolonged febrile seizures. Study of these seizures is important, because they are common, illustrative of seizures—and of abnormal excitability—in the immature brain, and associated with subsequent epilepsy. Seizures generated in this model are evoked by hyperthermia, via mechanisms common to those of fever. The seizures are limbic in semiology and involve the hippocampal formation. When sustained for ~20 minutes, these experimental complex febrile seizures result in transient neuronal injury but no cell death. The seizures induce enduring structural, molecular, and functional changes in the hippocampal formation, including altered expression of the hyperpolarizationactivated cyclic nucleotide gated cation (HCN) channels. Threshold temperature for evoking the seizures provides a ready measure of excitability that is suitable for pharmaceutical screens, as well as for screening for the effects of genetic mutations/gene engineering on seizure susceptibility.
GENERAL DESCRIPTION OF THE MODEL: WHAT DOES IT MODEL? The model described here recapitulates the essential elements of prolonged febrile seizures in the human. Febrile seizures are the most common type of seizures in infants and young children, with a prevalence of 2–14% around the world (see Stafstrom, 2002 for a recent review). For short or simple febrile seizures, epidemiologic and prospective studies as well as retrospective analyses have suggested that seizures with duration of less than 10 (Annegers et al., 1987; Berg et al., 1997) or 15 minutes (Nelson and Ellenberg,
Models of Seizures and Epilepsy
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experimental febrile seizures provide a useful model for screening potential anticonvulsants for developmental epilepsies. Finally, genes that may lead to increased excitability might render the developing animal more susceptible to developing febrile seizures: the model provides a valuable instrument for screening epilepsy- or seizurepromoting genes. The model described here is suitable for investigating the five types of questions mentioned above. This chapter discusses the generation of either short or prolonged experimental febrile seizures in the immature rat, as well as adaptation of the model to the immature mouse, where it can be coupled to the power of mouse genetics.
Controls Induction of seizures using hyperthermia involves two variables: (1) hyperthermia and (2) hyperthermia-induced seizures. Therefore, to ascertain that any consequence of hyperthermic seizures is truly a result of the seizures rather than of the hyperthermia per se, hyperthermic controls must be used. These are generated by subjecting age-matched littermates to the same degree and duration of hyperthermia but preventing seizures using short-acting barbiturates (pentobarbital intraperitoneally; Dubé et al., 2000; Brewster et al., 2002). Fever Versus Hyperthermia
METHODS OF GENERATION OF EXPERIMENTAL PROLONGED FEBRILE SEIZURES Procedural Issues: Animal Species and Age, Controls, Fever Versus Hyperthermia Species A model of experimental prolonged febrile seizures was first developed in the immature rat (Baram et al., 1997; Toth et al., 1998, Dubé et al., 2000) then adapted successfully to several strains of mice (Dubé et al., 2005a). Age In the human, febrile seizures occur between ~3 months and ~5 years of age with a peak incidence at 18 months (Nelson and Ellenberg, 1981; Hauser, 1994). Comparing the development of the hippocampal formation between humans and rodents indicates that the first year of human life may be equivalent to postnatal days 7–14 (P7–14) in the rat (Table in Avishai-Eliner et al., 2002). Therefore, an appropriate rat model of febrile seizures should use rats at a developmental stage at which human infants are most susceptible to febrile seizures. In addition, systematic analysis of the temperatures required to elicit hyperthermic seizures shows that this susceptibility is age-dependent, with a nadir of threshold temperature during the second week of life (Olson et al., 1984; Hjeresen and Diaz, 1988; Morimoto et al., 1990, 1991; Baram et al., 1997). We have elicited experimental febrile seizures in P6–17 rats and in P11–17 mice and have chosen to use P10–11 for rat and P14–15 in mouse for three reasons: (1) Consideration of hippocampal development (see preceding); (2) these ages fall at the nadir of threshold temperature, and, remarkably, these threshold temperatures are close to those required in normal children (Berg et al., 1992); and (3) the behavioral seizures at these ages are reliable, reproducible, and stereotyped.
Febrile seizures in humans are convulsions associated with fever. The model described here relies on hyperthermia rather than fever to evoke seizures. We think this is justified for three reasons: (1) Hyperthermia without fever also causes seizures in infants (in the setting of anticholinergics or theophylline overdose or hot water baths); (2) it is almost impossible to provoke true fever (>1°C increase of core or brain temperature) in infant rats (Heida et al., 2003). Importantly, fever and hyperthermia may utilize common mechanisms to elicit seizures: The pyrogenic cytokine IL-1 contributes to fever generation and, conversely, fever leads to IL-1 production within hippocampus (Takao et al., 1990; Ban et al., 1991; Cartmell et al., 1999; Gatti et al., 2002). These facts support the involvement of IL-1 in the mechanisms of both febrile and hyperthermic seizures. Others (Blake et al., 1994; Haveman et al., 1996) and our data (Dubé et al., 2005a) demonstrate that release and synthesis of IL-1 are governed primarily by the actual increase of temperature (hyperthermia) rather than other components of the febrile response. Thus, mechanisms by which fever and hyperthermia induce seizures may be similar, sharing cytokines as a key mediator (Rothwell and Luheshi, 1994, 2000; Gatti et al., 2002; Dubé et al., 2005a).
Procedures General Procedure Experimental prolonged febrile seizures are induced in 1–2 rats or mice at a time. Features of the paradigm that are common to both species will be discussed, followed by points that distinguish each specie. Procedures for EEG recordings will be found in the Monitoring section, following. Experiments are initiated at 8–11AM to minimize potential diurnal variability in seizure susceptibility. Pups are placed on a euthermic pad at least 15 minutes before the onset of hyperthermia. Baseline core (rectal) temperature is measured using a hypodermic needle probe (HYP2-21-11/2-TG-48-OSTM, Omega, Stamford, CT) connected to a temperature indicator (DP41-TC, Omega Stamford, CT). To
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Methods of Generation of Experimental Prolonged Febrile Seizures
prevent variability of baseline core temperature, pups are prevented from climbing on top of one another. Two pups are placed in a 3-liter jar (the hyperthermia chamber) fitted with a cloth pad taped to its bottom, to absorb excreta and prevent exposure to heated glass. The chamber is covered by a styrofoam lid with a central hole and placed in a Faraday cage. After measuring onset core temperatures, a regulated stream of moderately heated air is blown obliquely through the hole in the lid using a commercial adjustable hair dryer (Prostyler 1600W, 097RIR, Conair, set at medium). The goal is to increase core and brain temperatures ~2° C/minute until seizure onset, when seizure temperature threshold is measured. Times of hyperthermia onset and seizure onset are noted. In our hands, increasing the temperature at ~2° C/minute leads to a latency of 2–4 minutes and an excellent correlation of brain and core temperatures. Prolonged Experimental Febrile Seizure Protocol These are generated by maintaining hyperthermia (40–42° C) for 30 minutes, with seizure onset considered time 0. Once seizures commence, hyperthermia is continued, and the temperature measured every 2 minutes. If core temperature is >41.5° C, pups are removed to a cool metal surface for 2 minutes to prevent excessive heating. The cycle of warming for 2 minutes, temperature measure, and continued warming or time-out is maintained for a total of 30 minutes, resulting in seizures of ~24.1 minute (Table 1). The procedure yields seizure threshold temperatures of ~40.8° C and mean duration of hyperthermia of ~27.9 minute. Recovery Procedure Following hyperthermia pups are submerged (1 second) in room temperature water, hydrated orally (~0.1 ml water) using a 1 cc syringe, and transferred to a cool metal surface until their core temperature reaches 32–34° C. They are kept on a euthermic pad for 1 hour then returned to home cages. In our hands, rat and mouse dams receive the pups without difficulty and initiate grooming and nursing. We keep total separation time from the dams to less than 4 hours, and have observed no weight loss or growth retardation after the procedure.
Monitoring Temperature Human febrile seizures are typically elicited by temperatures higher than 38.5° C, and a study in Swedish children suggested a normal threshold temperature of 40.9° C (Knudsen, 1996). In addition, brain temperatures higher than 43° C may provoke neuronal injury (Burger and Fuhrman, 1964; Germano et al., 1996). Therefore, this model limits maximal temperatures to ~42° C, and importantly correlates the measured (core) temperature to that of brain. While the goal of the monitoring is to maintain core and brain temperatures in the 40–42° C range, it is not feasible to monitor brain temperature chronically in P10 rats (or P14 mice) because of the size of available implantable probes. Therefore, we have correlated brain and core temperatures in the model, under standard conditions of the velocity and temperature of the heating air stream (see following text). This is important, because core temperature may rise disproportionately to that of brain. Figure 1 shows a series of measurements of core- and brain-temperatures in individual rats. Baseline core values were measured at 5-minute intervals over a 25-minute period, and averaged 32.88 ± 0.07°C (n = 10) (Figure 1A). These temperatures were virtually identical to those obtained in rats upon removal from home cages (not shown). Under the same conditions, brain temperatures for an individual rat were also highly consistent (Figure 1B). Interestingly, mean euthermic (normal) brain temperatures were 2.8° C higher than mean core values. This divergence was temperature-dependent and approached zero at temperature ranges provoking seizures. Thus, at the onset of experimental prolonged febrile seizures (i.e., at threshold temperatures), brain temperatures averaged 40.7 ± 0.2° C (n = 29) and core temperatures were 40.88 ± 0.3° C (n = 31; Dubé et al., 2005b). These data indicate that, within the confines of the parameters recommended for this model, core measurements provide an adequate approximation of brain threshold temperatures for experimental febrile seizures. It is recommended that each laboratory perform initial experiments correlating brain and core temperatures under the conditions it uses. EEG Monitoring EEG monitoring is performed whenever the model is being set-up, then periodically and when a new species or
TABLE 1 Parameters of Seizures and of Hyperthermia in the Immature Rat Febrile Seizures Model Group parameters Experimental febrile seizures Hyperthermic controls
Maximal Temperature (°C)
Threshold temperature (°C)
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26
42.3 ± 0.047
n
Hyperthermia duration (min)
Seizure duration (min)
40.8 ± 0.06
27.9 ± 0.07
24.1 ± 0.1
—
28.4 ± 0.23
—
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Recovery Sedation in the hyperthermic control groups persists typically for ~1 hour; these rats are hydrated orally and handled as described in the preceding for the other groups: Following hyperthermia, pups are removed to a euthermic pad and hydrated. They are returned to home cages when fully awake and recovered. Little evidence of dehydration is found in the hyperthermic control and experimental prolonged febrile seizures groups (<3% loss of body weight). Ease of development and reliability are discussed in the limitations section (IV). Occurrence of Spontaneous Seizures Whereas we have previously published on the absence of spontaneous seizures in this model, these observations were based on daytime intermittent observation and EEG recording (Dubé et al., 2000). Using nocturnal simultaneous videoEEG recordings, there is preliminary evidence for the occurrence of spontaneous seizures (epilepsy) in a subgroup of the rats that had sustained experimental febrile seizures. These studies are ongoing.
CHARACTERISTICS/DEFINING FEATURES Behavioral Features
FIGURE 1 Correlation of brain and core temperatures in the euthermic immature rat. A: Core temperatures of individual rats are tightly clustered within and among individuals. Diamonds denote means ± standard errors of five measurements obtained over 25 minutes from individual rats kept on a euthermic pad. (Values for rat #1 were too clustered to visualize error bars.) B: Brain temperatures, obtained under the same conditions by directing the probe through the cranial suture to the dura. These temperatures were on average 2.8° C higher than core ones (modified from Dubé et al., 2005b, with permission).
mouse strain is added. Methods for implanting electrodes in limbic structures of immature rodents have been published (Baram et al., 1992; Dubé et al., 2000, 2005a). See EEG features of the seizures in the section following. Behavior Behavior is determined for each 2-minute epoch within the 30 minutes of hyperthermia. The characteristic behaviors for mice and rats are described in Behavioral Features, following.
The initial seizure behaviors in both immature rats and mice consist of acute sudden arrest of the hyperthermiaevoked running and other types of hyperactivity. Freezing (altered consciousness?) is seen in both species, followed rapidly by oral automatisms. This sequence at seizure onset is typical for human and animal seizures of limbic origin. The subsequent course of the seizures differs somewhat between immature rats and mice. In rats, freezing (Racine stage 0) is followed by oral automatisms (chewing/biting, Racine stage 1) and often forelimb clonic movements (Racine stage 3) (Racine, 1972). A typical behavior, which can be used for consistent recording of threshold temperature, is the sudden chewing/biting of an extremity. Later in the seizures, tonic body flexion may occur and may indicate an eventual propagation of the seizure to the brainstem. In mice, onset of experimental febrile seizures (when seizure threshold temperature is measured) is heralded by sudden immobility, with reduced response to stimulation (altered consciousness), and often with facial automatisms (chewing, vibrissae movements). Tonic body flexion is not observed in mice.
Electrographic Seizures During development of the model, to investigate the epileptic nature of the behavioral seizures provoked by
Characteristics/Defining Features
hyperthermia and determine their origin and location within the brain, electrophysiologic recordings from multiple brain sites have been conducted in rats and mice. As mentioned above, initial seizure behaviors in both species consist of acute sudden cessation of hyperthermia-evoked hyperactivity, with freezing and oral automatisms, a typically limbic behavior. Indeed, bipolar electrode recordings from basal amygdala, dorsal hippocampus, and frontoparietal cortex of freely moving pups (Baram et al., 1992, 1997; Dubé et al., 2000) suggested the onset of EEG spike-trains in hippocampus and amygdala that coincided with the immobility and oral automatisms, with no change or flattening of cortical EEG activity. The ictal EEG activities during experimental febrile seizures in the P10–11 rat have been published (Dubé et al., 2000; Brewster et al., 2002). They consist of trains of spikes and spike-waves with progressively increasing amplitude in hippocampal and amygdala leads, with variable progression to the cortex. The EEG patterns in the mouse are quite similar and are shown below (Figure 2).
Neuropathology In mature hippocampus, seizure-induced alteration of brain excitability and development of TLE are generally believed to require death of specific populations of hippocampal neurons (Houser, 1999).
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Experimental prolonged febrile seizures in immature rodent lead neither to acute neuronal death, as determined using the in situ end labeling technique (Toth et al., 1998), nor to long-term neuronal death, evaluated up to 3 months after the seizures (Bender et al., 2003a). This neuronal sparing includes populations that have been shown to be the most vulnerable to seizures-induced cell death in adult models of limbic seizures, including specific subpopulations of interneurons (Houser and Esclapez, 1996; Buckmaster and Dudek, 1997) and mossy cells (Sloviter, 1994). However, experimental prolonged febrile seizures induce transient neuronal injury in limbic structures including hippocampus, amygdala, and perirhinal cortex, visualized using silver staining (Toth et al., 1998), but not Fluoro-Jade (Dubé et al., 2004). The distribution of argyrophilic neuronal injury observed in this model overlaps the structures involved in TLE, including hippocampal pyramidal cells, lateral basal and central nuclei of amygdala, and the perirhinal cortex, and persists up to 2 weeks. Because argyrophilia (but not Fluoro-Jade) targets cytoskeletal proteins, the results using these two methods implicate these proteins in the transient changes evoked by experimental febrile seizures. Among other potential structural processes that may be provoked by seizures and may contribute to the epileptogenic process, prolonged febrile seizures do not modify granule cell neurogenesis in the immature hippocampal
FIGURE 2 Hippocampal EEG recordings from immature mouse (P14): Low amplitude baseline trace with variable rhythms is replaced by epileptiform discharges (trains of spikes and spike-and-waves) evoked by hyperthermia. The behavioral correlates of the EEG seizure consist of sudden behavioral arrest associated with facial limbic automatisms. Calibration: vertical, 50 mV; horizontal, 2 seconds.
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formation (Bender et al., 2003a). Similarly, sprouting is not prominent after these seizures (Bender et al., 2003a).
Imaging Using serial magnetic resonance imaging (MRI) on a 4 Tesla scanner, prolonged experimental febrile seizures were found to increase T2 signal in limbic structures in 75% of animals at 24 hours and in 87.5% of the seizure group a week later. These involved dorsal hippocampus, amygdala, piriform cortex and the medial ventroposterior thalamic nucleus (Dubé et al., 2004). The T2 signal changes were not accompanied by evidence of neuronal injury or death in these regions (assessed using Fluoro-Jade), but may indicate other cellular pathologic processes that promote epileptogenesis.
Molecular Changes Seizure-evoked hippocampal hyperexcitability was apparent by a week after the seizures and persisted long term (Dubé et al., 2000; Chen et al., 1999, 2001), indicating the occurrence of profound molecular changes. The spectrum and sequence of molecular changes evoked by experimental prolonged febrile seizures in this model have not yet been fully explored. In addition, the molecular events that have already been elucidated are complex and fall outside the scope of this chapter. The interested reader is referred to: Toth et al., 1998; Chen et al., 1999, 2001; Brewster et al., 2002, 2004; Bender et al., 2003a, b; Santoro and Baram, 2003; Dubé et al., 2005a. Briefly, within hours after seizures, regulation of calcium (Ca2+) entry was altered as a consequence of transient down-regulation of GluR2 expression and the formation of Ca2+-permeable AMPA receptors (Pellegrini-Giampietro et al., 1997; Eghbal-Ahmadi et al., 2001) that also permitted Zn2+ accumulation in CA3 neurons (Yin et al., 2002). Perhaps as a consequence of these events, the transcription of other specific channels was altered, starting already by 24–48 hours (Brewster et al., unpublished): The mRNA levels of the hyperpolarization-activated cyclic nucleotide-gated cation channel type 1 were reduced, whereas HCN2 channel gene expression was enhanced (Brewster et al., 2002). These mRNA levels were followed by a “molecular switch” of HCN1/HCN2 ratios also at the protein level (Brewster et al., 2005), and should promote hyperpolarization-evoked rebound neuronal firing (Chen et al., 2001; Santoro and Baram, 2003), i.e., enhanced hippocampal excitability. It is notable that expression of HCN channels is altered also in the “sclerosed” hippocampus of humans with severe TLE (and typically a history of early life seizures; Bender et al., 2003b). These expression changes, consisting of increased HCN1, may potentially be neuroprotective (Bender et al., 2003b; Santoro and Baram, 2003).
Response to Antiepileptic Drugs/Usefulness in Screening Drugs In human febrile seizures, phenobarbital, valproate, and the benzodiazepines are effective in controlling febrile seizures while other anticonvulsants including phenytoin and carbamazepine are ineffective (e.g., Knudsen, 2002). This anticonvulsant efficacy profile is recapitulated in the model of febrile seizures (Dubé and Baram, unpublished data). Therefore, the model should be useful for screening pharmaceutical agents as potential anticonvulsants in the developing brain.
Genetic Influence Using this model, febrile seizures have been generated in rats and several strains of mice. Seizures are evoked in virtually all immature rodents (P10–11 rat; P14–15 mice) regardless of genetic background. However, genetic background affects the threshold temperatures for eliciting the seizures: temperatures resulting in seizures in, e.g., C57BL mice (39.7 ± 0.3° C, n = 17) are significantly lower than those in mice of the 129/Sv strain (41.3 ± 0.2° C, n = 21; Dubé et al., 2005a); furthermore, genetic manipulation of the interleukin 1 gene receptor led to significant elevation in threshold temperature, implicating this gene in the mechanisms of febrile seizure generation (Dubé et al., 2005a). Therefore, the model is suitable for testing the effects of gene(s) of interest on neuronal excitability and seizure susceptibility in the developing brain.
LIMITATIONS As implied in the previous paragraphs, the setup of this model simply does not require costly equipment. However, it does require certain procedures in each laboratory to validate the nature of the evoked seizures, the absence of hyperthermia-induced injury, and the correlation of brain and core temperatures: 1. Because routine monitoring involves only core temperature, maintaining consistent conditions after verifying the correlation of core and brain temperatures is important. This involves using the same hair dryer with the same setting, held at a constant distance and angle. Rigorous training of personnel and periodic correlation of core and brain temperatures are recommended. 2. To avoid direct hyperthermic injury and agonal, terminal seizures, temperatures should be carefully controlled and maintained at 40–42° C. Specifically, those higher than 43° C may lead to direct injury. As an additional requirement, a hyperthermic control group (with hyperthermia but without seizures, achieved by barbiturate pre-admin-
References
istration) is required in studies assessing the effects of the seizures. 3. Mice have a somewhat wider spectrum of seizure behaviors. In addition, genetic background has a profound effect on susceptibility to these seizures (see preceding section, Genetic influence). Therefore, attention to controls of appropriate genetic background when assessing the consequence of any gene alteration is necessary. While mortality rate is nil, prevention of morbidity requires maintaining distance from the source of heated air, avoiding excessive temperatures, and using padded hyperthermia chambers. These precautions, as well as rinsing the chamber with room temperature water between rounds, eliminate burns, particularly of the paws.
Reproducibility This model of prolonged febrile seizures is highly reproducible. In our hands more than 99% of the rats develop prolonged seizures, and threshold temperatures in over 500 rats have been in a narrow range. In addition, to our knowledge at least a dozen laboratories around the world have applied the model to both rats and mice. Because this is not an extreme model (in comparison, for example, to status epilepticus, that kills 100% of a given neuronal population), a degree of interanimal variability is unavoidable (Brewster et al., 2002; Bender et al., 2003a; Dubé et al., 2005a). The molecular, structural, and imaging changes are evident in >87.5% (MRI; Dubé et al., 2004)— >90% (HCN channel changes) of rats, but not in 100%. Therefore, size of experimental groups (n) larger than 6 is recommended.
INSIGHTS INTO HUMAN DISORDERS This model provides insight into febrile seizures and their mechanisms as well as their potential consequences. It provides a model of epileptogenesis in the developing brain with molecular, physiologic, and functional changes, as well as the onset of spontaneous ones (epilepsy). In addition, it provides a screen for manipulations aiming to modify excitability (seizure susceptibility) in the developing brain, be they genetic or pharmaceutical.
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Baram, T.Z., Hirsch, E., Snead, O.C. III, and Schultz, L. 1992. Corticotropin-releasing hormone-induced seizures in infant rats originate in the amygdala. Ann Neurol 31: 488–494. Baram, T.Z., Gerth, A., and Schultz, L. 1997. Febrile seizures: an appropriate-aged model suitable for long-term studies. Dev Brain Res 98: 265–270. Bender, R.A., Dubé, C., Gonzalez-Vega, R., Mina, E.W., and Baram T.Z. 2003a. Mossy fiber plasticity and enhanced hippocampal excitability, without hippocampal cell loss or altered neurogenesis, in an animal model of prolonged febrile seizures. Hippocampus 13: 399–412. Bender, R.A., Soleymani, S.V., Brewster, A.L., Nguyen, S.T., Beck, H., Mathern, G.W., and Baram, T.Z. 2003b. Enhanced expression of a specific hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) in surviving dentate gyrus granule cells of human and experimental epileptic hippocampus. J Neurosci 23: 6826–6836. Berg, A.T., Shinnar, S., Hauser, W.A., Alemany, M., Shapiro, E.D., Salomon, M.E., and Crain, E.F. 1992. A prospective study of recurrent febrile seizures. N Engl J Med 327: 1122–1127. Berg, A.T., and Shinnar, S. 1996a. Unprovoked seizures in children with febrile seizures: short-term outcome. Neurology 47: 562–568. Berg, A.T., and Shinnar, S. 1996b. Complex febrile seizures. Epilepsia 37: 126–133. Berg, A.T., Shinnar, S., Darefsky, A.S., Holford, T.R., Shapiro, E.D., Salomon, M.E., Crain, E.F. et al. 1997. Predictors of recurrent febrile seizures. A prospective cohort study. Arch Pediatr Adolesc Med 151: 371–378. Blake, D., Bessey, P., Karl, I., Nunnally, I., and Hotchkiss, R. 1994. Hyperthermia induces IL-1 alpha but does not decrease release of IL-1 alpha or TNF-alpha after endotoxin. Lymphokine Cytokine Res 13: 271– 275. Brewster, A., Bender, R.A., Chen, Y, Dubé, C., Eghbal-Ahmadi, M., and Baram, T.Z. 2002. Developmental febrile seizures modulate hippocampal gene expression of hyperpolarization-activated channels in an isoform- and cell-specific manner. J Neurosci 22: 4591–4599. Brewster, A., Bernard, J.A., Gall, C.M., and Baram, T.Z. 2005. Formation of heteromeric hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the hippocampus is regulated by developmental seizures. Neurobiol Dis 19: 200–207. Buckmaster, P.S., and Dudek, F.E. 1997. Neuron loss, granule cell axon reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J Comp Neurol 385: 385–404. Burger, F.G., and Fuhrman, F.A. 1964. Evidence of injury by heat in mammalian tissues. Am J Physiol 206: 1057–1061. Cartmell, T., Southgate, T., Rees, G.S., Castro, M.G., Loweinstein, P.R., and Luheshi, G.N. 1999. Interleukin-1 mediates a rapid inflammatory response after injection of adenoviral vectors into the brain. J Neurosci 19: 1517–1523. Cendes, F., Andermann, F., Dubeau, F., Gloor, P., Evans, A., Jones-Gotman, M., Olivier, A. et al. 1993. Early childhood prolonged febrile convulsions, atrophy and sclerosis of mesial structures, and temporal lobe epilepsy: an MRI volumetric study. Neurology 43: 1083–1087. Chen, K., Baram, T.Z., and Soltesz, I. 1999. Febrile seizures in the developing brain results in persistent modification of neuronal excitability in limbic circuits. Nat Med 5: 888–894. Chen, K., Aradi, I., Thon, N., Eghbal-Ahmadi, M., Baram, T.Z., and Soltesz, I. 2001. Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability. Nat Med 7: 331–337. Dubé, C., Chen, K., Eghbal-Ahmadi, M., Brunson, K.L., Soltesz, I., and Baram, T.Z. 2000. Prolonged febrile seizures in immature rat model enhance hippocampal excitability long-term. Ann Neurol 47: 336– 344. Dubé, C., Vezzani, A., Behrens, M., Bartfai, T., and Baram, T.Z. 2005a. Interleukin 1b contributes to the generation of experimental febrile seizures. Ann Neurol 57: 152–155.
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Dubé, C., Brunson, K.L., Eghbal-Ahmadi, M., Gonzalez-Vega, R., and Baram, T.Z. 2005b. Endogenous neuropeptide Y prevents recurrence of experimental febrile seizures by increasing seizure threshold. J Mol Neurosci 25: 275–284. Dubé, C., Hon, Y., Nalcioglu, O., and Baram, T.Z. 2004d Serial magnetic resonance imaging after prolonged experimental febrile seizures: altered T2 signal does not signify neuronal death. Ann Neurol 56: 709–714. Eghbal-Ahmadi, M., Yin, H., Stastrom, C.E,. Tran, K., Weiss, J.H., and Baram, T.Z. 2001. Altered expression of specific AMPA type glutamate receptor subunits after prolonged experimental febrile seizures in CA3 of immature rat hippocampus. Soc Neurosci Abstr 31: 684.6. French, J.A., Williamson, P.D., Thadani, V.M., Darcey, T.M., Mattson, R.H., Spencer, S.S., and Spencer, D.D. 1993. Characteristics of medial temporal lobe epilepsy: I. Results of history and physical examination. Ann Neurol 34: 774–780. Gatti, S., Vezzani, A., and Bartfai, T. 2002. Mechanisms of fever and febrile seizures: putative role of interleukin-1 system. In Febrile Seizures Ed. T.Z. Baram, and S. Shinnar. pp. 169–188. San Diego: Academic Press. Germano, I.M., Zhang, Y.F., Sperber, E.F., and Moshe, S.L. 1996. Neuronal migration disorders increase susceptibility to hyperthermia-induced seizures in developing rats. Epilepsia 37: 902–910. Hamati-Haddad, A., and Abou-Khalil, B. 1998. Epilepsy diagnosis and localization in patients with antecedent childhood febrile convulsions. Neurology 50: 917–922. Hauser, W.A. 1994. The prevalence and incidence of convulsive disorders in children. Epilepsia 35: S1–S6. Haveman, J., Geerdink, A.G., and Rodermond, H.M. 1996. Cytokine production after whole body and localized hyperthermia. Int J Hyperthermia 12: 791–800. Heida, J.G., Teskey, G.C., and Pittman, Q.J. 2003. Experimental febrile convulsions in the rat and their effects on the development of kindling induced epilepsy in adulthood. Soc Neurosci Abstr 303.12. Hjeresen, D.L., and Diaz, J. 1988. Ontogeny of susceptibility to experimental febrile seizures in rats. Dev Psychobiol 3: 261–275. Houser, C.R., and Esclapez, M. 1996. Vulnerability and plasticity of the GABA system in the pilocarpine model of spontaneous recurrent seizures. Epilepsy Res 26: 207–218. Houser, C.R. 1999. Neuronal loss and synaptic reorganization in temporal lobe epilepsy. Adv Neurol 79: 743–761. Knudsen, F.U. 1996. Febrile seizures—treatment and outcome. Brain Dev 18: 438–449. Knudsen, F.U. 2002. Practical management approaches to simple and complex febrile seizures. In Febrile Seizures Ed. T.Z. Baram, and S. Shinnar. pp. 273–304. San Diego: Academic Press. Morimoto, T., Nagao, H., Sano, N., Takahashi, M., and Matsuda, H. 1990. Hyperthermia-induced seizures with a servo system: neurophysiological roles of age, temperature elevation rate and regional GABA content in the rat. Brain Dev 12: 279–283.
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27 Repetitive Seizures in the Immature Brain* QIAN ZHAO AND GREGORY L. HOLMES
Procedures
Epilepsy is a condition characterized by recurrent seizures. To mimic the clinical condition in immature animals, investigators have primarily used electrical kindling (see Chapter 30), electroshock, and chemoconvulsants. The majority of recent studies in young animals using chemoconvulsants have employed flurothyl and pentyelentetrazol. Less commonly repetitive doses of pilocarpine and kainic acid have been used to induce recurrent bouts of status epilepticus. All four chemoconvulsants and electroshock will be discussed.
The typical way that flurothyl is administered is through inhalation. Adult rats are placed in a plastic container (17.5 ¥ 10 ¥ 10.5 inches) that allows them to turn, rear, and move about, while immature rats (
FLUROTHYL The volatile agent flurothyl (bis-2, 2, 2-triflurothyl ether) is a potent and rapidly acting central nervous system stimulant that produces seizures within minutes of exposure (Truitt et al., 1960). Because of its reliability and reproducibility, it is frequently used to induce recurrent seizures in young animals.
What Does It Model? Flurothyl is used to model recurrent generalized tonicclonic seizures (Moshé et al., 1994).
Methods of Generation Animal Issues Flurothyl can be administered repeatedly to rat and mice pups of both genders. *Grant sponsors: NIH NINDS; Grant numbers: NS27984 and NS44295.
Models of Seizures and Epilepsy
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Characteristics/Defining Features Behavioral Effects of Flurothyl
FIGURE 1 Diagram of chamber used in inducing seizures with flurothyl. Liquid flurothyl is dripped onto filter paper suspended from the top of the plastic chamber. It evaporates and is inhaled by the rats. The chamber is made of clear plastic.
used to induce clonic and tonic seizures, respectively (latency x rate of infusion) (Xu et al., 1992). Since the infusion rate is usually kept constant some investigators simply report latencies to either the myoclonic or tonic seizures. Because the seizure ends within 30 seconds of placing the animals in room air the investigator has control of the duration and severity of the seizures. For example, by maintaining the animal in the testing chamber with continuous administration of flurothyl status epilepticus can be elicited (Sperber et al., 1999). For the purpose of recurrent seizures the rats are usually removed from the chamber once the tonic phase begins. Flurothyl may be given once or multiple times daily to immature rats (Wasterlain, 1977; Dwyer and Wasterlain, 1984; Huang et al., 1999; Sogawa et al., 2001). Our laboratory routinely induces five seizures per day over 5 to 10 days with each seizure separated by at least two hours (Holmes et al., 1998; Huang et al., 1999; de Rogalski Landrot et al., 2001). We have found that more frequent daily seizures results in unacceptable weight loss. Rat pups subjected to 25 to 50 seizures using a protocol of five seizures per day thrive and are healthy. However, even with five seizures per day weight loss is common (Cha et al., 2001). Recurrent flurothyl seizures can also delay sexual maturation in female rats (Bhanot and Wilkinson, 1984). Control rats are placed in the flurothyl chamber but not exposed to any of the drug. To help control for differences in weight between controls and flurothlyl-treated rats we separate the control rats from the dam for the same duration of time. Monitoring Flurothyl recurrent seizures are easy to induce and are reproducible. Following the seizures the rats are placed in separate cage and kept warm via a heating blanket. Control rats are handled in a similar fashion. The pups are not returned to the dam until they are actively moving.
There are age-related differences in the clinical features of flurothyl-induced seizures (Sperber and Moshé, 1988). Gatt and colleagues (Gatt et al., 1993) administered flurothyl to rats at postnatal days 5, 10, 20, 30, and 70 and monitored both behavioral and EEG changes. In all age groups flurothyl inhalation initially resulted in agitation with increased exploring of the testing chamber. Swimming movements were prominent in P5 pups, followed by abrupt onset of tonus. Clonic seizures were not fully developed before P10. Behavioral features of the seizures did not change after P20 and consisted of myoclonic jerks followed by forelimb clonus, wild running, loss of posture, and severe tonic posturing. Urinary and fecal incontinence and salivation were common. When flurothyl is administered repeatedly in rat pus similar behavioral features are noted. In rat pups recurrent flurothyl seizures in rats below the age of 10 days result in agitation, swimming movements, and tonic activity with clonic activity emerging at P8–P10. Recurrent seizures do not accelerate or delay the age-related behavioral features of the seizures. During the tonic phase of the seizures cyanosis can occur. McCabe et al. (McCabe et al., 2001) performed blood gas measurement in rat pups (P4 or P5) and adult rats during flurothyl seizures. During the tonic phase of the seizure pO2 levels dropped to approximately 35 mm Hg and O2 saturations dropped to approximately 50% in rat pups. In adult rats the pO2 fell to approximately 50 mm Hg and the O2 saturations to approximately 60%. Mild acidosis was seen in both the immature and mature rats. In both age groups blood gas measurements returned to baseline within 5 minutes of the end of the seizures. While we have not done blood gases in immature rats that have had recurrent seizures, the degree of cyanosis does not appear to be influenced by number of prior seizures. The rats recover quickly from the seizures. Within 5–10 minutes of the end of the seizure the rats are upright and walking in the cage. Within 30 minutes they appear to be behaving normally. However, in a study examining duration of postictal impairment of memory, Boukhezra et al. (Boukhezra et al., 2003) found that rats did not return to their baseline performance in the Morris water maze, a measure of visual-spatial memory, for 40 minutes. For this reason we typically allow two hours between seizures. The mortality rate with acute flurothyl seizures is quite low. Rats rarely die if they are removed from the chamber at the onset of the tonic seizure and placed in room air. For both rat pups and adult rats the mortality rate is typically <5% (Huang et al., 1999; Sogawa et al., 2001).
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EEG Features There have been no studies systematically studying EEGs during recurrent flurothyl-induced seizures in immature animals. Neuropathology Recurrent flurothyl seizures during the first two weeks of life results in no discernible cell (Holmes et al., 1998; Liu et al., 1999; Riviello et al., 2002). When the animals are studied as adults there is extensive synaptic reorganization of the axons and terminals of the dentate granule cells, the mossy fibers (Holmes et al., 1998; Huang et al., 1999). The sprouting of mossy fibers occurs in both the molecular region of the dentate granule cells as well as the CA3 hippocampal subfield. Recurrent flurothyl seizures during early development can alter the formation of new neurons. McCabe et al. (2001) studied the extent of neurogenesis in the granule cell layer of the dentate gyrus over multiple time points following a series of 25 flurothyl-induced seizures administered between P0 and P4. Rats with neonatal seizures had a significant reduction in the number of the thymidine analog 5-bromo2¢-deoxyuridine-5¢-monophosphate-(BrdU) labeled cells in the dentate gyrus and hilus compared to the control groups when the animals were sacrificed either 36 hours or two weeks after the BrdU injections. The reduction in BrdUlabeled cells continued for six days following the last seizure. BrdU-labeled cells largely co-localized with the neuronal marker neuron-specific nuclear protein and rarely co-localized with the glial cell marker glial fibrillary acidic protein (GFAP), providing evidence that a very large percentage of the newly formed cells were neurons. Immature rats subjected to a single seizure did not differ from controls in number of BrdU-labeled cells. In comparison, adult rats undergoing a series of 25 flurothyl-induced seizures had a significant increase in neurogenesis compared to controls. This study indicates that following recurrent seizures in the neonatal rat there is a reduction in newly born granule cells. These seizure-induced decreases in neurogenesis is consistent with prior studies by Wasterlain and Plum (1973) who concluded that recurrent neonatal seizures, while not causing cell death, resulted in reduced cell number.
Genetic/Molecular Changes Sogawa et al. (2001) exposed rat pups starting at postnatal day P0 to 45 flurothyl-induced seizures over a 9-day period of time. Brains were evaluated for cell loss, mossy fiber distribution, and AMPA (GluR1) and NMDA (NMDAR1) subreceptor expression at P20 or P35. GluR1 expression was increased in CA3 at P20 and NMDAR1 was increased in expression in CA3 and the supragranular region of the dentate gyrus at P35. These findings demonstrated that
the alterations in cognition and seizure susceptibility were paralleled by sprouting of mossy fibers and increased expression of glutamate receptors. Bo et al. (2004) investigated the effects of a single and six flurothyl seizures administered daily starting on P6 on protein expression of the NMDA receptor (NR) subunits, NR1, 2A, 2B, 2C, and 2D, in the cerebral cortex and hippocampus by Western blotting analysis when the animals were sacrificed as adults. NR subunit expression in the cerebral cortex and hippocampus of the rats with single seizure was similar to those in the control rats. In the recurrentseizure group, the protein expressions of NR1, NR2A and NR2B in the cerebral cortex and NR2A in the hippocampus was significantly decreased, but NR2C protein expression in the cerebral cortex and hippocampus significantly increased. Changes in GABA receptor expression has also been reported following recurrent flurothyl seizures (Ni et al., 2004). Both single and recurrent flurothyl seizures in neonatal rats sacrificed at P7 or P75 showed significantly reduced polypeptide levels of the GABAA alpha1 receptor subunit.
Behavioral Consequences of the Seizures Sprague-Dawley rats subjected to a series of recurrent seizures during the first weeks of life have cognitive impairment when the animals are studied during adolescence or adulthood. In a series of studies (Huang et al., 1999; Liu et al., 1999; Sogawa et al., 2001), we have used the Morris water maze, a measure of visual-spatial memory (Morris et al., 1982b; 1986; 1989) to assess cognitive function following neonatal seizures. In this test, animals are placed in a 2 meters in diameter tank filled with water (Figure 2). Four points on the rim of the pool were designated north (N), south (S), east (E), and west (W), thus dividing the pool into four quadrants (NW, NE, SE, SW). An 8 ¥ 8 cm Plexiglas platform, onto which the rat can escape, is positioned in the center of one of the quadrants, 1 cm below the water surface. Figure 2 provides examples of the swimming path for rats during the various phases of testing. On day 1 each rat is placed in the pool for 60 seconds without the platform being present; this free swim enables the rat to become habituated to the training environment. On days 2–5 rats are trained for 24 trials (six trials a day) to locate and escape onto the submerged platform. For each rat, the quadrant in which the platform is located remains constant, but the point of immersion in the pool varies between N, E, S, and W in a quasi-random order for the 24 trials, so that the rat is not able to predict the platform location from the point at which it is placed in the pool. The latency from immersion into the pool to escape onto the platform is recorded for each trial and the observer also manually records the route taken by the rat to reach the platform. On mounting the platform, the rats are given a 30 second rest
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probe trial is a measure of the strength of spatial learning (Jeltsch et al., 2001). Following neonatal seizures, there is impairment of visual spatial memory in the Morris water maze, with rats subjected to recurrent flurothyl seizures during the neonatal period requiring longer times to find the platform than controls (Neill et al., 1996; Holmes et al., 1998; Huang et al., 1999; Liu et al., 1999). This impairment occurs when the rats are tested either during adolescence or when fully mature. Recurrent seizures are critical in causing the cognitive impairment. When only a single flurothly seizure is induced at P6 no impairment in water maze performace occurs (Bo et al., 2004). Animals subjected to recurrent flurothyl seizures between P15–20 also have impairment of auditory discrimination (Neill et al., 1996).
Effects on Seizure Susceptibility
FIGURE 2 Example of swimming path during water maze testing. 1, Habituation without platform; 2–5, Swiming path during the four days of training. 6, Probe test. See text for details.
period, after which the next trial is started. If the rat does not find the platform in 120 seconds, it is manually placed on the platform for a 30 second rest. At the start of each trial, the rat is held facing the perimeter and dropped into the pool to ensure immersion. One day after completion of the last latency trial (Day 6) the platform is removed and animals are placed in the water maze in the quadrant opposite to where the platform had previously been located. The path and time spent in the quadrant where the platform had been previously placed is recorded. In this part of the water maze, the probe test, normal animals typically spend more time in the quadrant where the platform had been previously located than in the other quadrants. The Morris water maze is a test of hippocampaldependent spatial memory (Morris et al., 1982; Morris, 1984), the closest parallel to episodic memory in humans (Jeltsch et al., 2001; Spiers et al., 2001). The testing procedure used during the 4 days of locating the hidden platform provides a measure of spatial reference memory, while the
Recurrent flurothyl seizures in immature rats do not lead to spontaneous recurrent seizures. However, animals subjected to multiple flurothyl-induced seizures demonstrate a kindling phenomenon with a decrease latency to forelimb clonus (Liu et al., 1999). In addition, recurrent flurothyl seizures are associated with a reduced seizure threshold when examined at an older age (Holmes et al., 1998; Sogawa et al., 2001). The basis for the reduced seizure threshold following recurrent flurothyl seizures has been addressed using in vitro recordings (Villeneuve et al., 2000). Intracellular recordings of CA1 and CA3 pyramidal neurons from neonatal flurothylinduced seizures revealed impairment in spike frequency adaptation. In addition, the afterhyperpolarizing potentials following a spike train were markedly reduced when compared with controls. In contrast, no significant alterations in the firing properties of CA3 pyramidal neurons were found. It was concluded that neonatal seizures lead to persistent changes in intrinsic membrane properties of CA1 pyramidal neurons. These alterations are consistent with an increase in neuronal excitability and may contribute to the behavioral deficit and epileptogenic predisposition observed in rats that experienced repeated neonatal seizures.
Response to Antiepileptic Drugs/ Usefulness in Screening Drugs There have been no studies examining the effects of antiepileptic drugs on recurrent flurothyl-induced seizures.
Limitations The flurothyl inhalation model is easy to use and produces reliable seizures with a low mortality rate. The model has been well characterized in the immature rat.
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Pentylenetetrazol
PENTYLENETETRAZOL Pentylenetetrazol (PTZ) is a tetrazol derivative (Stone, 1970) that has been shown to have convulsant actions in mice, rats, cats, and primates, presumably by impairing GABA-mediated inhibition by an action at the GABA receptor (Olsen, 1981; Ramanjaneyulu and Ticku, 1984). PTZ is best known for its use in screening antiepileptic drugs (Kupferberg, 2001).
What Does It Model? The behavioral and EEG manifestations of PTZ-induced seizures in rodents suggest that the drug is a model of generalized seizures, both absence and generalized tonicclonic seizures. When used to induce recurrent seizures PTZ has been used to model generalized tonic-clonic seizures.
Animal Issues PTZ can be administered serially to immature rats and mice.
Characteristics/Defining Features Behavioral/Clinical Features The behavioral expression of PTZ-induced motor seizures is age-dependent (see Chapter 11). Clonic motor seizures are not regularly observed in rat pups before the third postnatal week, and they are not fully developed until the fourth postnatal week. In contrast, myoclonic jerks and generalized tonic-clonic seizures are observed throughout development. The dose needed to induce clonic seizures in 50% of animals (CD50) is almost constant after the third postnatal week; however, the CD50 for tonic-clonic seizures progressively increases with age. The latency to the onset of tonic-clonic seizure is shortest during the second and third postnatal week following a single dose of 100 mg/kg administered subcutaneously, suggesting that the rats in this age group have a lower seizure threshold than other age groups. Weller and Mostofsky (1995) also found that compared to P18, P28, and P60 rats, P10 rats had a more rapid onset to generalized tonic-clonic seizures. Serial injections of PTZ do not alter the behavioral features of the seizures.
EEG Features Procedures PTZ can be given intravenously, intraperitoneally, or subcutaneously. Seizures are usually induced by a single systemic administration; both the dose and route of administration determine the latency to seizure onset and behavioral manifestations. When given in sufficient amounts, PTZ can result in status epilepticus (Pereira, de Vasconcelos et al., 1992; Nehlig and Pereira de Vasconcelos, 1996). In addition, PTZ can be administrated repeatedly to immature rats (Holmes et al., 1999; Huang et al., 2002b). In a study of the effects of 15 daily PTZ seizures starting at P1, P10, or P60, Holmes et al. (1999) used a daily dosage of 40 mg/kg intraperitoneally. The authors noted that pilot studies demonstrated a mortality rate exceeding 50% with both 45 and 50 mg/kg while 30–35 mg/kg failed to produce consistent seizures. Because of variations in dose requirements in various strains of rodents, it is recommended that pilot dose-response trials be administered before determining an appropriate dose. When used to induce recurrent seizures investigators have given PTZ once or twice daily (Holmes et al., 1999; Huang et al., 2002a; 2002b). Monitoring PTZ recurrent seizures are easy to induce and are reliable. Following the seizures the rats are placed in separate cage and kept warm via a heating blanket. Control rats are handled in a similar fashion. The pups are not returned to the dam until they are actively moving.
There have been no studies systematically studying EEGs during recurrent PTZ-induced seizures. Neuropathology Holmes et al. (1999) evaluated the effects of 15 daily PTZ-induced convulsions in immature rats beginning at P1, 10, or 60. In addition, another group of P10 rats were subjected to twice daily seizures for 15 days. Both supragranular and terminal sprouting in the CA3 hippocampal subfield was assessed in Timm-stained sections by using a rating scale and density measurements. Prominent sprouting was seen in the CA3 stratum pyramidale layer in all rats having 15 daily seizures, regardless of the age when seizures began. Based on Timm staining in control P10, P20, and P30 rats, the terminal sprouting in CA3 appeared to be new growth of axons and synapses as opposed to a failure of normal regression of synapses. In addition to CA3 terminal sprouting, rats having twice daily seizures had sprouting noted in the dentate supragranular layer, predominately in the inferior blade of the dentate Cell counting of dentate granule cells, CA3, CA1, and hilar neurons, with unbiased stereological methods demonstrated no differences from controls in rats with daily seizures beginning at P1 or P10, whereas adult rats with daily seizures had a significant decrease in CA1 neurons. Similarly, Huang et al. (2002b) induced daily PTZ seizures for five consecutive days starting at P10. When the animals were sacrificed at P35 or P60 there was no cell loss observed. However, PTZ-treated rats exhibited more Timm staining in the CA3 subfield than the controls. These
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studies demonstrate that there are age-specific changes in the brain following recurrent seizures.
Behavioral Consequences of the Seizures Huang et al. (2002b) induced daily PTZ seizures for five consecutive days starting at P10. At P35 and P60 rats were tested for spatial memory using the Morris water maze task. PTZ-treated rats showed significant spatial deficits in the water maze at both P35 and P60. There were no differences in seizure threshold, motor balance, or activity level during the open field test. Genetic/molecular changes The have been no genetic or molecular changes described following recurrent PTZ seizures. Effects on Seizure Susceptibility Holmes et al. (1999) found that recurrent PTZ seizures were associated with a reduced seizure threshold when the animals were re-challenged with PTZ at an older age. However, Huang et al. (2002b), using flurothyl to assess seizure susceptibility, found that five PTZ seizures starting at P10 did not alter subsequent seizure susceptibility. Spontaneous seizures are not observed following recurrent PTZinduced seizures in rat pups (Huang et al., 2002b). Response to Antiepileptic Drugs/Usefulness in Screening Drugs There have been no studies examining the effects of antiepileptic drugs on recurrent PTZ-induced seizures. Limitations The recurrent PTZ model is easy to use and produces reliable seizures with a low mortality rate. The model has been well characterized in the immature rat.
RECURRENT STATUS MODELS General Description of the Model Both kainic acid (KA) and pilocarpine have been used to induce recurrent episodes of status epilepticus in the immature brain.
Methods of Generation Animal Issues Both KA and pilocarpine can be administered repeatedly to rat pups of both genders. Procedures To induce serial bouts of status epilepticus KA and pilocarpine are given intraperitoneally. The dose of both KA (Stafstrom et al., 1992) and lithium-pilocarpine are highly age dependent (Hirsch et al., 1992; Cilio et al., 2003). Monitoring Following the seizures the rats are placed in separate cage and kept warm via a heating blanket. Control rats are handled in a similar fashion. The pups are not returned to the dam until they are actively moving.
Characteristics/Defining Features Behavioral/Clinical Features Repeated injections of both KA and pilocarpine will produce episodes of recurrent status epilepticus. When KA is given serially beginning at P20 the rats initially become immobile then progress to facial twitching, salivation, myoclonic jerks, and eventually four limb clonus with rearing (Sarkisian et al., 1997). Recurrent pilocarpine injections staring at P7 result in scratching, body tremors, mastication, clonic movements of the forelimbs and head bobbing before progressing into tonic and clonic activity (Priel et al., 1996; dos Santos et al., 2000). Differences between the response to recurrent KA and pilocarpine induced seizures have been described. Rats given KA at P20, P22, P24, and P26 demonstrated progressively longer onset latencies and decreased severity with each subsequent seizure (Sarkisian et al., 1997). In contrast, the investigators found that KA serially administered to adult rats caused severe seizures after each of the 4 injections. A similar decrease in seizure severity has not been found in rats receiving multiple pilocarpine on P7, P8, and P9 (dos Santos et al., 2000; Santos et al., 2000). Rather the seizures appeared to escalate in intensity with the recurrent doses of pilocarpine. The reason for these differences is not clear. However, the ages of the rats receiving KA were different from those receiving pilocarpine.
What Does It Model? Both KA and pilocarpine model status epilepticus that begins as partial seizures and then become secondarily generalized.
EEG Features Recordings from the hippocampus during KA-induced recurrent status epilepticus have shown rhythmic spikes and
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Electroshock Seizures
sharp waves (Sarkisian et al., 1997; Mikati et al., 2003). Using recurrent KA injections Sarkisian et al. (1997) found that epileptiform EEG changes were most prominent after the first KA injection with a progressive decrease in activity with each subsequent injection.
Limitations There have been few studies using recurrent status epilepticus in immature rats. The role of strain, age, and chemoconvulsant are important variables that need further study. Additional work is necessary to determine reproducibility of the seizures in the immature brain.
Neuropathology No pathological lesions have been reported following recurrent KA-induced status epilepticus induced between P20 and P26 (Sarkisian et al., 1997). Santos et al. (2000) found no major long-term pathological changes in rats undergoing three episodes of status epilepticus using pilocarpine on P7–9 when the animals were sacrificed as adults. However, an increased number of TUNELpositive nuclei were present in both the hippocampus and the thalamus in animals undergoing recurrent status epilepticus.
Behavioral Consequences of the Seizures Wistar rat pups (P7–9) subjected to three consecutive episodes of status epilepticus induced by systemic pilocarpine injections were studied at P60 in the inhibitory stepdown avoidance test, skinner box, rota-rod, open field and elevated plus-maze (dos Santos et al., 2000). Rats subjected to repetitive bouts of status epilepticus had increased spontaneous exploratory activity, learning impairment, and reduced anxiety. However, Sarkisian et al. (1997), using four serial KA episodes of status epilepticus in Sprague-Dawley rats, found no deficits in spatial learning retention following the status. Conversely, adult rats subjected to four bouts of status epilepticus with KA had severe impairment in the water maze (Sarkisian et al., 1997).
Effects on Seizure Susceptibility EEG recordings made at the age of 30, 60 and 90 days following three bouts of pilocarpine-induced status epilepticus showed the occurrence of several episodes of spikes and polyspikes appearing simultaneously in hippocampus and cortex (dos Santos et al., 2000). However, only three isolated spontaneous seizures were observed during the whole period of observation. The long-term effects of recurrent KA-induced status epilepticus in rat pups have not been described.
Response to Antiepileptic Drugs/Usefulness in Screening Drugs There have been no studies examining the effects of antiepileptic drugs on recurrent status epilepticus with KA or pilocarpine.
ELECTROSHOCK SEIZURES General Description of the Model Electroshock or electroconvulsive seizures are induced by administration of electrical currents to the ears on rodents.
What Does It Model? Electoctrochock has been used to model recurrent generalized tonic-clonic seizures in immature rats by some investigators (Wasterlain and Plum, 1973; Jorgensen et al., 1980; Dwyer and Wasterlain, 1982; Bhanot and Wilkinson, 1984).
Methods of Generation Animal Issues Electroshock seizures can be administered repeatedly to rat pups. Procedures Seizures are induced by administration of a 150 Volts or 100 mA, 1 sec, alternating current through saline-soaked compresses applied to the ears (Wasterlain and Plum, 1973; Jorgensen et al., 1980; Dwyer and Wasterlain, 1982). Investigators have administered one or two seizures per day (Wasterlain and Plum, 1973; Dwyer and Wasterlain, 1982). Monitoring Following the seizures the rats are placed in separate cage and kept warm via a heating blanket. Control rats are handled in a similar fashion. The pups are not returned to the dam until they are actively moving. Most experimental animals lag behnd their paired littermates in body growth following the electroschock seizures (Wasterlain and Plum, 1973).
Characteristics/Defining Features Behavioral/Clinical Features Immediately after the passage of electric current, experimental animals of all ages experience a strong tonic exten-
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sion lasting 5–20 seconds followed by clonic movements of all four limbs. Before P12 rats have a clonic phase that is brief and poorly developed (Dwyer and Wasterlain, 1982). The clonic phase increases progressively in duration as the animal gets older. Following the seizure the animals are in state of postictal depression and are immobile. Typically the rats take 30 minutes to recover.
EEG Features EEGs have not been recorded in immature rodents undergoing repeated electroconvulsive seizures. Neuropathology Wasterlain and Plum (1973) compared the effects of 10 daily electroconvulsive seizures on rats from P2–11, P9–18, and P19–28 days. Animals receiving neonatal (P2–11) or infantile seizures (P9–18) had significantly smaller brains than controls. In addition, neonatal seizures reduced total brain DNA, RNA, protein and cholesterol. The authors interpreted these findings to indicate a reduction of cell number, but not cell size, in rats with neonatal seizures. In a similarly designed study Dwyer and Wasterlain (1982) administered serial electroconvulsive seizures to rats during the neonatal period (P2–P11) or late suckling period (P9–P18). To assess the effects of the seizures on myelin accumulation the investigators measured cerebroside and proteolipid protein. Both were reduced following the seizures and preceded DNA reductions suggesting that reduced myelin content may result from curtailed growth of cellular process. Whereas cerebellar deficits in cerebroside content returned to normal by P30, forebrain cerebroside deficits appeared to persist or even progress after treatment. Reductions in synaptic proteins and the glial enzyme glutamine synthetase have also been found following recurrent seizures in the immature brain (Jorgensen et al., 1980).
Limitations Somewhat surprising, there have been few recent studies using the electroconvulsive model in immature rats. The method does result in reproducible generalized seizures in immature rats.
Conclusions Limitations The use of all the models described above allow the investigator to mimic recurrent seizures in children. However, it is important to recognize that these recurrent seizures models do not mimic human epilepsy which con-
sists of spontaneous recurrent seizures. In the case of the animal models described above a chemoconvulsant or electrical stimulation is repeatedly administered to a normal rat whereas in human epilepsy there is a pathological process that results in spontaneous recurrent seizures. Despite the differences between evoked seizures in the models and spontaneous seizures in the human, animal models of recurrent seizures provide information about the pathological and behavioral consequences of seizures in the immature brain. As described above, it is known that recurrent seizures in the immature brain can result in alterations in neurogenesis, expression of glutamate receptors, and synaptic connectivity as well as changes in behavior. What is not known is whether the morphological changes noted in the immature brain following serial seizures is responsible for seizure-induced cognitive deficits. Furthermore, whether the observations noted in animal models are relevant to the human condition is unclear. Finally, when dealing with immature rats it is important to carefully consider factors other than seizures that may influence their results. For example, in a study examining the role of maternal deprivation in seizure-induced injury Huang and colleagues (2002a) divided rat pup into four groups according to whether the rat pups were subjected to maternal deprivation or neonatal seizures. Rats in the maternal deprivation group underwent daily separation from their dams from P2 to P9; rats in the PTZ-treated group were subjected to PTZ-induced recurrent seizures from P10 to P14; rats in the maternal deprivation plus PTZ-treated group were subjected to maternal deprivation from P2 to P7 followed by serial seizures from P10 to P14. The investigators found that after PTZ administration, rats with a history of maternal deprivation had more intense impairment than did rats without maternal deprivation or rats with neonatal seizures alone. Rats with maternal deprivation plus PTZ-induced recurrent seizures exhibited poorer visual-spatial learning compared with rats with either maternal deprivation or PTZ-induced recurrent seizures alone. This study nicely demonstrates the need for investigators to be vigilant in considering variable other than seizures when interpreting their results. Need for Future Development The current models of recurrent seizures do not adequately model human epilepsy. A model of recurrent spontaneous seizures in immature animals is needed. In addition, childhood epilepsy is characterized by a wide variety of behavioral and EEG features. The need for adequate animal models is particularly important for the severe forms od childhood epilepsy. For example, there are no animal models of early infantile epileptic encephalopathy with suppressionburst (Ohtahara syndrome), infantile spasms (West syndrome), severe myoclonic epilepsy of infancy (Dravet
References
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Pereira de Vasconcelos, H., el Hamdi, G., Vert, P., and Nehlig, A. 1992. An experimental model of generalized seizures for the measurement of local cerebral glucose utilization in the immature rat. II. Mapping of brain metabolism using the quantitative [14C]2-deoxyglucose technique. Brain Res Dev Brain Res 69: 243–259. Priel, M.R., dos Santos, N.F., and Cavalheiro, E.A. 1996. Developmental aspects of the pilocarpine model of epilepsy. Epilepsy Res 26: 115– 121. Ramanjaneyulu, R., and Ticku, M.K. 1984. Interactions of pentamethylenetetrazol and tetrazol analogues with picrotoxin sit of the benzodiazepine-GABA receptor-ionophore complex. Eur J Pharmacol 98: 337–345. Riviello, P., de Rogalski, I, Holmes, G.L. 2002. Lack of cell loss following recurrent neonatal seizures. Brain Res Dev Brain Res 135: 101–104. Santos, N.F., Marques, R.H., Correia, L., Sinigaglia-Coimbra, R., Calderazzo, L., Sanabria, E.R., and Cavalheiro, E.A. 2000. Multiple pilocarpine-induced status epilepticus in developing rats: a long-term behavioral and electrophysiological study. Epilepsia 41(Suppl 6): S57–S63. Sarkisian, M.R., Tandon, P., Liu, Z., Yang,Y., Hori, A., Holmes, G.L., and Stafstrom, C.E. 1997. Multiple kainic acid seizures in the immature and adult brain: ictal manifestations and long-term effects on learning and memory. Epilepsia 38: 1157–1166. Sogawa, Y., Monokoshi, M., Silveira, D.C., Cha, B.H., Cilio, M.R., McCabe, B.K., Liu, X. et al. 2001. Timing of cognitive deficits following neonatal seizures: relationship to histological changes in the hippocampus. Brain Res Dev Brain Res 131: 73–83. Sperber, E.F., Haas, K.Z., Romero, M.T., and Stanton, P.K. 1999. Flurothyl status epilepticus in developing rats: behavioral, electrographic, histological and electrophysiological studies. Develop Brain Res 116: 59–68. Sperber, E.F., and Moshé, S.L. 1988. Age-related differences in seizure susceptibility to flurothyl. Dev Brain Res 39: 295–297.
Spiers, H.J., Burgess, N., Maguire, E.A., Baxendale, S.A., Hartley, T., Thompson, P.J., and O’Keefe, J. 2001. Unilateral temporal lobectomy patients show lateralized topographical and episodic memory deficits in a virtual town. Brain 124: 2476–2489. Stafstrom, C.E., Thompson, J.L., and Holmes, G.L. 1992. Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures. Brain Res Dev Brain Res 65: 227–236. Stone, W.E. 1970. Convulsant actions of tetrazole derivatives. Pharmacology 3: 367–370. Truitt, E.B., Ebersberger, E.M., and Ling, A.S.C. 1960. Measurement of brain excitability by use of hexaflurodiethyl ether (Indoklon). J Pharmacol Exp Ther 129: 445–453. Velisek, L., Kubova, H., Pohl, M., Stankova, L., Mares, P., and Schickerova, R. 1992. Pentylenetetrazol-induced seizures in rats: an ontogenetic study. Naunyn Schmiedebergs Arch Pharmacol 346: 588–591. Villeneuve, N., Ben-Ari, Y., Holmes, G.L., and Gaiarsa, J.L. 2000. Neonatal seizures induced persistent changes in intrinsic properties of CA1 rat hippocampal cells. Ann Neurol 47: 729–738. Wasterlain, C.G. 1977. Effects of neonatal seizures on ontogeny of reflexes and behavior. An experimental study in the rat. Eur Neurol 9: 9–19. Wasterlain, C.G. 1978. Neonatal seizures and brain growth. Neuropaediatrie 9: 213–228. Wasterlain, C.G. 1976. Effects of neonatal status epilepticus on rat brain development. Neurology 26: 975–986. Wasterlain, C.G., and Plum, F. 1973. Vulnerability of developing rat brain to electroconvulsive seizures. Arch Neurol 29: 38–45. Weller, A., and Mostofsky, D.I. 1995. Ontogenetic development and pentylenetetrazol seizure thresholds in rats. Physiol Behav 57: 629–631. Xu, S.G., Garant, D.S., Sperber, E.F., and Moshe, S.L. 1992. The proconvulsant effect of nigral infusions of THIP on flurothyl-induced seizures in rat pups. Brain Res Dev Brain Res 68: 275–277.
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28 The Kindling Phenomenon DAN C. MCINTYRE
Wada and Osawa, 1976). Such animals are then quite undeniably epileptic.
INTRODUCTION Since its initial discovery by Graham Goddard (1967), and its later naming by Goddard et al. (1969), the kindling phenomenon has been employed extensively as a chronic model of temporal lobe epilepsy (TLE). Kindling initially presents itself as a simple phenomenon, whereby provocation of focal seizures (most commonly using focal electrical brain stimulation) induces a clear progressive change in response over daily repetitions. The progression begins on the first day with a brief, low frequency electrographic afterdischarge (AD) at the electrode tip, which is associated with little behavioral response. However, the complexity of the phenomenon begins to appear as the response evolves over days, resulting eventually in the triggering of long, high frequency ADs associated with strong convulsive responses (Goddard et al., 1969). This progression is readily apparent from all limbic and most forebrain stimulation sites, but it is most dramatic from temporal lobe structures, such as the amygdala and adjacent cortices, including the piriform, perirhinal, insular, and entorhinal cortices (McIntyre et al., 1999). It is the daily progressive increase in response severity in both the EEG and behavior that defines kindling. From this brief description, it should be apparent that kindling initially models focal partial seizures. However, with daily repetitions, and the recruitment from the focus that precipitates the spread of seizures, kindling comes to model complex partial seizures with secondary generalization— particularly as consciousness is lost during these more complex events (McIntyre, 1970, 1979). Ultimately, after many days of triggering kindled seizures by the experimenter, seizures begin to appear spontaneously, i.e., without application of the kindling stimulus (e.g., Pinel et al., 1975;
Models of Seizures and Epilepsy
METHODS OF GENERATION Animal and Surgical Preparation All known species of animals tested to date show the kindling phenomenon. In this chapter, however, I will only describe the necessary preparations for kindling in the rat. The procedures commonly employed in kindling studies using young cats and infant rats are presented in Chapters 29 and 30, respectively. To electrically (versus chemically) kindle an animal, the animal must first be surgically implanted chronically with one or more intracranial electrodes. Such an implantation procedure can vary slightly between laboratories and surgeons, but most aspects of the procedure are quite constant between investigators. In presenting a surgical method to accomplish this goal, I will highlight the stimulation/ recording system of Molino and McIntyre (1972), which is commonly used by many investigators. Stereotaxic Surgery Correct placement of electrodes into the desired neuroanatomic regions requires the use of a stereotaxic instrument. The basic procedures for stereotaxic use in rodents are available elsewhere (Skinner, 1971; Cooley and Vanderwolf, 1977; Moore, 1981), so I will only describe specific aspects of the procedures that are critical to a successful outcome for chronic indwelling electrodes. The stereotaxic
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instrument is designed to rigidly hold the head of the animal in a fixed position relative to a carrier device that contains the electrode to be implanted. Determining the location of the brain site for electrode implantation is accomplished using a stereotaxic atlas. In the case of the rat, the atlas most frequently used is that of Paxinos and Watson (1998). Differences between atlas-based procedures are of two major kinds. The first procedural difference involves the reference point used for moving the micromanipulator that holds the electrode. That reference point is either (1) the interaural line between the two auditory meatii or (2) the bregma suture on the dorsal surface of the skull. The second procedural difference is based on whether the skull is positioned with its dorsal surface completely horizontal and parallel to the base plate of the stereotaxic instrument, or whether the anterior pole of the skull is raised or lowered relative to the posterior pole. Differences in the angle of inclination of the skull, thus, will determine what structures in the anterior-posterior plane will be impaled and implanted by the advancing electrode. Interaural Line vs. Bregma The original brain atlases used the interaural line as the reference point for positioning electrodes in the brain. More commonly bregma is now used. Bregma, of course, is the intersection point of the midline or sagittal suture with the anterior or coronal suture. In most brain atlases, both coordinate procedures are available for use. Our preference for bregma stems from its high visibility, its ease of zeroing with the electrode tip, and its greater accuracy during surgery involving rats of varied sizes (Cooley and Vanderwolf, 1977; Whishaw et al., 1977), particularly involving neuroanatomic sites in the anterior half of the brain (Paxinos and Watson, 1998). Skull Inclination The angle of inclination of the skull presented in most atlases is the horizontal plane, i.e., when the toothbar is adjusted so that the bregma and lambda sutures are in the same plane, and the skull is parallel to the base of the stereotaxic instrument (Swanson, 1993; Paxinos and Watson, 1998). In other atlases, the tooth bar is elevated 5 mm above the interaural line (Pellegrino et al., 1979), which allows for a slightly different approach to deep structures, where the insulated sides of the indwelling electrode will remain permanently secured as the electrode is cemented into position in the deep structure (e.g., in the amygdala). Of course, it is critically important that one uses the skull inclination position that corresponds to the atlas under consideration. Surgical Procedures In anticipation of surgery, electrodes need to be prepared or purchased. A description of our electrode construction
used in preparation for surgery and kindling studies with rats has been presented earlier by Molino and McIntyre (1972) and more recently by Mary Ellen Kelly (1998). The implantation of those electrodes requires anesthetizing the rat, a surgical approach to the brain, implantation and securing of electrodes to the skull, and postsurgical recovery. These various procedures are described below. Anesthesia Typical implantation of a single bipolar electrode requires about 1 hour. Anesthesia, of course, must be maintained across that period of time. Frequently used as an anesthetic is sodium pentobarbital, which is generally administered by injection into the peritoneal cavity (IP). Although the dose can vary with the strain of rat, typically in adult males (between 220 and 500 g), 55–60 mg/kg is adequate to achieve a surgical level of anesthesia. One can also use inhalant anesthetics like halothane. We, however, prefer to use halothane (or isothane) as an adjunct in the event that the primary anesthetic did not present an adequate level of anesthesia for surgery. It is important to make sure that an adequate air exchange or scavenger system is in place with inhalants, since repeated exposure to low levels of halothane can have toxic side effects. Aseptic Surgery Although in years past, rats were extremely hearty and resistant to pathogens in the face of surgical treatments, rats these days appear to be much less so. Indeed the change in sensitivity to pathogens appeared at about the time all breeders began producing specific pathogen free, or SPF, rats. Presumably heartiness of the rats against the pathogens has been lost. The use of aseptic procedures during surgery considerably reduces the likelihood of pathogen exposure (to the likes of Staphylococcus epidermidis), and the development of “skull rot”; the latter is characterized by skull softening and eventual disintegration, which invariably results in the loss of the headcap assembly with its intracranial electrodes. Electrode Implantation The procedures we use in the implantation of our stimulating/recording electrodes are enumerated below. Similar descriptions and detailed approaches to stereotaxic surgery with pictorial examples also are given by Cooley and Vanderwolf (1977) and Skinner (1972). Critical to this activity is the proper placement of the rat in the head holder of the stereotaxic instrument. This usually involves successful insertion of the two ear bars into the external auditory meatii of the rat. Although the insertion is very easily achieved by well-trained personnel, the novice usually experiences considerable difficulty. Thus it is best to involve experienced instructors when training the novice in this procedure to reduce both the physical stress to the rat from repeated
Methods of Generation
attempts with the ear bars and psychological stress to the novice. 8. 1. Sterilization of the instruments and disposable materials, like gauze pads and cotton swabs, is performed before surgical procedures are effected. This can be achieved with any number of devices that meet National Institutes of Health guidelines. Liquid disinfectants also are generally used when the instruments are reused during surgical procedures. 2. Electrodes can either be purchased or manufactured. They can also either be bipolar or monopolar in their construction. Many variations in electrode attributes are detailed elsewhere (Kelly, 1998). We prefer bipolar electrodes of our own construction and our own headcaps (Molino and McIntyre, 1972), which allows us up to 4 intracranial stimulation/recording electrodes and the required animal ground electrode per rat. In that assembly, insulated wires of various diameters can be used, which are typically 100 to 200 mm. The construction of those electrodes for surgical implantation is detailed by Molino and McIntyre (1972) and Kelly (1998). 3. Once the electrodes have been generated and have been sterilized (or placed in liquid disinfectant for ~15 minutes), they are positioned singly in the electrode holder of the stereotaxic instrument and adjusted to be in the vertical plane in preparation for implantation. 4. Now is the appropriate time to generate the anesthetized rat. After anesthesia instillation, when the rat no longer gives evidence of a flinch to a strong tail pinch, the fur on its head should be shaved with an electric clipper. The shaved area should extend from immediately behind the eyes to just behind the ears. 5. The rat can then be positioned in the ear holders of the stereotaxic instrument. It is critical to have the animal’s head held correctly to obtain both bilateral symmetry and the correct angle of inclination. If the ear bars are properly positioned in the external auditory meatii of the rat, the snout of the rat will move very little laterally but will easily rotate up and down in a vertical arc, pivoting around the ear bars. The teeth are then positioned in the tooth-restraining device, without overtightening, as the latter can inappropriately raise the nose and change the inclination of the head. 6. Now place a drop of sterile solution into each eye and begin cleaning and disinfecting the incision area with a combination of three different solutions (hibitaine, 70% alcohol, and bridine). First swab the incision area with hibitaine, followed by alcohol, and finally a small amount of bridine. 7. The incision down the midline of the scalp is now made in a single stroke using a surgical scalpel (#10 is preferred). The incision extends fully from the shaved area
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behind the eyes to the area behind the ears. However, it should not extend past the dorsal surface of the skull. This incision now allows for access to the skull surface and the important surgical reference points on the skull, including bregma, the midline suture, and lambda. For complete access, however, the periosteum (the thin membrane covering the skull) must be removed. Although some surgeons merely scrape the periosteum to the sides of the skull with the scalpel, we prefer to remove it with a careful but rapid dissection of its perimeter. Using two of more hemostats, now more broadly expose the skull surface. Using sterile cotton swabs, wipe the skull surface to remove blood or other debris. At this point, we apply to the skull a few drops of penicillin G (~400 IU/ml) and allow it to remain in situ a few minutes before rinsing with sterile saline. Next it is time to prepare the holes for the electrode(s) and skull screws. Based on the coordinates derived from the stereotaxic atlas, the electrode in its holder is positioned over the skull, directly above the structure to be implanted, according to the anterior/posterior and medial/lateral coordinates dictated by the atlas. The electrode tip is then gently lowered to a position immediately above the skull, and its anticipated position on the skull is marked on the skull with a fine-tipped marker. The electrode is then moved away from the area, and the hole to accommodate the electrode is then drilled using a bit that is clearly larger than the electrode. The drilling is done in repeated short vertical movements, ensuring that the skull is fully penetrated, but extra care is taken to ensure that the drill does not pass thought the skull into the dura mater. Once the hole is drill satisfactorily, the dura is then cut with a sterile hypodermic needle; this will allow the electrode to easily penetrate the brain without being displaced or deformed by the relatively tough dura. This exercise is repeated for each electrode to be implanted. After drilling the holes for the electrodes, the holes for the stainless steel jeweler screws are generated (e.g., ~1.6 mm o.d. and 3.2 mm long; Stoelting, #51457). We prefer to secure the headcap assembly with 6 screws. The drill bit used to make those holes is appropriate to the size of the screws to be employed (typically 0.6– 1.0 mm o.d.). The screw holes are placed as broadly as possible on the skull to create the most stable and secure headcap platform (much like the stability of a table with broadly vs narrowly placed legs). When drilling the screw holes, avoid all suture areas, as many blood vessels pass beneath them (e.g., superior sagittal sinus below the midline suture). Also avoid the muscles on the lateral edges of the skull. After carefully cleaning the skull, turn the screws into their holes. Be sure to only pass the screw into the skull
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Chapter 28/The Kindling Phenomenon
by a few full rotations to avoid its appearance below the skull where it would depress the dura mater and/or enter the brain. With the jeweler screws securely in place, including the ground screw and its attached wire, again approach the drilled electrode hole with the electrode in its holder. The depth reading for implantation of the electrode can be based on either a skull surface measurement or interaural line coordinate measurements (described in the atlas). From the metric scale on the electrode carrier, one can easily determine the depth the electrode must pass ventralward to be implanted in the desired structure. Once the electrode is lowered to the determined depth, it is cemented in place with dental acrylic (e.g., dental cement kit, Stoelting, Plastics One). This is accomplished by mixing a small amount of the powder with the liquid solvent (methylmethacrylate) until it becomes viscoid. Then with a small spatula, this paste is applied to the skull/electrode interface and nearby screws. Be sure to avoid getting the cement prematurely into any other electrode holes. The cement is then allowed to dry (~5 minutes) before proceeding to subsequent electrodes. When the electrode is secured, remove the electrode holder, and repeat the procedure if other electrodes are involved. If using the McIntyre connector, carefully insert the amphenol pins into the headcap using a needle nose hemostat. Then, the exposed electrode wires are placed gently between the headcap assembly and the dried cement surface, and the entire wire assembly and bottom third of the headcap are artistically covered with cement. If a commercially available headplug and single electrode is employed, it now can be secured to the skull with a covering of cement in an identical fashion to the multiple electrodes in the McIntyre connector. Following this operation, the rat is removed from the stereotaxic instrument and is placed on either a warm heating pad or under a warming light until evidence of its recovery is clear. Analgesics can now be administered, if desired. In addition, a subcutaneous dose of penicillin G procaine (22,000 units/kg; IM) should be administered to minimize possible subsequent infections.
Postoperative Care We allow our rats 7–10 days postoperative recovery before beginning a kindling study. During this period and all remaining time, the rats are housed singly, since excessive grooming and aggressive interactions with paired rats will damage the headcap. Within 24 hours of surgery, daily handling of the rat begins. This handling will reduce stress on the rat during the kindling phase, and will permit easy headcap connections and disconnections. Similar handling can also be very calming for the rats when performed for several days before surgery.
Kindling Procedures Basic Procedure One can electrically kindle a rat with a very modest amount of equipment. After surgical implantation of bipolar electrodes in a highly sensitive and epileptic-prone forebrain structure like the amygdala or hippocampus, a brief electrical current of sufficient intensity (and other particular characteristics described below) will trigger a local seizure or AD, which begins the process of epileptogenesis. Stimulus Parameters The efficacy of numerous stimulus parameters on the genesis of kindling has been investigated by a number of researchers over the years (e.g., Goddard et al., 1969; Racine, 1972a; Racine et al., 1973; Corcoran and Cain, 1980). Basically, however, if an AD is triggered by the stimulus configuration, kindling will occur (Racine, 1972a). Although stimulation at intensities below threshold for triggering an AD will lower the AD threshold over time, kindling will not proceed without the triggering of frank ADs. Typically either sine- or biphasic square-wave pulses are delivered through the implanted electrode using a constant current stimulator at tetanizing frequencies (10–150 Hz, but most commonly 60 Hz) for 1 or more seconds. The intensity of the stimulus selected for kindling is usually done in one of two ways. One can first determine the AD threshold, which is the stimulus intensity that will trigger an AD that outlasts the stimulus itself for a specified length of time (we use a minimum criterion of 2 seconds or longer). Thus on all subsequent stimulus applications, the stimulus intensity is presented at that threshold value. A different approach is to present a much higher stimulus intensity that will trigger a maximal response in all recipients. For example, many investigators kindle the dorsal hippocampus at an intensity of 400 mA, which is about 10 times its AD threshold value. This approach largely ignores questions about the seizure sensitivity of the local circuitry at the electrode tip and how it might be altered during the kindling process. Low-frequency stimulation (~3 Hz) can also kindle a rat, and can do so fairly rapidly, but the current intensity of the stimulus (in excess of 1 mA) must be extremely high (Corcoran and Cain, 1980; 1981) and the duration quite long (many seconds). This effect, however, has only been reported for square wave stimulation and has not been seen with sine waves. Thus, it is quite plausible that the lowfrequency square kindling effect is based on the fact that square waves are created by aggregates (averages) of sine waves (based on a single fundamental frequency like 3 Hz), which are composed of many frequencies in the highfrequency range. In doing so, and because of the high intensity necessary to kindling with the low-frequency square wave, much power is assigned to those higher frequencies, tetaniz-
Methods of Generation
ing wave forms. As a result, it might be that the high current intensities presented simultaneously to those high-frequency waves actually generate the low-frequency kindling effect. Afterdischarge Threshold (ADT) The ADT is a very important feature of the focal seizure network, yet, as indicated above, many investigators do not assess it either before or after their applied treatments. During an assessment of the ADT, other important focal characteristics can be observed, including the duration of the focal AD and its recruitment of other structures (assuming one has placed recording electrodes elsewhere in the brain). The most common technique for determining the ADT is by using some form of an ascending method of limits. In Racine’s (1972a) original procedure, one begins with a low intensity like 10 mA. If this stimulus does not trigger an AD, the intensity is doubled for the next trial. This procedure of doubling continues until an AD is triggered. At this point, to more carefully determine the real ADT in Racine’s procedure, one next selects a stimulus intensity that is halfway between the intensity that triggered the AD and the immediately previous value that did not. Thus one is slowly titrating the current intensity to a more precise threshold value. One difficulty with this procedure is that the elicitation of an AD often makes the network more refractory to subsequent AD events and will artificially and temporarily raise the ADT. Because of this elevation, Racine suggested that the rats should be stimulated every second day to circumvent the problem. This titration procedure, of course, creates a fairly lengthy ADT protocol. We use a different, much more expedient ADT assessment method. It also begins with a low intensity like 15 mA. The stimulus intensity is then incremented by predetermined values once ever minute until an AD is triggered. For example, intensities of 15, 25, 35, 50, 75, 100, 150, 200, 250, 300 mA peak-to-peak are delivered until an AD is triggered, which then ends the procedure. This ADT assessment is often employed at the beginning of the experiment and then again at its conclusion. The stimulus that is selected initially as the kindling stimulus is usually the value that triggered the initial ADT response. Importantly, ADTs in most limbic sites (excluding the granule cells in the dentate gyrus), and particularly cortical structures, continue to drop during the kindling process (or following long periods of implantation alone; Löscher et al., 1993). In such circumstances, one can track these changes. Instead of administering daily kindling stimulation at the ADT value, rats can be stimulated first at an intensity one increment below that ADT value. If that lower intensity is insufficient to trigger an AD that day, then 1 minute later the next higher value is administered, until an AD is triggered (Mohapel et al., 1996). In this way, the ADT is quickly redetermined on a daily basis.
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Interstimulus Intervals The interstimulus interval (ISI) or the rate at which the kindling stimulus is applied to the animal, considerably influences the speed of epileptogenesis (Freeman and Jarvis, 1981). In our original paper describing kindling, we examined the ISI variable and observed that as the ISI shortened and approached 20 minutes, the efficacy in amygdala kindling was substantially reduced (i.e., the kindling rate lengthened). We felt that the best ISI using the minimum numbers of stimulation to produce the kindled state was ~24 hours (Goddard et al., 1969). This conclusion was later modified by Racine et al. (1973), who found that with repeated provocation of ADs, the ADTs often rose, and, that in our original report, since we did not initially record EEG activity, we likely stimulated many times below threshold for AD, which gave a false minimally effective ISI. In their reassessment of ISIs and kindling rate with EEG recordings, they found that kindling above threshold for ADTs at intervals of 1–2 hours was the minimum ISI for most efficient amygdala kindling (Racine et al., 1973). Age of the animal is also an important variable in the application of ISIs to the ease of kindling (Moshé 1981). To this end, Moshé et al. (1983) kindled the amygdala of rat pups with an ISI of 15 minutes; this interval, of course, is totally ineffective in producing amygdala kindling in adult rats. These kindling procedures with rat pups are described in detail in Chapter 30. The structure to which the kindling stimulus is applied further affects the rate of epileptogenesis (see Monitoring the Progressive Development of Kindling, following). In the context of ISIs, short intervals are very poorly able to kindle the amygdala, as indicated above, but, with modification to other aspects of the stimulus configuration can result in fairly “rapid kindling” from the hippocampus, as described by Lothman and Williamson (1994). In reality, rapid kindling of the hippocampus in 1 day with short ISIs is characterized by progressively heightened excitability during the repeated presentations, but the kindled state is not achieved during that treatment, as it would be with longer ISIs over days. To achieve the kindled state following rapid kindling, the kindling stimulus must be applied again in some fashion over several more days. The genetics of the rat also importantly affect kindling sensitivity to ISIs, where rats that are more naturally seizureprone (Fast kindlers) will benefit in their kindling rate from exposure to the kindling stimulus at shorter ISIs, while naturally seizure-resistant rats (Slow kindlers) will not (Elmér et al., 1998; Shin et al., 2004). The mechanism(s) of resistance to kindling and short ISIs is not known but likely involves recruitment of inhibitory mechanisms. Indeed, the provocation of kindled seizure activity results in a variety of postseizure inhibitory effects.
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One obvious EEG correlate of the postseizure inhibitory effects is the postictal spike (nonrhythmic spiking that occurs after the rhythmic or ictal event). When the ictal events are triggered frequently in an amygdala-kindled rat (i.e., with short ISIs), the triggering of full kindled convulsive seizures is greatly diminished, while the concomitant incidence of postictal spiking significantly increases (Engel and Ackermann, 1980). This diminishment in ictal activity with short ISI stimulation is likely related to a change in the normal balance of excitation and inhibition in the kindled network in favor of the inhibition (Engel et al., 1981). In rats, like the Slow kindling rat strain (Racine et al., 1999; McIntyre et al., 1999), the trigger of postictal spiking is more provocative and much longer-lasting than in the Fast kindling strain. Thus genetic variation in a population of rats (and perhaps humans) should result in individuals who are highly varied in their seizure proclivity, including seizure genesis, postictal effects, and susceptibility to ISIs. Clearly vulnerability of the recipient to ISI treatments varies with structure, age, and expressed genes.
MONITORING THE PROGRESSIVE DEVELOPMENT OF KINDLING The progressive development of kindling epileptogenesis, resulting eventually in the kindled state, is quite easy to monitor. The daily repetition of the kindling stimulus results in both electrographic and behavioral advancements, which lead first to the kindled state, and then second, at a much later time, to spontaneous seizures. The term kindling is applied to the process of epileptogenesis, independent of whether stimulus is applied to different limbic sites. Such ubiquitous application gives the impression that kindling proceeds in a homogeneous fashion in those different sites; however, this is not true. Each site has its own specific profile of kindling development. Outlined briefly below are some of the unique features of kindling in different parts of the forebrain.
Amygdala Kindling We originally described the behavioral sequence that results from daily amygdala kindling largely without the benefit of EEG recordings (Goddard et al., 1969). In that description, the first few stimulations generally were without behavioral consequence, except for an occasional arrest of movement. However, as automatisms begin to develop, first the eyelid ipsilateral to the kindling electrode closes during the stimulus application; this response is followed a few stimulations later by chewing and head bobbing movements. As the stimulation trials progress, the focal seizure begins to generalize and provoke mild unilateral forelimb clonus. Soon thereafter, the clonus becomes bilateral and progres-
sively stronger with rearing, and then, later, with rearing with falling. Racine (1972b) has detailed this behavioral progression during amygdala kindling with his now classic five point rating scale. The scale classifies initial oralimentary movements and head bobbing as stages 1 and 2, respectively. With the appearance of the mild forelimb clonus, stage 3 is recorded. The subsequent addition of rearing to this sequence is named stage 4, followed by a loss of balance and falling, which defines stage 5. Although these stage-5 seizures are clonic-tonic-clonic seizures, involving all 4 limbs, the rat must be suspended to clearly see the hindlimb involvement (Chen et al., 1996). The rate at which the progression proceeds through the 5 stages is determined by a number of variables, including the strain of rat (see Table 1) and the placement of electrodes within the amygdala (e.g., Mohapel et al., 1996; McIntyre et al., 1999). When kindling from the amygdala, the electrographic progression of AD is also quite clear. The initial stimulation triggers a simple, short mono- or biphasic AD of low frequency, which propagates poorly to the contralateral hemisphere (Racine, 1972a). Stage-1 behavioral arrest usually accompanies this response. With repetitions, the AD increases in complexity (waveform, frequency, duration) and projection more forcefully to distant neuroanatomic sites. The behavioral sequelae, of course, are evolving progressively with these more provocative electrographic seizures. A typical kindling progression in AD expression and behavior is indicated for the amygdala in Figure 1. If the kindling stimulus continues to be applied to the focus after the development of stage 5 seizures, the behavioral sequence evolves further into more severe seizures, stages 6–8, described by Pinel and Rover (1978); they suggested these stronger seizures represent progressively
TABLE 1 The typical progression of motor seizures stages (from left to right) using Racine’s Scale (1972b) during kindling from five different brain structures in rats that were bred to be either seizure-prone (Fast rats) or seizureresistant (Slow rats) to amygdala kindling. Note that the Fast rats skip the partial seizure stages (1,2) in all structures, while Slow rats show the more “typical” progression in the amygdala and piriform cortex but require many more trials than normal outbred rats Kindled Structure
Fast rats
Slow rats
Amygdala
3,4,5
1,2,3,4,5
Piriform Cortex
3,4,5
1,2,3,4,5
Perirhinal Cortex
4,5
3,4,5,1,2
3,4,5
3,4,5,1,2
4,5
3,4,5,1,2
Dorsal Hippocampus Frontal Cortex
Monitoring the Progressive Development of Kindling
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B C
Stimulation FIGURE 1 Progressive development of EEG seizure activity in the two paired amygdalae, right (top) and left (bottom), during amygdala kindling. A, first afterdischarge triggered by stimulation of the left amygdala. Note the large monophasic, low-frequency response, which propagates poorly to the contralateral side. B, fifth stimulation triggers a more complex response, which projects contralaterally with greater strength. C, first stage-5 convulsion (between the filled circles) on the 15th stimulation. Note that the afterdischarge continues for many seconds after the termination of the convulsive seizure.
greater involvement of brainstem circuits. With further kindling, these more severe seizures can be followed in many days or even months by the appearance of spontaneous seizures (Pinel et al., 1975), i.e., where convulsive seizures now occur independent of the kindling stimulus. The spontaneous occurrence of seizures happens to rats that were kindled from the amygdala, but also with protracted kindling from other neuroanatomic sites, including the frontal and posterior cortex, perforant path or entorhinal cortex (Pinel, 1981; Michael et al., 1998).
Kindling from Other Forebrain Sites Although kindling proceeds from other limbic or forebrain sites in a manner not dissimilar to the amygdala, there are distinct and important differences. Considering the adjacent piriform and perirhinal cortices, both exhibit ADTs that are much higher than those of the amygdala (McIntyre et al., 1999). Like the amygdala, the piriform cortex follows the 5 stages of seizure development described by Racine (1972b), but the rate of development is usually faster than the amygdala (Table 1). The evolution of the kindling AD in the piriform area is also similar to the amygdala. The perirhinal cortex, on the other hand, not only kindles faster than any other structure in the forebrain, its progres-
sion rarely follows Racine’s stages (McIntyre et al., 1993). Indeed, the first seizures triggered from the perirhinal area are usually convulsive stage 3–5 responses (Table 1). The nonconvulsive stages (1–2), if they occur at all, appear later in the kindling protocol, after the expression of the forelimb convulsion (McIntyre et al., 1993). This makes sense based on the functional anatomy of the limbic networks. As the frontal motor cortex appears to be important in the expression of early convulsive seizures (Kelly et al., 1999) and the perirhinal area densely innervates that frontal cortical mantel (McIntyre et al., 1996), a seizure originating in the perirhinal cortex should result in convulsive forelimb expression very rapidly, as though the experimenter was directly activating the frontal motor cortex itself. The only difference between perirhinal cortex kindled seizures and direct activation of seizures from frontal cortex by focal stimulation is that direct frontal cortex stimulation usually triggers trial one convulsive seizures that are clonic-tonic-clonic in form and quite short (10–12 seconds), with a strong tonic phase and short clonic phases, compared to much longer and more clearly clonic perirhinal or limbic kindled seizures (McIntyre, 1970; Burnham, 1978; McIntyre et al., 1993). With many repetitions, the directly activated frontal cortical seizures will transform (kindle) into more typical limbic seizures, as they acquire a significant lengthening of the
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second clonic phase (Table 1). In doing so, frontal seizures also acquire the memory disrupting or amnestic properties of typical limbic or amygdala kindled seizures (McIntyre 1970, 1979). Kindling from the hippocampus, regardless of its dorsal or ventral aspect, will produce a convulsion profile that is generally quite similar to the amygdala, with some exceptions (Racine et al., 1977). Yet, the hippocampal AD profile is quite different from the amygdala or other areas, as it is characterized by a primary discharge of ~20–30 seconds followed by an isoelectric period of ~20–30 seconds and a secondary or rebound AD of ~10 seconds (Burnham, 1975; Grace et al., 1990; Leung, 1987; Racine et al., 1977; McIntyre and Kelly, 1993). This profile is never seen in the perirhinal cortex and only occasionally in the amygdala. In addition, during the kindling process, this same AD profile reoccurs daily with little progression until the seizure appears to more strongly recruit the temporal cortices (e.g., amygdala and the perirhinal areas). At this time, the seizure stage usually advances to its convulsive form, and the hippocampal AD complexity changes as the seizure discharge in the temporal cortical structures appears to return to the hippocampus, where it is recorded in the EEG. Such an EEG example is shown in Figure 2.
Hippocampal kindling is also often associated with the early appearance of “wet dog shakes” (WDS); these responses occur during seizure discharges fairly early in the kindling process and well before any of Racine’s motor stages. The WDS progressively disappear during the course of kindling as the convulsive expression of the rat begins to express Racine’s motor stages. Interestingly, rats exhibiting high frequencies of WDS often kindle at much slower rates than rats infrequently showing that behavior. Such relative slowness in kindling is also true of rats expressing high levels of interictal or postictal spiking (e.g., Engel and Ackermann, 1980). Although these observations are generally true of the hippocampus, they are much more evident when the kindling site is in the amygdala. It is very clear that kindling occurs best in neuroplastic areas (Goddard et al., 1969; Racine, 1978; Morimoto et al., 2004). If the area does not show such properties, it likely will not show the progressive development that defines kindling. For example, the hypothalamus shows little evidence of kindling per se. However, if a site in the hypothalalmus is driven at a sufficient intensity, it will project activity to other areas that are more neuroplastic and can be readily kindled (Cullen and Goddard, 1975). Certainly much of the brainstem and cerebellum shows no evidence of kindling (Goddard et al., 1969).
A
B Stage-5 Seizure FIGURE 2 EEG response in the amygdala (AM) and the kindled ipsilateral dorsal hippocampus (DH) following the kindled stimulation delivered to the DH on the trial before the first stage 5 response (A), and the next trial (B), which triggered the first stage-5 convulsion (between the two filled circles). Open arrowheads indicate application of the kindling stimulus to the DH. Note, in A, that the primary afterdischarge in the DH is followed by an isoelectric period and then a secondary afterdischarge with relatively little projection to the ipsilateral amygdala. By contrast, in B, a similar primary discharge in the DH recruited the amygdala, which triggered a stage-5 seizure. The high voltage amygdala response during the convulsion appears to project back into the DH in the isoelectric period to change slightly its presentation, with little change to the secondary discharge.
Monitoring the Progressive Development of Kindling
Kindling Transfer and Antagonism Once the kindled network has been established and stage5 convulsive seizures can be elicited on a regular daily basis, the kindling of a second site usually proceeds at a much faster rate than if kindling of the first or primary site had been omitted. This phenomenon, termed positive transfer (Goddard et al., 1969; McIntyre and Goddard, 1973; Burnham, 1975), occurs throughout the forebrain between structures in both the ipsilateral and contralateral hemispheres. The positive transfer phenomenon likely is also the basis of the “mirror” focus phenomenon often described by Morrell (reviewed by McIntyre and Poulter, 2001). Positive transfer, however, does not always occur between kindled sites. If two sites during kindling are stimulated concurrently, for example, stimulating the amygdala and septum on alternate days, one site progressively kindles at a normal rate (the amygdala), while seizure genesis in the other site (septum) is suppressed. This phenomenon has been called “kindling antagonism” (Burchfiel and Applegate, 1989), and it appears to make use of natural inhibitory mechanisms recruited by the seizure activity from the more dominant site (Applegate et al., 1986).
Neuropathology As there is often considerable evidence of neuropathology in human epilepsy and some animal model systems, one might assume that this also would be the case with epilepsy induced by kindling. Yet this is not the case. Clearly, there are many ways in which the balance between excitation and inhibition can be altered in the brain to result in the expression of seizures. For example, there can be (1) the loss of inhibitory interneurons, or (2) increased sprouting of excitatory fibers onto principal cells, or (3) a change in glia numbers and varieties that alter the metabolic and ionic environment of the network—all of which can tip the balance in favor of excitation. In addition, all of these various changes occur when epilepsy develops after the brain has been subjected to the severe seizures of experimental status epilepticus (Morimoto et al., 2004). By contrast, neuronal loss, sprouting, and glial alterations produced during kindling are far more circumspect. As a result, kindling dramatically tells us what is not necessarily lost or altered during the process of epileptogenesis (McIntyre et al., 2002). In fact, most researchers find very little evidence of neuron loss as a consequence of kindling using either a normal kindling protocol with a relatively modest number of triggered seizures (e.g., Tuunanen and Pitkanen, 2000; Morimoto et al., 2004) or after many stimulations and the development of spontaneous convulsions (Michalakis et al., 1998). The few exceptions to that statement include Cavazos et al. (1994), who reported progressive reductions of neuron numbers in the CA1-3 region of the hippocampus, the
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dentate hilus, entorhinal cortex, and endopiriform nucleus following the elicitation of 150 stage-5 seizures. Although Racine’s group also reported an apparent loss of hilar cells following kindling (Spiller and Racine, 1994), they later found the apparent “loss” of neurons was due to an expansion of the hilar area (Adams et al., 1998), and that correction for the expansion suggested that the hilar neuron numbers actually were not altered by kindling. An actual loss of neurons, however, was suggested by Lindvall’s group (Bengzon et al., 1997), where daily kindling resulted in apoptosis of some dentate granule cells. Of course, the loss of some cells during kindling would go unnoticed, since the seizures also increase synaptogenesis in those same areas (e.g., Bengzon et al., 1997; Parent et al., 1998; see the following). The expansion of the hilus during kindling, as well as other areas of the brain, can readily be associated with astrocyte hypertrophy. Such hypertrophy is accompanied by reorganization of the astrocytic cytoskeleton early in the kindling process, which persists for many weeks in the hippocampus, amydgala, and piriform cortex following the last seizure (Khurgel and Ivy, 1996). Thus kindling is not benign, and by altering the number of glial cells, a considerable change in the functioning of both the glial and neuronal networks can easily occur. Although kindling does not result in a large loss of neurons, there is a clear kindling-induced reorganization of neuronal circuitry, as presumably new connections are being formed. This reorganization is based on both synaptogenesis engendered by kindling (Parent et al. 1998) and sprouting of fibers from pre-existing axons. In the latter case, Sutula et al. (1988) have shown sprouting in kindled brains represented as a change in pattern of Timm staining of the mossy fibers in the inner molecular layer of the dentate gyrus. Such kindling-induced sprouting also appears in stratum oriens of CA3 in the dorsal hippocampus following amygdala kindling (Represa and Ben-Ari, 1992). The sprouted changes likely reflect the creation of new circuits, involving both feedforward excitation and inhibition, which speculatively may or may not impact epileptogenesis. Nonetheless, the kindled brain is certainly a different brain from one that is naïve to the kindling process.
Imaging and Metabolic Changes The neuronal networks associated with kindling can be visualized using a variety of metabolic and other markers. Using a modification of the 14C-2-deoxyglucose autoradiography technique, Engel et al. (1978) were the first to show increased glucose uptake in the stimulated amygdala and many of its projection structures during partial seizures (stages 1–2), while generalized seizures (stages 3–5) were associated with more widespread and bilateral uptake. These same general patterns were also seen during amygdala
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kindling using the metabolic marker c-fos (Dragunow et al., 1988; Teskey et al., 1991; Hosford et al., 1995; Foster et al., 2004). Although both techniques indicate increased metabolic activity associated with seizures, they do not discriminate as to whether the neuronal substrate of that activity is primarily excitatory or inhibitory in nature, as both kinds of neurons (as well as glia) require glucose to operate. In any case, such markers do define the cells and networks that are using the greatest amounts of energy during seizures, which then suggests functional relationships.
Genetic/Molecular Changes Kindling also results in a large number of molecular changes. Some of those changes certainly underwrite the altered excitability that is kindling, while others are likely unrelated to the network excitability changes, but reflect functional alterations in other systems that impact different behaviors and/or personality attributes. These changes include many of the neurotransmitter systems; this can involve the altered production of transmitters per se, presynaptic release and postsynaptic receptor mechanisms, glia transport mechanisms, second messenger mechanisms, as well as alterations to neurotrophin and neuropeptide mechanisms, and many other outcomes. Clearly kindling is complicated. Many of these molecular changes have been reviewed recently elsewhere (e.g., Morimoto et al., 2004) and will not be revisited here. In addition, the long-lasting changes that create the kindled state must be associated with genetic alterations to their control mechanisms. The search for those mechanisms and their controlling genes continues to occupy the attention of many epileptologists and researchers (e.g., Gloor, 1989; Mody, 1993; Schwarzer et al., 1996; Chiasson et al., 1997; Mody, 1999; Cole, 2000; Klitgaard and Pitkanen, 2003)
Response to Antiepileptic Drugs (AEDs) Because kindling so closely models the clinical phenomenology of complex partial seizures with secondary generalization, both in its etiology and its pharmacology, it has been widely accepted as a model of temporal lobe epilepsy (TLE) (Sato et al., 1990; Löscher, 1999, 2002). Other commonly used models for this purpose have been the poststatus epilepticus (SE) models. Happily the pharmacologic response of the SE models and kindling are remarkably similar (Löscher, 2002). As kindling is much less neurologically insulting than the SE models, it should assume a preferred position for drug testing of antiepileptic compounds against chronic seizure disorders, especially TLE. Its importance in that regard stems from the fact that the chronicity of epilepsy almost certainly subscribes to several mechanisms that differ from those of acute seizures. Thus the more appropriate models for drug screening for epilepsy should
be aware of this likelihood (White, 2003). Indeed, kindling has been able to corroborate and duplicate the efficacy of many well-known AEDs and led to the actual discovery of one drug that had escaped identification by the acute models (Löscher et al., 1998; Löscher, 2002; Klitgaard and Pitkanen, 2003).
LIMITATIONS TO KINDLING The main limitation to using the kindling procedure is its labor-intensive nature. If one is only interested in an animal model that develops spontaneous seizures, then such a result can be more simply achieved using a variety of other treatments that are less labor intensive. Such alternative procedures include SE as well as various genetic models (Löscher, 2002). In the case of SE, depending upon which precise model is employed (e.g., kainic acid, pilocarpine, electrical, etc.), the protracted seizure induced by the treatment first causes considerable brain damage, which is followed days or weeks later by spontaneous seizures. However, it is unknown whether the brain damage that follows SE is causative of the subsequent spontaneous seizures or is merely a bystander effect. As the kindling procedure also produces spontaneous seizures (after many stimulations) and is associated with very little brain damage (Michael et al., 1998; Löscher, 2002), the brain damage engendered by SE is likely not causative of the spontaneous seizures, and may confound interpretation of causation. Certainly, if one is interested in studying the process of epileptogenesis, particularly in an extended form (where it is not necessary to achieve spontaneous seizures as the end point), then kindling is ideal. Kindling is very easy to induce once the animal is surgically prepared, and the genesis of the seizures is completely under experimenter control. Further, kindling is not associated with life-threatening physical insults like those that threaten the animal during and after SE. As a result, mortality during kindling studies is typically zero; by contrast, the animals in SE models are extremely vulnerable and morbidity is often high, especially in older animals. The kindling model is also very easily replicated; indeed, when using either inbred strains (e.g., mice) or those previously subjected to genetic selection (rats or other animals), the variability in kindling profiles between animals is very low (McIntyre et al., 1999). Kindling in developing young cats and in rats is described in Chapters 29 and 30, respectively.
INSIGHT INTO HUMAN EPILEPSY The pragmatics of kindling as a research tool are clearly seductive, especially as the phenomenon satisfies all of the important features proposed by Wada (1976) of a good
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References
experimental model of human epilepsy. Those features include (1) precise experimental control over the site of seizure induction, (2) an ability to create an epileptic site without producing gross pathology, (3) good control over the initiation of individual seizures and their progressive development, (4) easy induction of a seizure by an identified stimulus, (5) eventual development of spontaneous seizures that mimic electroclinical patterns in humans and (6) persistence of the epileptic state for long periods. Although kindling clearly fulfills these various criteria, there have been those who have questioned its validity as a model of human complex partial seizures. One of those criticisms is that there is little published evidence for kindling in humans. The reasons for this, however, should be fairly obvious. If one intentionally kindles patients in North America, a legal liability would likely be an inescapable spectre. Yet there are two reports that have been bold enough to suggest kindling in humans with implanted intracranial electrodes (Monroe, 1982; Sramka et al., 1977), as well as a number of studies describing recurrent spontaneous seizures in schizophrenic patients after receiving repeated electroconvulsive stimulation (Sato et al., 1990). Indeed, as indicated previously (McIntyre et al., 2002), several neurologists have described to me (DCM) that they have seen the kindling process in their patient with deep electrodes and brain stimulation, but would not consider formally reporting that effect. One of the researchers, after observing a progressive increase of ADs over days using tetanic stimulation, chose to continue the stimulation protocol, but at an intensity below threshold for the triggering of ADs and seizures. Certainly, it would be grossly illogical, and perhaps arrogant, to assume that humans are the only animal that does not show some degree of kindling progression when confronted with brain stimulation in neuroplastic, seizure-prone areas. An aspect of kindling that is particularly interesting and important for epilepsy is its progressive nature. In that context, kindling might be best viewed as a model of human epilepsies that are progressive in nature, modeling those patients whose seizures clearly become more frequent over time and progressively more refractory to treatment (Engel and Shewmon, 1991). However, there may be an unrecognized progressive nature to seizure development in all humans, as most patients generally present to the clinic with seizures that developed previously (and progressed?) into clinical events. In comparing this observation in humans with the events of experimental kindling, the period of time in which the behavior and electrographic seizures are most progressive in the rat is during the early trials from the first focal seizures to the early convulsive responses (see Figure 1). During that period, ADs progressively grow into their full “mature” form, including frequency, complexity, and duration. A similar early period in humans might be one containing progressive preclinical seizures; as a result, possibly
progressive preclinical seizures would go unrecognized and unrecorded. Indeed, focal seizures restricted to the hippocampus of humans are not associated with any behavioral response (and thus are preclinical), and go undetected until they “progress” sufficiently to recruit other temporal lobe or cortical structures, where they become clinically significant. Again, to this same point, once experimental kindled seizures are fully developed over many trials, most animals exhibit quite stable seizure profiles for relatively long periods of time. Thus, during this apparently stable period, kindling does not seem to be progressive at all, perhaps like the human forms that do not appear to be progressive! These “stable” kindled seizures would now only show a progressive character if examined over much longer periods of time (where stage 6–8 seizures might be finally observed). Lastly, to this same point, when kindled seizures are triggered frequently (perhaps like those humans who have frequent seizures), a variety of seizure phenotypes are observed, including different forms and duration. This is particularly true when considering variation in the genetic background of the rat (McIntyre and Poulter, 2001; Shin et al., 2004). Such possible genetic underpinnings associated with variability in seizure expression profiles and progression in humans is not known, but might be expected based on various animal model systems (e.g., McIntyre et al., 1999). In recent times, the value of kindling has become quite apparent in the pharmaceutical industry where drug screening is taking place. Since kindling has been able to identify important new antiepileptic compounds (e.g., levetiracetam) that have been missed by other seizure assessment tools, it will likely remain near the top of researchers’ armament list in the employment of drug discovery.
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Cavazos, J.E., Das, I., and Sutula, T.P. 1994. Neuronal loss induced in limbic pathways by kindling: evidence for induction of hippocampal sclerosis by repeated brief seizures. J Neurosci. 14: 3106–3121. Chen, L., Noffel, M., Cottrell, G.A., Hwang, P.A., and Burnham, W.M. 1996. Amygdala kindled convulsions in suspended rats. Exp Neurol. 141: 347–349. Chiasson, B.J., Hong, M.G., and Robertson, H.A. 1997. Putative roles for the inducible transcription factor c-fos in the central nervous system: studies with antisense oligonucleotides. Neurochem Int. 31: 459–475. Cole, A.J. 2000. Is epilepsy a progressive disease? The neurobiological consequences of epilepsy. Epilepsia. 41 (Suppl 2): S13–S22. Cooley, R.K., and Vanderwolf, C.H. 1977. Stereotaxic Surgery in the Rat: A Photographic Series. London, Ontario: University of Western Ontario. Corcoran, M.E., and Cain, D.P. 1980. Kindling of seizures with lowfrequency electrical stimulation. Brain Res. 196: 262–265. Corcoran, M.E., and Cain, D.P. 1981. Kindling with low-frequency stimulation: generality, transfer, and recruiting effects. Exp Neurol 73: 219–232. Cullen, N., and Goddard, G.V. 1975. Kindling in the hypothalamus and transfer to the ipsilateral amygdala. Behav Biol. 15: 119–131. Dragunow, M., Robertson, H.A., and Robertson, G.S. 1988. Amygdala kindling and c-fos protein(s). Exp Neurol. 102: 261–263. Elmér, E., Kokaia, M., Kokaia, Z., McIntyre, D.C., and Lindvall, O. 1998. Epileptogenesis induced by rapidly recurring seizures in genetically fast but not slow kindling rats. Brain Res. 789: 111–117. Engel, J. Jr., Wolfson, L., and Brown, L. 1978. Anatomical correlates of electrical and behavioral events related to amygdaloid kindling. Ann Neurol. 3: 539–544. Engel, J. Jr., and Ackermann, R.F. 1980. Interictal EEG spikes correlate with decreased, rather than increased, epileptogenicity in amygdaloid kindled rats. Brain Res. 190: 543–548. Engel, J. Jr., Ackermann, R.F., Caldecott-Harzard, S., and Kuhl, D.E. 1981. Epileptic activation of antagonistic systems may explain paradoxical features of experimental and human epilepsy: a review and hypothesis. In Kindling 2. Ed. Wada, J.A. pp. 193–217. New York: Raven Press. Engel, J. Jr., and Shewmon, D.A. 1991. Impact of the kindling phenomenon on clinical epileptology. In Kindling and Synaptic Plasticity. Ed. Morrell, F. pp. 195–210. Boston: Birkhauser. Foster, J.A., Puchowicz, M.J., McIntyre, D.C., and Herkenham, M. 2004. Activin mRNA induced during amygdala kindling shows a spatiotemporal progression that tracks the spread of seizures. J Comp Neurol. 476: 91–102. Freeman, F.G., and Jarvis, M.F. 1981. The effect of interstimulus interval on the assessment and stability of kindled seizure thresholds. Brain Res Bull. 7: 629–633. Goddard, G.V. 1967. Development of epileptic seizures through brain stimulation at low intensity. Nature 214: 1020–1021. Goddard, G.V., McIntyre, D.C., and Leech, C.K. 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol. 32: 295–330. Gloor, P. 1989. Epilepsy: relationships between electrophysiology and intracellular mechanisms involving second messengers and gene expression. Can J Neurol Sci. 16: 8–21. Grace, G.M., Corcoran, M.E., and Skelton, R.W. 1990. Kindling with stimulation of the dentate gyrus. I. Characterization of electrographic and behavioral events. Brain Res. 509: 249–256. Hosford, D.A., Simonato, M., Cao, Z., Garcia-Cairasco, N., Silver, J.M., Butler, L., Shin, C. et al. 1995. Differences in the anatomic distribution of immediate-early gene expression in amygdala and angular bundle kindling development. J Neurosci. 15: 2513–2523. Kelly, M.E. 1998. The kindling model of temporal lobe epilepsy. In Neuropharmacology Methods in Epilepsy Research Ed. S.L. Peterson, and T.E. Albertson. pp. 41–75. Boca Raton: CRC Press.
Kelly, M.E., Battye, R.A., and McIntyre, D.C. 1999. Cortical spreading depression reversibly disrupts convulsive motor seizure expression in amygdala kindled rats. Neurosci. 91: 305–313. Khurgel, M., and Ivy, G.O. 1996. Astrocytes in kindling: relevance to epileptogenesis. Epilepsy Res. 26: 163–175. Klitgaard, H., and Pitkanen, A. 2003. Antiepileptogenesis, neuroprotection, and disease modification in the treatment of epilepsy: focus on levetiracetam. Epileptic Disord. 5 (Suppl 1), S9–S16. Leung, L.S. 1987. Hippocampal electrical activity following local tetanization. I. Afterdischarges. Brain Res. 419: 173–187. Löscher, W., Horstermann, D., Honack, D., Rundfeldt, C., and Wahnschaffe, U. 1993. Transmitter amino acid levels in rat brain regions after amygdala-kindling or chronic electrode implantation without kindling: evidence for a pro-kindling effect of prolonged electrode implantation. Neurochem Res. 18: 775–781. Löscher, W., Honack, D., and Rundfeldt, C. 1998. Antiepileptogenic effects of the novel anticonvulsant levetiracetam (ucb L059) in the kindling model of temporal lobe epilepsy. J Pharmacol Exp Ther. 284: 474–479. Löscher, W. 1999. Animal models of epilepsy and epileptic seizures. In Antiepileptic Drugs. Handbook of Experimental Pharmacology. Ed. Eadie, M.J., and Vajda, F. pp. 19–62. Berlin: Springer. Löscher, W. 2002. Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res. 50: 105–123 Lothman, E.W., and Williamson, J.M. 1994. Closely spaced recurrent hippocampal seizures elicit two types of heightened epileptogenesis: a rapidly developing, transient kindling and a slowly developing, enduring kindling. Brain Res. 649: 71–84. McIntyre, D.C. 1970. Differential amnestic effects of cortical vs amygdaloid elicited convulsions in rats. Physiol Behav. 5: 747–753. McIntyre, D.C. 1979. Effects of focal vs generalized kindled convulsions from anterior neocortex or amygdala on CER acquisition in rats. Physiol Behav. 23: 855–858. McIntyre, D.C., and Kelly, M.E. 1993. Are differences in dorsal hippocampal kindling related to amygdala-piriform area excitability? Epilepsy Res. 14: 49–61. McIntyre, D.C., Kelly, M.E., and Armstrong, J.N. 1993. Kindling in the perirhinal cortex. Brain Res. 615: 1–6. McIntyre, D.C., Kelly, M.E., and Staines, W.A. 1996. Efferent projections of the anterior perirhinal cortex in the rat. J Comp Neurol. 369: 302–318. McIntyre, D.C., Kelly, M.E., and Dufresne, C. 1999. FAST and SLOW amygdala kindling rat strains: Comparison of amygdala, hippocampal, piriform and perirhinal cortex kindling. Epilepsy Res. 35: 197–209. McIntyre, D.C., and Poulter, M.O. 2001. Kindling and the Mirror Focus, In Brain Plasticity and Epilepsy: A Tribute to Frank Morrell. Ed. J. Engel, Jr., Schwartzkoin, P.A., Moshé, S.L., and Lowenstein, D.H. pp. 387–404. San Diego: Academic Press. McIntyre, D.C., Poulter, M.O., and Gilby, K. 2002. Kindling: some old and some new. Epilepsy Res. 50: 79–92. Michael, M., Holsinger, D., Ikeda-Douglas, C., Cammisuli, S., Ferbinteau, J., DeSouza, C., DeSouza, S. et al. 1998. Development of spontaneous seizures over extended electrical kindling. I. Electrographic, behavioral and transfer kindling correlates. Brain Res. 793: 197–211. Mody, I. 1993. The molecular basis of kindling. Brain Pathol. 3: 395– 403. Mody, I. 1999. Synaptic plasticity in kindling. Adv Neurol. 79: 631–643. Mohapel, P., Dufresne, C., Kelly, M.E., and McIntyre, D.C. 1996. Differential sensitivity of various temporal lobe structures in the rat to kindling and status epilepticus induction. Epilepsy Res. 23: 179–187. Molino, A., and McIntyre, D.C. 1972. Another inexpensive headplug for the electrical recording and/or stimulation of rats. Physiol Behav. 9: 273–275.
References Monroe, R.R. 1982. Limbic ictus and atypical psychoses. J Nerv Ment Dis. 170: 711–730. Moore, R.Y. 1981. Methods for selective-restrictive lesion placement in the central nervous system. In Neuroanatomical Tract Tracing Methods. Ed. L. Heimer, and M.J. Robarts (Chapter 2), New York: Plenum Press. Morimoto, K., Fahnestock, M., and Racine, R.J. 2004. Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog Neurobiol. 73: 1–60. Moshé, S.L. 1981. The effects of age on the kindling phenomenon. Dev Psychobiol. 14: 75–81. Moshé, S.L., Albala, B.J., Ackermann, R.F., and Engel, J. Jr. 1983. Increased seizure susceptibility of the immature brain. Brain Res. 283: 81–85. Parent, J.M., Janumpalli, S., McNamara, J.O., and Lowenstein, D.H. 1998. Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett. 247: 9–12. Paxinos, G., and Watson, C. 1998. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press. Pellegrino, L.J., Pellegrin, A.S., and Cushman, A.J. 1979. A Stereotaxic Atlas of the Rat Brain. New York: Plenum Press. Pinel, J.P.J., Mucha, R.F., and Phillips, A.G. 1975. Spontaneous seizures generated in rats by kindling: a preliminary report. Physiol Psychol. 3: 127–129. Pinel, J.P.J., and Rovner, L.I. 1978. Experimental epileptogenesis: kindlinginduced epilepsy in rats. Exp Neurol. 58: 190–202. Pinel, J.P.J. 1981. Kindling induced experimental epilepsy in rats: cortical stimulation. Exp Neurol. 72: 559–569. Racine, R.J. 1972a. Modification of seizure activity by electrical brain stimulation. I. Afterdischarge threshold. Electroencephalogr Clin Neurophysiol. 32: 269–280. Racine, R.J. 1972b. Modification of seizure activity by electrical brain stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol. 32: 281–294. Racine, R.J., Burnham, W.M., Gartner, J.G., and Levitan, D. 1973. Rates of motor seizure development in rats subjected to electrical brain stimulation: Strain and interstimulus interval effects. Electroencephalogr Clin Neurophysiol. 35: 553–556. Racine, R.J., Rose, P.A., and Burnham, M.W. 1977. Afterdischarge thresholds and kindling rates in dorsal and ventral hippocampus and dentate gyrus. Can J Neurol Sci 4: 273–278. Racine, R.J. 1978. Kindling: the first decade. Neurosurgery 3: 234–252. Racine, R.J., Steingart, M., and McIntyre, D.C. 1999. Development of kindling-prone and kindling-resistant rats: selective breeding and electrophysiological studies. Epilepsy Res. 35: 183–195.
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29 Kindling Kittens and Cats MARGARET N. SHOUSE
eral, tripolar leads are also aimed at the locus ceruleus (LC: P2–3, L2.5, H-2.5–3.0). Amygdala and LC leads are usually attached to 18–21 g cannulae, with the tips extending 5 mm below for microinfusion studies. Important surgical issues are:
METHODS OF GENERATION Female kittens reach puberty at ~7–9 months and males within 1 year (Inglis, 1980). Amygdala kindling has been performed in weaned, prepubertal kittens between 2.5 and 6.5 months of age (0.7–2.3 kg) and in adults 1 year old (3.0–5.2 kg) at initial afterdischarge (AD) (Shouse et al., 1990; Shouse, Langer, and Dittes, 1990; Shouse, Dittes, Langer, and Nienhuis, 1992). Aseptic stereotaxic neurosurgery is performed 1 to 2 weeks earlier using a Kopf stereotaxic frame for dogs and cats and isoflurane anesthesia. Kittens can be resistant to anesthesia, so that it may be necessary to increase isoflurane from 2% to 4% in the young. Twenty-to-40 surface and depth electrodes can be implanted. Stereotaxic coordinates for preadolescents are extrapolated from atlases for cats <35 days of age (Rose and Goodfellow, 1973) and older cats weighing >3.5 kg (Snider and Niemer, 1961). Coordinates are typically 1 mm closer to the middle of the brain on all stereotaxic planes for younger animals than for adults. The implant is designed to study epilepsy, sleep, and microinfusion effects. All cats have bilateral, tripolar electrodes in the basolateral nucleus or the juncture between the central and basolateral nuclei for amygdala kindling (A10–11, L9–10, H-4–6); jeweler’s screws threaded into the bone over the motor cortex (A25–27, L6 and 8) for cortical EEGs as well as above the orbit to record eye movements (electrooculograms, or EOGs) and into the intact frontal sinus for indifferent leads; stainless steel wires inserted into the neck to record muscle tone (electromyograms, or EMGs). We also implant unilateral or bilateral tripolar electrodes in the lateral geniculate nucleus (LGN) of the thalamus to record ponto-geniculo-occipital (PGO) spikes. Unilateral or bilat-
Models of Seizures and Epilepsy
1. The tentorium in cats is a bone. To implant electrodes in the medulla or pons, one usually employs an electrode carrier (manipulator) slanted from posterior to anterior at a 33° angle so that the electrodes will enter the brainstem beneath the posterior edge of the tentorium. However, the topography of the tentorium is so variable that one frequently still has to drill through it. This poses a significant survival risk only for kittens. Use of an anterior-to-posterior slant at ~ a 13° angle usually allows placements in the medial-lateral part of the dorsal pons without colliding with the tentorium and does not jeopardize postoperative recovery in kittens. 2. To help stabilize the implant in kittens, we prepare two metal clamps improvised from stainless steel rods (~1 mm in width). A 2-cm long section is cut, and 3–4 mm ends are bent for insertion into small holes drilled in the frontal and parietal bones on the lateral edges of the implant. The clamps are oriented longitudinally and, together with the rest of the implant, are secured to the skull using acrylic glue. Sterile cement significantly reduces risk of acute and chronic postoperative infection. 3. Kittens can be quite active and self-injurious postoperatively and must be kept quiet for ~10 hours. We give 20–40 mg of sodium phenobarbital, IM or IV, and 0.03 mg buprenorphine, IM, just after extubation and repeat buprenorphine every 30 minutes to 3 hours, as needed. Physical restraint may also be necessary.
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Kindling Electrical kindling procedures are initiated 1–2 weeks after surgery. Electrical stimulation is applied through two adjacent leads in the amygdala or through an amygdala lead paired to an indifferent. Stimuli consist of a 1-second train of 1-ms biphasic square waves at 60 Hz. A standard adult cat kindling protocol involves one stimulus per day. At first, initial AD threshold is established by a method of limits procedure in which the first stimulus is 100 mA. Each day thereafter, stimulus amplitude is increased by 100 mA per day until the first AD is detected and then reduced by 100 mA per day until no AD is evoked. Initial AD threshold is defined as the minimal stimulus intensity (mA) needed to elicit AD (Shouse et al., 1990; Shouse, Langer, and Dittes, 1990; Shouse, Dittes, Langer, and Nienhuis, 1992; Shouse, Bier, Langer et al., 1996). The animal is then stimulated at initial AD threshold until kindled, defined by the first evoked generalized tonic-clonic convulsion or GTC (stage 6 seizure). Postkindling threshold is established using the same method of limits procedure as at the beginning kindling except that the endpoint is a stage 6 seizure (Inglis, 1980; Rose and Goodfellow, 1973; Sato and Nakeshima, 1975; Shouse and Ryan, 1984; Shouse et al., 1990; Shouse, Langer, and Dittes, 1990; Shouse, Dittes, Langer, and Nienhuis, 1992; Shouse, Bier, Langer et al., 1996; Shouse, Scordate, and Farber, 2004, submitted). The kindling procedure was modified to deal with the high AD thresholds in young kittens (<5 months old) and is now used in all age groups (Shouse, Scordato, and Farber, 2004). Multiple stimulations per day are used to establish AD thresholds at the beginning of kindling. The initial stimulus is 100 mA and the increment is 100 mA. The interstimulus interval (ISI) is 1 minute instead of the 24-hour ISI typically used in adults. Threshold is defined as the stimulus intensity required to elicit the first AD. Afterward, once daily stimulation at initial AD threshold is used to obtain the first GTC. After initial kindling, threshold is re-established in 1 day, as at the beginning of kindling, except that the endpoint is a stage 6 seizure. In animals without AD at 2 mA, the increment is increased from 100 mA to 1, 5, or 10 mA. In one animal without AD at 40 mA, we resorted to bilateral amygdala stimulation (Shouse, Scordato, and Farber, 2004).
Post-Kindling Follow-Up During follow-up, stage 6 seizure thresholds may be reestablished every work day. The procedure is the same as at the end of kindling. Adjustments in stimulus intensity may be needed (e.g., 25 mA vs. 100 mA increments) to maximize threshold differences in sleep vs. waking states or after microinfusions, e.g., Shouse, da Silva, and Sammaritano, 1997. The mean duration of follow-up is 4 months. Initially, follow-up was briefest in young kittens (3 days to 2 months)
due to disruption of daily postkindling threshold tests by spontaneous seizures and/or from deaths attributed to undetected convulsive status epilepticus, defined as more than one GTC within 5 minutes or by a GTC lasting longer than 5 minutes. Extended monitoring and treatment, as described in the following, have resolved these problems.
Monitoring Ten to 20 minute routine EEGs with or without behavioral video monitoring are performed every workday. Periodic 8–24 hour split-screen polygraphic-behavioral video recordings may be performed before, during, and after kindling for assessment of sleep states and/or when spontaneous seizures are detected. The addition of 24 hour video in the vivarium significantly improves detection of spontaneous GTCs as well as the timely treatment of frequent GTCs (>1 per hour) and convulsive status epilepticus. The safest, long-lasting (~12 hour) treatment is sodium pentobarbital (35 mg/kg, SC. with atropine at 0.2–0.4 mg/kg, IM). Vivarium videotapes of group-housed cats (n = 4 per cage) also permit assessment of social behaviors (Shouse, Scordato, and Farber, 2004).
Amygdala Kindling Development in Kittens and Cats Youngest kittens (2.5–5.0 months old) can display very high AD thresholds at the beginning of kindling (mean = 3.9 mA, range = 0.5–20 mA) when compared to older preadolescents (5.5–6.5 months old, mean = 1.2, range = 0.3–4.0 mA) and adults 12 months (mean = 1.1, range = 0.1–1.5 mA). Most older kittens and adults have initial AD thresholds less than 2 mA. Cats of all ages show a significant reduction in thresholds at the end of kindling when compared to their own initial AD thresholds, but thresholds in the youngest kittens do not usually decline to the levels of older animals until 2–4 months after kindling (Shouse et al., 1990; Shouse, Scordato, and Farber, 2004). Once initial AD threshold has been elicited, the youngest kittens kindle most easily, as evidenced by accelerated kindling rates, defined as the number of ADs needed to elicit the first stage 6 seizure or GTC. Young kittens require only about 14 ADs to the first GTC when compared to an average of 24 ADs in older kittens and adults. Accelerated kindling in the young may be attributed to rapid progression from the “focal” or partial seizure stages 1 and 2, which involve unilateral then bilateral eye, nose, and/or cheek twitches, to the so-called generalized seizure stages (stages 3–6). Stage 3 is a complex-partial seizure characterized by lip-smacking or chewing, autonomic signs such as salivation and head clonus with the body rotating to the side contralateral to the kindling electrode. The cat then proceeds to circling (stage 4), jumping or rocking (stage 5), and finally displays a
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Other Characteristics and Defining Features
violent GTC usually lasting 1.5–2.5 minutes. Young kittens are likely to exhibit spread of AD to thalamic, cortical, or brainstem sites while still showing localization-related clinical seizure signs (e.g., ipsilateral eye blinks). Kittens who show only generalized AD during kindling can have focal amygdala onset spontaneous GTCs, and, conversely, kittens with focal amygdala AD during kindling may have only extrafocal, multifocal, or apparent primary generalized onset seizures (Shouse, Scordato, and Farber, 2004).
olds can remain low up to a year after kindling without intervening evoked seizures. (Wada and Sato, 1974). Spontaneous seizures, on the other hand, may remit or recede, particularly the infrequently detected seizure types. Spontaneous GTCs are infrequently seen >72 hours after an evoked seizure (Shouse et al., 1990; Shouse, Scordato, and Farber, 2004, submitted).
Spontaneous Seizures after Kindling
OTHER CHARACTERISTICS AND DEFINING FEATURES
Young kittens are more likely than older kittens and adults to develop spontaneous epilepsy, defined by seizures occurring 1 hour after an evoked seizure. Nearly 60% of kittens <5 months old develop spontaneous epilepsy, when compared to 20% of older preadolescents and 6% of adults. The youngest kittens usually develop spontaneous epilepsy shortly after kindling (<1 month), particularly when 5 per week threshold trials are performed just after kindling. Older kittens and adults require many more evoked GTCs prior to onset (mean = 39 ± 1) than do younger animals (mean = 12.9 ± 2) (Shouse et al., 1990; Shouse, Scordato, and Farber, 2004, submitted). Four seizure types have been documented by split-screen EEG and behavior recordings. Of these, only GTCs were detected in all three age groups. The other seizure types were registered exclusively in the youngest kittens, two-thirds of which have multiple seizure types. The two most frequently detected seizure types resemble either stage 3 or stage 6 evoked kindled seizures. The other two seizure types are less frequently detected and differ entirely from evoked EEG and clinical seizure manifestations. One atypical seizure type consists of focal, subclinical seizures. These are characterized by repetitive brief (3–10 seconds) or long-lasting (1.5 minutes) EEG seizures recorded in the kindled amygdala or in the ipsilateral LGN, do not have clinical accompaniment, and appear to last 1 or 2 days. The other atypical seizure type has been dubbed a “catnip” seizure because the animal looks disoriented during the episodes. They consist of nearly continuous multifocal or generalized EEG seizure discharge, sometimes with overt clinical signs (e.g., fast pulse, staring, purring, and jackknife spasms, like infantile spasms) and sometimes with comparatively little noticeable clinical accompaniment. EEG discharge is so pervasive that waking and slow wave sleep (SWS) EEGs are indistinguishable for periods lasting 3 hours to 2 days. The seizures stop only when the animal enters REM sleep or is alerted by physical manipulation (Shouse et al., 1990; Shouse, Scordato, and Farber, 2004, submitted).
Remission Kindled cats of all ages are considered to display a permanent reduction in seizure susceptibility because thresh-
Young kittens also differ from older animals by showing more frequent, higher density convulsive seizure clusters, defined as more than one GTC per day or at least one GTC on two consecutive days. The mean daily GTC incidence per cluster in the youngest kittens (5.3 ± 0.9) is over twice that of older animals. Dense seizure clusters may end in convulsive status epilepticus, although episodes of convulsive status can occur at any time after kindling (Shouse, Scordato, and Farber, 2004, submitted). Dense seizure clusters are reliably associated with transient behavioral sequelae (Shouse, Scordato, and Farber, 2004, submitted), which occur without detected EEG seizure discharges and last 3–30 days after the end of the cluster (Table 1). These behaviors are life-threatening because the kittens do not actively seek out food or water. This may not be obvious during casual observation or sporadic health checks so timely review of videotapes is mandatory. Specific behaviors may include atonic or drop attacks, always include reduced mobility and social isolation, often include “psychic blindness,” which refers to a visual agnosia in which the significance of common items such as food versus. toys is not discriminated, and in several cases hypersensitivity to all sensory modalities (e.g., a GTC can be precipitated by sudden loud noise). Table 1 also lists odd,
TABLE 1 Behavior Disorders Associated with Amygdala Kindling in Cats Age at initial AD (months)
Transient behaviors (sequelae) associated with dense stage 6 seizure clusters
Persistent behaviors with or without spontaneous seizures (interictal or post-ictal)
2.5–5.0
Atonia; Reduced mobility; Social isolation; “Psychic” blindness; ≠ sensory sensitivity
Moderate to severe: Temperamental (docile-hostile); Sexual (assault, disinterest, dysmenhorrhea); Post-ictal bulimia
5.5–6.5
None
None to moderate
≥12
None
None to moderate
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persistent behaviors that are seen most often after kindling in kittens but can occur at any age regardless of onset of spontaneous epilepsy (Shouse, Scordato, and Farber, 2004, submitted). The most notable are extreme fluctuations in temperament and signs of sexual dysfunction, such as frequent homosexual assaults. Some of these transient or persistent behavior disorders listed in Table 1 are reminiscent of Kluver-Bucy syndrome16 and others of Landau-Kleffner (Stefanatos, Kinsbourne, Wasserstein, 2002). Either disorder may be associated with temporal lobe epilepsy, and all clinical signs have been associated with amygdala dysfunction (Baron-Cohen et al., 2000).
Sleep Amygdala-kindled cats resemble humans with temporal lobe epilepsy (TLE) with respect to the timing of evoked or spontaneous seizures in the sleep–wake cycle and to sleep disorders (Shouse, Langer, and Dittes, 1990; Shouse, Dittes, Langer, and Nienhuis, 1992; Shouse, da Sliva, and Sammaritano, 1997). Kindled seizure thresholds are lowest in NREM sleep and highest in REM sleep regardless of age. Similarly, epileptic kittens display nearly all spontaneous GTCs in NREM sleep or in REM transitions. Focal subclinical or partial-complex seizures may occur at any time in the sleep–wake cycle, but other generalized seizures, including GTCs and “catnip seizures,” are least likely to occur in REM (Shouse, Langer, and Dittes, 1990; Shouse, Dittes, Langer, and Nienhuis, 1992). Kindled cats also show the same kinds of sleep disorders and sleep deprivation effects seen in human TLE. This includes sleep fragmentation (frequent stage shifts, wakefulness after sleep onset, prolonged REM onset latencies) and activation of seizures after sleep deprivation (Shouse, da Silva, Sammaritano, 1997). Cats are ideal subjects for investigating sleep-related seizure mechanisms. Dissociative manipulations have been used in feline epilepsy models, including amygdala kindling (see Shouse, da Silva, and Sammaritano, 1997), to differentiate the epileptogenic, physiologic characteristics (components) of NREM sleep from the antiepileptic components of REM. Systemic atropine administration creates an NREM sleep-like EEG pattern with sleep spindles throughout the sleep–wake cycle, presumably by blocking the cholinergic generators of waking and REM sleep EEG desynchronization. The generators are thought to originate in the nucleus basalis and the pedunculopontine tegmentum. Atropine also abolishes protection against EEG seizure discharge propagation in waking and REM without affecting the suppression of clinically evident motor seizures in REM. In contrast, a lesion of the glutaminergic and cholinoceptive subceruleus region, which generates profound lower motor inhibition in REM, creates a syndrome of REM sleep
without atonia. The lesion also selectively abolishes protection against clinical motor accompaniment in REM without affecting EEG seizure discharge propagation. The findings suggest that the burst-pause cellular discharges underlying the global EEG synchronization of NREM are also conducive to EEG seizure discharge propagation, while the presence of tone permits clinically evident seizures in NREM. Conversely, the asynchronous cellular discharge patterns of waking and REM are not conducive to EEG seizure discharge propagation, and the profound lower motor neuron inhibition of REM sleep appears to block clinical motor accompaniment during this state (Shouse, da Silva, and Sammaritano, 1997). Forebrain vs. hindbrain seizures. Kindling subcortical forebrain sites is usually easier than kindling neocortex regardless of age. Examples of deep forebrain sites other than amygdala that have been kindled in adult cats are hippocampus (Sato and Nakashima, 1975) and lateral geniculate nucleus of thalamus (Shouse and Ryan, 1984). Brainstem kindling (dorsal raphe nucleus) has been performed in adult cats and is characterized by seizure-induced NREM sleep onset (Fernandez-Guardiola et al., 1982). We have not conclusively documented spontaneous, brainstem onset seizures in cats or kittens.
Neuropathology Global cell loss associated with prolonged status epilepticus may be seen in older cats but has not been seen in young kittens in spite of more frequent, similar duration episodes. Minimal tissue damage is seen around the amygdala electrode tips in kindled kittens regardless of onset or severity of spontaneous epilepsy or of behavioral anomalies (Shouse et al., 1990; Shouse, Scordato, and Farber, 2004, submitted). Observations in adult cats and kittens are limited to gross estimates based upon light microscopy, whereas similar age-related observations have been quantified in detailed anatomic studies of kindled rodents (see appendix, this volume: kindling rat pups) (Haas et al., 1998).
Response to Anti-Epileptic Agents Kindled cats provide a good model for studying effects of systemic anti-epileptic drugs on epilepsy, sleep, and its interaction. Microinfusion of receptor agonist and antagonists reliably influences thresholds in cats (see Shouse, da Silva, and Sammaritano, 1997), and effects can potentially be studied on spontaneous seizures in kittens. The feasibility of microinfusion is now being studied in humans with intractable epilepsy. The feline model may prove useful for screening agonist/antagonists for this new treatment alternative.
References
SUMMARY: ADVANTAGES AND LIMITATIONS Kittens and cats are easily kindled. Mortality issues related to surgery and to spontaneous epilepsy in kittens have been resolved. Adults rarely show spontaneous seizures, but young, weaned kittens are quite prone to spontaneous, catastrophic multifocal epilepsy. Kindling in adult cats may provide a model of TLE with secondary generalization and allows for behavioral, sleep and drug studies. Kindled cats may be preferable to rodents in that seizure types, such as complex partial seizures (e.g., lip-smacking) and generalized tonic-clonic seizures better resemble clinically evident seizures in humans and also exhibit the same distribution of seizures in the sleep–wake cycle. Some of the neural mechanisms of sleep are better described in cats than in rodents, making cats particularly well suited for studies of sleep-related seizure mechanisms, whereas rodents are much more suitable for circadian rhythm studies. Amygdala-kindled kittens provide an even better model for TLE, sleep epilepsy, and drug effects than adults because kittens display several spontaneous seizure types. Kindled kittens may also be preferable to rodents for some developmental epilepsy studies because the multiple seizure types and behavior disorders can closely resemble the complications seen in childhood seizure disorders, such as Landau-Kleffner, West syndrome, and possibly even Lennox-Gastaut. There are presently no animal models of these disorders. Combined EEG and behavioral monitoring is still discontinuous in cats so that the incidence and diversity of spontaneous seizure types likely remains underestimated. Studies in cats or kittens with abnormal brains or with a genetic predisposition to epilepsy have not been performed.
References Baron-Cohen, S., Ring, H.A., Bullmore, E.T., Wheelright, S., Ashwin, C., and Williams, S.C.R. 2000. The amygdala theory of autism. Neurosci Biobehav Rev. 24: 355–364.
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Fernandez-Guardiola, A., Jurado, J.L, Calvo, J.M., Barrados, J.A., and Barragan, L.A. 1982. Effects of pharmacological manipulations on raphe nuclei kindling and sleep. In Sleep and Epilepsy. Ed. Sterman, M.B., Shouse, M.N., and Passouant, P. New York: Academic Press. Haas, K.Z., Sperber, E.F., Benenati, B., Stanton, P.K., and Moshe, S.L. 1998. Idiosyncrasies of limbic kindling in developing rats. In Kindling 5. Ed. Corcoran, M.E., and Moshe, S.L. pp. 15–24. New York: Plenum Press. Inglis, J.K. 1980. Introduction to Laboratory Animal Science and Technology. New York: Pergamon. Rose, G.H., and Goodfellow, E.F. 1973. A Stereotaxic Atlas of the Kitten Brain: Coordinates of 104 Selected Structures. University of California, Los Angeles: Brain Information Service/Brain Research Institute. Sato, M., and Nakeshima, T. 1975. Kindling: secondary epileptogenesis, sleep and catecholamines, Can J Neurol Sci. 3: 439–446. Shouse, M.N., and Ryan, W. 1984. Thalamic kindling: Electrical stimulation of the lateral geniculate nucleus (LGN) produces photosensitive grand mal seizures. Exp Neurol 86: 18–32. Shouse, M.N., King, A., Langer, J., Vreeken, T., King, K., and Richkind, M. 1990. The ontogeny of feline temporal lobe epilepsy: kindling a spontaneous seizure disorder in kittens. Brain Res 525: 215–224. Shouse, M.N., Langer, J., and Dittes, P. 1990. Spontaneous sleep epilepsy in amygdala kindled kittens: a preliminary report. Brain Res 535: 163–168. Shouse, M.N., Dittes, P., Langer, J., and Nienhuis, R. 1992. Ontogeny of feline temporal lobe epilepsy, II: Stability of spontaneous sleep epilepsy in amygdala-kindled kittens. Epilepsia 33: 789–798. Shouse, M.N., Bier, M., Langer, J. et al. 1996. The alpha-2 agonist clonidine suppresses seizures, whereas the alpha-2 antagonist idazoxan promotes seizures in amygdala-kindled kittens: A comparison of anygdala and pontine microinfusion effects. Epilepsia 37: 709–717. Shouse M.N., da Silva A.M., and Sammaritano M. 1997. Sleep. In Epilepsy: A Comprehensive Textbook. Ed. Engel, J.P.J. Jr, and Pedley, T.A. pp. 1929–1942. Philadelphia: Lippencott-Raven Publishers. Shouse, M.N., Scordato, J.C., and Farber, P.R. 2004. Ontogeny of feline temporal lobe epilepsy in amygdala kindled kittens: An update. Brain Research 1027: 126–143. Snider, R.S., and Niemer, W.T. 1961. A Stereotaxic Atlas of the Cat Brain. Chicago: University of Chicago Press. Stefanatos, G.A., Kinsbourne, M., and Wasserstein, J. 2002. Acquired epileptiform aphasia: A dimensional view of Landau-Kleffner Syndrome and the relation to autistic spectrum disorders. Child Neuropsychology 8: 195–228. Varon, D., Pritchard, P.B., Wagner, M.T., and Topping, K. 2003. Transient Kluver-Bucy syndrome following complex partial status epilepticus. Epilepsy & Behavior 4: 348–351. Wada, J.A., and Sato, M. 1974. Generalized convulsive seizures induced by daily stimulation of the amygdala in cats. Neurology 24: 565–574.
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30 Electrical Kindling in Developing Rats ARISTEA S. GALANOPOULOU AND SOLOMON L. MOSHÉ
METHODS OF GENERATION
Procedures Electrode Implantation
Animal Issues
Electrode implantation is done at least 1 day prior to kindling in P7–8 Sprague-Dawley rats (Baram et al., 1993; Baram et al., 1998) and 2 days prior to testing in P9 or older rats (Baram et al., 1993; Baram et al., 1998; Moshé, 1981; Moshé et al., 1981). The recovery period needs to be shorter in infant rats than in adults, to avoid misplacement of the electrode tip due to the ongoing head growth. P6–10 rats are anesthetized under halothane anesthesia (Baram et al., 1993; Baram et al., 1998), whereas P12 or older rats are injected with a mixture of ketamine (70 mg/kg IP) and xylazine (6 mg/kg IP) (Moshé 1981; Moshé et al., 1981). When deeply anesthetized, rats are placed on an infantile rat stereotaxic apparatus. The tooth bar is set at 3.5 mm. Bipolar twisted wire electrodes are targeted through a burr hole, to the basolateral nucleus of the amygdala unilaterally. In P6–10 pups, bipolar electrodes with a wire diameter of 0.1–0.15 mm and vertical inter-tip distance of 0.5–1 mm have been used (Baram et al., 1993; Baram et al., 1998). In older pups, insulated electrode with wire diameters 0.23–0.35 mm (MS 303/1 or MS 303/2, Plastic One, Roanoke, VA, USA) can be used (Moshé 1981; Haas et al., 1998). The coordinates used to target each structure are based on the stereotaxic coordinates included in the atlas by Paxinos (Paxinos et al., 1994; Paxinos and Watson, 1997) but they need to be adjusted according to the age, strain, source, or weight of the rats. Table 1 presents published coordinates that can be used as starting points when targeting the amygdala or other sites.
Because of the rapid growth of infant rats, kindling stimulations must be delivered within a finite period of time, usually 48 hours. The first protocol of amygdala kindling in infantile rats (P15–18) was published by Moshé in 1981 using 1 hour interstimulus intervals (ISI) with 60 Hz alternating current stimulations. However, even shorter (15 minute) ISI were able to effect successful kindling, due to the lack of a refractory period (Moshé, 1981; Moshé et al., 1981; Moshé and Albala, 1983; Haas et al., 1990). With this paradigm, kindling can occur within 1 day. This is not the case with adult rats. Irrespective of the ISI, kindling once induced is permanent and persists into adulthood either in the original kindling site or the contralateral site (Moshé and Albala, 1982). Subsequently, several other studies adopted the 15 minute ISI kindling protocol and succeeded in kindling rats as young as P7 (Baram et al., 1993; Baram et al., 1998), and covering all the stages of development, i.e., the infantile (P8–P20), juvenile (P21–P34), and pubertal period (P35–P40) (Dahl et al., 1988; Ojeda et al., 1986). These studies revealed several age-related differences in kindling parameters, expression, and consequences. Briefly, age-related differences have been found in the presence of a refractory period, the afterdischarge (AD) threshold (ADT), kindling rate, behavioral manifestations of kindled seizures, the interaction between 2 foci kindled concurrently, and neuropathologic and metabolic sequelae.
Models of Seizures and Epilepsy
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Copyright © 2006, Elsevier Inc. All rights of reproduction in any form reserved.
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Chapter 30/Electrical Kindling in Developing Rats
In this lab, electrodes are usually inserted at a 0° angle in reference to the vertical plane (Table 1A). If additional electrodes or cannulae need to be inserted, the electrodes can be placed at a different angle, with appropriate adjustment of the coordinates (Table 1B). Electrodes are then fixed to the skull with 2 or 3 screws and dental acrylic. The pups are then returned to the litter. We prefer to keep the mother separated from the infants till all the pups are operated. We then cover the pups with the shavings already present in the cage to cover up any obnoxious smells acquired during the procedure. When pups are ready to kindle, they are placed in a test box and are connected to the stimulating and recording apparatus via a connector cable (Plastics One; MS 303 series). Recording of the EEG is done before and after each stimulation. Kindling The first step is to determine the ADT. A 60 Hz sinusoidal current is delivered for 1 second. Stimulations can be delivered as frequently as every 2 minutes, until an AD is elicited.
Once an AD is observed, the ISI is kept at 15 minutes or as otherwise stated in the selected protocol (Table 2). If determination of ADT is an endpoint in the experimental design, a protocol using small increments of stimulation amplitude may be used. For example, stimulations can be started at 30 mA and increased by 30 mA in each subsequent trial until an AD is obtained (Moshé et al., 1981). The current can then be decreased by 15 mA. If an AD is evoked, the current can further be increased by 5 mA increments until the lowest current that can generate an AD is reached (Moshé et al., 1981). In most cases, however, ADT determination simply serves the purpose of ensuring that all subsequent stimulations will be above ADT to achieve successful kindling. In such cases, the starting stimulus intensity can be set at 100 mA and progressive increments by 100 mA are applied till an AD is recorded. Generally, rats not responding to 500 mA stimulation with an AD are excluded, as they have been shown to be refractory to kindling (Moshé SL, unpublished data). Determination of the ADT is done the same day as kindling.
TABLE 1 Age- and Site-Specific Coordinates for Targeting Limbic Structures for Kindling A. Insertion at 00 angle Age (days)
Tooth bar (mm)
Antero-posterior in reference to bregma (mm)
Lateral (mm)
Depth (mm)
Reference
-1.5 to -1.8 -1.5
3.5 3.5
6.5–7.4 8.8
(Moshé, 1981) (Moshé, 1981)
Basolateral nucleus of the amygdala 7–14 -3.5 ≥32 -3.5 Dorsal hippocampus 13–14
-3.5
-3.2
2.7 to 3
2.8–3
(Haas et al., 1990)
Ventral hippocampus 7–21 28
0 5
-3 to -2.9 -3.6
3.7–4.9 4.9
3.7–4.9 4.9
(Michelson and Lothman, 1991)
Deep endopiriform cortex 13 -3.5
-1.2
3.1
5.5
(Sperber et al., 1998)
Piriform cortex 13
-1.2
3.7
6
(Sperber et al., 1998)
-3.7
B. Angled coordinates
Age (days)
Tooth bar (mm)
Basolateral nucleus of the amygdala 14 -3.5 Dorsal hippocampus 13–14
-3.5
Angle
Antero-posterior in reference to bregma (mm)
Lateral (mm)
Depth (mm)
Reference
150 forward
+5
3.5
7.4
(Haas et al., 1992)
150 backward
-4
3
2.9
(Haas et al., 1992)
Stereotaxic coordinates for targeting the basolateral nucleus of the amygdala, the dorsal or ventral hippocampus, the deep endopiriform cortex, and the piriform cortex in developing Sprague-Dawley rats. Insertion is routinely made at a 00 angle, in reference to the vertical plane (Table 1A). If additional electrodes need to be placed, angled coordinates can be used (Table 1B). Further adjustments may need to be made according to the specific strain, source, or weight of the rats.
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Methods of Generation
TABLE 2 Age and Site-Specific Protocols of Kindling in Developing Rats Site Amygdala (basolateral nucleus)
Age P7–12 P15–18 P30–35
Protocol
Reference
1 sec pulse, 60 Hz monophasic current, 400 mA, 15 min ISI 1 sec pulse, 60 Hz sinusoidal current, 400 mA, 15 min ISI 1st day: 20 stimulations; 2nd day: 10 stimulations 1 sec pulse, 60 Hz sinusoidal current, 400 mA, 60 min ISI 1st day: 20 stimulations; 2nd day: 10 stimulations
(Baram et al., 1993) (Moshé et al., 1983) (Moshé, 1981) (Moshé et al., 1981) (Holmes and Weber, 1983)
Deep endopiriform nucleus
P15–16
1 sec pulse, 60 Hz sinusoidal current, 400 mA, 15 min ISI, 1st day: 20 stimulations; 2nd day: 10 stimulations
(Sperber et al., 1998)
Piriform cortex
P15–16
1 sec pulse, 60 Hz sinusoidal current, 400 mA, 15 min ISI, 1st day: 20 stimulations; 2nd day: 10 stimulations
(Sperber et al., 1998)
Dorsal hippocampus
P15–17
(Haas et al., 1992)
P30
1-sec, 400 mA. 60 Hz, sinusoidal current, every 15 min 1st day: 20 stimulations; 2nd day: 10 stimulations 10-sec, 10 Hz or 60 Hz, 1000 mA, 5 min ISI
Ventral hippocampus
P7–28
10-sec, 20 Hz, every 30 min for 9 hours per day, 2 days
(Michelson and Lothman, 1991)
Perforant pathway
P14–60
10-sec, 60 Hz, bipolar square wave pulses, twice the ADT, every 30 min
(Trommer et al., 1994)
When the ADT is determined, the amplitude of the stimulation current used for kindling can be set at 100 mA higher than the ADT and kept the same throughout the kindling protocol. In most protocols, however, amplitude can be set at 400 mA, which is sufficient to kindle rats at all ages and in most sites. To kindle the amygdala, stimulations (1 second, 60 Hz sinusoidal, 400 mA) are delivered every 15–20 minutes, to a maximum of 20 stimulations the first day. Kindling continues until pups develop three consecutive bilateral/generalized seizures (i.e., stage 3.5 to stage 7; Table 3). A measure of how fast kindling proceeds is the kindling rate, i.e., the number of stimulations required to achieve the first of three consecutive bilateral seizures (stage 3.5 to stage 7, Table 3). Between stimulations, pups are returned to their dam. After 10 stimulations, pups are rested for 1 hour before resuming kindling. The following day, pups are again kindled up to 10 times, or until they develop three consecutive stage 3.5–7 seizures. A total of 30 stimulations over a 2-day period can elicit stage 6 or 7 repeated seizures, which last for 100–120 seconds (Sperber et al., 1992). Postictal refractoriness can be measured an hour after the last kindled seizure, delivering eight repetitive 1-second stimulations, 400 mA, 60 Hz pulses, every 2 minutes, and comparing the AD duration and the kindling stage (Moshé and Albala, 1983). When kindling permanence or the long-term effects of kindling are studied, it is necessary to remove the original electrodes to avoid misplacement of the tip due to continuing head growth as well as infectious/inflammatory complications. New electrodes may then be re-inserted at a later stage if needed in the experimental design (Moshé and Albala, 1982). The sensitivity of different brain regions to kindling is, however, different. Attempts to kindle the dorsal hippocam-
(Holmes and Thompson, 1987)
TABLE 3 Behavioral Manifestations of Kindling in Infantile Rats Stage
Behavior
0
Behavioral arrest
1
Mouth clonus
2
Head bobbing
3
Unilateral forelimb clonus
3.5
Alternating forelimb clonus
4
Bilateral forelimb clonus
5
Bilateral forelimb clonus with rearing and falling
6
Wild running and jumping with vocalizations
7
Tonus
The currently used seizure scales describing the progression of seizures during kindling of infantile pups are based on the modified infantile scale initially proposed by Moshé (Haas et al., 1990; Haas et al., 1998). Age- and site-related variations may be observed as discussed in section IIA. (Reproduced from Haas et al., 1990, with permission from Elsevier.)
pus, piriform cortex, deep endopiriform nucleus (“area tempestas”) and amygdala in P15–17 pups, using the same experimental protocol, showed that the deep endopiriform nucleus kindled the fastest (Sperber et al., 1998; Haas et al., 1998). The different sensitivity to kindling in certain cases necessitated the use of site-specific protocols, as summarized in Table 2. Modifications may thus need to be made in the duration and frequency of the stimulation as well as the ISI. In the study by Michelson and Lothman, kindling of the ventral hippocampus of P7–28 rats with 5 minute-ISI resulted in erratic kindling, whereas efficiency was improved with ISIs of 30 minutes (Michelson and Lothman,
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Chapter 30/Electrical Kindling in Developing Rats
1991). These differences may suggest the existence of a relative refractory period in the ventral hippocampus at this age.
CHARACTERISTICS AND DEFINING FEATURES Monitoring ADT/Kindling Rates Age- and site-related differences in ADTs have been reported. In the amygdala, the highest ADT occurs at P15–18 and declines thereafter till adulthood (Moshé et al., 1981). Kindling rate is the lowest at P7–8 rats (Baram et al., 1998), highest at P35, and intermediate at the other ages (Moshé et al., 1981). The effects of age on ADTs and kindling rates may differ in other brain sites (Holmes et al., 1987; Trommer et al., 1994; Lee et al., 1989; Haas et al., 1998). For instance, although dorsal hippocampus kindles as fast as the amygdala and piriform cortex in 2-week-old pups, in adults hippocampal kindling proceeds at a slower rate than in the other brain sites (Haas et al., 1998; Lee et al., 1989; Moshé 1981; Moshé et al., 1981). Behavioral/Clinical Features Kindled seizures are semiologically different in infants than in adults. This prompted the adoption of a modified seizure scoring scale, based on the seizures observed in P15–18 rats undergoing amygdala kindling (Table 3) (Haas et al., 1990). The main differences from the adult scale include the appearance of alternating forelimb clonus in the same or consecutive seizures (stage 3.5), which is indicative of bilateral or generalized seizures. The incomplete myelination of the corpus callosum has been postulated to be a reason for the development of asynchronous bilateral motor manifestations (Haas et al., 1998; Gravel and Hawkes 1990; Kristensson et al., 1986). Another distinguishing feature of kindling of the immature rats is the early appearance of severe kindled seizures. Following the development of stage 5 seizures, P15 rats also develop more severe seizures, consisting of wild running with jumping and vocalizations (stage 6 seizures) and sometimes tonus (stage 7 seizures) (Haas et al., 1990). This may occur early on, after only less than 30 stimulations. In contrast, in adult rats more than 100 stimulations may be required to induce similarly severe seizures (Haas et al., 1998). The incidence of stage 6 or 7 seizures depends upon the stimulated site. Sixty to 80% of pups kindled at the amygdala, hippocampus, or area tempestas develop severe seizures, whereas none of those kindled at the piriform cortex do (Haas et al., 1990; Sperber et al., 1998). In P7–9 pups, alternating clonus and rearing were rarely observed (Baram et al., 1993). Furthermore,
P7–9 kindled rats rarely develop bilateral clonic seizures or rearing and progress from unilateral clonic seizures to tonic seizures (Baram et al., 1998). The kindled seizures in peripubertal rats are semiologically similar to those observed in adults, and therefore the adult scale can be used (Racine, 1972) (see also Chapter 28 on “The Kindling Phenomenon”). Similar scales have also been adopted in studies of kindling at other sites (Sperber et al., 1998; Haas et al., 1998; Michelson and Lothman, 1991; Holmes and Thompson, 1987; Lee et al., 1989) (Table 3). P15–17 pups are also more prone to develop recurrent kindled seizures and status epilepticus than adult rats (Moshé and Albala, 1983). The absence of a postictal refractory period in infant rats has been proposed to be a factor contributing to this increased propensity of the immature brain to develop status epilepticus or recurrent kindled seizures (Moshé and Albala, 1983). Kindling Antagonism In contrast to adult kindled rats, P15–17 pups do not exhibit kindling antagonism (Haas et al., 1998). Concurrent kindling of the amygdala with either the contralateral amygdala or the contralateral or ipsilateral hippocampus, using alternating stimulations at each site, accelerated the kindling at each site, with pups manifesting more severe, and occasional spontaneous, seizures (Haas et al., 1998; Haas et al., 1990; Haas et al., 1992). These findings suggest that the immature brain is not yet ready to suppress the development of multiple kindling foci, explaining therefore the higher incidence of multifocal seizures in the immature brain (Haas et al., 1992). Spontaneous Seizures Amygdala kindling may lead to the appearance of spontaneous seizures. In the study by Baram’s group, spontaneous seizures were defined as those observed at least 4 minutes after the last stimulation and typically after stage 3.5–5 seizures. Spontaneous seizures in that study persisted for 2–3 hours, till sacrifice. The investigators found that spontaneous seizures appeared in 25–50% of kindled P7–12 pups (Baram et al., 1998). Spontaneous seizures resembling stage 6 seizures (wild running, jumping, and vocalizations) have also been reported in P15–16 pups kindled with alternating stimuli at the ipsilateral amygdala and dorsal hippocampus (Haas et al., 1992). There are, however, no long–term monitoring studies to determine whether and how long spontaneous seizures continue to occur till adulthood. Persistence of Kindling Kindling of limbic structures during the infantile period appears to leave persistent alterations in the brain, through adulthood. P15–18 rats kindled to manifest either general-
Characteristics and Defining Features
ized or focal seizures can be re-kindled faster during adulthood, whether stimulation is done at the ipsilateral or contralateral amygdala (Moshé and Albala, 1982; Moshé and Albala, 1983). For instance, rats that were fully or partially kindled at P18 required only 3–6 stimulations respectively to re-kindle in adulthood, as opposed to 10–13 stimulations needed in controls (Moshé and Albala, 1982). AD duration was also significantly longer in adult rats that were previously kindled in infancy and their seizures were more severe (Moshé and Albala, 1982).
Neuropathology Histologic examination of kindled rats has not demonstrated significant histopathologic differences compared to same-age rats implanted but not kindled (Moshé and Albala, 1983). Nissl staining of brain sections from rats kindled at P15–17 did not reveal significant cell loss at the CA3c or CA1 hippocampal regions, 2 weeks following the last kindled seizure (Haas et al., 2001). Similarly, there was no significant mossy fiber sprouting in Timm-stained hippocampal sections from these rats (Haas et al., 2001), in agreement with the studies supporting the relative resistance of the immature brain to seizure-induced pathologic changes. These studies demonstrate that kindling in pups is permanent in the absence of overt histologic changes, albeit only studied in amygdala and hippocampus. The development and neuropathologic consequences of kindling, however, are enhanced in pups that have a pre-existing neuronal migration disorder. Germano et al. showed that P15 pups with experimentally-induced neuronal migration disorder, produced by transplacental injection of methylazoxylmethanol acetate, had lower ADTs, faster kindling rates, and longer ADs the second day of hippocampal kindling. In addition, kindled-seizure induced damage was observed at the CA3 sectors bilaterally (Germano et al., 1998).
Neuroimaging Increased deoxyglucose uptake in rhinencephalic structures, but not in basal ganglia or neocortex, was observed in P15–16 albino rats with stage 6 and 7 kindled seizures (Ackermann et al., 1989). The increased deoxyglucose accumulation in the hippocampus was not associated with concomitant increase in glucose accumulation (Sperber et al., 1992). It has been proposed that the rapid propagation and increased severity of seizures at this age may reflect the immaturity of subcortical structures involved in seizure control (Haas et al., 1998).
Genetics and Molecular Changes Kindling has been described mainly in immature Sprague-Dawley rats (Moshé, 1981; Moshé et al., 1981;
375
Baram et al., 1993; Baram et al., 1998), but also in weanling (3-week old) Wistar pups (Kawahara et al., 1989). It is the experience of this lab (Moshé SL, unpublished observations) that the development of kindling in Sprague-Dawley pups obtained from different sources may vary, suggesting that exogenous or genetic factors influence this process. The existence of strain differences in the development of kindling has been well documented in adult Fast and Slow rats, a crossbreeding of Wistar and Long-Evans rats (Xu et al., 2004; McIntyre et al., 1999). Similar differences also exist in immature pups. During amygdala kindling, P15–16 Fast and Slow rats exhibit an elevated ADT and require longer ISIs (at least 45 minutes) compared to Sprague-Dawley pups (Veliskova et al., 2004) . Fast P15–16 pups kindle faster than Slow pups (Veliskova et al., 2004; Moshé et al., 1981). There are no studies about the effects of specific genetic or molecular alterations on the development of kindling or describing molecular changes resulting from kindling in developing rats. Furthermore, there are no definitive studies describing sex differences in kindling in developing rats, as all of the published data have been obtained from mixed litters, not analyzed according to sex.
Response to Antiepileptic Drugs/Usefulness in Screening Drugs Kindling has been used as a model to test the effects of a variety of conditions and drugs on the susceptibility of the developing brain to seizures and the development of the kindled state. To test whether a drug may alter sensitivity to kindling, drugs can be administered prior to the first daily kindling stimulus. It is also possible to study whether a drug may alter the establishment of the kindled state, by administering it once the rat is fully kindled. In these studies, the effects of drugs or pathologic states, such as hypoxia/ischemia, can be tested in regard to their ability to alter ADT, kindling rate, AD duration, and seizure severity. Only few antiepileptic drugs have so far been tested as to their ability to alter the development of kindling in immature pups. Diazepam (Albertson et al., 1982) and gabapentin (Lado et al., 2001) inhibited the development of kindling in immature rats. Acute and chronic ACTH treatment inhibited the development of the kindled state (Holmes and Weber, 1986), but had no effect once the kindled state was already established (Thompson and Holmes, 1987). Furthermore, other drugs and compounds not included among the conventional antiepileptics have also been tested. For instance, progesterone-like substances (Holmes and Weber, 1984), NMDA receptor antagonists (Trommer and Pasternak, 1990; Holmes et al., 1990), the GABAB receptor agonist baclofen (Wurpel, 1994), and the calcium channel blockers verapamil and nimodipine (Wurpel and Iyer, 1994) also inhibited kindling in developing rats. Estrogens had no effect on the development of kindling (Schultz-Krohn et al., 1986).
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Baclofen also suppressed the severity and duration of established kindled seizures and increased postictal refractoriness (Wurpel, 1994).
LIMITATIONS Ease of Development/Reliability Despite a higher ADT, amygdala kindling is produced faster in infantile rats compared to adults. The short ISI kindling protocol reliably kindles rats of most ages, although the success rate may differ according to the specific age and site. Amygdala kindling of P15–18 rats with 15 minute ISI kindled 100% of the pups (Moshé et al., 1983). In younger pups (P7–8) the success of inducing AD with this protocol falls to 50% (Baram et al., 1993).
Mortality Mortality during the kindling process is insignificant. If rats are allowed to grow to adulthood, a higher than expected mortality has been observed in kindled (40%) versus implanted but not kindled rats (20%) (Moshé and Albala, 1982).
WHAT’S IT GOOD FOR? Kindling of immature rats has been shown to be a reliable method of assessing susceptibility to the development of seizures and establishing a state of increased seizure susceptibility that persists till adulthood. Although spontaneous seizures, the principal feature of the epileptic state, have been reported, there are no studies documenting the persistence of these spontaneous seizures till adulthood. This may be due to excessive animal costs and the difficulty of maintaining intracerebral electrodes in growing animals. Therefore, kindling in developing rats can probably be used as a model of epileptogenesis but not as a model of spontaneous epilepsy. It is a useful method to assess the effect of a variety of drugs or conditions on several aspects of seizure susceptibility, i.e., ADT, kindling rates, recurrent triggered bilateral seizures, or postictal refractoriness. Kindling can also be used to study the consequences of repetitive seizures in the developing brain and persistence of any seizure-induced deficits.
Acknowledgements This work was supported by NIH NINDS grants NS20253 and NS048856 (SLM), and NS45243 (ASG).
References Ackermann, R.F., Moshé, S.L., and Albala, B.J. 1989. Restriction of enhanced [2-14C]deoxyglucose utilization to rhinencephalic structures in immature amygdala-kindled rats. Exp Neurol 104: 73–81.
Albertson, T.E., Bowyer, J.F., and Paule, M.G. 1982. Modification of the anticonvulsant efficacy of diazepam by Ro-15-1788 in the kindled amygdaloid seizure model. Life Sci 31: 1597–1601. Baram, T.Z., Hirsch, E., and Schultz, L. 1993. Short-interval amygdala kindling in neonatal rats. Brain Res Dev Brain Res 73: 79–83. Baram, T.Z., Hirsch, E., and Schultz, L. 1998. Short interval electrical amygdala kindling in infant rats: the paradigm and its application to the study of age-specific convulsants. In Kindling 5, vol. 48. Ed. Corcoran, M.E., and Moshé, S.L. pp. 35–44. New York: Plenum Press. Dahl, K.D., Jia, X.C., and Hsueh, J.W. 1988. Bioactive follicle-stimulating hormone levels in serum and urine of male and female rats from birth to prepubertal period. Biol Reprod 39: 32–38. Germano, I.M., Sperber, E.F., Ahuja, S., and Moshé, S.L. 1998. Evidence of enhanced kindling and hippocampal neuronal injury in immature rats with neuronal migration disorders. Epilepsia 39: 1253–1260. Gravel, C., and Hawkes, R. 1990. Maturation of the corpus callosum of the rat: I. Influence of thyroid hormones on the topography of callosal projections. J Comp Neurol 291: 128–146. Haas, K.Z., Sperber, E.F., Benenati, B., Stanton, P.K., and Moshé, S.L. 1998. Idiosyncrasies of limbing kindling in developing rats. In Kindling 5, vol. 48. Ed. Corcoran, M.E., and Moshé, S.L. New York: Plenum Press. Haas, K.Z., Sperber, E.F., and Moshé, S.L. 1990. Kindling in developing animals: expression of severe seizures and enhanced development of bilateral foci. Brain Res Dev Brain Res 56: 275–280. Haas, K.Z., Sperber, E.F., and Moshé, S.L. 1992. Kindling in developing animals: interactions between ipsilateral foci. Brain Res Dev Brain Res 68: 140–143. Haas, K.Z., Sperber, E.F., Opanashuk, L.A., Stanton, P.K., and Moshé, S.L. 2001. Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling. Hippocampus 11: 615–625. Holmes, G.L., and Thompson, J.L. 1987. Rapid kindling in the prepubescent rat. Brain Res 433: 281–284. Holmes, G.L., Thompson, J.L., Carl, G.F., Gallagher, B.S., Hoy, J., and McLaughlin, M. 1990. Effect of 2-amino-7-phosphonoheptanoic acid (APH) on seizure susceptibility in the prepubescent and mature rat. Epilepsy Res 5: 125–130. Holmes, G.L., and Weber, D.A. 1983. Increased susceptibility to pentylenetetrazol-induced seizures in adult rats following electrical kindling during brain development. Brain Res 313: 312–314. Holmes, G.L., and Weber, D.A. 1984. The effect of progesterone on kindling: a developmental study. Brain Res 318: 45–53. Holmes, G.L., and Weber, D.A. 1986. Effects of ACTH on seizure susceptibility in the developing brain. Ann Neurol 20: 82–88. Kawahara, R., Matsuda, K., Ishida, A., Takeshita, H., Okubo, I., Tanaka, T., Sakamoto, T. et al. 1989. Amygdaloid kindling in weanling rat and rekindling upon maturization. No To Shinkei 41: 135–141. Kristensson, K., Zeller, N.K., Dubois-Dalcq, M.E., and Lazzarini, R.A. 1986. Expression of myelin basic protein gene in the developing rat brain as revealed by in situ hybridization. J Histochem Cytochem 34: 467–473. Lado, F.A., Sperber, E.F., and Moshé, S.L. 2001. Anticonvulsant efficacy of gabapentin on kindling in the immature brain. Epilepsia 42: 458–463. Lee, S.S., Murata, R., and Matsuura, S. 1989. Developmental study of hippocampal kindling. Epilepsia 30: 266–270. McIntyre, D.C., Kelly, M.E., and Dufresne, C. 1999. FAST and SLOW amygdala kindling rat strains: comparison of amygdala, hippocampal, piriform and perirhinal cortex kindling. Epilepsy Res 35: 197–209. Michelson, H.B., and Lothman, E.W. 1991. An ontogenetic study of kindling using rapidly recurring hippocampal seizures. Brain Res Dev Brain Res 61: 79–85. Moshé, S.L. 1981. The effects of age on the kindling phenomenon. Dev Psychobiol 14: 75–81. Moshé, S.L., and Albala, B.J. 1982. Kindling in developing rats: persistence of seizures into adulthood. Brain Res 256: 67–71.
References Moshé, S.L. and Albala, B.J. 1983. Maturational changes in postictal refractoriness and seizure susceptibility in developing rats. Ann Neurol 13: 552–557. Moshé, S.L., Albala, B.J., Ackermann, R.F., and Engel, J., Jr. 1983. Increased seizure susceptibility of the immature brain. Brain Res 283: 81–85. Moshé, S.L., and Ludvig, N. 1988. Kindling. In Recent Advances in Epilepsy, vol.4. Ed. Pedley, T.A., and Meldrum, B.S. pp. 21–44. New York: Churchill Livingstone. Moshé, S.L., Sharpless, N.S., and Kaplan, J. 1981. Kindling in developing rats: variability of afterdischarge thresholds with age. Brain Res 211: 190–195. Ojeda, S.R., Urbanski, H.F., and Ahmed, C.E. 1986. The onset of female puberty: studies in the rat. Recent Progress in Hormone Research 42: 385–442. Paxinos, G., Ashwell, K.W.S., and Tork, I. 1994. Atlas of the Developing Rat Nervous System. San Diego: Academic Press. Paxinos, G., and Watson, C. 1997. The Rat Brain in Stereotaxic coordinates. San Diego: Academic Press. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32: 281–294. Schultz-Krohn, W.A., Thompson, J., and Holmes, G.L. 1986. Effect of systemic estrogen on seizure susceptibility in the immature animal. Epilepsia 27: 538–541. Sperber, E.F., Stanton, P.K., Haas, K., Ackermann, R.F., and Moshé, S.L. 1992. Developmental differences in the neurobiology of epileptic brain damage. Epilepsy Res Suppl 9: 67–81.
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Sperber, E.F., Veliskova, J., Benenati, B., and Moshé, S.L. 1998. Enhanced epileptogenicity of area tempestas in the immature rat. Dev Neurosci 20: 540–545. Thompson, J., and Holmes, G.L. 1987. Failure of ACTH to alter transfer kindling in the immature brain. Epilepsia 28: 17–19. Trommer, B.L., and Pasternak, J.F. 1990. NMDA receptor antagonists inhibit kindling epileptogenesis and seizure expression in developing rats. Brain Res Dev Brain Res 53: 248–252. Trommer, B.L., Pasternak, J.F., Nelson, P.J., Colley, P.A., and Kennelly, J.J. 1994. Perforant path kindling alters dentate gyrus field potentials and paired pulse depression in an age-dependent manner. Brain Res Dev Brain Res 79: 115–121. Veliskova, J., Asnis, J., Lado, F.A., and McIntyre, D.C. 2004. Development of kindling in Immature fast and slow kindling rats. In Kindling 6 Ed. Corcoran, M.E., and Moshé, S.L. Series: Advances in Behavioral Biology, vol. 5, in press. New York: Plenum Press. Wurpel, J.N. 1994. Baclofen prevents rapid amygdala kindling in adult rats. Experientia 50: 475–478. Wurpel, J.N., and Iyer, S.N. 1994. Calcium channel blockers verapamil and nimodipine inhibit kindling in adult and immature rats. Epilepsia 35: 443–449. Xu, B., McIntyre, D.C., Fahnestock, M., and Racine, R.J. 2004. Strain differences affect the induction of status epilepticus and seizure-induced morphological changes. Eur J Neurosci 20: 403–418.
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31 Chemical Kindling MARY E. GILBERT AND JEFFREY H. GOODMAN
trations of convulsant drugs, systemically or directly into the brain, require many fewer stimulations to reach generalized seizures when subsequently challenged in a standard electrical kindling paradigm (see Cain, 1986; Wasterlain et al., 1989; Uemura and Kimura, 1990). Subthreshold chemical and electrical stimulation appear to activate similar anatomic pathways, such that a common seizure circuitry gradually evolves with repetition of either stimulus type. Strong evidence of chemical kindling is provided when facilitation of subsequent electrical kindling can be demonstrated. The savings and bidirectional transfer that occurs between chemical and electrical kindling is also taken as evidence that the same neurochemical and anatomic substrates are utilized in these two models. Studies have invoked the use of chemical kindling paradigms to investigate the chemical and molecular substrates of electrical kindling, to study basic mechanisms of epileptogenesis, and as a screening tool for anticonvulsant drugs.
Epilepsy is a clinical disorder manifest by recurrent selfsustaining paroxysmal bursts of abnormal electrical brain activity. Kindling is the progressive development of seizures in response to a previously subconvulsant stimulus administered in a repeated and intermittent fashion (Goddard et al., 1969). Kindling has been well studied as an epilepsy model of complex partial seizures with secondary generalization. Traditionally, kindling has been induced by application of brief, low-intensity trains of electrical stimulation and is the topic of another chapter in this book (see Chapter 28). Chemical kindling is similar to electrical kindling in that the gradual development of electrographic and behavioral seizures occurs with repeated stimulation, but in this case a chemical rather than an electrical stimulus is the source for activation of the neural circuitry. As with electrical kindling, the main features of chemical kindling are the progressive development of behavioral seizures, a reduction in seizure threshold, and a maintained heightened sensitivity to the seizure-inducing stimulus. Chemical kindling can be induced by direct intracerebral administration of low doses of excitatory agents (Table 1) or, more routinely, by repeated systemic administration of convulsant agents at subthreshold concentrations (Table 2). Increased sensitivity after initial kindling with one agent that generalizes to other treatments capable of inducing epileptiform discharge is another aspect of the kindling phenomenon (Cain, 1986; Gilbert, 1992; Wasterlain et al., 1985; 1989). Rats kindled by traditional focal electrical stimulation of the amygdala are rendered permanently more susceptible to convulsions induced by other seizure-inducing treatments (e.g., alcohol withdrawal, chemoconvulsant administration) (Racine, 1978). Similarly, rats chemically kindled by repeated administration of subthreshold concen-
Models of Seizures and Epilepsy
WHAT DOES IT MODEL? Unlike most other experimental epilepsy models, kindling is progressive and persistent and creates a permanently enhanced seizure susceptibility in the absence of major destruction of tissue. Kindling is well suited as a model of clinical epilepsy because its chronicity and its electrographic and behavioral manifestations closely mimic partial complex seizures in humans. Electrical and chemical kindling specifically model partial complex epilepsy with secondary generalization. This type of epilepsy is regarded as the most refractory to antiepileptic drug therapy (Loscher, 1993; McNamara, 1984). As a result, pharmacologic devel-
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TABLE 1 Repeated administration of chemicals directly into the brain at subthreshold concentrations leads to the progressive development of kindled seizures Intracerebral Excitatory Amino Acids glutamate/aspartate NMDA Opiates b-endorphin met-enkephalin Cholinergic Agonists carbacol acetyl-b-methylcholine muscarine pilocarpine physostigmine Cyclic Nucleotides cAMP GABA Antagonists b-carboline FG-7142 pentylenetetrazol picrotoxin bicuculline
Site/Species
Interval
Reference
amygdala/rat
48 hr
Mori and Wada, 1987; Croucher and Bradford, 1989; Croucher et al., 1995
amygdala, hippocampus/rat
48 hr
Cain and Corcoran, 1985; Tanaka et al., 1989
amygdala, hippocampus, ventricle/rat
24–48 hr
Cain, 1983; Girgis, 1981; Morin et al., 1983; Vosu and Wise, 1975; Wasterlain et al., 1985; Wasterlain and Fairchild, 1985
amygdala/rat
48 hr
Yokoyama et al., 1989
amygdala/rat
48–96 hr
Cain, 1982; 1987; Corda et al., 1990; Little et al., 1987; Morin et al., 1983; Uemura and Kimura, 1988; 1990
TABLE 2 Repeated systemic administration of a variety of chemoconvulsants at subthreshold concentrations leads to the progressive development of behavioral seizures Systemic Administration GABAergic Antagonists PTZ beta carboline (FG7142) picrotoxin bicuculline Neurotoxicants Dieldrin Endosulfan Lindane Trimethylolpropane Local Anesthetics cocaine lidocaine
Route/ Species
Interval
No. of Treatments
References
ip/ rat, mouse, guinea, pig
24–96 hr
12–127
Cain, 1981; 1987; Diehl et al., 1984; Getova et al., 1998; Karler et al., 1989a; Lewin et al., 1994; Lin et al., 1998; Little et al., 1987; Mason and Cooper, 1972; Pinel and Weidmann, 1989; Vindrola et al., 1984
ip, oral/ rat
24–72 hr
12–30
Gilbert, 1992; 1995a; Joy et al., 1980; Lin et al., 1998
ip, sc/ rat, mouse
24 hr
6–19
Abel and Carney, 1993; Clark et al., 1992; Lekic, 1988; Karler et al., 1989b; Stripling and Ellinwood, 1977; Weiss et al., 1989
IP, intraperitoneal; PTZ, pentylenetetrazol; SC, superior colliculus
opment of new antiepileptic therapies have been based on mechanisms underlying kindling, and preclinical screening tests have incorporated the kindling model. Recently a model of absence seizures has been described using repeated timed injections of low doses of pentylenetetrazol (PTZ) or g-hydroxybutyrate that shares some features with chemical kindling paradigms (Hu et al., 2001; Wong et al., 2003). Unlike chemical kindling, however, repeated
doses are given within a brief period of time (i.e., one hour). The maximal total dose of PTZ administered would be convulsive if delivered as a single bolus. By contrast, intermittent administration of low doses induces a nonconvulsive state of behavioral arrest and immobility, accompanied by intermittent bursts of spike and wave cortical discharges, that closely parallel cortical absence seizures. As with kindling, a heightened responsivity to gen-
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Methods of Generation
eralized spike and wave cortical discharge is maintained upon subsequent challenge with a subconvulsant dose of PTZ, and this enhanced sensitivity occurs in the absence of overt structural damage.
RELATIONSHIP TO ILAE CLASSIFICATION Using the International League Against Epilepsy (ILAE) classification from 1981, electrically kindled seizures are considered a model of complex partial seizures with secondary generalization (Bradford, 1995; Fisher, 1989; McNamara, 1984). The ILAE recently revised this terminology to focal seizures with secondary generalization (Engel, 2001). This designation for electrical kindling can also be used for chemically kindled seizures induced by intracerebral microinjection of a convulsant agent. Chemical kindling that results from the systemic administration of the convulsant may be different since the whole brain is exposed. Since in these models there may be more cortical involvement, especially with PTZ, it has been suggested that this type of kindled seizure more closely models primary generalized epilepsy (Ono et al., 1990).
METHODS OF GENERATION Rodents are the predominant species in which chemical kindling studies have been assessed. Young adult male inbred strains of rats and mice are the subjects of choice, but chemical kindling has also been demonstrated with rabbits (Girgis, 1981) and guinea pigs (Diehl et al., 1984). In a typical chemical kindling experiment, animals are injected on an intermittent schedule with a drug with convulsant properties, at a concentration that is below the convulsant threshold for all animals. Subjects are observed for behavioral manifestations of hyperexcitability and seizure activity. The rate of development of chemical kindling can be variable, dependent upon the drug, the dose, the route of administration, the duration of treatment, and the interval between successive administrations. Kindling development can be characterized by the number of animals exhibiting clonic seizures following a certain number of treatments, the mean number of treatments required to induce clonic seizures, or the mean seizure score after a defined number of treatments.
Intracerebral Infusions Studies employing direct intracerebral introduction of a chemoconvulsant to limbic or cortical regions typically report electrographic as well as behavioral characterizations of the seizure response. In the majority of reports, the injection site is the amygdala, to permit direct comparison to
electrical kindling paradigms that have routinely used the amygdala as the primary kindling focus (see Table 1). Chemoconvulsants are infused through a permanently implanted cannula at a slow rate to avoid perfusion-induced neuronal damage at the infusion site. Electrical activity from that site is simultaneously monitored by way of an electrode mounted to the sleeve of the injector cannula. This form of chemical kindling most closely resembles the electrical kindling procedures.
Systemic Administration of Chemoconvulsants Systemic administration of subthreshold concentrations of convulsive agents is the most common form of chemical kindling. The procedures are straightforward. Animals are dosed intravenously, ip, sc, or by gavage, at 24–72-hour intervals and assessed for behavioral signs of seizure. Electrographic monitoring of brain activity to detect seizure discharge has been performed (e.g., Ono et al., 1990) but is not routine in paradigms where chemical kindling is induced by repeated systemic administration of a convulsant. As such, these paradigms do not provide direct electrographic confirmation of subclinical manifestations of seizure discharge. Rather, the gradual appearance of behavioral indices of seizure activation is relied upon for evidence of kindling. These assessments are more subjective and less quantitative than electrographic assessments but are less labor-intensive and have been reliably used to determine pro- and anticonvulsant properties of a variety of experimental manipulations. Parameters that can be recorded in chemical kindling studies that rely on behavioral observations include the severity of seizure response, frequency of a particular behavioral response within a given recording period (typically 30–60 minutes, depending on the drug and the route of administration), latency to onset of behavioral manifestations, duration of behavioral response, and the number of drug treatments required to induce a convulsive response. A variety of convulsive agents, many of which are summarized in Table 2, with distinct actions on the central nervous system have been used to induce chemical kindling by systemic administration. The most common chemical kindling agent is PTZ, although other drugs that interfere with GABAergic function are also effective kindling agents. PTZ is a selective blocker of the chloride channel coupled to the GABAA receptor and is associated with a reduction in GABA-mediated neurotransmission in the central nervous system. The advantage of PTZ is its relatively shallow doseresponse function such that a dose can be identified which is subthreshold for convulsions in all animals. As a result, a gradual emergence of seizure responsiveness is readily identifiable, and with sufficient repetitions, the majority of animals progress to clonic-seizures. In animals kindled with PTZ, enhanced susceptibility to convulsions is maintained
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for several weeks upon challenge with a low dose of PTZ or a variety of other inhibitors of central GABAergic function (e.g., the chloride channel blocker picrototoxin, the benzodiazepine receptor ligands FG-7142 and RO15-4513, and the GABA synthesis inhibitor isoniazid) (Corda et al., 1991; 1992; Little et al., 1986; 1987; Nutt et al., 1982). Intermittent administration with intervals of 2–3 days appears to produce most reliable kindling with PTZ. Corda et al. (1991) report enhanced kindling rates when PTZ is administered three times per week relative to daily injections of 30 mg/kg, ip. Twice daily administration at half the dose, however, was ineffective. More robust behavioral seizure progression with thrice weekly versus daily administration has also been reported for chemical kindling induced by the cyclodiene pesticides dieldrin, endosulfan, and lindane (Gilbert, 1992b; 1995; Joy et al., 1982).
orously assessed with other agents. Metabolic concerns have been addressed by measuring brain concentrations of PTZ. Brain bioavailability of PTZ did not differ between single and repeated administrations of subconvulsant doses of PTZ, and no alterations in blood-brain barrier were induced by PTZ (Sierra-Paredes et al., 1989). The rapid distribution and relatively sustained activation of circuitry accompanying systemic administration of convulsant drugs is an important distinction to be kept in mind when generalizing across findings from electrical and chemical kindling models. In contrast to focal electrical stimulation in traditional kindling paradigms, systemic administration of a chemoconvulant such as PTZ provides ready access to all brain areas, allowing for the simultaneous activation of all seizure circuitry.
BEHAVIORAL/CLINICAL FEATURES METHODOLOGIC CONSIDERATIONS Interpretations of chemical kindling can be confounded by several methodologic issues that lead to apparent but spurious kindling (see Gilbert, 1994; Nutt et al., 1982; Wasterlain et al., 1989). Increased seizure severity may result from an accumulation of the chemical in the body; aging may lead to increased sensitivity to convulsant agents; increases in body weight over the course of the dosing period may result in higher absolute target doses with a constant delivered dose; or alterations in pharmacokinetics may lead to increased plasma and brain levels and account for an augmented behavioral response. Therefore, a number of criteria should be met in a chemical kindling experiment prior to concluding that repeated exposure to a chemical produces kindling: 1. The chemical kindling process should begin with an initially subconvulsive dose of the chemical of interest in order to monitor the gradual emergence of a behavioral response with repeated dosing. 2. There must be a maintained behavioral/electrographic responsiveness to the initially subconvulsive dose upon challenge following a washout period of sufficient duration to permit elimination of the chemical from the body. 3. Repeated chemical exposure inducing chemical kindling should demonstrate positive transfer to electrical kindling. Just as transfer occurs between kindling sites, if chemical kindling has occurred, a significant savings in electrical kindling development must follow in the absence of further exposure. These criteria have been met for some GABAergic agents, most notably PTZ (e.g., Cain, 1981; DeSarro et al., 2000; Sierra-Paredes et al., 1989), but have not been as rig-
Seizures evoked by repeated administration of PTZ originate in the forebrain, and, in well kindled animals, some elements of hindbrain seizures eventually develop. Many published reports on chemical kindling have invoked the 5 stage scale of motor seizure progression of Racine (1972) to characterize chemical kindling. Modifications to this seizure scoring system have been introduced to more closely capture the subtle progression of behavioral transitions that occurs during repeated exposure to subthreshold doses of chemoconvulsants (e.g., Table 3). PTZ has been reported to produce kindling to fully generalized convulsions (Corda et al., 1991; Ito et al., 1977; Mason and Cooper, 1972). In the behavioral scoring scheme described by Corda et al. (1991), the most severe seizure type described includes elements of tonic extension and status epilepticus. In actuality, the mean seizure score reported in these studies after 50 daily injections did not exceed generalized tonic-clonic seizures (Racine’s Stage 5). Others have reported that intermittent myoclonic jerking represents the maximal seizure response associated with PTZ despite a prolonged treatment (Pinel and Cheung; 1977; Nutt et al., 1982). Variability associated with chemical kindling development is influenced by the drug, the dose, the interval, and the route of administration. Doses ranging from 25–45 mg/kg of PTZ have been used for PTZ kindling, the more rapid development typically occurring with the higher doses. However, many of these studies report clonic seizures in a subset of animals with the initial administration and, as such, are not truly representative of the kindling process (e.g., da Silva et al., 1998; Ito et al., 1977). Interpretation of effects of experimental manipulation under investigation on the epileptogenic process can be compromised under these circumstances. Mortality associated with PTZ kindling is not commonly reported, but severe seizures and death may be more prevalent with convulsants with a steep dose–response curve. As
Imaging the Seizure Network
TABLE 3 Seizure ranking scheme to identify subtle transitions in seizure severity with low level dose administration of pesticides with chemical kindling potential. Modified from Gilbert (1995). Similar patterns of seizure development have been described for more prototypic kindling agents. Some agents, however, produce more severe seizures with repeated clonic-tonic convulsions and death Severity Score
Behavioral Manifestation
0
Immobility, rearing, locomotion, burrowing, grooming, sleeping, chewing food, boli, or bedding
1
Jaw clonus, sustained rapid chewing in absence of food, boli, or bedding. Twitching of ears and eyes, extended periods of immobility, and staring
2
MCJ: 1–2 incidences of abrupt flexion and extension of the forelimb, head, and neck musculature
3
Multiple MCJ: more than 2 MCJ over the 30–60 minute observation period
4
Prolonged MCJ: MCJ is extended in time and accompanied by a rapid burst of chewing, facial twitching, or brief clonic motion of the forelimbs (2–5 seconds). Facial automatisms associated with MCJ are reminiscent of Stage 2 kindled seizures of Racine scoring scoring scheme. Straub tail.
5
Clonic Seizures: Unilateral or bilateral forelimb clonus that endures for more than 10 seconds. Reminiscent of Racine Stage 3–4 seizures
6
Multiple clonic bouts: More than 1 clonic seizure episode over the observation period MCJ, myoclonic jerk
with electrical kindling, increased sensitivity to subthreshold levels of chemoconvulsants is maintained for several weeks. Corda et al. (1991) report enhanced sensitivity to subthreshold PTZ one year after completion of chronic treatment. With electrical kindling, spontaneous seizures do result after very significant numbers of stimulations (see Racine, 1978; Sato et al., 1990). The occurrence of spontaneous seizures has not been investigated with chemical kindling. Given the commonality of substrates apparently shared by kindling induced by electrical or chemical means, it is not unreasonable to suspect that spontaneous seizures may also accompany chemical kindling with sufficient repetition.
ELECTROGRAPHIC (EEG) FEATURES Although many published reports have recorded brain activity during the development of chemical kindling, this is more the exception than the rule. Dependent on dose, interval and potency of the chemoconvulsant, behavioral signs begin to appear after 5–10 injections. Isolated spikes
383
may be evident in cortical or limbic electrographic recordings upon initial injections. Brief discharges appear, some accompanied by behavior, others not. Robust clonus is accompanied by spike-and-wave discharges in limbic areas and the cortex. Figure 1A is a recording from the amygdala from a naive animal administered a low dose of the pesticide lindane (10 mg/kg, po). Lindane is a member of the cyclodiene class of pesticides that has antagonistic action at the GABA receptor site (Hulth et al., 1978; Llorens et al., 1990). No behavioral signs of seizure were evident and two spike-and-wave discharges were recorded during this 1.5 hour observation period. Figure 1B is a recording from an animal 1 month after the last 30 administrations of a 10 mg/kg dose of lindane. In contrast to the initial electrographic and behavioral response, this administration of lindane evoked multiple bouts of clonic seizure activity accompanied by afterdischarges and intermittent periods of spike-and-wave discharge (Gilbert, 1995).
IMAGING THE SEIZURE NETWORK The rapid, activity-dependent induction of protooncogene c-fos has been widely applied as a marker of spatially distributed, multi-synaptic networks in a variety of experimental paradigms. The spatial pattern of c-fos expression during electrical kindling identifies the propagation of seizure activity and maps the behavioral expression of seizures with a high degree of anatomical and cellular resolution (Applegate et al., 1998; Burchfield et al., 1998; Clark et al., 1991). Increased expression of c-fos mRNA is observed in limbic structures early in the preclonus stages of kindling, whereas bilateral activation of limbic and cortical sites accompanies fully-generalized electrically-kindled seizures (Applegate et al., 1998; Burchfield et al., 1998; Clark et al., 1991). This approach has been applied to a much more limited extent in chemical kindling paradigms. C-fos mRNA expression is increased in the cortex, caudate, septum, amygdala and hippocampus bilaterally in animals kindled with PTZ or cocaine (Erdtmann-Vourliotis et al., 1998; Clark et al., 1992). In PTZ-kindled animals expressing fully kindled generalized tonic-clonic seizures, the pattern of c-fos expression is indistinguishable from acute seizures induced by a convulsive dose of PTZ (Erdtmann-Vourliotis et al., 1998). Behavioral manifestations of myoclonic jerks early in the process are common in chemical kindling, and suggest cortical involvement. However, unlike electrical kindling, c-fos expression was not elevated over basal levels in brains of PTZ-kindled animals with myoclonic jerks and mild forelimb clonus (Erdtmann-Vourliotis et al., 1998), or in animals administered repeated subthreshold doses of cocaine that failed to exhibit behavioral seizures (Clark et al., 1992). Thus it appears that although brain sites participating in generalized convulsions induced by chemical kindling can
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FIGURE 1 Electroencephalogram (EEG) recorded from the amygdala in a rat administered the organochlorine pesticide lindane (10 mg/kg, po), for the first time (A). A representative 30-second sample of EEG was extracted from the 30-minute pre-dosing baseline. EEG and behavior were monitored for 1.5 hours after dosing and time indicators are placed along the tracing (m = minute post-dose). Two high-amplitude spikes are evident in the EEG (arrowheads) approximately 40 minutes after dosing. No behavioral signs were observed. In contrast, the EEG record in B is from an animal with a history of 30 administrations of 10 mg/kg of lindane. This record was taken upon challenge with the same dose of lindane after a 4-week nondosing period to demonstrate both enhanced behavioral and electrographic responsiveness and maintained susceptibility to a previously subthreshold dose. As in A, a pre-dose period was collected and behavior and EEG monitored after dosing. Clear behavioral and electrographic evidence of seizure discharge is present. Interictal spike and wave discharges were accompanied by immobility (im) and myoclonic jerks (MCJ), and sustained ictal discharge was coincident with clonic seizure (Stage 5 seizure from Table 3). Additional periods of rebound afterdischarges associated with immobility followed the convulsion. (Modified and reprinted with permission from Gilbert, 1995.)
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Advantages and Limitations
be identified using this technique, the use of c-fos expression is not suitable to track the anatomical loci involved in epileptogenesis induced by chemical kindling may not be possible.
NEUROPATHOLOGY For a number of years, the general consensus was that electrical kindling and kindling-induced seizures did not induce cell death. The objective of early investigations was to identify neuroanatomic changes responsible for the kindling process, rather than an examination of the mechanisms of epilepsy-induced neuronal damage. Numerous studies reported no evidence of overt cell loss or reactive gliosis after electrical kindling (see Racine, 1978 for review). More recently several studies have identified anatomic changes that may accompany electrical kindling. Sutula and colleagues have focused on kindling-induced changes in the hippocampus, specifically in the dentate gyrus. They have reported evidence of neuronal cell loss in the dentate hilus (Cavazos and Sutula, 1990; Cavazos et al., 1994; Kotloski et al., 2002) and synaptic reorganization of the mossy fibers into the inner molecular layer (Cavazos et al., 1991; Sutula et al., 1988). This group has recently reported that inhibitory neurons immunoreactive for cholecystokinin and the GABA transporter GAT-1 are particularly vulnerable to kindled seizures, which could explain the decrease in inhibition present after excessive kindling (Sayin et al., 2003, but see Spiller and Racine, 1995). Recently von Bolen et al. (2004) reported a decrease in neuronal density in the amygdala and cortex of amygdala-kindled rats. There is also evidence that kindled seizures can induce dentate granule cell loss by apoptosis (Bengzon et al., 1997; Pretel and Piekut, 1997) and increase the area of the dentate hilus (Bertram and Lothman, 1993; Adams et al., 1997). It has been hypothesized that the increase in hilar area after kindling is due to an increase in reactive gliosis (Adams et al., 1998). Despite these observations, the nature and magnitude of structural changes and whether they occur as cause or consequence of seizure activity remains a subject of debate (Cavazos et al., 1994; Khrugel et al., 1995; Spiller and Racine, 1995). The neuropathology associated with chemical kindling has not been as extensively studied as the changes associated with electrical kindling. Anatomic changes after intracerebral injection of convulsant agents into a limbic site can be expected to cause similar pathologic changes to what has been reported for electrical kindling, except for the potential of additional damage at the injection site. The systemic administration of a convulsant such as PTZ has the additional complication that the entire brain is exposed. There are several reports of neuronal loss and synaptic reorganization in animals kindled by the systemic administration of PTZ. Neuronal cell loss was observed in the hippocampus of PTZ-kindled rats (Franke and Kittner, 2001; Pohle et al.,
1997) accompanied by an increase in the immunoreactivity of glial fibrillary acid protein (GFAP), a marker of astrocytes (Franke and Kittner, 2001). The increased staining for GFAP suggests an increase in reactive gliosis. In addition to neuronal cell loss and gliosis, PTZ kindling has been reported to induce an increase in mossy fiber sprouting (Golarai et al., 1992) and sprouting in CA1 and the subiculum of the rats (Cavazos et al., 2004).
ADVANTAGES AND LIMITATIONS Chemical kindling is an easy paradigm to implement in even the sparsest of laboratories. Kindling with PTZ is reliable and robust and does not require the implantation of chronic indwelling electrodes. The phenomenon is highly reproducible across laboratories and species, as evidenced by the multitude of papers that have been published using the PTZ model. Mortality is low and with sufficient repetitions at appropriate intervals the majority of animals sustain generalized motor seizures. Some of the attractive features for implementation of this model, however, come at a cost. In the absence of EEG recordings, assessment of seizure activity must rely solely on behavioral indicators. Compared to electrical kindling, this model provides much less experimental control in the timing of evocation of seizures, and the absence of EEG recordings does not permit evaluation of seizure parameters that are not associated with clinical signs. In electrical kindling paradigms, the threshold for inducing a brief electrographic event, duration of the focal seizures, and progressive development of the spectral features of the evoked afterdischarge can be readily determined and comprise additional quantitative experimental parameters upon which to evaluate treatment-mediated effects. In electrical kindling paradigms, the afterdischarge event is immediate upon delivery of the stimulus. Following systemic administration, there is a delay from the time of drug delivery to the onset of clinical signs of seizure due to the requirement for drug absorption and distribution. Behavioral seizures manifest once sufficient plasma and brain concentrations have been attained and persist until clearance or pharmacologic tolerance has occurred. Additionally, in early stages of electrical kindling, seizures are typically limited spatially and temporally by the stimulation site and the focal evoked afterdischarge. Unlike an electrical 1–2 second afterdischarge-evoking stimulus train, systemic administration of a chemical stimulus is not a punctate event. Rather, the seizure initiating stimulus is widely dispersed, activating circuitry throughout the brain and remaining active until metabolic clearance from body. Behavioral observations should continue until it can be reasonably assumed that brain levels of inducing stimulus have declined and no seizure activity is likely to occur.
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In its primary use as a screening tool for anticonvulsant drugs, the issue of potential interaction between experimental drug of interest and the chemical used to induce the seizure must be considered. Direct competition at the brain target site in addition to more indirect peripheral metabolic interaction can complicate interpretation of results and possibly contribute to differences in findings derived from electrical versus chemical kindling (e.g., Schmidt, 1987; Wang et al., 1993). It might appear that chemical kindling may be aptly suited for the investigation of epilepsy in young animals. Developmental studies using electrical kindling paradigms are plagued with the need to secure chronic headsets on the thin skulls of immature animals and to complete the kindling procedure over a restricted timeframe to ensure the electrode stability. As such, stimulation intervals are kept short to allow fully kindled seizures to manifest before rapid brain growth in the developing organism alters the position of the stimulating electrode and consequently the kindling focus. Chemical kindling does not require an indwelling electrode and thus dramatically simplifies the procedure in young animals, but the interval between chemical stimulations must be carefully selected to eliminate simple accumulation of drug as the cause of seizure induction. Similarly, ontogenetic differences in peripheral and central pharmacokinetic and pharmacodynamic processes (e.g., drug uptake, metabolism, enzyme induction, receptor up and down regulation) also complicate interpretation if kindling is to be compared across different age groups. Perhaps it is worthy of note that in our literature searches this paradigm has thus far not been actively engaged in developmental studies.
INSIGHTS INTO HUMAN DISORDERS Although one of the primary applications of chemical kindling has been as a screening tool for antiepileptic drugs (Table 4), chemical kindling procedures have also been utilized to elucidate the underlying mechanisms of the epileptogenesis; to evaluate the impact of a history of seizures on cell function; and to assess the long-term functional consequences of a history of seizures.
Mechanisms of Epileptogenesis Mechanisms of epileptogenesis have been studied in three different kinds of chemical kindling paradigms. The first entails the direct intracerebral administration of agents to activate neurotransmitters believed to be altered in epilepsy (Cain, 1986; Gilbert, 1992c; Pohle et al., 1997; Wasterlain et al., 1985; 1989). This approach most closely parallels the procedures adopted with traditional electrical kindling. Excitatory amino acids, cholingeric agonists, GABA antagonists are among the substances applied to discrete limbic sites in a manner analogous to focal electri-
cal stimulation (Table 1). Electrographic and behavioral assessment revealed the progressive development of seizure activity with repeated intracerebral administration. The generality, persistence, and overlap of neural substrates between chemical and the traditional electrical kindling paradigms have been evaluated by transfer studies. Facilitation of electrical kindling following chemical kindling and, vice versa, chemical kindling following electrical kindling is evidence of commonality of seizure circuitry. Other studies utilized systemic administration of a variety of substances and expanded the range of chemical classes that exhibit chemical kindling potential. Some of these are summarized in Table 2. A different but related approach originally used in electrical kindling paradigms but which readily transferred to chemical kindling involved the use of pharmacologic probes to slow (anticonvulsant) or accelerate (proconvulsant) the rate of kindling development. These studies differ from chemical kindling described in the preceding sections in that the proconvulsant or anticonvulsant drug of interest is co-administered with the chemical kindling agent. This application has spawned by far the most avid use of chemical kindling procedures—as a screening tool for anticonvulsant therapies. The simplicity, reliability, and sensitivity of the chemical kindling model has been exploited to determine the efficacy of therapeutic drugs to treat epilepsy. Some of these studies are summarized in Table 4. In another application, the chemical kindling paradigm has been used to assess the potential neurotoxicity of some environmental contaminants. Many pesticides produce a pronounced increase in central nervous system excitability when presented to humans and laboratory animals in acutely toxic quantitites (Hayes, 1991). Several classes of pesticides have been demonstrated to be proconvulsant when administered during the development of electrical kindling (Gilbert, 1988; 1992; Gilbert and Mack, 1989; Gilbert et al., 1990; Joy, 1982; 1985; Joy et al., 1983; Wurpel et al., 1993). The ability of xenobiotics to slow or facilitate the kindling process reveals that even at low doses, these toxicants possess neuroactive properties and can interfere with processes of synaptic plasticity. Some pesticides of the cyclodiene class have also been shown to act as kindling agents themselves. In the absence of electrical stimulation, repeated low-dose administration promotes the development of seizures and a persistent increase in seizure susceptibility (Gilbert, 1992; 1995; Joy et al., 1980).
What Does Epilepsy Do to the Brain? Cellular changes in kindled brains occur at many levels and reflect the complexity of the phenomenon. Although distinctions between chemical and electrical kindling paradigms have been identified, many parallels exist in cellular
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TABLE 4 Examples of anticonvulsant drugs tested using the chemical kindling with the pentylenetetrazol or b-carboline (FG 7142). Actions on the development of kindling (epileptogenesis) as well as kindled state have been assessed Drug
Development
State
x x x x x x x nt nt nt nt x ne/x nt ne x
x x x x x x ne x x x x nt ne/x x ne x
nt x x x
x nt nt nt
deSarro et al., 2000; Georgiev et al., 1995 Getova et al., 1998
x x ne ne
nt nt nt nt
Becker et al., 2001 de Sarro et al., 1999 Stephens and Turski, 1993 Stephens and Turski, 1993
x x
nt x
Stephens and Turski, 1993 Corda et al., 1992; Karler et al., 1989b; Krug et al., 1998
Nootropics Meclofenoxate Methylglucamine ootate Naftidrofuryl Piracetam Pyritinol Vinpocetine
nt nt nt ne nt nt
x x x x/ne x x
Angiotension Function Angiotension II Lorsartan (DuP 753) PD 123319
nt nt nt
x x x
Georgiev et al., 1996
Neuroactive Steroids Ganaxolone
x
nt
Beckman et al., 1998; Gasior et al., 2000
Purine Function 2-chloradenosine
x
x
Stephens and Weidmann, 1989
x x x/f
nt x nt
Han et al., 2000 Homayoun et al., 2002 Singh et al., 2003; but see Suzuki et al., 2001
x x
x x
Becker et al., 1999 Grecksch et al., 1999
GABA Function GABAA/Benzodiazepine Alphaxalone Clonazepam CL 218 872 Diazepam Di-n-propylacetate (Dekapine) Ethosuximide Flumazenil Gabrene Medazepam Metatolylcarbamide Nipecotic Acid Phenobarbital Sodium Valproate Succinic Acid THIP ZK 93 426 GABAB Baclofen CGP 36742 CGP 56433 CGP 61334 Glutamate Function AMPA AMPA CFM-2 CNQX g-D-GAMS NMDA AP-7 MK 801
Antioxidants 7-Nitroindazole Cyclosporine A FK506 Opioids Enadoline Naltrindole
References
Hansen et al., 2004 Stephens and Weidmann, 1989 Stephens and Weidmann, 1989 Becker et al., 1994; Hansen et al., 2004 Becker et al., 1994; Georgiev et al., 1991 Stephens and Weidmann, 1989; Karler et al., 1989a Hansen et al., 2004 Georgiev et al., 1995 Lazarova-Bakarova et al., 1990 Georgiev et al., 1995 Georgiev et al., 1995 Becker et al., 1995; daSilva et al., 1998 Stephens and Weidmann, 1989; Gasior et al., 2000 Yue et al., 2002 Hansen et al., 2004 Stephens and Weidmann, 1989
Schmidt, 1990
Schmidt, 1990; Pohle et al., 1997 Schmidt, 1990
(continues)
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TABLE 4 Drug Other Antiepilepsirine s BR16A Cannabidiol Carbamazepine Centella asiatica Cysteamine Dipropylacetate Ethanol Felbamate Histidine/Histamine Linalool Panax ginseng Phenytoin
(Continued)
Development
State
nt nt x x/ne x x x x x x x x ne
x x x ne nt x nt x x x nt nt ne
References
Wang et al., 1993 Kulkarni and George, 1995 Karler et al., 1989a Stephens and Weidmann, 1989; Weiss et al., 1989 Gupta et al., 2003 Assouline et al., 1985; Barkai et al., 1990 Becker et al., 1995 Fischer and Kittner, 1998 Giorgi et al., 1996 Zhang et al., 2002; Chen et al., 2002 Elisabetsky et al., 1999 Gupta et al., 2001 Krug et al., 1998; Stephens and Weidmann, 1989
x, protective effect was observed; ne, no effect; nt, not tested; f, indicates facilitation rather than protection against kindling development. AP7, 2-amino-7-phosphono-heptanoic acid; CFM-2, 1-(4¢-aminophenyl)-3-,5-dihydro-7,8-demethyoxy-4H-2,3-benszodiazepin-4-one; CGP 36742, 3-aminopropyl-n-butyl-phosphinic acid; CGP 56433, [3- -(S)-[3-(cyclohexylmethyl) hydroxyphosphinyl]-2-(S)-hydroxypropyl]-amino]ethyl]benzoic acid; CGP 61334, [3- [3[(diethoxymethyl)hydroxyphosphinyl]-propyl-amino meth yl]-benzoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; g-D-GAMS, g-D-glutamylaminomethylsulphonic acid; THIP, 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol
changes that accompany kindling by either means. Some brain changes are transient, the direct result of an acute seizure event. Others are adaptive or a consequence of a history of seizure activity. Other differences between the brains of naive and kindled animals are those that underlie the pathophysiology of epilepsy. Assessments of receptors, neuromodulators, neuropeptides, neurotransmitters, gene expression, and ion channel function have been performed and the brains of kindled animals contrasted with those of vehicle-injected controls. The contribution of these differences to the development and maintenance of the kindled state has been evaluated. Alterations in expression of genes encoding glutamate and GABAA receptors accompany electrical and chemical kindling. Glutamate and GABAA binding, chloride uptake, and GABAA-receptor expression are modified in various brain regions of rodents kindled by treatment with PTZ, FG 7142, or lidocaine (Abel and Carney, 1993; Becker et al., 1999; Corda et al., 1992; Follesa et al., 1999; Lewin et al., 1989; 1994; Little et al., 1987). Changes in neuropeptide concentrations and gene expression, functional measures of cellular hyperexcitability, and altered neuronal responsivity to glutamate agonists have been reported in cortical and amygdala neurons of chemically kindled animals (Barkai et al., 1994; Neugebauer et al., 2000; Vindrola et al., 1983; 1984).
What are the Functional Consequences of Epilepsy? Temporal lobe epileptics suffer from impairments in cognitive abilities and a predisposition to psychopathology in
the form of clinical depression and anxiety. Deficits in cognition and affect are evident in epileptic patients during seizure-free periods, far removed from the acute ictal event (e.g., Aarts et al., 1984; Flor-Henry, 1991; Gallassi et al., 1988; Hermann et al., 1987; Mikulka and Freeman, 1984). One of the properties that makes kindling an attractive epilepsy model is the opportunity to investigate behavioral sequelae that persist well beyond the ictal episodes and overlap those seen in epileptic patients. Impairments in cognitive performance and increases in anxiety have been demonstrated in animals as a consequence of electrical kindling (e.g., Adamec, 1990; 1999; Adamec and McKay, 1993; Depaulis et al., 1997; Helfer et al., 1996; Kalynchuk et al., 1998a; 1998b; Leung et al., 1990; 1991; Peele and Gilbert, 1992; Robinson, 1992; Rosen et al., 1996). There has been much less use of the chemical kindling paradigm in this regard, and the limited available data has produced mixed results. Deficits in shuttle box avoidance behavior and passive avoidance learning have been reported in rats and mice following PTZ kindling (Becker et al., 1994; 1995; 1999; Pohle et al., 1997; Voigt and Morgenstern, 1990). Daily administrations of subthreshold concentrations of the GABAA receptor antagonist bicuculline directly into the amygdala induced progressive augmentation of physiologic indices of fear (i.e., blood pressure and heart rate) (Sanders et al., 1995). Consistent with the results of Sanders and colleagues (1995), perturbations in defensive and offensive behavior were observed in partially and fully PTZ-kindled rats (Franke and Kittner, 2001). Reductions in exploratory behavior and increased freezing in open field test persisted up to 10 weeks after completion of kindling. However,
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References
Taylor et al. (1988) failed to find changes on a variety of tests of anxiety following kindling with the inverse benzodiazepine receptor agonist FG-7142. Inconsistency in the findings of behavioral impairments resulting from chemical kindling may be the result of the bluntness of the tools to evaluate subtle impairments in cognition and affect in rodents, the lack of a specific focus of kindling site, and variability in implementation of the procedures across laboratories. A crucial parameter dictating the clinical utility of a new antiepileptic drug is the therapeutic index, an estimate of the margin between a dose that is clinically effective in protecting against seizure outbreak and the adverse effects that may accompany that level of seizure protection. Typical side effects of antiepileptic medications include sedation, hypnosis, anxiolytic symptoms, muscle relaxation, amnesia, and anesthesia. Epileptic animals can be more vulnerable to these side effects than healthy naive controls (Klitgaard et al., 2002). Cognitive and behavioral impairments associated with an epileptic condition can also be further exacerbated by antiepileptic drug therapy. As such, preclinical evaluations of drug efficacy should include assessments of side effects in epileptic animals, for which chemical kindling paradigms may be well suited.
SUMMARY Chemical kindling can be induced via central or systemic administration of a number of different agents. Intracerebral administration of the convulsant agent comes closest to mimicking traditional electrical kindling paradigms. Like electrical kindling, chemical kindling is a model of complex partial seizures in patients with epilepsy and shares many features with electrical kindling. As with electrical kindling, chemical kindling is induced by the repeated, spaced administration of subthreshold stimulation, but by a drug rather than an electrical current. Repeated drug administration is accompanied by progressive changes in behavioral and electrographic seizure activity. Once fully kindled, increased seizure susceptibility is maintained, such that subsequent administration of the agent at previously subthreshold levels will result in a fully developed behavioral seizure. Despite these parallels, several key differences between the two kindling models also exist. These differences are more significant when the chemical kindling process is initiated by systemic administration of the agent. The convulsant action of a systemically administered agent depends on its rate of absorption and concentration in the brain. Unlike an electrical stimulus which has a short duration and localized effect, this form of chemical kindling results in the entire brain being exposed to the convulsant agent for a relatively long period of time. This type of chemical kindling more closely models primary generalized epilepsy due to cortical involve-
ment and can result in a different pattern of neuropathology than occurs with electrical kindling. An important advantage of this model is that it is relatively easy to set up if an electrographic index of seizure activity is not required. As a result, it is well suited for testing of the efficacy of new anticonvulsant agents.
Acknowledgment The information in this document has been funded in part by the US Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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32 Kindling, Spontaneous Seizures, and the Consequences of Epilepsy: More Than a Model THOMAS P. SUTULA AND JEFFREY OCKULY
multiple species, including primates, has obvious clinical implications for epilepsy and its consequences. As a chronic model, kindling has had a major influence on understanding the consequences of repeated brief seizures and has contributed to the increasing appreciation that the effects of seizures are not benign. Kindling is a fundamental property of diverse neural circuits in species extending from amphibians to primates. As a robust phenomenon of activitydependent, seizure-induced plasticity, which represents the neurobiology underlying network synchronization, kindling should be regarded as more than a mere model of epilepsy.
The phenomenon of kindling was first recognized in 1968 by Graham Goddard during the course of experiments investigating the effects of repeated hippocampal electrical stimulation on learning in rats. In these experiments, repeated electrical stimulation of a variety of brain pathways, which was anticipated to have minimal long-term effects, unexpectedly evoked gradually evolving seizures and a permanent increase in seizure susceptibility. Goddard described the evolving, progressive, and permanent features of the stimulus-evoked phenomenon as “kindling” and recognized its potential significance for the development of epilepsy (Goddard, 1969; Goddard et al., 1969). Kindling is a robust phenomenon of seizure-induced plasticity that is commonly studied as a chronic model of epilepsy. Induction of epilepsy by kindling is initiated by periodic stimulation that evokes episodic network synchronization accompanied by brief behavioral seizures. Repeated episodes of network synchronization evoked by kindling induce gradually progressive functional and structural alterations in neural circuits that increase seizure susceptibility and eventually result in the emergence of recurring spontaneous seizures, the defining feature of epilepsy. Kindling has been extensively studied as a model of temporal lobe epilepsy, yet in many respects its advantages and distinctiveness compared with other chronic models remain misunderstood. It is now recognized that kindling induces a predictable sequence of evolving molecular and cellular alterations at every level of biological organization in neural circuits, from gene transcription to patterns of neuronal connectivity. Although the slowly evolving progression of kindling to spontaneous seizures has led some to question its relevance as a model for human epilepsy, the permanence of molecular and cellular changes induced by kindling in
Models of Seizures and Epilepsy
KINDLING: A PHENOMENON OF SEIZURE-INDUCED PLASTICITY AND EPILEPTOGENESIS The term kindling refers to the progressive, permanent increase in susceptibility to evoked and spontaneous seizures induced by repeated episodes of network synchronization and is a robust, long-term form of activitydependent plasticity. The process of epilepsy induction by kindling is initiated by periodic application of a brief stimulus that evokes repetitive epileptic spikes (an afterdischarge, or AD) accompanied by a brief behavioral seizure. The physiologic events underlying the AD include network synchronization and repetitive neuronal discharges. With repeated stimulations, the duration of the evoked ADs and behavioral seizures gradually increase, the strength of the stimulus required to evoke network synchronization decreases, and the result is an overall increase in susceptibility to additional seizures (Goddard, 1969; Goddard et al., 1969). The induced increase in susceptibility to additional seizures is permanent,
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and eventually spontaneous seizures emerge as a manifestation of a chronic epileptic state (Michalakis et al., 1998; Pinel and Rovner, 1978; Sayin et al., 1974; Wada et al., 1974, 1975, 1978). Kindling can thus be regarded as a process of gradually evolving seizure-induced plasticity and epileptogenesis that eventually culminates in emergence of spontaneous seizures and establishment of a permanent epileptic state.
KINDLING: A MODEL OF TEMPORAL LOBE EPILEPSY Kindling is most commonly used as a chronic model of temporal lobe or “limbic” epilepsy because it is particularly easy to evoke progressive seizures by stimulation of hippocampal and limbic pathways. In addition, the behavioral features of the evoked seizures resemble partial limbic seizures evolving into secondary generalization, as observed in human temporal lobe epilepsy. Limbic kindling has features that provide attractive opportunities for the study of temporal lobe epilepsy that can be distinguished from chronic models of temporal lobe epilepsy induced by kainic acid or pilocarpine, which are initiated by severe status epilepticus accompanied by pronounced limbic damage and relatively rapid emergence of spontaneous seizures. In contrast, kindling is an incrementally induced, gradually evolving chronic epileptogenic process. Repetition of the initial brief evoked seizures induces subtle but cumulative neuronal loss and a variety of cellular alterations in neural circuits that eventually result in the emergence of spontaneous seizures (Sayin et al., 2003). The distinctive anatomic and temporal differences between chronic models induced by an initial episode of status epilepticus and the gradual induction process of kindling to spontaneous seizures provide opportunities to study rapidly and slowly evolving epileptogenic processes, which are potentially relevant to the diverse syndromes of human temporal lobe epilepsy (Sutula, 2004).
permanently increased seizure susceptibility (Racine, 1972a, 1972b). The most common sites used for induction of kindling by electrical stimulation include the amygdala, perforant path, dorsal hippocampus, olfactory bulb, and perirhinal cortex. The range of electrical stimulation parameters that induce ADs and evolving kindled seizures was empirically characterized by Goddard and colleagues in the original description of kindling (Goddard et al., 1969). High-frequency trains of stimulation (e.g., 30–100 Hz) are generally most effective, but any combination of stimulus frequency and intensity that evokes AD can induce kindling. The typical stimulus train is a 1- to 2-second train of 1-msec bipolar pulses delivered at a frequency range of 50 to 100 Hz. Bipolar pulses are generally used to minimize the potential for tissue damage at the stimulation site as a result of unidirectional current flow. The behavioral seizure manifestations that accompany the evoked ADs follow a predictable progression in rodents and typically are classified from stages I through V according to a scale defined by Racine) (Figure 1). The initial stages I and II are focal or “partial” seizures as defined in the International Classification of Seizures for classification of human seizures (Gastaut, 1970). The class III–V seizures in the Racine classification are partial seizures with secondary generalization. During kindling of rodents, the initial focal seizures (classes I–II) evolve into bilateral clonic movements accompanied by loss of postural tone (the defining feature of class V). As an animal gradually progresses from class I to class V or experiences repeated class V seizures, each evoked seizure reliably recapitulates the sequence of behavioral manifestations of a partial class I seizure evolving into a class V secondary generalized tonicclonic convulsion. As the loss of postural tone that defines a class V seizure is an easily and reliably recognized manifestation, the milestone of a class V seizure has proven to be a reproducible and accurate measure of kindling progression in different laboratories.
ELECTRICAL KINDLING METHODS
SUSCEPTIBILITY TO KINDLING: A GENERAL PROPERTY OF NEURAL PATHWAYS
Kindling is most commonly induced by repeated electrical stimulation, but any experimental method that repeatedly evokes network synchronization and brief ADs in neural circuits induces the progressive functional alterations that define kindling (Cain, 1983; Golarai et al., 1992). This feature indicates that kindling is a form of activitydependent, seizure-induced plasticity, and it should not be regarded merely as a model for epilepsy induction by a specific form of focal stimulation. Repeated brief episodes of network synchronization, manifesting as ADs, are an absolute requirement for kindling progression and induction of
Kindling can be induced by stimulation delivered to pathways in diverse regions of the central nervous system (Goddard et al., 1969). In addition to hippocampal and limbic pathways, other subcortical pathways, neocortex, and brainstem circuitry demonstrate a capacity for kindling. This observation is consistent with the view that neural circuitry is generally susceptible to seizure-induced plasticity. There are notable and reliable differences in the rate at which animals develop class V seizures as a result of repeated stimulus trains that depend on the location of stimulation. Limbic pathways develop class V seizures relatively rapidly com-
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Permanence of Kindling
FIGURE 1 Comparison of the classification of kindled seizures and the International Classification of Seizures. Classes I and II are focal or partial seizures. Class III seizures with unilateral forelimb clonus are partial seizures, but they may be secondary generalized when forelimb clonus is bilateral. The class IV and V seizures in the Racine classification are partial seizures with secondary generalization. During kindling, the initial focal seizures (classes I–II) evolve into bilateral clonic movements accompanied by loss of postural tone (the defining feature of class V). During the progression from class I to class V or during repeated class V seizures, each evoked seizure reliably manifests the behavioral features of a partial class I seizure evolving into a class V secondary generalized tonic-clonic convulsion.
pared with neocortical and other subcortical structures, and extrahippocampal limbic structures such as the amygdala, olfactory bulb, and perirhinal cortex appear to progress to the stage of class V seizures particularly rapidly (Goddard et al., 1969). The number of ADs required to reach the milestone of the first class V seizure varies according to the stimulation site, but the reasons for the site differences are not well understood. It has been suggested that the rate of development of class V seizures depends on access of the stimulated site to motor pathways underlying motor manifestations of the seizures. Characterization of the pathways that undergo progressive cellular alterations during the behavioral progression of kindling from different sites remains a subject of interest for understanding the mechanisms and functional consequences of repeated seizures.
underlying kindling is the repeated occurrence of ADs or episodes of network synchronization. The cumulative consequences of repeated seizures induced by one form of network activation or stimulation are retained or “transferred” when another form of stimulation is sequentially applied (Cain, 1983). “Transfer” occurs between different stimulation locations, and the “transfer” of kindling processes is bidirectional between different modes of activation. The phenomenon of transfer demonstrates that common neurobiological mechanisms underlie the consequences of repeated network synchronization evoked by a variety of methods, supporting the view that kindling is a neurobiological phenomenon of plasticity and should be regarded as more than a chronic model for epileptogenesis.
CHEMICAL KINDLING AND “TRANSFER” EFFECTS
PERMANENCE OF KINDLING
Kindling can also be induced by repeated applications of chemical agents that evoke ADs (Cain, 1983; Golarai et al., 1992). As with electrical stimulation, kindling by repeated chemical application also induces gradually increasing duration of evoked ADs and behavioral seizures, a reduction in the threshold or strength of stimulation required to evoke ADs, and permanently increased seizure susceptibility. The permanently increased susceptibility to epileptic events induced by repeated chemical or pharmacologic stimulation is sometimes referred to as “chemical kindling” and can be induced by a variety of agents with diverse pharmacologic mechanisms of action such as carbacol, acetylcholine, bicuculline, lidocaine, cocaine, and pentylenetetrazol. Because the pharmacologic actions of these agents are clearly distinct, the observation that these diverse agents can induce kindling supports the view that the common mechanism
Goddard’s pioneering experiments revealed that animals experiencing kindled seizures retained increased susceptibility to seizures throughout their lifetime. The neuronal and circuit alterations induced by repeated seizures are long lasting and essentially permanent (Goddard et al., 1969). Animals that achieve the stage of a class V seizure are generally regarded as “kindled,” but this designation is arbitrary because there is a continuing evolution of progressive structural and functional changes in neurons and neural circuits with repeated seizures (Michalakis et al., 1998; Sayin et al., 2003). Once an animal reaches the stage of the first class V seizure, most, but not all, repeated stimulus trains continue to evoke class V seizures. The life-long susceptibility to class V seizures in response to the stimulus trains that initially evoked minimal or mild class I behaviors is a manifestation of the permanent progressive functional reorganization induced by kindling. It is less clear that alter-
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ations induced during earlier stages (classes I–IV) are permanent because some of the alterations induced during the early stages kindling, such as increases in the N-methyld-aspartate (NMDA) component of the evoked synaptic currents, although long lasting (about 3 months), are not permanent (Sayin et al., 1999).
ADVANCED STAGES OF KINDLING: EMERGENCE OF SPONTANEOUS SEIZURES Repeated episodes of network synchronization evoked by kindling induce gradually progressive functional and structural alterations in neural circuits that eventually result in the emergence of recurring spontaneous seizures (Sayin et al., 2003), the defining feature of epilepsy. In this respect, kindling and chronic models that begin with initial status epilepticus (e.g., kainic acid, pilocarpine, and models of initial intense electrical stimulation that evoke status epilepticus) induce the common outcome of spontaneous seizures, albeit with distinctive time courses. Kindling is only infrequently continued to the advanced stage of spontaneous seizures, which are observed in rodents after about 90 to 100 evoked class V seizures (Sayin et al., 2003). In most studies kindling is typically pursued only until a series of a few class V seizures have been evoked. The specific molecular and cellular alterations in hippocampal circuits that are associated with the emergence of spontaneous seizures in various chronic models are of interest; as noted, the variety of chronic models provides opportunities to study temporally distinctive evolving processes of epileptogenesis that are potentially relevant to the diverse syndromes of human temporal lobe epilepsy.
SPECIES SUSCEPTIBILITY Goddard’s initial observations revealed progressive effects of repeated evoked seizures in cats and nonhuman primates in addition to rodents, and subsequent studies have demonstrated that kindling can be induced in a broad range of species extending from amphibians to primates (Cain and Corcoran, 1980; Morrell and Tsuru, 1976; Wada et al., 1974, 1975, 1978; Xiao-Ping et al., 2004). In each of these species, the primary defining features of the kindling phenomenon (i.e., gradual induction of progressive, increasing, and permanent susceptibility to seizures) are observed. Long-term studies have confirmed that the process of kindling in primates eventually results in the emergence of recurring spontaneous seizures, the defining feature of epilepsy (Wada et al., 1975, 1978). Notable differences across species include the observation that progression to spontaneous seizures is typically longer in primates than other mammals (Wada et
al., 1975, 1978), which some have suggested argues against potential relevance of kindling in human epilepsy. Although this may be a relevant consideration for some cases of human epilepsy, many patients with epilepsy experience hundreds of seizures, and in any case the slowly progressive cumulative effects of repeated network synchronization may contribute to consequences of poorly controlled seizures. More generally, the susceptibility of neurons and neural circuits across a broad range of species to the progressively evolving effects of repeated network synchronization supports the view that kindling is not merely a chronic model of epilepsy but rather is a potentially relevant neurobiological phenomenon for understanding the range of consequences that can result from poorly controlled seizures.
EFFECTS OF TIMING OF REPEATED AFTERDISCHARGES: INTERSTIMULUS INTERVALS AND “MASSED” STIMULATION EFFECTS Repeated episodes of network synchronization during evoked ADs are an absolute requirement for the induction of permanent long-term increases in seizure susceptibility by kindling. Goddard observed that progressive kindling in response to periodic stimulation that evoked AD was reduced when stimulus trains were applied with interstimulus train intervals shorter than once daily, an observation that he referred to as a massed stimulation effect. An interstimulus train interval of 24 hours or longer appeared to be optimal for inducing gradually progressive effects. Progressive kindling effects were diminished when interstimulus intervals were shortened. This “massed” stimulation effect can be partially overcome by increasing the duration of stimulus trains; seizure susceptibility can be permanently increased when longer stimulus trains are delivered at shorter intervals (e.g., 10-second trains at 30-minute intervals for 6 hours), which has been referred to as rapid kindling (Lothman and Williamson, 1994), but there are differences in decay rate and permanence of the induced effects. At the extreme of very short interstimulus train intervals (e.g., 10-second trains of 20-Hz pulses delivered once per minute for 24 hours) (Sloviter, 1983, 1987), the initial evoked events have features of status epilepticus rather than brief seizures and, not surprisingly, have different long-term effects: The outcome in terms of neuronal loss, other cellular circuit alterations, and functional susceptibility to additional seizures and their consequences vary in these models (Nairismagi et al., 2004; Pitkanen et al., 2002). The neurobiology underlying these systematic, reproducible differences in acute and long-term effects of network synchronization as a function of intersynchronization interval has not been systematically characterized.
Progressive Functional and Structural Alterations Induced by Kindling
EFFECTS OF DEVELOPMENT AND AGING ON KINDLING Kindling is most commonly induced in adult rats, but the phenomenon of kindling is also observed during postnatal development. Long-term increases in seizure susceptibility can be induced by repeated evoked seizures in preweanling rat pups, but prominent age-dependent differences are apparent in the induction methods and in the long-term consequences of repeated seizures during early postnatal development (Haas et al., 2001; Kubova et al., 2004; Lynch et al., 2000; Moshe, 1981a, 1981b; Moshe and Albala, 1982; Trommer et al., 1994). For example, the massed stimulation effect, as observed with short interstimulus intervals in adult rats, is not apparent in rat pups, which develop permanent increases in seizure susceptibility in response to stimulus trains delivered at interstimulus train intervals as short as 15 minutes (Moshe and Albala, 1982). Other studies in juvenile rats demonstrate that there are distinctive age-dependent differences in kindling-induction processes and outcomes (Michelson and Lothman, 1991). These observations indicate that kindling should not be regarded as a single unitary process but rather more generally as the neurobiological processes of plasticity accompanying repeated network synchronization, and these processes have age-dependent differences. Differences in kindling-induction processes that have been at least preliminarily characterized in preweanling and juvenile rat pups (postnatal days PN 10–30) and more extensively in adult rats (generally 90 or more days of age) have not been systematically examined in age groups from 30 to 90 days. Difficulties in pursuing systematic kindling studies in developing rat pups include problems of maintaining chronic implanted electrodes and head stages during periods of skull growth. Whereas these limitations in postnatal rat pups have precluded conventional application of stimulation once or twice daily for long periods, as in adults, studies that have systematically induced repeated seizures during early life and examined the long-term consequences in adulthood have detected robust age-dependent differences (Kubova et al., 2004; Lynch et al., 2000). In contrast to the effects of repeated seizures and status epilepticus in adult and preweanling rats, which induce progressive increases in seizure susceptibility, both status epilepticus and a few brief febrile seizures during the early postnatal period induce long-term decreases in seizure susceptibility in adulthood, accompanied by reduction in capacity for long-term potentiation (LTP), enhanced inhibition, and memory disturbances (Lynch et al., 2000; Sayin and Sutula, 2002). Comparatively less study has been done of how aging and senescence might affect kindling and seizure-induced plasticity (Chiba et al., 1994; Grecksch et al., 1997). In a study that investigated pentylenetetrazol-evoked kindling in 24-
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month-old senescent rats, diminished seizure expression and kindling capacity were observed in aging rats, but impairments in cognitive functions induced by kindling appeared more severe as a function of age (Grecksch et al., 1997). It thus appears from studies of kindling across the lifespan that the progressive phenomenon of kindling in adulthood is only one example of a range of systematic age-dependent consequences of repeated network synchronization and seizures.
PROGRESSIVE FUNCTIONAL AND STRUCTURAL ALTERATIONS INDUCED BY KINDLING Goddard speculated in 1969 that kindling might induce structural alterations “within or between neurons.” It is now recognized that kindling induces a predictable sequence of evolving molecular and cellular alterations at every level of biological organization in neural circuits, from gene transcription to patterns of neuronal connectivity. The initial seizures evoked by kindling in adults induce alterations in synaptic transmission and activation of signal transduction pathways, which are followed by a sequence of gene expression that eventually results in morphologic reorganization of neurons and neural circuits accompanied by functional and behavioral changes. It should be emphasized that the alterations described in the following section may show pronounced age dependence.
Single and Repeated Seizures to Alter Synaptic Transmission and Induce Gene Expression The brief episodes of network synchronization during seizures evoked by kindling induce alterations in both excitatory and inhibitory synaptic transmission. These seizureinduced alterations in synaptic transmission have been particularly well described in granule cells of the dentate gyrus, where a single episode of network synchronization accompanying an AD increases both excitatory and inhibitory synaptic transmission. An initial seizure evoked by kindling increases the NMDA receptor-dependent component of excitatory synaptic transmission in granule cells of the dentate gyrus (Mody and Heinemann, 1987; Mody et al., 1988). This seizureinduced increase in NMDA-dependent synaptic transmission increases seizure susceptibility as a result of the voltage-dependent characteristics of NMDA receptors, which contribute only minimally to synaptic transmission at membrane potentials in the range of -60 to -75 mV because of voltage-dependent block of the channel by Mg2+. During seizures or ADs, when the membrane potential depolarizes
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to -30 to -40 mV and the Mg2+ block is relieved, the receptor becomes permeable to both Na+ and Ca2+, which results in a slowly developing, long-duration synaptic current and repetitive spike discharges that propagate into CA3. The effect of the seizure-induced activation of NMDA receptors on spiking by granule cells of the dentate gyrus is noteworthy because granule cells normally fire only single action potentials, even when inhibition is reduced or blocked (Lynch et al., 2000), which contributes to “filtering” properties of the dentate gyrus (Behr et al., 1998; Lynch et al., 2000). By increasing the NMDA-dependent synaptic transmission, single or repeated kindled seizures increase propagation of spike discharges into CA3, thereby reducing the filtering of spike input into the hippocampus by the dentate gyrus. The effects of seizure-induced increases in NMDAdependent currents on granule cell spike generation and propagation of inputs into CA3 are relatively long lasting (about 3 months), but they are not permanent and diminish by 3 months after the last seizure (Sayin et al., 1999). These observations support the view that seizure-induced increases in NMDA-dependent synaptic transmission are epileptogenic and furthermore imply that seizure control for periods of 3 months could have beneficial effects on seizure susceptibility in pathways of the dentate gyrus and hippocampus. The initial seizures evoked by kindling also induce a sustained increase in GABAA-dependent inhibition in the dentate gyrus, as demonstrated by a variety of physiologic measurements (Buhl et al., 1996; Nusser et al., 1998; Oliver and Miller, 1985; Otis et al., 1994; Stringer and Lothman, 1989; Tuff et al., 1983). The increase in inhibition evoked by seizures during the early stages of kindling appears paradoxical given the gradually increasing susceptibility to additional seizures induced by kindling. In advanced stages of kindling, inhibition in the dentate gyrus is reduced in association with the emergence of spontaneous seizures (Sayin et al., 2003). It should be noted that alterations in synaptic transmission induced by kindling vary depending on location, as demonstrated by reduction of inhibition observed in CA1 during early stages of kindling (Michelson et al., 1989; Rempe et al., 1995; Titulaer et al., 1995). The NMDA receptor–dependent increase in Ca+2 permeability induced by kindled seizures increases intracellular Ca+2 and activates other second messengers and signaltransduction pathways. An initial episode of neural synchronization exceeding about 30 seconds in the hippocampus induces alterations in transcription of early immediate genes (Shin et al., 1990) and initiates a cascade of gene transcription contributing to long-term, potentially permanent neuronal and circuit alterations (Hughes et al., 1998). Antagonism of the NMDA receptor significantly blocks the progression of kindling and seizure-induced morphologic reorganization, suggesting that NMDA-dependent gene expression is important in the sequence of events leading to long-term alterations
induced by repeated seizures (Sutula et al., 1996). Evidence shows that the neurotrophins, including BDNF, NT3, NT4, and the their tyrosine kinase receptors in the trkA,B family, are among the genes that may be contributing to long-term functional effects of kindling (Adams et al., 1997; Elmer et al., 1997; Kokaia et al., 1995; Osehobo et al., 1999; Rashid et al., 1995; Van der Zee et al., 1995; Xiao-Ping et al., 2004).
Apoptosis and Neuron Loss Induced by Repeated Kindled Seizures Kindling induces progressive neuronal loss, which has been detected by a variety of techniques, including TUNEL staining for apoptotic neurons, silver impregnation for degenerating cells, and stereologic methods for counting neuronal numbers. Both single and repeated seizures evoked by kindling induce apoptotic neuronal death in the hilus of the dentate gyrus (Bengzon et al., 1997; Zhang et al., 1998); and, after repeated seizures, apoptosis is observed in CA1, the subiculum, and the neocortex (Pretel et al., 1997). With repeated seizures, cumulative neuronal loss can eventually be detected by stereologic methods in a variety of locations, presumably as a consequence of repeated seizure-induced apoptosis (Cavazos and Sutula, 1990; Cavazos et al., 1994; Dalby et al., 1998; Haas et al., 2001; Kotloski et al., 2002). The cumulative neuronal loss has been observed in the hilus of the dentate gyrus and in the hippocampal subfields CA1 and CA3 in a pattern resembling classic human hippocampal sclerosis (Cavazos et al., 1994; Kotloski et al., 2002). The distribution of neuronal loss induced by kindling depends on the site of stimulation (Kotloski et al., 2002).
Neurogenesis Induced by Repeated Kindled Seizures Repeated seizures evoked by kindling also induce cell proliferation in the dentate gyrus (Bengzon et al., 1997; Nakagawa et al., 2000; Parent et al., 1998), as indicated by labeling with bromodeoxyuridine (BrdU), a marker for recent DNA replication. Most newly born cells differentiate into neurons (Bengzon et al., 1997; Parent et al., 1998), but some also differentiate into glia. Neurogenesis has been detected after only partial seizures evoked by kindling (Bengzon et al., 1997; Nakagawa et al., 2000). Pilocarpineinduced status epilepticus also induces neurogenesis, and the newly born neurons in this chronic model appear to integrate into local networks and are activated by recurring seizures (Scharfman et al., 2002).
Axon Sprouting Induced by Repeated Kindled Seizures The recognition of mossy fiber sprouting in the dentate gyrus of kindled rats in 1988 demonstrated that even brief
Kindling-Induced Cumulative Neuronal Loss and Memory Deficits
seizures induce long-lasting structural reorganization in neural circuits (Sutula et al., 1988) and was a milestone for epilepsy research. Mossy-fiber sprouting is also induced in association with hippocampal damage induced by status epilepticus in numerous chronic models of epilepsy and in the human epileptic temporal lobe (Cronin and Dudek, 1988; Houser et al., 1990; Nadler et al., 1980; Sutula et al., 1989). Sprouting of mossy-fiber axons is easily detected by Timm histochemistry, which labels synaptic terminals of mossy fibers because of their high zinc content. The Timm method demonstrated that mossy-fiber sprouting develops after only a few brief seizures evoked by kindling, progresses with repeated seizures, and is permanent (Cavazos et al., 1991; Sutula et al., 1988). The sprouted mossy-fiber axons extend their terminal field into the inner molecular layer of the dentate gyrus (Cavazos et al., 1991; Sutula et al., 1988) and into the infrapyramidal zone in CA3 (Represa and Ben-Ari, 1992; Represa et al., 1989). Detailed anatomic studies using track tracing methods and examination of individual axon arbors have revealed that sprouted mossy-fiber axons also undergo substantial reorganization within the hilus of the dentate gyrus and along the dorsal-ventral hippocampal axis (Sutula et al., 1998). The sprouted axon terminals induced in the dentate gyrus by kindling form asymmetric spinous synapses consistent with recurrent excitatory circuits (Cavazos et al., 2003), and more than 98% of the synapses formed by sprouted mossy-fiber axons in pilocarpine-treated rats are with other granule cells (Buckmaster et al., 2002). Seizures induced by kindling and other methods also induce sprouting in CA1 (Cavazos et al., 2004; Esclapez et al., 1999; Smith and Dudek, 2002). The observation that sprouting is progressively induced by repeated brief kindled seizures indicates that even brief seizures are sufficient to induce remodeling of neural circuits and suggest that poorly controlled seizures in human epilepsy may induce progressive sprouting and synaptic reorganization. The functional effects of the recurrent excitatory circuits formed by mossy-fiber sprouting are subtle in normal physiologic conditions, but short-latency connections consistent with monosynaptic transmission between blades of the dentate gyrus in rats treated with kainic acid (Lynch and Sutula, 2000), and paired recordings of granule cells have revealed formation of functional recurrent excitatory circuits between individual granule cells in rats treated with pilocarpine (Scharfman et al., 2003). Robust evidence for recurrent excitation reliably emerges in the dentate gyrus of kindled rats or rats with mossy-fiber sprouting induced by kainic acid or pilocarpine when inhibition is reduced or in conditions where the extracellular environment is altered (Cronin et al., 1992; Hardison et al., 2000; Lynch and Sutula, 2000; Patrylo and Dudek, 1998; Wuarin and Dudek, 1996). The functional effects of mossy-fiber sprouting and seizureinduced recurrent excitatory circuits are conditionally
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expressed or unmasked during episodic synchronization when inhibition is reduced or the extracellular environment is altered by activity-dependent increases in [K+]o. These observations suggest that recurrent excitation may be unmasked in reorganized circuits with progressive mossyfiber sprouting by activity-dependent increases in [K+]o accompanying network synchronization evoked by kindling stimulation, and could contribute to the progressive features of kindling.
Glial and Astrocytic Proliferation Induced by Repeated Seizures Astrocytes and glia undergo proliferation in response to repeated seizures evoked by kindling. Glial fibrillary acidic protein (GFAP) mRNA and protein levels are increased by kindled seizures, which cause glial cell hypertrophy and proliferation (Hansen et al., 1990; Khurgel and Ivy, 1996) and microglial activation (Szyndler et al., 2002). The significance and functional effects of glial alterations induced by kindling have not been extensively investigated but could contribute to progressive aspects of kindling. For example, seizure-induced alterations such as impaired [K+]o buffering could contribute to activity-dependent changes in the extracellular environment and unmask recurrent excitation in excitatory circuits formed by mossy fiber sprouting.
KINDLING-INDUCED CUMULATIVE NEURONAL LOSS AND MEMORY DEFICITS The apoptosis and cumulative neuronal loss induced in the hilus of the dentate gyrus, CA1, and CA3 by kindling progressively increases as a function of the number of seizures. Damage to hippocampal circuitry has been implicated in memory dysfunction by human and experimental studies, so it is not surprising that kindled seizures, which induce hippocampal damage, also induce memory deficits (Sutula et al., 1995). Memory deficits in kindled rats have been observed in a variety of behavioral tasks, including the radial arm maze and Morris water maze (Gilbert et al., 2000; Hannesson et al., 2001a, 2001b; Huang et al., 2002), and can clearly be dissociated from acute effects of recurring seizures; these deficits appear to be long lasting, probably permanent (Kotloski et al., 2002; Sutula et al., 1995). In the radial arm maze, errors indicating memory dysfunction become apparent after about 30 evoked class V seizures, increase as function of the number of previous seizures, and are associated with the progressive neuronal loss in the hippocampus that resembles hippocampal sclerosis (Kotloski et al., 2002; Sutula et al., 1995). Because kindling induces pathway- and region-specific patterns of neuronal loss that depend on the number of evoked seizures, kindling is poten-
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tially useful as a model for investigation of the behavioral consequences of relatively subtle but pathway-specific lesions of the hippocampus and limbic regions.
KINDLING-INDUCED INTERNEURON LOSS AND EMERGENCE OF SPONTANEOUS SEIZURES Kindling induces spontaneous seizures after about 90 to 100 evoked class V seizures, and seizure-induced cellular alterations accompanying this emergent property, which defines epilepsy, have been characterized (Sayin et al., 2003). The apoptosis and progressive cumulative neuronal loss that are consequences of repeated evoked kindled seizures eventually reduce GABAergic interneurons in the dentate gyrus. Significant reduction in GABAergic interneurons becomes detectable after about 90 to 100 evoked class V seizures, in association with the emergence of spontaneous seizures, and includes reduction of specific subclasses of interneurons labeled by cholecystokinin (CCK) and the neuronal GABA transporter GAT-1 (Sayin et al., 2003). These subclasses project GABAergic axon terminals onto the cell bodies and axon initial segments and thereby contribute to axosomatic and axoaxonic inhibition regulating the propagation of action potentials into the mossy fiber axons of granule cells and CA3. Other interneuron subclasses projecting axodendritic terminals in the dentate gyrus are relatively spared. Loss of these subclasses of inhibitory interneurons and GABAergic synaptic terminals is accompanied by a reduction in inhibition as measured by reduced amplitude and duration of evoked inhibitory postsynaptic currents and loss of paired pulse inhibition, which presumably contribute to the emergence of spontaneous seizures (Sayin et al., 2003). The pattern of subclass specific interneuron loss observed in the dentate gyrus of kindled rats experiencing spontaneous seizures resembles neuropathological alterations that have been observed in resected human epileptic hippocampus, where loss of interneurons labeled by CCK, parvalbumin, and GAT-1 has been observed (Zhu et al., 1997).
THE SEQUENCE OF CELLULAR AND FUNCTIONAL ALTERATIONS INDUCED BY REPEATED KINDLED SEIZURES The sequence of seizure-induced alterations in the hippocampus and dentate gyrus evoked by limbic kindling are summarized in Figure 2. These seizure-induced alterations have been described in considerable detail in hippocampal circuits undergoing progressive modification during limbic kindling, but they have not been extensively studied in other regions. With the susceptibility of neocortical, subcortical, and brainstem pathways to kindling, however, and the recog-
FIGURE 2 The sequence of structural and functional alterations induced by repeated kindled seizures in adult rats beginning with the initial evoked seizure and progressing to the stage of spontaneous seizures, which are observed after about 90 to 100 evoked seizures. Because epilepsy is defined as recurring spontaneous seizures, the period of gradually evolving cellular alterations preceding the milestone of 90 to 100 evoked class V seizures can be regarded as a process of seizure-induced epileptogenesis, and the period after 90 to 100 class V seizures represents fully developed epilepsy.
nition that activity-dependent plasticity is a general property of neural circuits, it seems probable that similar processes of seizure-induced structural and functional remodeling are likely to be occurring in pathways throughout the nervous system. The sequences of gene expression that underlie the progression of kindling have not been characterized, but evidence from strains of “fast-kindling” and “slowkindling” rats indicates that genetic background exerts a profound influence on susceptibility to kindling (Racine et al., 1999; Racine et al., 2003).
KINDLING AS AN EXPERIMENTAL MODEL OF EPILEPSY: COMPARISON TO OTHER MODELS For nearly 40 years, kindling has been used as an experimental model of epilepsy; yet in many respects its advantages and distinctiveness compared with other models remain relatively misunderstood, and some of its features have been critically regarded as limitations.
Limitations of Kindling as an Experimental Model The limitations of kindling as a model, primarily the protracted time course for induction of spontaneous seizures and the uncertainty about the relevance of its slowly developing time course to human epilepsy, have sometimes been regarded as potential disadvantages compared with chronic experimental models that begin with status epilepticus and evolve more rapidly into spontaneous seizures. Other prac-
Kindling as a Form of Activity-Dependent Plasticity: Implications for Epilepsy and Its Consequences
tical disadvantages of kindling include laborious handling and stimulation procedures, the potential for loss of chronic head plugs and electrodes, and the costs associated with the protracted time course for induction. These practical limitations contribute to the relatively infrequent use of kindling to the advanced stage of spontaneous seizures. The requirement for chronic implanted electrodes is a significant limitation in developing animals because of skull fragility and growth, and is a major reason why kindling is usually pursued in adult rats rather than in rat pups or in mice. The difficulty of pursuing kindling in mice is a potential disadvantage given the contemporary opportunities for study of epileptogenesis in strains of transgenic mice (Xiao-Ping et al., 2004).
Advantages of Kindling as an Experimental Model Although the requirement for chronic implanted electrodes and the prolonged time course are practical challenges for the use of kindling as an experimental model, many other features of kindling offer particular advantages for the study of epileptogenesis. Compared with models with initial status epilepticus, the induction process of epileptogenesis by kindling is easily controlled, reliably measured, and has minimal mortality. These features of kindling allow seizure number to be used as a variable in experimental design, a potentially advantageous feature that has been exploited for examining seizures as a cause of molecular and cellular alterations and for assessment of the consequences of seizures on neurons, neural circuits, and higher-order functions. The ability to measure accurately and reproducibly the rate of kindling has been particularly useful for assessing how experimental manipulations (e.g., lesions or drugs) might modify the rate of development of epileptic susceptibility (epileptogenesis) (Sutula et al., 1996). In the kindling paradigm, susceptibility to epileptogenesis can be measured as the number of repeated seizures required to evoke a class V seizure or to reach more advanced milestones, such as 30, 50, 75, or 100 or more evoked seizures. Measuring the effects of a lesion or other manipulations on the progression of epileptogenesis is considerably more problematic in models induced by initial severe status epilepticus and injury processes (e.g., kainic acid or pilocarpine) that are difficult to control or manipulations that cause lesions, such as cortical undercutting or alumina application. In these chronic models, both the initial induction process and the lesion may be variable, and outcome measures of epileptogenesis, such as latency to onset of seizures, seizure duration and severity, are also subject to variability and difficulties of accurate measurement. In contrast, the gradual development and progression of epileptogenesis by kindling can be reliably quantified by
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both electrographic measures (AD number and duration) and behavioral measures (seizure stages and number of class V seizures). The sequence of progression of evoked behavioral seizures from class I to class V is associated with increasing duration of evoked ADs, which can be accurately measured in the region of the stimulating-recording electrode. The AD duration is a direct measure of network synchronization, and thus the cumulative AD duration provides a direct measure of the total duration of epileptic activity experienced by an animal and can be easily and accurately determined. This feature of kindling is potentially valuable in that the number and total duration of seizures (cumulative network synchronization) can be evaluated as a variable contributing to other outcome measures, such as memory loss or circuit reorganization. Experimental design incorporating numbers and duration of seizures is more difficult in status models in which accurate quantification of duration and recurrence of initial seizures is problematic because of the continuous electrographic abnormalities and the sometimes subtle electrographic and behavioral manifestations of ongoing status epilepticus.
KINDLING AS A FORM OF ACTIVITYDEPENDENT PLASTICITY: IMPLICATIONS FOR EPILEPSY AND ITS CONSEQUENCES Although Goddard emphasized that kindling was potentially important for studying the development of epilepsy, the permanence of kindling was initially regarded as an example of information storage in neural pathways that might be potentially relevant to mechanisms of learning and memory formation. With the recognition that kindling induces memory disturbances and cognitive dysfunction in a variety of behavioral tasks, it is now clear that kindling is not a model of memory, even if kindling and other forms of activity-dependent synaptic plasticity, such as long-term potentiation (LTP) are processes of information storage in the neural circuitry that may share some common molecular and cellular mechanisms. In contrast to the relatively short-term modifications in function induced by LTP-LTD, the permanence of the seizure-induced alterations induced by kindling is a robust example of long-term, activity-dependent plasticity and is a fundamental and distinctive property of neurons and neural circuits. Goddard’s observation of the permanence of activity-dependent phenomenon of kindling and the subsequent recognition of other forms of synaptic plasticity (LTP and LTD), lesion-induced plasticity, and cortical map plasticity contributed to the contemporary view that neurons and neural circuits are capable of robust short-term and permanent structural and functional modification across every level of biological organization, from genes and molecules to neurons, networks, and behaviors.
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As a model for the induction of epileptogenesis, kindling is a laborious and slowly developing process, but nevertheless it has distinctive advantages in that progression to epileptogenesis and spontaneous seizures are more easily and reliably measured than in rapidly developing chronic models. Although some have argued that the protracted time course of kindling is of limited relevance for human epilepsy, the permanence of changes induced by repeated network synchronization evoked by kindling in multiple species has obvious clinical implications for epilepsy and its consequences. As a chronic model, kindling has had a major influence on understanding the consequences of repeated seizures, and it has contributed to the increasing appreciation that the effects of seizures are not benign (Sutula, 2004; Sutula and Pitkanen, 2002; Sutula et al., 2003). As a robust phenomenon of activity-dependent, seizure-induced plasticity observed in diverse pathways of the nervous system in species ranging from amphibians to primates, kindling is a fundamental property of neural circuits. It represents the neurobiology underlying network synchronization and should be regarded as more than a mere model of epilepsy.
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33 Tetanus Toxin Model of Focal Epilepsy JOHN G.R. JEFFERYS AND MATTHEW C. WALKER
usually are provoked by hypoxia, suggesting that the toxin is restricted to spinal cord and brainstem.
GENERAL DESCRIPTION OF MODEL Tetanus toxin is one of the oldest methods of inducing chronic experimental epilepsies. It was first described by Roux and Borrell (1898). When injected into the brain, the toxin binds avidly to neural tissue, resulting in a long-term epileptic state with properties determined by the location of injection. In its modern form, the model involves the stereotaxic injection of a minute dose of toxin into the brain, where it establishes an epileptic focus which discharges spontaneously over a long period. The toxin is cleared from the brain within a few days (Mellanby, 1989), but its epileptogenic effect persists much longer, in many cases resulting in spontaneous seizures recurring for the rest of the life of the animal. Even after intrahippocampal injection in the adult, where seizure remission usually occurs after a few weeks, brain function remains permanently disrupted, leading, for instance, to impairments of learning and memory. The toxin is secreted by the bacterium Clostridium tetani. It is one of the most potent toxins found in nature: Less than a mg is sufficient to kill a human. In natural pathogenesis, bacteria enter through a wound, the toxin is taken up by peripheral nerves, and it enters the central nervous system (CNS) via retrograde axonal transport along motor nerves to the spinal cord. In the CNS it crosses synapses to inhibitory interneurons in the spinal cord and brainstem, where it cleaves synaptobrevin (Schiavo et al., 1992a), a protein that is involved in, and probably essential for, transmitter release. This leads to the spastic paralysis of “lockjaw” reflex spasms and autonomic instability; these often prove fatal (Farrar et al., 2000). It is not known whether the toxin enters the cortex in tetanus, but cortical seizures are rare in tetanus and
Models of Seizures and Epilepsy
WHAT CLASSES OF EPILEPSY DOES INTRACRANIAL TETANUS TOXIN MODEL? When injected directly into the brain, tetanus toxin produces epileptic foci centered on the injection site. It therefore models partial/focal epilepsies, with the specific subtype determined by the location at which the toxin is injected. The two main models we describe here use injections either into the hippocampus, which models complex partial seizures (temporal lobe or limbic epilepsy), or into the neocortex, which models focal neocortical epilepsy, secondary generalized seizures, and epilepsia partialis continua. Other sites can be injected, with behavioral consequences related to the normal function of the region (Kryzhanovsky et al., 1976; Mellanby et al., 1999b).
METHOD OF GENERATION Animal Issues (Species, Strain, Age Specificity, Gender) The toxin will induce epileptic activity in any vertebrate species. Roux and Borrell (1898) described seizures in rats, mice, guinea pigs, and rabbits. Subsequently it has been used in larger species, including dogs (Carrea et al., 1962) and cats (Brooks et al., 1962; Darcey et al., 1987; Louis et al., 1990). However, we restrict our discussion to the rat because it is by far the most common species currently used. Strain
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and sex do not seem to have much impact on the induction of epileptic foci, but age does. Injections made into the rat hippocampus at postnatal day (PN)10 produce a very different time course from those made at or later than PN20 (see later discussion).
Experimental Procedures The toxin’s considerable toxicity has several practical consequences. Perhaps most important is the need for stringent safety controls, including vaccination of personnel, and possibly measurement of antibody titers. Tetanus toxoid vaccination is straightforward and is part of vaccination programs within the general population. To be adequately vaccinated, boosters are usually required at least every 10 years. Care needs to be taken in the disposal of contaminated items, especially sharps; fortunately, autoclaving and chemical treatments are very effective. The toxin is relatively fragile: the chains can separate if solutions are agitated too vigorously, and so should be handled relatively gently. Like many proteins, tetanus toxin binds avidly to many surfaces. Using an inert protein such as bovine serum antigen (BSA) or gelatin at a low concentration (0.2%) in the solution can help reduce the loss of toxicity. Because tetanus toxin can be destroyed by bacterial contamination, aseptic technique is appropriate. It is heat labile, so refrigeration is essential. Freezing and thawing of solutions cause loss of toxicity, but in practice a single cycle of freezing and thawing has a relatively small effect; so aliquoting a stock solution into amounts suitable for one day’s injections, freezing, and then thawing them as required works well. Aliquots would typically contain much less than a human lethal dose, which has advantages from the safety point of view. In most studies the toxin is injected stereotaxically into the brains of animals under general anesthesia. The volume should be kept small, to 1 ml or less, and the injection needle should be left in place for several minutes, to minimize losses of toxin through the needle track. Typical injection sites for the neonatal intrahippocampal model are 2.4 mm caudal and 2.9 mm lateral from bregma and 2.95 mm below cortical surface (Lee et al., 2001). For the adult version, the injections can be made either into a ventral site 3.0 mm anterior to the interaural axis, 4.8 mm lateral, and 2.0 mm above interaural on the De Groot atlas (Hawkins et al., 1987; Mellanby et al., 1977), or into a dorsal site 2.8 mm caudal to bregma, 3.5 mm lateral, and 3.0 to 3.5 mm below cortical surface on the Pellegrino, Pellegrino, and Cushman atlas (Jeffreys, 1989; Jefferys et al., 1987). The ventral site would be about 6 mm caudal to the bregma and 7 mm below cortical surface on the latter coordinate system. The extent, monitoring, and reliability of the neocortical seizure model depend on the site of injection; we have found
that injection into the motor cortex (1.1 mm ventral, 2.5 mm lateral from the bregma) achieves the most reliable and reproducible seizure model in rats. Although some studies have favored repeat dosing at weekly intervals (Louis et al., 1990), we have found that single injection of 25 to 50 ng in 0.5 ml has a reliable outcome (Nilsen et al., 2005); this single dose is, however, greater than the total dose reported to have been used in cats (Louis et al., 1990). The difference in dose could be related to differences in species, in toxin toxicity, or toxin application (epidural in cats, intracortical in rats). Alternatively, the work in cats could involve a kindling-type phenomenon with repeated doses. In our experience special care with toxin placement and toxin batch effects needs to be taken when setting up the model.
Monitoring The neonatal and adult intrahippocampal models result in secondarily generalized seizures, which can easily be recognized visually or by time-lapse video. These motor seizures underestimate the number of electrographic seizures by a substantial margin, particularly in the early stages of the model (Hawkins et al., 1987); electrical recording is therefore the most reliable approach to quantify the spontaneous epileptic activity (Finnerty et al., 2000, 2001, 2002). Monitoring of neocortical seizures can be difficult because the clinical manifestation can be difficult to detect. This may be more so with injection into the sensory cortex, in which clinical manifestations can be undetectable (e.g., sensory auras). Thus we have found that injection into the motor cortex gives us a reliable and easily monitored seizure manifestation. Monitoring of these neocortical seizures is best achieved through intracortical electroencephalography (EEG) with simultaneous electromyography (EMG) to monitor the clinical seizure manifestation. Using the preceding coordinates, the seizure manifestation consists of facial twitching, which can be monitored by using an EMG electrode placed subcutaneously over the facial musculature (although ideally placed contralateral to toxin injection, ipsilateral EMG placement is adequate to detect seizure activity).
Ease of Development and Reliability The model is very reliable. The main risk comes from poor batches of toxin and from damage to the toxin during preparation, storage, or use. Both the hippocampal and neocortical models depend on accurate stereotaxic placement of the toxin. We have found that when our protocols are used all animals exhibit seizures. With neocortical injection, dose is critical because a high dose will result in rapid onset of secondary generalized seizures, whereas too low a dose results in no recordable seizures (Brooks et al., 1962). With
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hippocampal injection, dose also is important, but there is a reasonable safety margin. Too low a dose fails to induce seizures, whereas doses two orders of magnitude greater than those typically used (Jefferys, 1989; Jeffreys et al., 1987; Mellanby et al., 1977) can cause substantial lesions and high mortality (Bagetta et al., 1990).
Spontaneous Seizures After intrahippocampal injections, generalized seizures certainly occur spontaneously. They occur up to 30 times per day (Jefferys et al., 1995, 1998), and typically last less than 2 minutes. They usually start with behavioral arrest and perhaps vibrissal twitching, followed by forelimb myoclonus and ultimately rearing and falling, rather like a stage 5 kindled seizure. After the seizure ends, there is often a period of quiescence and then grooming or exploration. Intracranial monitoring reveals partial seizures that are always bilateral when induced in dorsal hippocampus, with neither side leading consistently. Frequencies of bursts within each seizure start relatively fast and then slow progressively during the seizure, down to about 16 Hz, which usually is the prelude to secondary generalization (Finnerty et al., 2000, 2002). The generalized motor component is often a relatively short fraction of the total duration of the electrographic seizure. Intrahippocampal injection of tetanus toxin at PN10 triggers a very different sequence of events (Benke et al., 2004; Lee et al., 1995). During the first week, seizures occur frequently, typically about 1 per hour (but up to 7 per hour), varying in duration from a few seconds to several minutes and often associated with wet-dog shakes and wild running. Seizure frequency usually peaks near day 2 and then declines over the week following injection. After the first week the generalized seizures remit, but interictal spiking continues. When these rats become adults, they can exhibit unprovoked seizures, but the incidence appears to be low (Lee et al., 1995, 2001). The seizures observed in the intracortical model depend on site of injection and dose. Injections into sensory cortex produce a model with frequent interictal spikes, variable motor seizures, and occasional generalized seizures occurring a few days after injection and continuing for at least 7 months (Brener et al., 1991). Injection into the motor cortex produces a model in which focal motor seizures, epilepsia partialis continua, and occasional secondary generalized seizures can occur (Louis et al., 1990; Nilsen et al., 2005). The occurrence and delay to onset of seizures depend on dose, but using our protocol we reliably had consistent seizures from 2 weeks to at least 5 months consisting of focal seizures with electroclinical features typical of epilepsia partialis continua, including discrete focal motor seizures.
Remission In the version of the model induced by intrahippocampal injection in adult, most (~90%) rats do gain remission after 6 to 8 weeks. The remaining approximately 10% continue to have seizures indefinitely. However, none of the rats returns to normal. They all have enduring impairments of learning and memory (George et al., 1982; Jefferys et al., 1987; Mellanby et al., 1999a) and a loss of the aversion that rats normally have to novel objects (Mellanby et al., 1977, 1999a). They also retain enduring changes in neuronal structure and function, including reduced synaptic responses and changes in several intrinsic neuronal properties (Brace et al., 1985; Vreugdenhil et al., 2002). Changes in axonal projections are outlined here in the section on plasticity. Remission in the adult intrahippocampal version of this model could be an active protective process, given that the hippocampus will remain abnormal in many respects, a minority of these rats will remain epileptic, and higher proportions of rats will remain permanently epileptic in the neonatal intrahippocampal and adult neocortical versions of the model. When tetanus toxin is given intrahippocampally in the neonate, the initial seizures stop within about 1 week, but remission from behavioral seizures appears to be less common than when it is given to adults (Jiang et al., 1998; Lee et al., 1995). As with the adult model, learning is impaired many months after the injection of toxin (Lee et al., 2001). In both adult and neonatal models, this finding shows that neither gross neuronal lesions nor the presence of anticonvulsant drugs is necessary for problems in learning and memory associated with temporal lobe epilepsy. Both adult and neonatal models also show that continued electrographic epileptiform activity is not required for these cognitive impairments; in the adult model most of the rats have no epileptic electrical activity, and in the neonatal model about half have none. When the toxin is injected into the neocortex, there is little sign of remission over periods of many months (Brener et al., 1991; Louis et al., 1990; Nilsen et al., 2005).
CHARACTERISTICS AND DEFINING FEATURES Clinical and Behavioral Features of Seizures The adult intrahippocampal version of the model results in Racine stage 5 seizures, which last less than 2 minutes. These behavioral seizures start with behavioral arrest and vibrissal twitching, often progressing to clonic forelimb movements and on to rearing and falling (Mellanby et al., 1977, 1981). There is no status epilepticus at any stage with
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the low doses used in most studies. The seizures start in the forebrain; partial seizures are contained within the hippocampal formation and associated structures. Electrographic seizures recorded from the hippocampus start either with a sudden prolonged burst of activity or with short bursts at 2 to 3 Hz, developing into faster burst discharges at about 20 Hz, which then slow to a bilaterally synchronous 9 to 16 Hz activity, which often was a prelude to secondary generalization. Finally the seizures often end with 2 to 5 Hz discharges (Finnerty et al., 2000, 2002). Seizures usually start 3 to 8 days after injection, with generalized seizures usually appearing a few days later (Finnerty et al., 2000, 2002; Hawkins et al., 1987). This correlates with studies of hippocampal slices in vitro, which generated no epileptic discharges until 3 days after injection and needed 7 days to produce spontaneous epileptic activity (Jefferys, 1989). These observations suggest the existence of a latent period, but unlike the pilocarpine and kainic acid models, described elsewhere in this volume, this latent period does not follow a period of status epilepticus. At their peak, seizures can occur up to 30 times a day or more, but for much of the time one to four times a day is more typical. Seizures rarely exceed 2 minutes in duration, which may explain the low mortality and the ability of the animals to maintain themselves in good condition. They do, however, become hyperreactive and may appear aggressive, particularly when handled with insufficient care or by unfamiliar experimenters (Mellanby et al., 1977). During the first week, neonatal intrahippocampal tetanus toxin induces very different behavioral seizures from the adult model. After a 1- to 2-day latent period, wild running seizures are common but are never seen when these rats become adult (Anderson et al., 1997), nor are they seen in rats that received intrahippocampal toxin as adults. The behavioral seizures reported in adults that had received intrahippocampal toxin as neonates included behavioral arrest, clonic activity, tonic posturing, and wet-dog shakes, of which forelimb myoclonus was the most common (Anderson et al., 1997). As in the adult version of the model, many electrographic seizures were not associated with detectable behaviors (Anderson et al., 1997; Hawkins et al., 1987; Mellanby et al., 1977). Injection of toxin into the motor cortex reliably induced a model in which seizure activity consistently occupied 60% of a 4-hour recording over a period of 4 weeks (Nilsen et al., 2005). After 5 months, seizure activity occupied about 40% of a 4-hour recording period. The latency to seizure occurrence was variable but occurred within a few days. Seizures manifested as behavioral arrest and myoclonic jerking lasting 1 to 15 minutes. The myoclonic jerking was not always discernable by eye, but it was evident on the EMG recording. These seizures were associated with a burst of fast EEG activity (>20 Hz) evolving into rhythmic spiking
at 1 to 2 Hz with higher (15-Hz) frequencies intermixed. The seizures were consistent with epilepsia partialis continua with frequent focal motor seizures. Higher doses of tetanus toxin into the motor cortex or repeated doses result in generalized seizures that can be fatal (Brooks et al., 1962; Louis et al., 1990). Injection into the sensory cortex gives a different clinical picture, with infrequent secondary generalized seizures but persistent interictal spiking for at least 7 months (Brener et al., 1991). This difference may be due to differences in the effects of tetanus toxin in sensory and motor cortex (Hagemann et al., 1999). Tetanus toxin applied to the motor cortex seems to result in a more localized and profound disruption of inhibition than does tetanus toxin applied to the sensory cortex, which results in a more diffuse disruption of inhibition (Brener et al., 1991; Hagemann et al., 1999). Neuropathology Very high doses of intracranial toxin do cause substantial cell death as well as a very high mortality rate (Bagetta et al., 1990, 1991). With lower doses of intrahippocampal toxin, cell loss (and mortality) is minimal in both adult and neonatal versions (Benke et al., 2004; Jefferys et al., 1992; Mellanby et al., 1977). In the neonatal version there is evidence of a dispersion of pyramidal cells from the tight laminar organization they normally have in rodents; this dispersion was not associated with gliosis, nor was it restricted to the tissue directly exposed to the toxin, as revealed by tagging with horseradish peroxidase (Lee et al., 2001). A small number of rats that received toxin as adults develop lesions in CA1, with incidences of 10% to 30% reported in different studies and some evidence of subtle differences between different colonies of rats (Jefferys et al., 1992; Shaw et al., 1990, 1994). Selective cell loss may occur slowly as the chronic epileptic syndrome continues, for instance, in hilar somatostatin-positive neurons (Mitchell et al., 1995), but most of the inhibitory interneurons survive (Najlerahim et al., 1992). This model has provided evidence on the long-standing debate on whether hippocampal sclerosis promotes epileptogenesis. In most studies of the tetanus toxin model, recurrent seizures occurred in the absence of hippocampal sclerosis. In an interesting study, hippocampal sclerosis caused by an episode of cerebral ischemia depressed rather than promoted epileptogenesis-induced by subsequent intrahippocampal tetanus toxin (Milward et al., 1999). Intracortical injection of tetanus toxin resembles intrahippocampal injection in that it results in minimal or no cell loss (Louis et al., 1990). There is little evidence of astrocytosis in the low-dose models described here. However, there is activation of microglia (Shaw et al., 1990, 1994) despite the absence of neurodegeneration.
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Plasticity In common with most models of temporal lobe epilepsy, mossy-fiber sprouting in the dentate gyrus occurs in the intrahippocampal model whether it is induced in adults (Mitchell et al., 1996) or in neonates (Anderson et al., 1999). In addition we found evidence of sprouting of CA1 pyramidal cell axons into the stratum radiatum (Vreugdenhil et al., 2002). In the intracortical models, no evidence has been found of axonal sprouting, mostly likely because it is more difficult to detect. The form of synaptic plasticity known as long-term potentiation (LTP) was investigated during the postseizure remission stage of the adult intrahippocampal tetanus toxin model as a potential basis for the enduring impairments of learning and memory found in this model. This was an attractive hypothesis given the absence of structural lesions and histopathology. Despite the significant and substantial impairments of learning and memory in the same rats, LTP of the perforant path to granule cell synapse was preserved; a reduction in the apparent excitability of the granule cells appeared a more likely basis for the impaired hippocampal function (Brace et al., 1985; Jefferys et al., 1987). The absence of cellular loss and the persistence of seizure and epileptiform activity despite clearance of the toxin in the neocortical models also suggest that long-lasting changes take place in local connectivity and neuronal function that have yet to be fully elucidated (Empson et al., 1993a).
Imaging and Metabolic Changes One study showed hypometabolism in the neocortical focus, as shown by quantitative 14C-deoxyglucose autoradiography (Hagemann et al., 1998), resembling the interictal hypometabolism associated with human focal epilepsy.
Genetic and Molecular Changes To date molecular changes have not been studied extensively. Glutamic acid decarboxylase (GAD) increases for the first approximately 2 weeks after intrahippocampal injection of tetanus toxin (Najlerahim et al., 1992), perhaps reflecting the recovery of g-aminobutyric acid (GABA) release during this period (Empson et al., 1993a; Whittington et al., 1994). There also were increases in the flip isoforms of GluR-1 and -2 glutamate receptor subunits (Rosa et al., 1999). Seizures induced by neocortical injection result in increased expression of immediate early genes at the focus and connected structures, but interestingly expression decreased in tissue surrounding the focus (Liang et al., 1997b). Other genes, including the GAD-67 and NMDA subunit NR1, showed a similar pattern of changes, whereas aCaMKII and GluR2 changed in the opposite direction (Liang et al., 1997a).
Response to Antiepileptic Drugs and Their Usefulness in Screening Drugs The hippocampal model has been used to study antiepileptic drugs. It responds to carbamazepine and lamotrigine (Doheny et al., 2002; Hawkins et al., 1985). The fact that the seizures are spontaneous makes this model resemble clinical epilepsy more closely than those models in which seizures must be evoked; however, this feature also complicates use of the model for screening because this use requires continuous, or at least long-term, monitoring, either by video recording (limited to secondary generalized seizures) or by intracranial recording (more labor intensive but able to detect both partial and generalized seizures). The variability of seizure frequency also requires some care, although in practice the numbers of animals needed to detect effects are relatively modest: groups of six sufficed for the study on lamotrigine (Doheny et al., 2002). The motor cortex model of epilepsia partialis continua is resistant to systemic administration of high-dose phenytoin or diazepam, and it mimics the drug resistance seen in human epilepsia partialis continua (Nilsen et al., 2005). High doses of tetanus toxin applied to motor cortex of cats results in focal and secondary generalized seizures that show variable response to phenobarbitone (Louis et al., 1990).
LIMITATIONS AND EASE OF USE All versions of the model discussed here are easy to implement. They do, however, depend on stereotaxic surgery and accurate location of the injection site, which requires skilled staff. Mortality is minimal at the low doses of toxin normally used, but it can be considerable with much higher doses (two orders of magnitude higher in the case of the intrahippocampal model). The epileptic syndromes are very reproducible, although some variation occurs between batches of toxin, probably because of variations in toxicity. Toxicity normally will have been assayed by the manufacturer; however, problems during transport can damage the toxin. It is possible to assay the toxin in the laboratory. The routine assay involves injection into the gastrocnemius muscle of the mouse and uses hindlimb paralysis as an endpoint (Lee et al., 1995; Mellanby et al., 1977); after paralysis appears, death will inevitably follow, and so the animals should be killed humanely at this stage. Given the steep dose-response curve, twofold serial dilutions are often used. However, these assays may not be essential. We use the manufacturer’s toxicity data for each new batch to determine the dose for the first small cohort of animals and to adjust the dose in the light of the epileptic foci induced in them if the response is too mild or too strong. The use of frozen aliquots means that such adjustments will be required only on the first use of a new batch.
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INSIGHTS INTO HUMAN DISORDERS Underlying Mechanisms Tetanus toxin is a zinc-dependent protease that cleaves synaptobrevin (Schiavo et al., 1992b) and hence blocks synaptic release. It comprises light and heavy chains; the former has the protease action, and the latter provides the binding, internalization, and transport properties (which have been exploited for tracing neuronal pathways). The whole toxin is essential for the model. Its relative selectivity for inhibitory neurons seems to be related to selective uptake rather than to any specific properties of its substrate in different classes of neuron (Bergey et al., 1983; Williamson et al., 1992). The toxin is cleared within a few days and is absent for most of the duration of these models (Mellanby, 1989). Its transient blockade of inhibitory neurotransmission appears to be able to induce the chronic epileptic foci characteristic of these models. Several factors may be involved in sustaining the epileptic focus in the long term, including reduced activation of inhibitory neurons, altered synaptic connectivity, and changes in intrinsic neuronal properties (Benke et al., 2004; Vreugdenhil et al., 2002; Whittington et al., 1994). There is evidence of a loss of inhibition in the neocortical epilepsy models (Brener et al., 1991; Empson et al., 1993a; Hagemann et al., 1999), the extent and degree of which depends on the precise cortical area injected; for instance, it was more widespread but less profound following injections into sensory than into motor cortex (Hagemann et al., 1999). In the intrahippocampal model, there is also evidence of a loss of GABA release and of inhibition (Empson et al., 1993b; Jefferys et al., 1991). Epileptic activity persists longer than the block of GABA release that presumably depends on synaptobrevin degradation (Empson et al., 1993b; Jefferys et al., 1991). Pharmacologically isolated, monosynaptic inhibitory postsynaptic currents (IPSCs) are depressed in parallel with the loss of GABA release, recovering over 2 to 4 weeks; but inhibitory responses evoked in the absence of drugs remain depressed indefinitely (Vreugdenhil et al., 2002; Whittington et al., 1994). This finding suggests that excitation of inhibitory neurons is disrupted in some way. The weakening of inhibition more than 5 months after injection was selective for feedback inhibition, with feed forward returning to normal levels, which suggests a relatively selective disruption of this component of the inhibitory circuitry (Vreugdenhil et al., 2002).
USEFULNESS FOR TREATMENT ASSESSMENT-DEVELOPMENT-SCREENING The spontaneous seizures found in this model in vivo allow exploration of the dynamics of seizures in the whole
animal and the role of long-range interactions between different structures in seizure initiation and generalization (Finnerty et al., 2000, 2001, 2002; Hawkins et al., 1987). The investigation of electrographic activity in the period preceding each seizure may prove useful for seizure prediction. The intrahippocampal model has already proved that it can be used for drug screening. Its sensitivity to levetiracetam (Doheny et al., 2002) shows that it is a good model for temporal lobe epilepsy, given that this drug failed the common screens but was identified by its efficacy in the kindling model (Loscher et al., 1993). The labor-intensive nature of such models makes them unlikely ever to provide a fast throughput screen for potential anticonvulsants. They do, however, provide a relatively realistic seizure syndrome for the evaluation of selected drugs and other novel treatments. Intracortical injection into the motor cortex is an ideal model for assessing drug responsiveness because of the frequent and consistent seizures. Thus the effects of single doses of an antiepileptic drug can be continuously monitored over a matter of hours (Nilsen et al., 2005). The resistance of this model to conventional antiepileptic drugs given systemically and the similarity of this model to human epilepsia partialis continua support its use for the assessment of new therapeutic approaches to this disabling condition. The highly focal nature of the motor seizures makes it an ideal model in which to study focal therapies; indeed, focal administration of the AMPA/kainate receptor antagonist, NBQX, can transiently halt seizure activity (Nilsen et al., 2004).
CONCLUSION Tetanus toxin provides a reliable means of inducing recurrent spontaneous epileptic seizures in selected structures, most often the hippocampus and neocortex. It differs from other common methods of inducing spontaneous focal seizures in that it does not start with status epilepticus and it does not have major cell loss as an early component of the syndrome. It has proved useful in showing that seizures can occur in the absence of gross pathology and that long-term behavioral complications of seizures need not depend on neuronal loss, ongoing epileptic activity, or the presence of antiepileptic drugs. More work is needed on the mechanisms of epileptic activity and on the basis of the seizure remission often found in the hippocampal version of the model.
Acknowledgments The work of the authors described here was funded by the Wellcome Trust.
References
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Hawkins, C.A., Mellanby, J., and Brown, J. 1985. Antiepileptic and antiamnesic effect of carbamazepine in experimental limbic epilepsy. J Neurol Neurosurg Psychiatry 48: 459–468. Hawkins, C.A., and Mellanby, J.H. 1987. Limbic epilepsy induced by tetanus toxin: a longitudinal electroencephalographic study. Epilepsia 28: 431–444. Jefferys, J.G.R. 1989. Chronic epileptic foci in vitro in hippocampal slices from rats with the tetanus toxin epileptic syndrome. J Neurophysiol 62: 458–468. Jefferys, J.G.R., Borck, C., and Mellanby, J. 1995. Chronic focal epilepsy induced by intracerebral tetanus toxin. Ital J Neurol Sci 16: 27–32. Jefferys, J.G.R., Evans, B.J., Hughes, S.A., and Williams, S.F. 1992. Neuropathology of the chronic epileptic syndrome induced by intrahippocampal tetanus toxin in the rat: preservation of pyramidal cells and incidence of dark cells. Neuropathol Appl Neurobiol 18: 53–70. Jefferys, J.G.R., and Mellanby, J. 1998. Behaviour in chronic experimental epilepsies. In Disorders of Brain and Mind. Ed. M.A. Ron, and A.S. David, pp 213–232. Cambridge: Cambridge University Press. Jefferys, J.G.R., Mitchell, P., O’Hara, L., Tiley, C., Hardy, J., Jordan, S.J., Lynch, M., et al. 1991. Ex vivo release of GABA from tetanus toxininduced chronic epileptic foci decreased during the active seizure phase. Neurochem Int 18: 373–379. Jefferys, J.G.R., and Williams, S.F. 1987. Physiological and behavioural consequences of seizures induced in the rat by intrahippocampal tetanus toxin. Brain 110: 517–532. Jiang, M., Lee, C.L., Smith, K.L., and Swann, J.W. 1998. Spine loss and other persistent alterations of hippocampal pyramidal cell dendrites in a model of early-onset epilepsy. J Neurosci 18: 8356–8368. Kryzhanovsky, G.N., Rechtman, M.B., Konnikov, B.A., and Sheikhon, F.D. 1976. An experimental vestibulopathy produced by the formation of a pathologically enhanced excitation generator in the vestibular nuclei. Bull Exp Biol Med 81: 142–145. Lee, C.L., Hannay, J., Hrachovy, R., Rashid, S., Antalffy, B., and Swann, J.W. 2001. Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy. Neuroscience 107: 71–84. Lee, C.L., Hrachovy, R.A., Smith, K.L., Frost, J.D.J., and Swann, J.W. 1995. Tetanus toxin induced seizures in infant rats and their effects on hippocampal excitability in adulthood. Brain Res 677: 97–109. Liang, F., and Jones, E.G. 1997a. Differential and time-dependent changes in gene expression for type II calcium/calmodulin-dependent protein kinase, 67 kDa glutamic acid decarboxylase, and glutamate receptor subunits in tetanus toxin-induced focal epilepsy. J Neurosci 17: 2168–2180. Liang, F., and Jones, E.G. 1997b. Zif268 and Fos-like immunoreactivity in tetanus toxin-induced epilepsy: reciprocal changes in the epileptic focus and the surround. Brain Res 778: 281–292. Loscher, W., and Honack, D. 1993. Profile of ucb L059, a novel anticonvulsant drug, in models of partial and generalized epilepsy in mice and rats. Eur J Pharmacol 232: 147–158. Louis, E.D., Williamson, P.D., and Darcey, T.M. 1990. Chronic focal epilepsy induced by microinjection of tetanus toxin into the cat motor cortex. Electroencephalogr Clin Neurophysiol 75: 548–557. Mellanby, J., George, G., Robinson, A., and Thompson, P. 1977. Epileptiform syndrome in rats produced by injecting tetanus toxin into the hippocampus. J. Neurol Neurosurg. Psychiat. 40: 404–414. Mellanby, J., Johansen-Berg, H., Leyland, R., and Milward, A.J. 1999a. The effects of tetanus toxin-induced limbic epilepsy on the exploratory response to novelty in the rat. Epilepsia 40: 1058–1061. Mellanby, J., Oliva, M., Peniket, A., and Nicholls, B. 1999b. The effect of experimental epilepsy induced by injection of tetanus toxin into the amygdala of the rat on eating behaviour and response to novelty. Behav Brain Res 100: 113–122. Mellanby, J., Strawbridge, P., Collingridge, G.I., George, G., Rands, G., Stroud, C., and Thompson, P. 1981. Behavioural correlates of an exper-
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imental hippocampal epileptiform syndrome in rats. J Neurol Neurosurg. Psychiatry 44: 1084–1093. Mellanby, J.H. 1989. Elimination of 125I from rat brain after injection of small doses of 125I-labelled tetanus toxin into the hippocampus. Neurosci Lett Suppl.36: S55. Milward, A.J., Meldrum, B.S., and Mellanby, J.H. 1999. Forebrain ischaemia with CA1 cell loss impairs epileptogenesis in the tetanus toxin limbic seizure model. Brain 122: 1009–1016. Mitchell, J., Gatherer, M., and Sundstrom, L.E. 1995. Loss of hilar somatostatin neurons following tetanus toxin-induced seizures. Acta Neuropathol (Berl) 89: 425–430. Mitchell, J., Gatherer, M., and Sundstrom, L.E. 1996. Aberrant Timm-stained fibres in the dentate gyrus following tetanus toxininduced seizures in the rat. Neuropathol Appl Neurobiol 22: 129– 135. Najlerahim, A., Williams, S.F., Pearson, R.C.A., and Jefferys, J.G.R. 1992. Increased expression of GAD mRNA during the chronic epileptic syndrome due to intrahippocampal tetanus toxin. Exp Brain Res 90: 332–342. Nilsen, K.E., Walker, M.C., and Cock, H.R. 2005. Characterisation of the tetanus toxin model of refractory focal neocortical epilepsy in the rat. Epilepsia 46: 179–187. Rosa, M.L.N.M., Jefferys, J.G.R., Sanders, M.W., and Pearson, R.C.A. 1999. Expression of mRNAs encoding flip isoforms of GluR1 and GluR2 glutamate receptors is increased in rat hippocampus in epilepsy induced by tetanus toxin. Epilepsy Res 36: 243–251.
Roux, E., and Borrel, A. 1898. Tétanos cérébral et immunité contre le tétanos. Ann Inst Pasteur 4: 225–239. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B.R., and Montecucco, C. 1992a. Tetanus and botulinumB neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359: 832–835. Schiavo, G., Poulain, B., Rossetto, O., Benfenati, F., Tauc, L., and Montecucco, C. 1992b. Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc. EMBO J 11: 3577–3583. Shaw, J.A.G., Perry, V.H., and Mellanby, J. 1990. Tetanus toxin-induced seizures cause microglial activation in rat hippocampus. Neurosci Lett 120: 66–69. Shaw, J.A.G., Perry, V.H., and Mellanby, J. 1994. MHC class II expression by microglia in tetanus toxin-induced experimental epilepsy in the rat. Neuropathol Appl Neurobiol 20: 392–398. Vreugdenhil, M., Hack, S.P., Draguhn, A., and Jefferys, J.G.R. 2002. Tetanus toxin induces long-term changes in excitation and inhibition in the rat hippocampal CA1 area. Neuroscience 114: 983–994. Whittington, M.A., and Jefferys, J.G.R. 1994. Epileptic activity outlasts disinhibition after intrahippocampal tetanus toxin in the rat. J Physiol (Lond) 481: 593–604. Williamson, L.C., Fitzgerald, S.C., and Neale, E.A. 1992. Differential effects of tetanus toxin on inhibitory and excitatory neurotransmitter release from mammalian spinal cord cells in culture. J Neurochem 59: 2148–2157.
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34 Kainate-Induced Status Epilepticus: A Chronic Model of Acquired Epilepsy F. EDWARD DUDEK, SUZANNE CLARK, PHILIP A. WILLIAMS, AND HEIDI L. GRABENSTATTER
reorganization occur in the dentate gyrus as well as in other areas. The kainate model is similar to other chronic epilepsy models (such as pilocarpine treatment and self-sustaining status epilepticus owing to repeated electrical stimulations) in which status epilepticus is followed by a latent period and then by a state of chronic spontaneous recurrent seizures. Although these models do have specific differences, the similarities suggest that a period of prolonged status epilepticus triggers common epileptogenic mechanisms. Kainate can be administered by intraperitoneal (IP), intravenous (IV), subcutaneous, intracerebroventricular, or intrahippocampal injections; treatments can be given as a single large dose (e.g., 8–15 mg/kg) or as repeated lower (2.5–5.0 mg/kg) doses, with the number of doses titrated until the development of status epilepticus. Our focus in this chapter is on systemic treatments (repeated low doses of kainate), which are effective in creating an animal with robust and frequent spontaneous seizures. Attention will also be given to models that employ focal intracranial injections, which have their own advantages and limitations. The treatment protocol appears to affect the reliability of the outcome and the nature of the epilepsy (e.g., the frequency of the spontaneous recurrent seizures). With respect to the specific methods, the overall goal of this chapter is to describe the main variants of the treatment protocols, provide information on how to use this model in a relatively efficient manner, and discuss some of the potential pros and cons. We then review briefly some of the histopathological and physiologic findings obtained using the model.
KAINATE-INDUCED STATUS EPILEPTICUS: A CHRONIC MODEL OF ACQUIRED EPILEPSY General Description of the Model The kainate model of epilepsy has been in use since it was reported that kainate induces repetitive seizures and neuronal damage in the hippocampus (Nadler et al., 1978) and amygdala (Ben-Ari and Lagowska, 1978), reminiscent of temporal lobe epilepsy (TLE) in humans. Subsequently this model has been widely studied, especially in regard to the chronic epileptic state produced after an apparent latent period following status epilepticus. It is now considered one of several animal models that, because of this acquired chronic epileptic state, may have particular advantages for studying the mechanism of injury-induced epileptogenesis and the basis for drug-resistant human epilepsies and for screening new drugs for these conditions (Stables et al., 2002, 2003). The kainate model has at least three major stages: (1) the initial hours-long episode of status epilepticus, (2) the daysto weeks-long seizure-free latent period, and (3) the gradual development and progressive increase in the frequency of recurrent spontaneous seizures. This final stage is generally permanent and serves as the defining property of the chronic epilepsy. Superimposed on these stages are the anatomic changes seen in the animals that parallel the acute and chronic neuropathological alterations reported in humans, including neuronal loss in the hippocampus, temporal lobe, and other brain structures. Axonal sprouting and synaptic
Models of Seizures and Epilepsy
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WHAT DOES THE KAINATE MODEL ACTUALLY MODEL? Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis The goal of most investigators using the kainate-treated rat as an animal model is to produce a rodent preparation that displays the general characteristics of human acquired or injury-induced epilepsy, including TLE. As defined by the International League Against Epilepsy (ILAE; see Wieser et al., 2004), mesial TLE with hippocampal sclerosis often has an “initial precipitating incident,” which is proposed to be the injury that eventually leads to the development of spontaneous recurrent seizures. The initial precipitating incident can include status epilepticus, complex febrile seizures, trauma, or hypoxia-ischemia. According to the ILAE Commission Report (Wieser et al., 2004), the neuropathological changes of TLE with mesial temporal sclerosis include (1) extensive neuronal loss and gliosis in the areas of CA1 and the hilus but also in other hippocampal regions to varying degrees; (2) synaptic reorganization, although not necessarily limited to the mossy fibers of the dentate gyrus; (3) dispersion of the dentate granule cells; and (4) extrahippocampal pathology. With respect to etiology, although status epilepticus can induce TLE in humans, many cases are not associated with any discernable initial precipitating injury, and yet they generally have histopathological damage in the temporal lobes and other areas, roughly similar to what occurs after kainate treatment. TLE with mesial temporal sclerosis has been proposed to be progressive, although definitive confirmation of this proposal is difficult, given the many possible confounding factors (Cole, 2000; Wieser et al., 2004).
Domoic Acid Poisoning The kainate-treated rat closely models domoic-acid poisoning, a rare condition that has been documented in humans and has many similarities to TLE. Domoic acid is structurally similar to kainate and is a natural toxin produced by certain phytoplankton, including the genus Pseudonitzachia, which has a worldwide distribution, including the waters of North America (Jeffery et al., 2004). The phytoplankton are ingested by shellfish, some of which accumulate the toxin. In 1987 a severe outbreak of amnesic shellfish poisoning occurred in Canada from contaminated mussels harvested from Prince Edward Island. More than a hundred people became acutely ill; 19 were hospitalized; 12 required intensive care because of seizures, coma, respiratory secretions, and unstable blood pressure; and 4 patients died (Perl et al., 1990). Patients requiring treatment in the intensive care unit also had mutism, purposeless chewing, and grimacing.
Teitelbaum and co-workers (1990) noted that all patients with seizures became unresponsive during the acute poisoning, and in three patients the seizures responded to IV diazepam and phenobarbital but not to phenytoin. Seizures declined over the next 8 weeks and were controlled by 8 months. The four fatalities from domoic acid poisoning had neuropathological lesions resembling the kainate model in that the most severely damaged areas included the hippocampus and amygdala (Teitelbuam et al., 1990). Cendes and coworkers (1995) described a patient whose acute intoxication included complex partial status epilepticus. After one seizure-free year, the patient developed TLE with complex partial seizures. Magnetic resonance imaging (MRI) revealed bilateral hippocampal atrophy. The patient died 2 years after the onset of the seizures; at that point, severe bilateral hippocampal sclerosis was detected. Several other areas were also damaged, including the amygdala and overlying cortex (Cendes et al., 1995). Thus the human case of domoic acid poisoning and the kainate model show clear parallels with respect to the initial precipitating injury (i.e., the toxin-evoked status epilepticus), a seizure-free latent period, the chronic occurrence of spontaneous recurrent seizures, and the characteristic neuropathological changes of TLE. Although domoic acid poisoning is rare, it demonstrates that humans can become chronically epileptic after domoic acid–induced status epilepticus by a mechanism probably similar to that by which rats become chronically epileptic in response to kainate-induced status epilepticus.
METHODS OF GENERATION OF THE MODEL The early work with kainate, and most of the more recent studies, can generally be grouped into two categories of treatment protocol: single, large, systemic injections (i.e., IP, IV, or subcutaneous) or single intracranial injections (e.g., intraventricular or intrahippocampal). One problem with the single-systemic-injection approach is that many animals die during or shortly after the treatment, although a few studies with kainate have apparently reduced this problem by stopping the resulting status epilepticus with a benzodiazepine (e.g., diazepam) after a specified amount of time (e.g., 30 minutes in Tremblay and Ben-Ari, 1984). This “anticonvulsant rescue,” which is routinely done with the pilocarpine model, would be expected to reduce the number of animals that die from the kainate-induced status epilepticus, but some animals may die before the benzodiazepine injection, and this approach may lead to a lower percentage of animals that develop robust and long-lasting convulsive status epilepticus and subsequent recurrent spontaneous seizures. Another major problem with single-injection protocols is that some animals do not experience convulsive status
Methods of Generation of the Model
epilepticus. The single large doses are usually about 10 mg/kg. For example, an IP dose of 9 mg/kg was used by Tremblay et al. (1984) and Pirttilä et al. (2001). Tauck and Nadler (1995) administered an IV dose of 11 mg/kg via the tail vein, and Cronin et al. (1992) used subcutaneous injections of 12 mg/kg. A critical issue with kainate, as with other chemoconvulsants, is a loss of potency with time; therefore, caution is advisable when using newly purchased kainate because it can lead to high mortality, and it is also important to be aware that “old” kainate may be less effective and lead to inadequate status epilepticus, particularly with the single-injection protocol. Intracranial (e.g., intraventricular and intrahippocampal) injections require anesthesia and sterotaxic techniques, and by their nature they lead to a more focal injury. Tauck and Nadler (1985) gave bilateral intraventricular injections in rats anesthetized with pentobarbital (55 mg/kg IP). The dose was 2.34 to 3.75 nmol of kainate in a 1-mL volume of artificial cerebrospinal fluid (ACSF) injected into each lateral ventricle over a 30-minute period. The authors noted that this protocol did not consistently cause as much damage as the intravenous injections (see preceding discussion). Bragin and co-workers (1999) injected 0.4 mg/0.2 mL kainate in normal saline unilaterally into the right posterior hippocampus of rats; Riban et al. (2002) injected 50 nl of a 20-mM solution of kainate in 0.9% NaCl (i.e., 1 nmol) into the right dorsal hippocampus of mice. In the study by Bragin and coworkers (1999), 20 rats underwent 4 weeks of electrographic and video monitoring 3 to 8 months after kainate treatment, and only eight rats were observed to have spontaneous seizures (i.e., 40%), although another rat was subsequently seen to have seizures. The likelihood of animal death during this protocol is highly dependent on the skill level of the investigators with the surgical techniques, but Riban and coworkers reported that 12 of 60 mice (i.e., 20%) died during the first 3 weeks after treatment. In a review of the early work, Nadler (1981) noted that when kainate is injected directly into a specific limbic region, the seizures begin in that area; when kainate is injected systemically, some of the first areas activated are the hippocampus and amygdala, and then seizure activity propagates to other limbic areas and the neocortex, the latter of which probably drives the motor seizures. Thus the intracranial approaches for kainate treatment lead to a more focal injury, and the subsequent epilepsy is also apparently more focal; however, the intracranialinjection approach is more difficult and time consuming than single systemic injections and, as discussed later herein, the proportion of animals that have been observed to generate spontaneous recurrent seizures and the frequency of the spontaneous recurrent seizures are comparatively low. Based on what was already known in the early 1990s and also on our own experience with a variety of protocols involving kainate and pilocarpine injections, we decided to develop a multiple low-dose protocol for systemic injections
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of kainate. This protocol was then used for a series of in vitro experiments on hippocampal slices aimed at testing hypotheses about the relative contributions of decreased inhibition versus increased recurrent excitation underlying graded versus all-or-none bursts in the CA1 area in kainatetreated rats (Meier and Dudek, 1996). In an earlier study using single high-dose injections (Meier et al., 1992), we observed inconsistent induction of status epilepticus, high mortality during and shortly after kainate treatment, and relatively weak and variable hippocampal damage. Thus we conducted a study using repetitive (i.e., 10 hourly doses) of 5 mg/kg kainate in saline over 9 hours in 100-g rats. Although a detailed quantitative comparison of the two protocols was not undertaken, our impression was that the repetitive low-dose protocol (Meier and Dudek, 1996) was more reliable and more effective than the single high-dose protocol (Meier et al., 1992).
Animal Issues (Species, Strain, Age, and Gender) Species and Strain Most experimental studies with animal models have used the standard laboratory rat (Ratus ratus); however, other species can be used, including mice (e.g., see Riban et al., 2002) and dogs (see Hasegawa et al., 2003). With respect to strain, we have consistently used the Sprague-Dawley rat, although experiments have been done using other strains. One potential problem with the kainate model in the mouse is that certain strains have been shown to be relatively ineffective for inducing neuronal damage and epileptogenesis, even though kainate may cause repetitive seizures (Shauwecker and Steward, 1997). Age Specificity In the studies from our laboratory and many others, the kainate model has been used primarily with young adult animals (i.e., approximately 200 g for rats); immature or aged animals are far less likely to generate a model with spontaneous recurrent seizures (e.g., Tremblay et al., 1984; Sperber et al., 1991). Immature animals (e.g., 1–3 weeks) generally have robust, repetitive seizures (i.e., profound status epilepticus) during kainate treatment, but relatively little neuronal death or mossy-fiber sprouting occurs, and the animals do not become obviously epileptic. On the other end of the time spectrum, aged rats (i.e., older than 1 year) are more likely to die during or after the kainate treatment; thus it has been difficult to generate a model of an aged rat with kainate-induced epilepsy (Peter Patrylo and Kevin Kelly, personal communication). Immature and aged animals have therefore not been as useful as young adults for generating animals with spontaneous recurrent seizures.
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Gender Differences Our laboratory and others have had success with both males and females (Hellier et al., 1998). Using electrically induced status epilepticus, Löscher and colleagues reported that females are more reliably rendered chronically epileptic (Brandt et al., 2004; Glien et al., 2001). It is possible that females will be more variable because of the differential effects of kainate and repetitive seizures on the brain during the estrous cycle, although this possibility requires further investigation, and there is an obvious need to have data in both genders.
Procedures Enhancing Survivability Several studies from our laboratory conducted during the late 1990s used the repeated low-dose protocol for kainate treatment, and the Methods and Results sections of these publications provide information on the protocol and outcomes (Buckmaster and Dudek, 1997a, 1997b; Hellier et al., 1998, 1999; Patrylo and Dudek, 1998; Wuarin and Dudek, 1996, 2001). The detailed procedures for systemic kainate treatment as used in our laboratory will be published (Hellier and Dudek, 2005). That article provides a step-by-step protocol for treating rats with kainate in a manner that aims to optimize the percentage of animals that experience status epilepticus and subsequently generate spontaneous recurrent seizures with histopathological abnormalities similar to mesial temporal sclerosis. Thus only a summary of the methods presently used in our laboratory are outlined here. Animals are weighed immediately before the treatment to ensure accurate dose calculations. If housed in pairs, they need to be permanently marked with a tattoo, identity chip, or ear punch (ear tags can be lost). Several steps can be taken to improve animal survival during the acute kainate treatment; these include (1) using a flat, disposable bed liner (instead of cob or woodchip bedding) to prevent choking on the chips during seizures; (2) using plastic cage lids (or inverted metal lids) to keep the animals from injuring themselves on the lid during seizures; (3) giving IP injections immediately lateral to the linea alba to avoid internal injuries (subcutaneous injections are also acceptable); (4) routinely using a new syringe and needle with each injection to prevent peritonitis; and (5) most importantly, titrating the dose of kainate for each rat based on the behavioral response to previous doses. Dose Titration The key feature to our approach of repeated IP injections is to monitor the response of each rat to the kainate treatment (or pilocarpine treatment; Glien et al., 2001) accord-
ing to the severity of previous seizure activity. Kainate injections are given every hour; the first dose is always 5 mg/kg, and subsequent doses may be a repeat of this dose, halfdoses (2.5 mg/kg), or withheld, depending on the seizure severity during the previous hour (see later). Saline-treated controls are given an equal volume of sterile normal saline at the same time as the kainate dose. Monitoring Seizures During repeated, low-dose kainate treatments, a critical factor is to monitor carefully the behavior of the animal throughout the treatment period. Similar to others, we use the Racine (1972) scale as a general description of the different classes of behavioral seizure activity. Class I (facial automatisms) and class II (head nodding) can be difficult to identify, particularly to differentiate from grooming or searching behavior; therefore we use only “wet-dog shakes” and the number of class III, IV, and V seizures as the criteria for deciding whether to administer additional injections. In class III seizures, the rat displays forelimb clonus and a lordotic posture. When a forelimb clonus continues to occur as the animal rears, this is defined as a class IV seizure. In class V seizures, the animal displays the same behaviors as in a class III or IV seizure, but in addition the rat falls over (or shows other evidence of a loss of the righting reflex). Thus class III, IV, and V behavioral seizures would generally be considered “motor seizures” or “convulsive seizures.” Some rats jump extensively throughout the cage; for this reason the cages need to be covered and obstacles temporarily removed (e.g., sharp-cornered metal feeders or water bottles). We record the volume and time of each injection and tally the types of seizure-like activity each hour throughout the treatment; the hourly assessment is used to determine whether the animals should receive additional kainate injections. The essential component of this titration strategy is to ensure that the rats do not receive so much kainate that they die or so little that they fail to exhibit adequate repetitive seizures to constitute an episode of status epilepticus. Dose adjustments are made based on several factors. Generally, if animals have only wet-dog shakes or only a few class III seizures during the previous hour, a full injection should be given. If the total number of class III/IV/V seizures is five to nine seizures per hour, the subsequent injection is reduced to 2.5 mg/kg. However, because reduction of the dose by 50% could lead to a premature cessation of status epilepticus, if the animal stops having motor seizures within 30 minutes, another 2.5 mg/kg can be given at that point. If a rat has had more than 10 class III/IV/V seizures per hour or begins to show excessive inactivity (e.g., is lethargic or obtunded) or hyperactivity (e.g., exaggerated jumping or running in circles), the next injection should not be administered until the subsequent assessment is made in the
Methods of Generation of the Model
following hour. Excessive inactivity or hyperactivity can be a warning sign that the animal will not survive additional doses of kainate, so usually no further kainate is given. We typically aim to ensure that the status epilepticus with repetitive convulsive motor seizures persists for longer than 3 hours, but occasional seizures have been observed up to 72 hours. Thus the 3-hour period represents a time of intense repetitive convulsive seizure activity, and yet seizures may continue for a day or two afterward, albeit infrequently (see Hellier et al., 1999). The total dose is usually about 20 to 25 mg/kg. Generally one expects that an animal will have a minimum of one obvious convulsive seizure per hour during treatment, but more typically the seizure number has generally ranged from 7 to 22 seizures in each 1-hour period. The multiple-injection protocol may last 6 to 8 hours, depending largely on when each animal begins to have motor seizures (i.e., a few hours may be required for the animal to have the first motor seizure). The care of animals after the 3+-hour period of status epilepticus is quite important in terms of optimizing survival and minimizing animal discomfort. For several days after the kainate treatment, we routinely inject warm lactated Ringer’s subcutaneously at approximately 1.5 to 2.5 ml/ 100 g of weight per rat. Typically one bolus is injected between the shoulder blades and another bolus of fluid in the flank (paralumbar fossa). Apple or banana slices (or other fruit) and softened rat chow (i.e., a mixture of warm water and rat chow in a low-profile metal bowl) are provided daily to aid recovery from hypoglycemia or dehydration until the rats regain weight and eat normally. In conclusion this is a labor-intensive protocol on the day of treatment, but if it is done carefully, it ensures a high level of survivability and a high percentage of animals will develop epilepsy.
Monitoring of Status Epilepticus Ease of Development This dose-titration approach to inducing status epilepticus has the advantage that convulsive status epilepticus occurs in almost all rats because each rat is given repeated injections until the targeted endpoint of prolonged status epilepticus. Dose titration minimizes the likelihood of giving a lethal dose, but the procedure is best done with two people working nearly continuously for a day. At least one of these individuals should always be monitoring the animals during treatment. The maximum number of animals that can easily be treated in a single session is approximately 12, including 6 to 8 kainate-treated animals and 4 to 6 saline controls. Because controls essentially never die during the treatment protocol, and because most experiments are more difficult with the epileptic animals, it is usually advisable to treat more animals with kainate than with saline.
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In principle, a single high-dose systemic treatment would be easier than the multiple low-dose protocol outlined previously, except one must consider the amount of effort per usable animal. Furthermore ethical considerations require that one attempt to minimize death from status epilepticus so that seizure monitoring and benzodiazepine therapy should be considered, which requires a level of effort that approximates the multiple low-dose protocol. The systemic kainate models do not require expertise in administering anesthesia, performing survival surgery, or using a stereotaxic device for electrode implantation or intraphippocampal or intraventricular injection. Therefore single-injection kainate protocols, either systemic or intracranial, appear to be less effective than the multiple low-dose protocol in generating animals with robust spontaneous recurrent seizures. Reliability As outlined already, when experiments with animal models of acquired epilepsy based on status epilepticus fail in terms of the development of a chronic epileptic state, the problem likely relates to one of two fundamental issues: lack of (or insufficient) status epilepticus or the animal’s death. One advantage of the titrated kainate protocol is that virtually all animals develop status epilepticus, few die, and most that survive develop recurrent spontaneous seizures. For example, in the study of Hellier and co-workers (1998), overall mortality was 15%, and 97% of the rats that had more than 3 hours of kainate-induced seizures during treatment were later observed to have spontaneous recurrent motor seizures. Of the rats that experienced less than 3 hours of seizures during kainate treatment, 85% still eventually developed spontaneous motor seizures (i.e., became epileptic). Furthermore it is important to emphasize that this study was based on only 6 to 8 hours per week of behavioral monitoring, so seizures may have occurred but were not detected in some kainate-treated animals. In a more recent series of studies on a cohort of 95 rats treated between April 2004 and March 2005 in our laboratory, 94% of the rats survived acute kainate treatment. This is in contrast to the single high-dose protocols (either IP or subcutaneous), during which more animals either die acutely or do not experience convulsive status epilepticus. The latter animals do not represent a true control group and thus are of limited usefulness because they may have experienced kainate-induced nonconvulsive status epilepticus or other neuronal injuries without having convulsive status epilepticus. Definition of the Type of Status Epilepticus The easiest analysis of status epilepticus is based purely on behavioral observations, although intrahippocampal and subdural recording electrodes can also be used (e.g., see Bragin et al., 2004; Hellier et al., 1999). An effective status
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epilepticus (i.e., in terms of subsequent development of chronic motor seizures) is many class III/IV/V convulsive seizures per hour for longer than 3 hours. However, single high-dose protocols may stop the status epilepticus after a shorter period (e.g., 30 minutes) to reduce mortality. Rats implanted with intrahippocampal and subdural electrodes show extensive electroencephalographic (EEG) “spikes” (Figure 1), which appear to represent synchronous postsynaptic potentials and population spikes (Hellier et al., 1999). Based on initial analyses of electrographic recordings during the status epilepticus, repetitive convulsive and nonconvulsive electrographic seizures occur in the hippocampal and cortical leads, although electrographic seizure activity and neuronal damage presumably occur in many areas of the brain. Thus the status epilepticus is defined as
FIGURE 1 Granule cell layer (dentate) and surface (EEG) recordings of nonconvulsive and convulsive seizures during kainate-induced status epilepticus. A: Nonconvulsive seizure during kainate treatment. In the expanded traces, seizure activity is shown on the electrode in the dentate gyrus (A3), but it is absent in the EEG trace (A4). B: A motor seizure (class IV) during kainate treatment. Epileptiform activity can be seen in the expanded dentate (B3) and EEG recordings (B4). Asterisks show truncated artifacts arising from “wet-dog” shakes. EEG, electroencephalography. (Reprinted with permission from Hellier, J.L., Patrylo, P.R., Dou, P., Nett, M., Rose, G., and Dudek, F.E. 1999. Assessment of inhibition and epileptiform activity in the septal dentate gyrus of freely-behaving rats during the first week after kainate treatment. J Neurosci 19:10053–10064, Figure 1, p. 10055.)
convulsive, regardless of whether the kainate was administered as a single injection or as several low-dose injections, although nonconvulsive electrographic seizures may also occur during this period (Hellier et al., 1999). This issue can be more difficult to analyze with intracranial-injection protocols because of the need for anesthesia, but some animals have convulsive seizures after recovery from the anesthesia used for intracranial injections of kainate.
CHARACTERISTICS AND DEFINING FEATURES OF CHRONIC EPILEPSY The ultimate defining feature of acquired epilepsy is the occurrence of spontaneous recurrent seizures after a brain injury. As outlined in a recent National Institutes of Health (NIH)-sponsored workshop on animal models of epilepsy (Stables et al., 2002), the most robust and reliable models of chronic epilepsy are thought to be those in which spontaneous recurrent seizures follow chemically or electrically induced status epilepticus. These models have potential for testing antiepileptic drugs (AEDs) and other therapies. Although this approach has numerous challenges, many can be minimized by having a robust model with many overt epileptic seizures. For the kainate model (and others based on chemically or electrically induced status epilepticus), the presence of a latent period until the first spontaneous seizure, the combination of nonconvulsive and convulsive seizures lasting 1 to 3 minutes that appear similar to complex partial seizures with secondary generalization, and the increase in seizure frequency over the course of epileptogenesis are characteristics that at least qualitatively model human TLE. In these animal models, the hippocampus and several surrounding structures show neuronal loss, reactive gliosis, and axonal sprouting similar to what has been seen in tissue from patients with TLE. This combination of features is the primary reason for considering the models based on status epilepticus to reflect a form of human acquired epilepsy, particularly TLE.
Behavioral/Clinical and Electrographic/Electroencephalographic Features of Spontaneous Recurrent Seizures Duration of Latent Period and Frequency of Seizures An important hallmark of acquired epilepsy is the time between an initial brain insult and the onset of spontaneous recurrent seizures (i.e., the latent period). A second clinical characteristic thought to be a fundamental property of epileptogenesis is a progressive worsening of the spontaneous seizures as a function of time after the injury. These two properties may involve similar mechanisms and thus
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be manifestations of the same process. Another important feature is obviously the frequency of spontaneous seizures, with the caveat that comparisons of an animal model with the human condition generally involve assessments of patients who have been treated continuously with AEDs since shortly after they started having seizures. Thus two issues ultimately need to be considered: how similar the seizure rates with and without comparable treatments are and what types of models and seizure frequencies are most useful in a practical sense for experimental studies on acquired epilepsy. Determination of the latent period is highly problematic because one risks missing the first seizure unless one monitors continuously (i.e., 24 hours/day, 7 days/week). For example, in a study of rats treated with repeated low-dose injections of kainate and then monitored behaviorally for approximately 6 to 8 hours per week (Hellier et al., 1998), seizure frequency increased with time after kainate treatment (Figure 2) and the apparent latency for the first spontaneous motor seizure was 77 ± 38 days (mean ± SD). However, 6 to 8 hours per week of monitoring is less than 5% of the hours in a week (i.e., 168 hours). When similarly treated rats were monitored 24 hour per day for 7 days after kainate treatment, no seizures were observed in 81% (n = 26) of the rats, but 19% (n = 5) did have seizures within the first week (Figure 3). Virtually all these rats later developed spontaneous recurrent motor seizures. Of the five rats in this cohort that showed spontaneous motor seizures during the first week, two had seizures on the first or second day but had no additional seizures for the rest of the week. Two other rats had their first motor seizure on day 7, and one rat had its first seizure on day 5 followed by multiple seizures on days 6 and 7. Thus, for most kainate-treated rats, the first detectable motor seizure did not occur during the first week after kainate treatment. For the few rats that had early seizures (e.g., had seizures within 2 days after kainate treatment but had no further seizures during that week), it is probable that these isolated early seizures were simply a residual effect of the kainate treatment rather than early episodes of chronic recurrent seizures. In a model based on intrahippocampal injections in mice, kainate treatment was followed by a latent period of approximately 2 weeks until ipsilateral paroxysmal discharges associated with behavioral arrest occurred (Riban et al., 2002). In summary, the latent period following kainate-induced status epilepticus is as brief as 1 to 2 weeks but may be longer. Problems associated with accurate determinations of the latent period are linked directly to how and when to measure seizure frequency. First, seizure frequency, when analyzed over a sufficiently long period (e.g., weeks or months), is clearly a function of the amount of time since the kainateinduced status epilepticus (Figure 2) (Hellier et al., 1998). The exact nature of this relationship depends on how the data are presented and analyzed, but it is likely a sigmoid or
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logistic function of time after kainate treatment. Second, the motor seizures do not always occur in a regular pattern, but instead they can occur in clusters or may include long seizure-free periods (e.g., see Figure 2 of Grabenstatter et al., 2005). For this reason, seizure frequency should be assessed over a relatively long time. These observations point to several possible problems in studying these models: (1) to conduct experiments on an animal with a high seizure frequency, one must generally wait several months after kainate treatment; (2) when seizure frequency is low, one must monitor more or integrate over a longer period to obtain an accurate measure of seizure frequency; and (3) the potential for false-negative outcomes is enormous unless one does extensive monitoring, particularly when the mean seizure frequency is low or the differences between control and experimental groups may be small (e.g., during AED testing, see later). As shown in Figure 2, although average motor seizure frequency has been documented to be as high as 0.7 seizures per hour (i.e., nearly a seizure every hour), this includes many episodes of clusters of seizures that can each last for several hours. Therefore seizure frequency increases with time after kainate-induced status epilepticus, and several caveats can impact measurements of seizure frequency in this and other models. The weeks-long duration of the latent period and the increase in the frequency of spontaneous recurrent seizures appear comparable to what has been observed in pilocarpine-treated rats using systemic injections. The measured latent period presumably depends on the exact model used (e.g., rat versus mouse; systemic versus intracranial injections), the endpoint (e.g., motor versus electrographic seizures), and particularly the amount and method of monitoring (e.g., continuous electrophysiologic recording versus intermittent or continuous behavioral observations). The available results, particularly when considered in conjunction with the data on seizure frequency, support the concept of a latent period, at least in regard to motor-seizure generation. One hypothesis is that a consistent but less severe status epilepticus than that described already leads to a longer latent period with a more gradual buildup in seizure frequency; this hypothesis is consistent with the views of many clinicians, but it needs further testing in animal models. Seizure Duration and Severity In humans both the tonic-clonic seizures and the complex partial seizures associated with TLE typically last 1 to 3 minutes. This distinguishes complex partial seizures from the comparatively benign absence seizures that may have behavioral similarities to complex partial seizures, but they last for substantially shorter periods (i.e., a few seconds). Although several potential descriptors of behavioral seizures in animals are available, the most widely used scheme in rats
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FIGURE 2 Effect of time, after kainate-induced status epilepticus, on the frequency of spontaneous seizures. The results are from kainate-treated rats (n = 47) that had at least one seizure every 30 days after their initial seizure and that were euthanized ≥4 months after kainate-induced status epilepticus. A: Seizure frequency as a function of time after kainate treatment. Fewer kainate-treated rats were available at later times because they were euthanized for experiments or died. B: Seizure frequency as a function of time after the onset of seizures. This graph shows the increase in seizure frequency independent of differences in the apparent latent period. C: Seizure frequency as a function of normalized epileptic period. The epileptic period was defined as the time between the onset of chronic seizures and death of the kainate-treated rat. This graph compensates for the preferential use of animals with high seizure frequency and for the increased likelihood of death in animals with high seizure frequency. D: Seizure frequency as a function of time after the onset of seizures for rats that survived for 7 months after kainate treatment (n = 7 rats). A square-root transformation of the data showed significantly higher seizure frequencies (p < 0.05, Student-Newman-Keuls test) at 4 to 7 months compared with 1 month after the onset of seizures (asterisks). Data include animals with 4 to 8 months of chronic seizures; values are mean and standard error of the mean. (Reprinted with permission from Hellier, J.L., Patrylo, P.R., Buckmaster, P.S., and Dudek, F.E. 1998. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 31:73–84, Figure 3, p. 78.)
is based on the work of Racine (1972). As discussed previously, seizures are ranked as class I to V; for most of our previous work, only class III, IV, and V seizures were defined as motor seizures. In rats with kainate-induced epilepsy, most motor seizures last for 1 to 3 minutes, but they can occur in clusters of several seizures per hour for many hours. As these clusters of seizures become more prolonged, it is
possible that they become more damaging than isolated seizures. We have also observed that some rats with kainateinduced epilepsy spontaneously undergo convulsive status epilepticus. Thus the kainate model of chronic epilepsy has seizures that include spontaneous nonconvulsive seizures, prolonged clusters of convulsive seizures, and spontaneous status epilepticus, all characteristics seen in humans.
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FIGURE 3 The relative lack of spontaneous motor seizures during the first week after kainate treatment. A: Continuous video monitoring (24 hours/day for 7 days) showed that 81% of kainate-treated rats did not have any spontaneous motor seizures during the first week after treatment, but the remaining 19% were observed to have one or more behavioral seizures. B: Two of five rats had one or two motor seizures within the first 27 hours after kainite treatment. C: The remaining three rats had their first motor seizure 5 to 7 days after treatment. (Reprinted with permission from Hellier, J.L., Patrylo, P.R., Dou, P., Nett, M., Rose, G., and Dudek, F.E. 1999. Assessment of inhibition and epileptiform activity in the septal dentate gyrus of freely-behaving rats during the first week after kainate treatment. J Neurosci 19:10053–10064, Figure 3, p. 10056.)
Neuropathology Neuron Loss and Reactive Gliosis The early work of Ben-Ari (Ben-Ari and Lagowska, 1978; Nitecka et al., 1984; Tremblay et al., 1984; see BenAri, 1985) and Nadler (Nadler et al., 1978; see Nadler, 1981) provided the background for the concept that kainate injections lead to a pattern of neuronal degeneration and gliosis that is similar to mesial temporal sclerosis. These workers reported that the loss of neurons in the hilus and CA3 area (but also in CA1) and associated gliosis is similar to alterations seen in tissue from patients with TLE. This earlier research (e.g., Nadler et al., 1978) also suggested that area CA3 is preferentially damaged compared with area CA1, which probably reflects the higher distribution of hippocampal kainate receptors in CA3 and is a difference from typical hippocampal sclerosis in humans, where CA1 is usually more damaged than CA3. The dentate gyrus and the CA2 area are relatively spared (as in humans). In the unilateral intrahippocampal kainate model, granule cell dispersion has been seen in the dentate gyrus (Bouilleret et al., 1999; Gouder et al., 2003; Suzuki et al., 1995), which is often observed in mesial TLE with hippocampal sclerosis (Wieser et al., 2004). Neuronal loss in the hilus and elsewhere in the hippocampus is generally greater in the temporal pole than in the septal pole of the hippocampus (Figure 4) (Buckmaster and Dudek, 1997a, 1997b), which is reminiscent of the damage to the anterior hippocampus in the human. Particu-
larly after systemic kainate treatments, neuronal damage can occur in many areas in addition to the hippocampus and amygdala, which are traditionally thought to be important in TLE. However, Margerison and Corsellis (1966) clearly pointed out the variability in neuronal loss across patients who died after being diagnosed and institutionalized with TLE; Wieser et al. (2004) cite similar findings. In particular, Margerison and Corsellis (1966) reported that damage was widespread throughout the brain, including areas such as the thalamus and cerebellum. Furthermore, some individuals with classic TLE had little or no neuronal loss in the hilus; in fact, 13% (7 of the 55 cases) had no discernible damage anywhere in the brain. The work of Margerison and Corsellis (1966) “emphasized the need to see beyond the temporal lobes and to take into account the possible importance of damage to other parts of the brain as well.” It could be argued that Margerison and Corsellis (1966) included patients who did not have true TLE based on current diagnosis standards. However, in another study, hippocampal sclerosis was not detected in 10 of 64 surgically removed hippocampi from patients with diagnosed and surgically cured mesial TLE (Williamson et al., 1993). Although tissue from 3 of the 10 had abnormal neurons or heterotopias, the remaining 7 (11%) had mild gliosis or the hippocampal tissue was normal. Thus, although the surgically removed hippocampii were important (in some way) for generating the seizures, hippocampal sclerosis was not required. Given these data with respect to animal models, it is important to remember that although the kainate model replicates
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FIGURE 4 Nissl staining of hilar neuron loss in a control rat and a rat with kainate-induced epilepsy. Sections near the septal pole of the hippocampus show some loss of dentate gyrus hilar neurons in a kainate-treated rat (B) compared with the control (A). Near the temporal pole, hilar neuron loss in the kainate-treated rat was severe (D) compared to control (C). m, molecular layer; g, granule layer; h, hilus; CA3, CA3 pyramidal cell layer. Bar = 500 mm. (Reprinted with permission from Buckmaster, P., and Dudek, F.E. 1997. Neuron loss, granule cell reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J Comp Neurol 385:385–404, Figure 1, p. 388.)
hippocampal sclerosis with respect to the neuronal loss and mossy-fiber sprouting, this alone does not make it an allencompassing model of human TLE, given the patients who have the disorder but do not have hippocampal sclerosis. An added issue in regard to the kainate model (and other rodent models) relating to human TLE is the substantial differences in the overall neuroanatomic organization of the human and rodent brains, which in turn could be expected to lead to differences in the pattern of histopathological injury observed in rodent models versus the actual human condition of TLE. Plasticity (Sprouting, and So On) In addition to a pattern of neuronal death and reactive gliosis in chronically epileptic tissue, other changes involving neurogenesis, the formation of new synaptic circuits, and the alteration of neurotransmitter receptors and ion channels are consequences of brain injury and a potential basis for chronic epileptogenesis. Considerable interest and controversy have centered on mossy-fiber sprouting, which was initially described as the presence of Timm’s stain in the inner molecular layer of the dentate gyrus in kainate-treated rats (Figure 5) (Nadler et al., 1980; see also Buckmaster, 2004; Dudek and Shao, 2004; Dudek et al., 2002; Nadler, 2003) and in tissue from humans with intractable TLE (Babb et al., 1991; de Lanerolle et al., 1989; Houser et al., 1990;
Sutula et al., 1989). Tissue from animals with spontaneous recurrent seizures after kainate treatment shows a timedependent increase in Timm’s stain in the inner molecular layer. Intracellular staining of dentate granule cells from several laboratories (e.g., Buckmaster and Dudek, 1997a, 1997b, 1999) have shown that the increased Timm’s stain represents the formation of new axonal collaterals in the inner molecular layer. Much research has been directed toward this phenomenon, primarily because many workers have considered the dentate gyrus to be important for epilepsy (e.g., Lothman et al., 1992; Sloviter, 1994). Also, Timm’s stain has focused attention on the dentate, although similar mechanisms could occur throughout the temporal lobe and other structures. Several recent studies provided evidence that axonal sprouting and the formation of recurrent excitatory circuits also occurs in CA1 pyramidal cells (e.g., Esclapez et al., 1999; Meier and Dudek, 1996; Perez et al., 1996; Shao and Dudek, 2004; Smith and Dudek, 2001). Thus rats with kainate-induced epilepsy, similar to other status epilepticus-based models, show axon sprouting and synaptic reorganization that likely occurs in many temporal lobe structures of animal models and humans. Imaging and Metabolic Changes MRI microscopy has been applied to fixed brains from kainate- and domoic acid–treated rats. Large cortical lesions
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FIGURE 5 Timm’s staining revealed more dark labeling in the granule cell layer and in the inner third of the molecular layer in kainate-treated rats. A: Hippocampal tissue from a control rat had little Timm’s stain (with light cresyl violet counterstaining) in the granule cell and inner molecular layers. B–E: Sections of the middle region of the septotemporal axis of the hippocampus from rats with kainate-induced epilepsy displayed different degrees of abnormal Timm’s staining in the granule cell layer (B the least, E the most). Arrows indicate regions of Timm’s staining in the inner molecular layer. M, molecular layer; g, granule layer; h, hilus; CA3, CA3 pyramidal cell layer. Bar = 500 mm. (Reprinted with permission from: Buckmaster, P. and Dudek, F.E. (1997) Neuron loss, granule cell reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J Comp Neurol 385:385–404, Figure 7, p. 395.)
were observed 12 hours after treatment, and lesion progression was detected at 24 and 72 hours, including damage to the hippocampus, amygdala, and pyriform cortex (Lester et al., 1999). Similar changes were observed in a long-term study of rats after kainate-induced status epilepticus (Pirttilä et al., 2001). Using magnetic resonance spectroscopic imaging (MRSI) with N-acetylaspartate in vivo, neuronal loss in the hippocampus, amygdala, and pyriform cortex was observed 3 days after kainate-induced status epilepticus (Ebisu et al., 1994). Similar findings were reported in an intra-amygdala kainate model in dogs using several MRI methods (Hasegawa et al., 2003). Histopathologic data cor-
roborated the changes in these studies. Nairismägi and coworkers (2004) followed changes in vivo after electrically induced status epilepticus for up to 4.5 months using multiparametric MRI. Overall this research suggests that MRI imaging methods will be useful for long-term monitoring during kainate-induced epileptogenesis. Genetic and Molecular Changes Seizures and status epilepticus produce many changes in the brain that occur on different time frames and in different areas; thus the potential for complexity in gene
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expression is enormous. For example, in an early study using differential cDNA cloning after kainate treatment, 52 cDNAs were identified as changed in the dentate gyrus alone, and the candidate plasticity-related genes were estimated to range from 500 to 1000 (Nedivi et al., 1993). Another study identified 362 up-regulated candidate plasticity-related genes and 41 down-regulated transcripts after kainate treatment (Hevroni et al., 1998). The challenge will be to identify those changes in gene expression that are necessary for epileptogenesis, which will require carefully selected controls to identify the critical factors (e.g., Goodenough et al., 1997). Some of the most-studied genes affected by seizures include immediate-early genes and growth factors. Immediate-early genes were some of the first identified as induced by seizures, and these changes were seen in several different animal models of epilepsy (for review, see Morgan and Curran, 1991). Gene induction after kainate-induced status epilepticus includes c-fos, fos B, jun B, and egr-1, fra-2 and c-jun; the last two may be more specific to damaged areas (Goodenough et al., 1997). Growth factor expression is also changed, including increases in mRNA levels for brainderived growth factor and nerve growth factor and decreases (or no change) in neurotrophin-3 (for review, see Gall, 1993). Many of the gene changes produced by kainate are also seen in other seizure models, and those changes found to be common to the different chronic models may help focus future research on epileptogenic mechanisms, rather than on toxin-specific responses.
Neurophysiology: Mechanisms of Epileptogenesis The use of animal models of TLE, such as the kainatetreated rat, has progressed over the last two decades in parallel with the development of increasingly sophisticated electrophysiologic analyses that use in vitro preparations (particularly hippocampal and neocortical slices) and chronic recordings from intact and freely behaving animals. The kainate-treated rat and comparable models have allowed investigators to examine detailed cellular and network mechanisms associated with and potentially causal for epileptogenesis by comparing in vitro electrophysiologic data from rats with kainate-induced epilepsy to salinetreated controls. The ability to have control tissue in the animal models is of course important in reference to human epilepsy because virtually all neurophysiologic experiments on human tissue have less-than-perfect controls, and some human slice experiments have no control tissue. Electrophysiologic experiments on slices from kainate-treated rats have focused on many different hypothetical mechanisms at molecular, cellular, and integrative levels; these include but are not limited to decreases in synaptic inhibition, increases in excitatory synaptic mechanisms (including new recurrent
excitatory circuits), and alterations of transmitter receptors and ion channels in many brain structures. All the research on these neurophysiologic issues obviously hinges on a comparison between an animal model that recapitulates some hypothesized component of human epilepsy and an appropriate control. To investigate and test a specific hypothesis about an actual mechanism (e.g., Is synaptic inhibition decreased, and if so, what is the mechanism? Does axon sprouting lead to new recurrent excitatory circuits?), not only does one need to make a valid comparison with a control, but one must usually use a combination of neurophysiologic methods and preparations to isolate and analyze one specific component of a mechanism from others (e.g., the loss of one class of interneurons). The challenge for the future will be to combine and integrate in vitro experiments on brain slices and acutely isolated neurons with other more intact preparations, including freely behaving animals with actual behavioral and electrographic seizures. The kainatetreated rat is one of the preparations—but only one—that has in the past (and presumably will in the future) allowed us to perform experiments that simply cannot be done ethically and practically with humans and their tissue. Our laboratory has studied several potential electrophysiologic mechanisms using animal models that are based on status epilepticus, but most of our research has focused on the hypothesis that epileptogenesis involves axon sprouting and the formation of new recurrent excitatory circuits. Our studies were initiated because (1) a series of histopathological reports on the dentate gyrus from patients with mesial TLE and hippocampal sclerosis showed Timm’s stain in the inner molecular layer, and (2) Tauck and Nadler (1985) developed preliminary electrophysiologic evidence supporting the hypothesis that this Timm’s staining pattern in the dentate gyrus reflects sprouting of the mossy-fiber axons and the formation of new recurrent excitatory circuits. Many electrophysiologic studies from several laboratories using brain slices support the hypothesis that these connections are functional and are excitatory (see Dudek et al., 2002; Dudek and Shao, 2003; Nadler, 2003), although in vivo electrophysiology studies with field potentials in anesthetized kainate-treated rats have argued that the new mossy-fiber collaterals synapse on dormant interneurons rather than other granule cells (Sloviter, 1992). The kainate-treated rat and other status epilepticus-based models allow rigorous quantitative experiments using whole-cell patch-clamp techniques in brain slices, which in turn permit experimental separation with pharmacologic techniques of gluatamatergic excitatory networks from g-aminobutyric (GABA)ergic inhibitory circuits so that one can study the respective synaptic currents of these two general mechanisms in isolation from each other. It is conceivable, however, that mossyfiber sprouting in rats with kainate-induced epilepsy differs from humans with mesial TLE and hippocampal sclerosis, but these experiments on the actual mechanism are difficult
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in human tissue (although see Gabriel et al., 2004). For this issue and many others, research on both human tissue and animal models may yield insights that would not be possible with either one alone. The work on mossy-fiber sprouting in animal models and human tissue has led to research on the question of whether this hypothetical cellular mechanism of epileptogenesis occurs in other brain areas in both human TLE and in animal models. The issue of the electrophysiologic consequences of mossy-fiber sprouting and its potential contribution to epileptogenesis is confounded by the differentiation between the potential importance of the cellular mechanism of axon sprouting and new recurrent excitatory circuits in any part of the brain (e.g., the CA1 area of the hippocampus) versus the importance of the dentate gyrus per se (i.e., the dentate has many other alterations that may occur along with axonal sprouting during epileptogenesis, such as loss of vulnerable interneurons and altered NMDA receptors). Rats with kainate- and pilocarpine-induced epilepsy have been used extensively to investigate this hypothesis. Several studies have shown epileptiform activity in the isolated CA1 area of these models, and this has been associated with
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increased axonal collaterals of CA1 pyramidal cells (e.g., Esclapez et al., 1999; Meier and Dudek, 1996; Perez et al., 1996; Smith and Dudek, 2001). It has been possible to differentiate the hypothetical mechanism of loss of inhibitory interneurons from the hypothetical mechanism of increased recurrent excitation by comparing the slices from normal control animals to those from rats with kainate- or pilocarpine-induced epilepsy after GABA-mediated inhibition was blocked pharmacologically (e.g., Smith and Dudek, 2001, 2002). By stimulating CA1 pyramidal cells in a relatively selective manner with focal flash photolysis of caged glutamate, the relative density of local excitatory circuits could be assessed without potential contamination by electrical stimulation of fibers of passage (Shao and Dudek, 2004). Using photostimulation via caged glutamate of CA1 pyramidal cells in hippocampal mini-slices containing only the CA1 area of rats with kainate-induced epilepsy versus age-matched saline-treated controls, it has been possible to provide relatively direct evidence for enhanced recurrent excitation in the CA1 area (Figure 6). Further studies with other techniques (e.g., dual whole-cell recordings) in the kainate-treated rat, other models, and human tissue will
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50 pA 200 ms FIGURE 6 Evidence for local excitatory connections in the CA1 area revealed by focal flash photolysis of caged glutamate in a slice from a rat with kainate-induced epilepsy. A: Diagram of the experimental protocol. Recordings were obtained from CA1 pyramidal cells (triangles) in an isolated mini-slice, and flash stimulations were presented at 150 to 200 mm intervals (circles) along the cell body layer. B: Repetitive flash stimulations (arrows, three to five flashes per site at 20-second intervals) in a slice from a kainate-treated rat consistently evoked a train of excitatory postsynaptic currents (EPSCs) in a CA1 pyramidal cell at two of five sites (sites 2 and 3 in A, 400 and 600 mm from the recorded CA1 pyramidal cell, respectively). Flashes at the other locations (1, 4, 5) did not evoke EPSCs (B1). (Reprinted with permission from Shao, L.-R., and Dudek, F.E. 2004. Increased excitatory synaptic activity and local connectivity of hippocampal CA1 pyramidal cells in rats with kainate-induced epilepsy. J Neurophysiol 92:1366–1373, Figure 5, p. 1370.)
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allow this cellular hypothesis about synaptic reorganization to be tested more directly in other areas besides the dentate gyrus. Using animal models and human tissue, anatomic staining of axonal pathways has led to evidence for the hypothesis that not only the dentate gyrus and the CA1 area but also other areas of the hippocampus undergo profound synaptic reorganization during epileptogenesis (Lehmann et al., 2000, 2001). The different animal models that are based on status epilepticus together may contribute to our understanding of human TLE because the models embody the variability seen in humans and also because common data across models suggest that a particular experimental result is not model specific and possibly reflects a fundamental mechanism of epileptogenesis that would also apply to the human brain. Electrophysiologic experiments with brain slices are useful for identifying cellular mechanisms, but they do not allow one to record actual epileptic seizures in the context of a typical animal or human. Seizures have been recorded with surface EEG or intrahippocampal electrodes in awake and freely moving rodents with kainate-induced epilepsy (e.g., Bouilleret et al., 1999; Bragin et al., 1999). Relatively little quantitative information, however, is available about the time course of development of interictal spikes and electrographic seizure activity in specific areas of the temporal lobe, and there is an obvious need to relate this type of in vivo electrographic data to results from in vitro preparations. A long-term goal with all chronic models, including the kainate-treated rat, is also to integrate results from anatomic studies (i.e., using histology as well as in vitro and in vivo imaging) with the in vitro and in vivo electrophsyiologic results.
testing of AEDs on spontaneous recurrent seizures in animals with chronic epilepsy. We have used both pilocarpine- and kainate-treated rats to test AEDs (Grabenstatter et al., 2005; Hernandez et al., 2000). Although we limit our discussion to rats with kainate-induced epilepsy, our comments presumably apply equally well to other chronic models. Among the possible problems encountered when testing AEDs in animals with spontaneous seizures are (1) the considerable variability in seizure frequency in different animals that have received apparently similar treatments and (2) the potential that the seizure frequency of an individual animal will gradually increase (or decrease) with time. One way to control for this variability is to design AED testing paradigms that allow animals to serve as their own controls while limiting interanimal variance. We have used a repeated-measures crossover design to study the effects of topiramate on spontaneous convulsive seizures (Grabenstatter et al., 2005). Topiramate reduced the frequency of spontaneous recurrent seizures in a dose-dependent manner (Figure 7), but it did not completely block them, even at high doses. Additional studies are required before one can conclude that rats with kainate-induced epilepsy are pharmacoresistant to topiramate because these studies were based only on the effects of single injections; thus it will be important to test more prolonged administration periods and to conduct studies with both behavioral and electrophysiologic analyses (i.e., chronic EEG recordings). Hypothetically, a new AED that is capable of blocking all convulsive seizures in the kainate model of TLE would be a potential treatment
Response to and Usefulness in Screening Antiepileptic Drugs As discussed, a recent NIH-sponsored workshop proposed that models using chronically epileptic animals might be advantageous for studying acquired epileptogenesis and pharmacoresistant seizures. The rationale is that if epileptogenesis involves receptors, channels, or circuits that are altered, it might be possible to develop drugs that target these abnormal mechanisms without disrupting normal processes. Support for this concept came from the study of Löscher and Hönack (1993), which reported that levetiracetam was not effective in traditional models (e.g., maximal electroshock, where seizures are evoked in normal mice), but it was effective in amygdala-kindled rats, a chronic model of partial limbic epilepsy. It was also acknowledged in the report from the NIH workshop that this hypothesis requires further testing (Stables et al., 2003), but the group’s consensus was that such studies should be undertaken. Although the chemoconvulsant models (e.g., kainate and pilocarpine) are costly and labor intensive, they allow
FIGURE 7 Dose-dependent effect of topiramate on the frequency of spontaneous seizures in rats with kainate-induced epilepsy. A repeatedmeasures analysis of variance (ANOVA) revealed that topiramate had significant effects (p < 0.05) relative to saline at the concentrations of 10 (p = 0.02), 30 (p < 0.0001), and 100 (p < 0.0001) mg/kg. (Reprinted with permission from: Grabenstatter, H.L., Ferraro, D.J., Williams, P.A., Chapman, P.L., and Dudek, F.E. 2005. Use of chronic epilepsy models in antiepileptic drug discovery: the effect of topiramate on spontaneous motor seizures in rats with kainate-induced epilepsy. Epilepsia, 2005, Figure 4.)
Insights Into Human Disorders
for patients with intractable epilepsy. Similar studies have been done on a potential new AED (RWJ333369) in the kainate model, and this drug was significantly more effective than topiramate (Grabenstatter et al., 2004).
LIMITATIONS: EASE OF PREPARATION, MORTALITY, REPRODUCIBILITY AND IMPROVEMENTS This chapter has focused on the repeated low-dose model of kainate-induced epileptogenesis because of its relative ease of preparation, low mortality, and good reliability. It allows consistent generation of chronically epileptic rats that possess many of the characteristics of mesial TLE (e.g., spontaneous recurrent seizures after a latent period and mesial temporal sclerosis). Similar to other chronic animal models based on induction of convulsive status epilepticus, the kainate model shows considerable variability in regard to the onset, frequency, and severity of the spontaneous recurrent seizures. Although one could argue that this reduces the validity of the model, considerable variability with respect to the seizures and neuropathology also exists in humans diagnosed with TLE (see Margerison and Corsellis, 1966; Wieser et al., 2004). Like the other models based on status epilepticus, it would be useful to know precisely how variations in the treatment protocol and the response of the animal to the treatment protocol affect the epileptogenic process. Although our laboratory has focused most of its research on the kainate model, we do not believe it has inherent scientific advantages over other models based on status epilepticus in terms of suitability for answering questions about acquired epilepsy. It appears that prolonged convulsive status epilepticus, regardless of how it is induced, can lead to a chronic epileptic state that has many similarities to TLE, including complex partial seizures and secondary generalization to tonic-clonic seizures. More studies, including a pharmacologic profile, are necessary to elucidate the clinical seizure type characterized by kainate-induced epilepsy.
INSIGHTS INTO HUMAN DISORDERS Underlying Mechanisms Studies from rats with kainate-induced epilepsy have provided information and insights into potential mechanisms of acquired epilepsy. Probably one of the most valuable aspects of this and the other models will be the identification of mechanisms that are common across models and thus would seem more likely to be associated with the fundamental mechanisms of epileptogenesis, as opposed to the effects of a specific toxin or stimulation protocol. One theme that does
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seem to emerge from studies of these models is that many potential cellular mechanisms are operative in many different structures. For example, the loss of some, but certainly not all, GABAergic interneurons during and potentially after status epilepticus has been seen in several models and structures, and thus seems likely to apply to human TLE. This result of a limited and selective loss of interneurons is in spite of the many different observations in which synaptically activated “inhibition” is increased, decreased, or unaffected. Axonal sprouting and new recurrent excitatory circuits have been found in several models and brain structures after various forms of injury, but it is unclear precisely how these reorganized circuits may contribute to epileptic seizures. Similarly, as discussed, synaptic reorganization of inhibitory circuits has been proposed to occur in several animal models and structures and may serve as a restorative mechanism. It is difficult if not experimentally impossible to identify and analyze these mechanisms without at least partially isolating the mechanism under investigation from other mechanisms that might confound an interpretation. At the same time, understanding the interactions of different hypothetical mechanisms during generation and propagation of spontaneous seizures will be an important goal that will require use of animal models such as the kainate-treated rat. The degree to which these different mechanisms contribute to the chronic epileptogenesis that occurs in kainate-treated rats versus other models will require further study; the degree to which this approach and these animal models will help us to understand human epilepsy will depend on the results of a coordinated analysis across these models and human patients with the disorder. Many analyses of tissue from patients with epilepsy (particularly from surgical tissue) and several subsequent hypotheses of epileptogenesis have focused on neuronal loss, including loss of interneurons and synaptic input to interneurons. Obviously the pattern and cause of neuronal loss are critical issues in epilepsy, and this information may provide clues to underlying mechanisms. For research on human tissue from autopsy cases and surgical resections, however, patients have nearly always had seizures for prolonged periods, and it is likely that one consequence of the long-standing epilepsy is additional neuronal death. Animal models, such as the kainate-treated rat, may allow an assessment of the time-dependent changes that are not possible in humans (other than with brain-imaging techniques). It is noteworthy that Margerison and Corsellis (1966) emphasized that some cases had no detectable neuronal damage. Similarly, although quantitative data are not available, most workers that have used both the pilocarpine and kainate models would agree that the former has more neuronal death in many areas (e.g., hilus, CA1 and elsewhere) but not necessarily more seizures. Thus one can speculate that other mechanisms may be more important in epileptogenesis.
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Experiments on animal models have spanned many different preparations, techniques, and conceptual levels, and several issues must be considered that may not be so obvious. One important consideration in any study on chronic epileptogenesis using the kainate model (or others) is the size and severity of the brain insult and the time after the insult; the available data suggest that these variables can have a huge impact on the outcome of experiments. Another critical issue is the level of analysis in relation to whether experiments deal more with the phenomenology of the preparation versus an actual experimental delineation of possible mechanisms that may be altered. This then leads to the differentiation between studying “hyperexcitability” (which may or may not underlie increased seizure susceptibility) versus the relatively unknown dynamic process by which seizures are actually initiated. Finally, decades of in vitro experiments on seizure models (as opposed to models of epilepsy) and more recent studies on transgenic mice suggest that many mechanisms can be altered to generate seizures, and thus chronic epilepsy after a brain injury may involve many different mechanisms, many with synergistic effects. It is possible, therefore, that a host of different physiologic mechanisms may contribute to the increased seizure susceptibility that arises after brain injury.
Usefulness for Treatment Assessment/Development/Screening The potential value of the kainate model for testing AEDs has been briefly described herein. The possibility that this and other status epilepticus-based models are pharmacoresistant to conventional AEDs deserves investigation, but this will be a major undertaking. Rats with kainate-induced epilepsy are potentially also valuable for conducting studies on strategies for blocking epileptogenesis. Again, it is not clear that the kainate-treated rat has any particular scientific value over other models with spontaneous recurrent seizures for these types of studies. For some experiments, it would be useful for all of these studies to know that ultimately all or nearly all of the animals will consistently develop chronic epilepsy. If a model has a highly variable fraction of animals that become epileptic with spontaneous recurrent seizures, it would be difficult to test antiepileptogenic treatments and to perform other studies concerning therapeutic approaches to epilepsy. Using the protocol we have outlined here, virtually every rat that experiences prolonged status epilepticus associated with kainate treatment develops chronic epilepsy that manifests as spontaneous recurrent seizures. In conclusion, the kainate model appears to parallel human mesial TLE with respect to several factors, including (1) many aspects of neuropathology (cell loss, sprouting, and granule cell dispersion); (2) seizure type (including seizures that could be considered complex partial seizures); (3) areas involved in seizure generation (e.g., the hippo-
campus); (4) and a clear pattern of epileptogenesis with an initial insult followed by a latent period, although shorter than in humans, to the eventual development of recurrent spontaneous seizures and then a progression that involves increased seizure frequency. Most of these parallels, however, are also seen in the other status epilepticus–based models. The extra effort and expense involved in using these chronic models will be justified if they provide unique insights into the acquired epilepsies that lead to an antiepileptogenic therapy or to better AEDs for pharmacoresistant patients. It is hoped that this chapter will help others to understand some of the advantages and disadvantages of using rats with kainate-induced epilepsy in their research programs.
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35 The Pilocarpine Model of Seizures ESPER A. CAVALHEIRO, MARIA G. NAFFAH-MAZZACORATTI, LUIZ E. MELLO, AND JOÃO P. LEITE
episode of SE as well as to a model of chronic spontaneous seizures that take place thereafter. Acute induction is characterized by long-lasting limbic SE associated with sustained electrographic discharges in limbic structures (Turski et al., 1983a, 1984). In these preparations, several parameters have been used to assess chronic epileptogenicity after the acute induction, such as thresholds for fluorothylinduced seizures, kindling facilitation, and response to paired electrical pulses. However, the best criterion for the epileptic condition is the occurrence of actual spontaneous seizures. As in temporal lobe epilepsy (TLE), in patients with prolonged febrile convulsions, spontaneous recurrent seizures occur after a seizure-free interval or apparently normal behavior and electrographic activity (Cavalheiro et al., 1991; Leite et al., 1990). Accordingly, the phenomenologic descriptions and pathological changes after SEinduced damage are critical for understanding the process of epileptogenesis.
GENERAL DESCRIPTION AND BACKGROUND The importance of cholinergic mechanisms for epilepsy was already hinted at by neurologists at the turn of the nineteenth century (Langley, 1901; Langley and Kato, 1915). The convulsant potential of acetylcholine and physostigmine was experimentally demonstrated in the electrocorticogram as early as the late 1930s (Brenner and Merrit, 1942; Chatfiled and Dempsey, 1943; Miller et al., 1938; Sjörtrand, 1937). In the following years, the use of acetylcholine mainly for in vitro preparations contributed to the understanding of electrophysiologic phenomena involved in epileptic spike discharges, neuronal depolarization, and burst generation (Benardo and Prince, 1982; Ferguson and Jasper, 1971; Krnejevic et al., 1970; McCormick and Prince, 1986). Finally in the 1980s it was demonstrated that periodic administration of carbachol effectively induces kindling (Wasterlain and Jonec, 1983) and that administration of high doses of pilocarpine may lead to the acute induction of status epilepticus (SE) and to the late onset of spontaneous seizures (Turski et al., 1983a, 1984). The pilocarpine model of epilepsy, as it is now called, thus emerged shortly after the kainate model (Ben-Ari et al., 1979) and currently represents the kindling model, one of the three most widely used animal models of epilepsy (Figure 1). Indeed, over the years, the particular features of the model, such as a large frequency of spontaneous recurrent seizures and the robust supragranular mossy-fiber sprouting, have made important contributions to its wider acceptance and use. The pilocarpine model of epilepsy relates both to a model of acute induced seizures culminating with a prolonged
Models of Seizures and Epilepsy
NATURAL HISTORY The administration of pilocarpine or lithium-pilocarpine to rodents induces ictal and interictal epileptic activity in hippocampal and cortical electrographic recordings, which are correlated with a sequence of behavioral alterations that includes akinesia, ataxic lurching, and facial automatisms, progressing to motor seizures and SE. After a single application of these drugs, SE can last from 6 to 12 hours. After spontaneous remission of SE, animals are comatose and both hippocampal and cortical recordings are depressed with high-voltage spiking activity. These events have been called the acute period of these models, and metabolic studies
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FIGURE 1 Number of published articles per 5-year period in the Institute for Scientific Information base using key words: MES and seizure; pilocarpine and seizure; kindl* and seizure; kain* and seizure. Note that from 1990 through 1994, the number of published papers using the MES model is almost the same as that using the pilocarpine model. This is in marked contrast with the number of published papers in the most recent 5year period. MES, maximal electroshock; Kindl, kindling; KA, kainic acid.
performed during this period have revealed increased glucose utilization, mainly in the hippocampus and other limbic structures, thalamus, and substantia nigra. The pattern of neuronal loss observed in these animals matches closely with the areas that are metabolically activated during SE. The similarity of cytopathology following pilocarpine and lithium-pilocarpine seizures to the tissue reaction to glutamate does not mean that the cholinergic system has excitotoxic potential. It is rather believed that the cholinergic system may play a role in triggering and maintaining seizure activity. An excessive synaptic release of glutamate leads rapidly to depolarization block and breakdown of membranes. A synaptic release of acetylcholine provides a sustained drive that keeps neurons firing (as in SE) without breaking down the membrane. Such a hypothesis sees the function of the cholinergic system in the maintenance of sustained seizure activity that drives excitatory mechanisms responsible for neuronal damage (e.g., glutamate/aspartate). Accordingly, experiments have shown that anticholinergic drugs could be active in prevention of sustained seizures but could not be useful for seizure arrest. Animals that survive the acute period of SE proceed to a latent “seizure-free” period with an apparently normal behavior except for some aggressiveness on manipulation and toward other rats if they are maintained in groups. This period lasts 1 to 8 weeks, depending on animal strain and method used to induce SE, and it ends with the occurrence of the first spontaneous seizure. The recurrence of spontaneous seizures during the “chronic period” may vary according to the model, ranging from one seizure per month to several seizures per day.
The evolution of spontaneous seizures follows the behavioral and electrographic stages of kindling. Although this aspect has been better characterized for the pilocarpine model, the progression from an initial brief limbic event to a more generalized motor seizure has also been described for the lithium-pilocarpine models. In most Wistar rats subjected to the pilocarpine model, the first seizure is normally characterized by paroxysmal discharges localized in the hippocampal or amygdala without changes in cortical recordings. Behavioral correlates of this seizure comprise an arrest reaction followed by eye blinking, chewing, and head nodding resembling kindling stages 1 or 2. Subsequent seizures show gradual synchronization of cortical and hippocampal records and longer duration of ictal events. Clonus of the forelimbs and rearing with falling as in kindling stages 4 or 5 are the hallmarks of such seizures. However, the progression of spontaneous seizures is variable in terms of the duration of paroxysmal discharges and the sequence of behavioral stages in every individual animal. Several seizure stages of similar or lesser intensity can be observed before a generalized motor convulsion is reached. Once the generalized convulsion similar to stage 5 kindled seizure occurs, the vast majority of the seizures that follow are also generalized. These observations made in the pilocarpine model of epilepsy suggest that the development of epilepsy as consequence of SE follows the stages of kindling.
METHODS OF GENERATION The acute model was originally described in rats (Turski et al., 1983a) and then in mice (Turski et al., 1984). Pretreatment of the animals with scopolamine prevented the development of convulsive activity caused by pilocarpine (Turski et al., 1983b). Later studies clearly demonstrated that activation of m1 muscarinic receptors is critical for the induction of SE by means of the systemic administration of pilocarpine (Hamilton et al., 1997). Pilocarpine effectively produces acute seizures in Wistar (Turski et al., 1983a) and Sprague-Dawley (Mello et al., 1993) rats. The initial characterization of the model in Swiss mice (Cavalheiro et al., 1996; Turski et al., 1984) was later followed by its description in C57Bl/6 (Borges et al., 2003; Shibley and Smith, 2002), CD1 (Shibley and Smith, 2002), CF1 (Borges et al., 2003), kinin B1 and B2 receptor knockout mice, and 129SV background strain (Aragañaraz et al., 2003). Administration to a rodent species, Proechimys sp., was also shown to be effective at triggering acute seizures (Fabene et al., 2001). Finally, systemic administration to the common marmoset (Callithrix jachus) also can promote acute seizures and SE (Mello et al., unpublished observations). Intracerebral administration of a number of cholinergic agents, most notably carbachol and bethanechol, has long been used to induce acute seizures (Turski et al., 1983b,
Methods of Generation
1983c; Wasterlain and Jonec, 1980). Pilocarpine can trigger acute seizures after intracerebroventricular (Croiset and De Wied, 1992) and intrahippocampal administration (Millan et al., 1992). In addition, the intrahippocampal administration of pilocarpine in rats is able to trigger spontaneous recurrent seizures in some of the injected animals and supragranular mossy-fiber sprouting ipsilateral to the injection in all animals (Furtado et al., 2002). Regarding age specificity, the pilocarpine model can be induced in rats starting from postnatal day (PN) 11 (Cavalheiro et al., 1987) to animals of approximately 12 months of age (Mello et al., unpublished observations). Administration of pilocarpine to rats from PN3 to PN6 leads to only minor electroencephalographic (EEG) changes characterized mostly by flattening of the background activity and occasional low-amplitude, low-frequency spikes with no clear seizure behavior (Cavalheiro et al., 1987). In 7- to 10day-old rats, more frequent spikes with greater amplitude could be seen in both cortex and hippocampus, together with infrequent spike and wave complexes but still without clear motor seizures. Intense behavioral signs evolving in some animals to limbic seizures and SE occurred when pilocarpine was administered to animals older than 11 days. Animals ranging from 15 to 21 days of age had the greatest sensitivity to pilocarpine-induced SE, which developed with a shorter latency and more often resulted in death compared with adult animals. The pattern of neuronal loss and ensuing plastic changes, however, is markedly less than that seen in adult animals (Cavalheiro et al., 1987). In contrast, induction of multiple episodes of SE in 7- to 9-day-old rat pups results in important plastic changes, leading to long-lasting epileptogenesis, as manifested by electrographic epileptiform discharges, behavioral deficits, and in vitro hyperexcitability of hippocampal networks (Santos et al., 2000). As for gender, most studies have been conducted with males, but female rats at both young ages (starting at PN7) or adult ages (usually 2 to 3 months of age) can have acute seizures induced by pilocarpine (Amado and Cavalheiro, 1998; Cavalheiro et al., 1987; Valente et al., 2002). Young animals usually have greater susceptibility to the acute induction of seizures by pilocarpine, often requiring doses 30% smaller than that used for adult animals. In contrast, young animals are less vulnerable to the insults caused by pilocarpine-induced SE as well as to the associated plastic changes (Cavalheiro et al., 1987; Santos et al., 2000). Susceptibility to the convulsant effects of pilocarpine increases during adult life: the threshold dose for generalized seizures is essentially constant from age 30 to 70 days, but it decreases thereafter, particularly beyond age 100 days (Patel et al., 1988). Older (and thus heavier) rats not only require a smaller dose of pilocarpine to develop SE, but they also have a greater mortality rate. Indeed, pilocarpinetriggered seizures in adult animals often have an intense tonic episode followed by respiratory arrest and death, even
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when produced by small doses of pilocarpine. In this sense, for experiments aimed at studying the functional and morphologic consequences of pilocarpine-induced SE, the ideal age range for rats is between 2 and 3 months of age at the time of status induction. Induction of SE with pilocarpine in female rats leads to an increased diestrus, suggesting an anovulatory estrous cycle and an inadequate luteal phase (Amado and Cavalheiro, 1998). In addition, pregnant and lactating rats show a clear effect of the hormonal changes that characterize such conditions: these animals have less frequent spontaneous seizures (Amado and Cavalheiro, 1998). As described, the susceptibility of different animals to pilocarpine-induced seizures is influenced by a number of different features. Indeed, even for the same animal species, strain, gender, and age (in this case 2- to 3-month-old male Wistar rats), the dose required to induce SE in a significant number of animals has ranged from 400 to 320 mg/kg (Mello and Mendez-Otero, 1996; Turksi et al., 1983a). Therefore, for anyone using the model, it might be interesting initially to assess the sensitivity of the animal batch. Original descriptions of the model already include the subcutaneous administration of methylscopolamine at the dose of 1 mg/kg to reduce the peripheral effects associated with the autonomic activation caused by pilocarpine. The remaining issues associated with the induction of SE depend on the intended use of the model. In this sense, the use of 400 mg/kg of pilocarpine will likely result in a 100% chance of induction of severe SE associated with a high mortality rate in the few hours after status onset. In addition, depending on the animal species and strain, this high dose may result in a number of animals having severe tonic seizures followed by respiratory arrest as a very early seizure manifestation. Seizures may result in less severe seizures associated with a greater survival rate with the reduction of the injected dose. This, however, results in a smaller number of the animals developing SE and thus with a less efficient use of the animals because often a greater initial number of animals will be required to achieve a given set of animals with SE per group. This side effect can be used to generate a control group that, despite being injected with pilocarpine, does not develop SE and thus may more effectively allow assessment of the effects associated with status induction as opposed to the effects associated with the mere administration of pilocarpine (Cavalheiro et al., 1991; Mello et al., 1993, 1996). For studies aimed at inducing SE in a great percentage of the animals and with a high long-term survival rate, the best strategy is to titrate the administered dose of pilocarpine (Longo et al., 2003). In such a case, administration of a pilocarpine dose yielding SE in about 40% of the animals is monitored for animals that do not develop clear seizures, with subsequent administration of 30% of the initial dose within 1 hour after initial administration. This procedure
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may be further continued for one or two additional doses and thereafter may produce results that are not as effective, with the remaining animals probably constituting a category of resistant animals. In the pilocarpine model, induction of SE is critical for the later development of spontaneous seizures. Pilocarpineinduced SE, however, is usually associated with a high mortality rate in the initial 3-day period. The mortality rate is effectively reduced by controlling the severity of the status episode. On the other hand, achieving complete control (remission) of SE in the first hour after its onset results in a large number of animals not developing spontaneous seizures (Lemos and Cavalheiro, 1995). A good survival rate associated with the development of epilepsy (i.e., spontaneous seizures) in all animals can be accomplished by titrating the severity of the SE (Mello et al., 1993). This can be accomplished, usually after 90 minutes of free-running status, by using various drugs effective at diminishing without actually abolishing it. Thus diazepam at a dose of 5 mg/kg or thionembutal at a dose of 25 mg/kg would serve as good alternatives for this purpose. Therefore both the dose used for the induction of SE and the duration of this episode should be carefully monitored to yield the greatest frequency of animals developing intense status episodes and surviving it to have animals with spontaneous epileptic seizures. In the hours after status induction, animals often become comatose and unable to care for themselves. Experiments requiring animals to survive 12 hours or longer after status induction usually require the animals to remain (and not return to the vivarium) in the laboratory overnight. Administration of saline, glucose, and electrolytes in general by the subcutaneous or IP route can be an alternative to minimize the loss of animals during this period. On the following day, some animals are already alert and able to care for themselves, whereas others are still in poor condition. For this latter group, hand feeding is an alternative that can have good results with the use of fruit juices mixed with sweetened milk and mashed rat-food pellets. In addition, leaving fruit slices in the floor cage might also be effective for faster recovery of the animals. For rats subjected to free-running SE with no use of antistatus medication, the chance of eventually developing spontaneous seizures is 100% (Cavalheiro et al., 1991; Leite et al., 1990). Controlling status 90 minutes after its onset as described already also has the same rate of efficacy for the development of spontaneous seizures (Mello et al., 1993). Earlier control of status, initiated 90 minutes before its induction, or its more complete control, achieved with higher doses of antistatus medication, are likely to affect the percentage of animals developing spontaneous seizures (Lemos and Cavalheiro, 1995). Once developed, spontaneous seizures do not remit for the animal’s life (Mello et al., 1993).
BEHAVIORAL AND CLINICAL FEATURES The sequential pattern of electrographic changes during the acute phase immediately after injection of pilocarpine is characterized by a significant theta rhythm that replaces the background activity in the hippocampus and low-voltage fast activity in the cortex. This activity progresses to highvoltage fast activity with spikes in the hippocampus. The spiking activity spreads to the cortex and evolves into electrographic seizures. Ictal periods recur every 3 to 5 minutes and finally lead to sustained discharges 50 to 60 minutes after the injection of pilocarpine (Turski et al., 1987a). This pattern of electrographic activity lasts for several hours and may evolve to a pattern of periodic discharges on a relatively flat background, very similar to the pattern described by Walton and Treiman in the lithium-pilocarpine model (Walton and Treiman, 1988). Within 24 to 72 hours afterward, the EEG is characterized by gradual normalization of hippocampal and cortical rhythms associated with spiking activity that predominates in the hippocampus and decreases in the subsequent days (Leite et al., 1990). The first spontaneous seizures are characterized by paroxysmal activity localized in the hippocampus without relevant changes in cortical recordings. During these episodes, animals are akinetic and present staring spells and eye blinking. These seizures are similar to stages 1 and 2 of the Racine scale for amygdala-kindled seizures (Racine, 1972). Subsequent seizures tend to be more prolonged and spread to the cortical electrodes. Therefore behavioral and electrographic features of these limbic seizures go on in a progression that somewhat resembles the evolution of kindling (Cavalheiro et al., 1991). Seizure frequency in the chronic period may vary considerably among epileptic rats, and several seizure patterns have been observed. Some pilocarpine-injected rats may present a low seizure frequency throughout several weeks or months; others may have daily seizures, and some may present clusters of seizures in short periods. Such variability in seizure frequency patterns may represent a drawback for behavioral or antiepileptic drug studies (AEDs). To assemble a homogeneous group, baseline monitoring and identification of rats with regular seizure frequency are necessary. It is important to stress that most behavioral or AED studies rely on video monitoring to establish seizure frequency. Therefore, class 3 to 5 limbic seizures are preferentially detected, and oligosymptomatic seizures (class 1 and 2) are frequently overlooked, which may be relevant because intractable complex partial seizures in humans, the equivalent of class 1 and 2 limbic seizures in rats, rarely generalize, even when AEDs are tapered during video EEG monitoring. High-resolution video capturing coupled with EEG is necessary to detect subtle behavioral seizures
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Neuropathology
correlated with, for example, focal ictal activity in the hippocampus or amygdala (Leite et al., 2002).
RESPONSE TO ANTIEPILEPTIC DRUGS AND THE USEFULNESS OF SCREENING TESTS Several drugs have proved to suppress or attenuate pilocarpine-induced SE efficiently when injected either before or right after pilocarpine administration. For this reason, benzodiazepines and barbiturates have been widely used to prevent the relatively high mortality associated with prolonged seizures (Leite and Cavalheiro, 1995; Mello et al., 1993). Attenuation of SE by both drugs after different duration times indicates that early SE suppression can prevent spontaneous recurrent seizures. In addition, neuron loss is prevented if treatment is established with 30 minutes of SE (Lemos and Cavalheiro, 1995). When administered before pilocarpine, clonazepam (90.35 mg/kg), phenobarbital (23.4 mg/kg), and valproic acid (286 mg/kg) prevent limbic seizures and protect against seizure-related neuron damage. Pretreatment with trimethadione (179 mg/kg) results in moderate protection against pilocarpine-induced seizures, whereas diphenylhydantoin (10–200 mg/kg) and carbamazepine (10–50 mg/kg) have no effect and do not prevent pilocarpine-induced brain damage (Turski et al., 1987b). The noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist MK-801, when given 20 minutes before pilocarpine (4 mg/kg) administration, has no effect in blocking the onset of pilocarpine-induced SE; however, MK-801 treatment prevents later spontaneous recurrent seizures and protects against CA1 pyramidal cells loss (Rice and DeLorenzo, 1998). Ketamine, another noncompetitive NMDA receptor antagonist, at a dose of 100 mg/kg IP, exerts a time-dependent protection of hippocampal cells and prevents late spontaneous seizures and special motor deficits in the Morris water maze (Hort et al., 1999). Drug testing in the latent period has a fundamental importance because several molecular changes occur in this phase and may contribute to the process of epileptogenesis (Loscher, 1998). In this scenario, a compound with efficacy of inhibiting the development of spontaneous seizures may be considered a true AED. A growing body of evidence suggests that there are differences between the mechanisms underlying the development of epileptogenesis and the mechanisms involved in maintaining the expression of seizures after they have established. Hence it is reasonable to assume that compounds that prevent epileptogenesis may be quite different from classic AEDs drugs (Leite et al., 2002; Loscher, 1998; Shinnar and Berg, 1996). Therefore drugs that regulate the expression of trophic factors involved in neuroprotection or plastic changes induced by damage may also have an antiepileptogenic effect.
Antiepileptic drugs that have efficacy to suppress pilocarpine-induced SE are not the same as those that are effective in controlling spontaneous limbic seizures. Diazepam, phenobarbital, valproic acid, and trimethadione protect against pilocarpine-induced SE, whereas phenytoin and carbamazepine are ineffective. In the chronic phase, carbamazepine and phenytoin are effective against spontaneous seizures. Valproic acid is also effective at large doses (600 mg/kg) and ethosuximide does not prevent spontaneous seizures (Leite and Cavalheiro, 1995).
NEUROPATHOLOGY Induction of status epilepticus by pilocarpine leads to severe widespread cell loss in several brain areas (Turski et al., 1983, 1984). Dying cells can be assessed via a number of different techniques that characterize a given biochemical or structural aspect of the degenerating cells. Differences in the biochemical and morphologic profile of dying cells may indicate whether the cell is suffering from an apoptotic or a necrotic degenerating process. As with a number of different pathological conditions, cell damage in the pilocarpine model has also been described in terms of its necrotic or apoptotic nature (Covolan et al., 2000a; Fujikawa et al., 1999; Turski et al., 1984). Rather, as recently indicated (Sloviter, 2002), classifying cell damage under these two categories might actually confuse and will therefore will be avoided here. A more fruitful perspective is to consider that the excitotoxic insult triggered by pilocarpine-induced SE leads both to immediate cell damage that takes place minutes to a few hours after its onset, but it also results in a protracted process of neurodegeneration that may take weeks or months to develop. The initial damage occurring a few hours after status onset is most intense in the superficial layers of some neocortical areas, hilus, endopiriform nucleus, piriform cortex, and claustrum (Covolan and Mello, 2000). After 8 hours of status onset, damage has further intensified in those areas and it also reaches the entorhinal cortex (where it was already present but to a lesser extent), amygdaloid nuclei, ventromedial nucleus of the hypothalamus, subiculum, and bed nucleus of the stria terminalis. Overall damage occurs in several distinct brain areas and has a specific time course in each different area (Covolan and Mello, 2000). Damage, however, is not restricted to the initial hours and days after status onset and tends progressively to involve other areas in the following months. To this end, damage to the thalamus is often found in animals killed many months after the onset of pilocarpine-induced SE, but it is notably less intense or even absent in some nuclei at shorter survival times (Covolan and Mello, 2000). In addition to cell loss, there is clear injury that results in both morphologic and functional pathology. Cell injury
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might be the result of damage that it is not intense enough to kill a given cell but is sufficient to cause protracted altered function. Evidence of altered cell morphology, probably as a result of cell injury, in the pilocarpine model has been provided mostly for the hippocampus (Covolan and Mello, 1996; Hamani et al., 1999; Isokawa and Mello, 1991; Longo et al., 2003). Altered distribution of dendritic spines in dentate granule cells (Isokawa and Mello, 1991) and distorted dendritic trees in putative g-aminobutyric acid (GABA)-ergic hippocampal interneurons (Hamani et al., 1999) are some of these changes. Additional morphologic changes are more likely to be reactive rather than a direct consequence of the initial insult. In this sense the emergence of axonal sprouting, the most notable being the supragranular mossy-fiber sprouting (Buckmaster et al., 2002; Mello et al., 1993; Okazaki et al., 1995), granule-cell dispersion (Mello et al., 1992), increased rate of neurogenesis (Covolan et al., 2000b; Parent et al., 1997), and granule-cell basal dendrites (Spigelman et al., 1998) characterize morphologic changes that are likely to represent a reactive response from the organism. As with any other lesional model, pilocarpine-induced SE does not uniformly damage different cell groups. Here again, so far this has been best studied in the hippocampus. Thus although damage is greater to principal cells, that is, the pyramidal and granule cells in the hippocampal complex, interneurons located in the other strata can also be damaged. Of these, most notably hilar mossy cells can be markedly damaged by pilocarpine-induced SE but with large variation in the extent of damage between different animals (Longo et al., 2003; Silva and Mello, 2000; see also Sloviter et al., 2004). Damage to GABAergic neurons is also extensive throughout the hippocampus (Obenaus et al., 1993). However, not all GABAergic neurons in the hippocampal complex are equally vulnerable, and specific populations in different strata have different loss rates (Hamani et al., 1999). In another study, however, the density of GAD65 mRNA-positive neuron profiles in layer III of the entorhinal cortex was similar in control and post-SE rats evaluated between 3 and 7 days after pilocarpine administration (Kobayashi et al., 2003). Similar evaluations so far have not been provided at the same extent to other brain areas, with the exception of a qualitative assessment of the neocortex (Silva et al., 2002). Glial pathology in the pilocarpine model has not received the same level of attention as neuronal pathology. Nevertheless, there have been descriptions regarding the proliferation of astrocytes, as shown by the increased expression of glial fibrillary acidic protein (GFAP) (Schmidt-Kastner and Ingvar, 1996) and other glial markers (Garzillo and Mello, 2002). In addition to glial proliferation, in the CA1 area of the hippocampal complex of animals subjected to pilocarpine-induced SE, glial cells adapt to permit rather large increases in extracellular potassium accumulation (Heine-
mann et al., 2000). Microglia and other markers of inflammatory tissue reaction have also shown to be present in the early phases after pilocarpine-induced SE.
CHEMICAL ALTERATIONS Partial or complex seizures, the main characteristic of TLE, have been related to important brain impact as well as to the eventual evolution of this syndrome. Thus different investigators have demonstrated that long-lasting seizures unchain a complex chemical cascade, triggering neurochemical alteration in neurons and glial cells. These immediate- or long-lasting events can modify the cellular environment through changes of ionic gradient across the cell membrane, alteration of gene expression (such as receptors, trophic factors, enzymes, proteins from cytoskeleton, protein from matrix), and phosphorylation of macromolecules. Furthermore, seizures can induce reactive gliosis, generated by cell death, induced by these long-lasting convulsions. These modifications promote synaptic remodeling, which can change the excitability of neurons from temporal structures, leading to the appearance of brain damage and a permanent hyperexcitability. Unfortunately, TLE is not an easily understandable brain dysfunction. The neurochemical alteration found in the brain of experimental animals as well as in human brain show high degree of complexity.
Neurotransmission The hippocampal formation seems to bee an important structure in TLE, and several researchers have reported neurochemical alterations in this structure. The hippocampus of rats submitted to the epilepsy model induced by pilocarpine shows increased utilization rate of norepinephrine (NE) and decreased utilization rate of dopamine during the acute, silent, and chronic period of this model. As reported, the utilization rate of serotonin was increased in only the acute phase (Cavalheiro et al., 1994). The NE depletion has been associated with increased seizure susceptibility, and NE release may be related to protection against seizure spread or initiation (Mason and Corcoran, 1979; MacIntyre and Edson, 1982; Mason and Corcoran, 1979). In contrast, in seizures induced by lithium plus pilocarpine, dopamine D2 antagonist reduces the threshold for convulsion and D1 antagonist prevents the convulsive activity, showing that dopaminergic receptors exert an opposite function on the regulation of convulsive activity in this model (Barone et al., 1991). According to Radley and Jacobs (2003), pilocarpineinduced SE increases cell proliferation in the dentate gyrus of adult rats via 5HT1A receptor-dependent mechanism because blockade of this receptor prevents the seizure-
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induced enhancement neurogenesis. However, this treatment did not blockade the development of mossy-fiber sprouting or the appearance of spontaneous seizures in the epilepsy model induced by pilocarpine. On the other hand, intrahippocampal infusion of DA and 5HT via microdialysis probe, in determined concentration, protected the brain from seizures induced by pilocarpine (Clinckers et al., 2004). Taken together, these dada associate monoaminergic pathways with the development of TLE. Concerning aminoacidergic neurotransmission, the acute phase of pilocarpine model was characterized by an increased glutamate release in the hippocampus (Cavalheiro et al., 1994; Costa et al., 2004). Hippocampal synaptosomes from animals presenting long-lasting SE (12 hours) still showed increased release of glutamate. However, the uptake of this amino acid is normal in animals presenting 12 hours of SE (Costa et al., 2004), suggesting an excitatory phenomenon, during the acute phase of pilocarpine model. In addition, Ormandy et al. (1989) have showed that MK-801, a noncompetitive NMDA receptor antagonist, produces an effective and dose-dependent anticonvulsant action on lithium-pilocarpine model, suggesting that activation of NMDA receptor plays an important role in SE and brain damage. Using microdialysis probes, Smolders et al. (1997) described a long-lasting increase in the release of glutamate in the rat hippocampus, also supporting the excitatory phenomenon. Indeed, when glutamate activates NMDA receptors, the intracellular Ca2+ raises inducing activation of lipases, proteases, and nucleases, killing the cell by necrosis or apoptosis or both (Melloni et al., 1989). The expression of proteins related to NMDA receptor is also modified in the pilocarpine model of epilepsy. Mint1 or X11 alpha plays an important role in vesicle synaptic transport toward the active zone at presynaptic site and also participates in the transport of NR2B subunit of NMDA receptor at the postsynaptic site. According to Scorza et al. (2003), this protein, which is expressed mainly in CA1 regions of control animals, had decreased levels 5 hours after SE onset and increased levels during the silent and chronic groups, suggesting that this protein is related to plasticity during epileptogenesis. In addition, Smolders et al. (2004) reported anticonvulsant effects of metabotropic glutamate receptors (mGlu1 and mGlu5) antagonist in this model and other investigators also showed increased expression of mGluR2/3 in stratum lacunosum moleculare of CA1 and dentate gyrus, 1 day after pilocarpine-induced SE (Tang et al., 2004). Funke et al. (2003) also found increased expression of mGluR1 in all hippocampal formation during the acute and silent periods, showing the participation of other glutamate receptors in excitatory phenomenon of TLE. According to Khan et al. (1999), the anticonvulsant action of diazepam against pilocarpine-induced seizures is associated with a prompt attenuation of extracellular hippocampal glutamate overflow,
without concurrent alteration of pilocarpine-induced increases in endogenous GABA levels. The silent phase of pilocarpine model is marked by an important unbalance between inhibition and excitation (Cavalheiro et al., 1994). The decreased concentration of GABA in the hippocampus during the silent period could suggest an increased release of this amino acid in attempt to control the tissue excitability. In contrast, the increased concentration of glutamate in the hippocampus could suggest a potential excitatory pathway of this structure, probably responsive for the appearance of spontaneous seizures. According to Silva et al. (2002), a diffuse decrease of parvalbumin, isoform 65 of glutamic acid decarboxylase (GAD65), and GABA transporter (GAT1) in the sensorimotor cortex pointed to specific neocortical disturbance in GABAergic inhibition, which could play a crucial role in seizure generation and expression on pilocarpine-treated animals. In addition, the alpha-5 subunit of GABAA receptor is also capable of substantial, prolonged down-regulation in pyramidal neurons from hippocampal formation of pilocarpine-treated animals (Houser et al., 2003). Thus, according to several authors, TLE has been related to excessive excitability in limbic structures, low function of inhibitory pathways, or the association between both events (Meldrum, 1991). Other pathways are also related to seizure control. Several lines of evidences suggest that cannabinoid compounds are anticonvulsant. The main extract of marijuana, the delta-9-tetrahydrocannabinol as well as cannabimimetics, completely abolished spontaneous seizures in the chronic phase of pilocarpine-induced epilepsy (Wallace et al., 2003). These investigators also suggest that endogenous cannabinoid tone modulates seizure termination and duration through activation of CB1 receptors, which was significantly increased throughout CA region of epileptic hippocampi. Other signaling pathway associated to seizure control is related to opioid receptors. Indeed, naloxone reduced the number of spikes in slices from epileptic rats and alfentanil induces epileptiform abnormalities in the hippocampal and cortical EEG of pilocarpine-treated rats. The potent action of alfentanil inducing epileptiform activity suggests a proconvulsive action of m-receptor opioids (Vieira et al., 2004).
Transduction Signal As consequence of neurotransmission alteration, the transduction signal through plasma membrane is also modified, changing neuronal metabolism and genes expression. Several studies have shown that short- or long-lasting seizures can modify the expression of some genes, mainly the transcription factors, such as c-fos and jun-B, c-Jun, and Jun D, which are related to genome control. The detection of c-fos, the immediate-early gene product, is thought to
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reflect, at least in part, in acute metabolic activation of specific brain regions. In addition, increased expression of c-fos was found in the hippocampus of rats submitted to longlasting seizures, induced by pilocarpine (Barone et al., 1993). According to Backer et al., (2003) a high number of differentially expressed genes (dentate gyrus, 400 genes; and CA1, 700 genes) was observed 3 days after SE induced by pilocarpine. Most of these up-regulated genes were associated with mechanisms of cellular stress and injury. Fourteen days after SE, numerous transcription factors and genes linked to cytoskeletal and synaptic reorganization were differentially expressed, and during the chronic phase, genes involved in various neurotransmission pathways were found differentially expressed in the hippocampus of these animals. In addition, Mudò et al. (1996) showed increased expression of brain-derived neurotrophic factor (BDNF), neuronal growth factor (NGF), and their receptors in the dentate gyrus 3 hours after pilocarpine-induced SE. Activation of growth factor receptors induces the autophosphorylation of these receptors and activation of different kinase proteins, including the phosphorylation of proteins on tyrosine residues, which are important in cell cycle and intracellular signaling mechanisms. These phosphotyrosine proteins (PTyP) of different molecular weights have been found to be increased in the hippocampus of rats during the early stages of pilocarpine-induced SE (Funke et al., 1998; Jope et al., 1991), showing that several intracellular events could undergo modifications during long-lasting seizures, mainly in CA3 region. Carnevalli et al. (2004) also found extensive phosphorylation of alpha subunit of translation initiation factor 2 (eIF2-alpha) in mice submitted to SE induced by pilocarpine, showing that the synthesis of proteins is promptly modified in the hippocampus of these animals. Results emerged from intracerebral infusions of BDNF (Larmet et al., 1995; Scharfman et al., 2002) and from transgenic mice overexpressing BDNF showed enhanced response of these animals to epileptogenic stimuli (Croll et al., 1999). According to Poulsen et al. (2002), organotypic hippocampal slices exposed to 5 mM pilocarpine for up to 7 days displayed increased BDNF expression, which is correlated with increased neuropeptide Y immunoreactivity, known to accompany seizure activity. Pilocarpine-treated animals also exhibited increased immunoreactivity against neuropeptide Y in regions of mossy-fiber terminals, in the dentate gyrus inner molecular layer, entorhinal cortex, amygdala and sensorimotor areas (Lurton and Cavalheiro, 1997). The increased expression of growth factors is also related to mitogen-activated protein kinase (MAPK) activation. After binding an agonist, trk receptors phosphorylate themselves on cytoplasmic domains on tyrosine residues, which become docking sites for intracellular signaling proteins. The adaptor proteins associate themselves with a specific
site in trk receptors, activating a signaling pathway involving Ras, Raf, MAPK1, MAPK2, Mek 1, and Mek 2. As consequence, the transcription factors and the regulation of gene expression are modified. As reported by Garrido et al. (1998), several limbic structures showed increased levels as well as increased phosphorylation of MAPKs (ERK1 and ERK2), which are important during the induction of the de novo synthesis of several proteins. According to He et al. (2002), trk-B undergoes phosphorylation in the mossy-fiber pathway and CA3 stratum oriens of the hippocampus during epileptogenesis. Other intracellular signaling pathways may also be modified during epileptogenesis. Levels of the neuromodulin or growth-associated phosphoprotein (B-50 or GAP-43), which is activated by protein kinase C (PKC), are modified in the hippocampus of rats in the pilocarpine epilepsy model. GAP-43 has been related to processes underlying cell proliferation in the fetal human brain and is correlated specifically with differentiation and outgrowth of axons (Kanazir et al., 1996). The levels of this protein increased in the inner molecular layer of the dentate gyrus (regions associated with the mossy-fiber sprouting), during the acute, silent, and chronic periods, in rats submitted to pilocarpine-induced epilepsy (Naffah Mazzacoratti et al., 1999). According to several researchers, the GAP-43 activation may be also induced by glutamate, acting on the NMDA receptor, because blockade of this receptor by MK801 prevents the GAP-43 expression as well as the mossy-fiber sprouting (McNamara and Routtenberg, 1995). In addition, Tang and colleagues (2004) showed expression of different isoforms of PKC in the rat hippocampus after pilocarpine-induced SE, mainly in CA1 and the dentate gyrus. Stimulation of cholinergic muscarinic receptors by pilocarpine activates the phospholipase C to effect hydrolysis of inositol-containing phospholipids. This hydrolysis results in the formation of two active second messengers: lipidsoluble diacylglycerol (DG) and water-soluble inositol 1-45 triphosphate (IP3). IP3 diffuses into the cell and increases free intracellular calcium concentration by releasing this ion from nonmitochondrial intracellular stores (Berridge and Irvine, 1989). IP3 is sequentially dephosphorylated to inositol monophosphate and inositol and used in the resynthesis of phospholipids. Hirvonen and co-workers (1993) found decreased cerebral inositol and increased inositol-1phosphate, showing increased turnover of this phospholipid in the hippocampus of pilocarpine-induced epilepsy. The subsequent excessive brain activation, characterized by prolonged amplification of brain PI turnover, may result in a widespread neuronal injury. In addition, glutamate-induced simultaneous facilitation in PI signaling may also contribute to the production of reactive oxygen species, exceeding the neutralizing capacity of the neuronal glutathione, resulting in an oxidative stress-driven neuronal injury (Hirvonen et al., 1993).
Chemical Alterations
Inflammatory Mediators in Epileptogenesis During long-lasting seizures activation of inflammatory processes may occur. Reactive gliosis, such as astrocytes, and microglia appears as tardy form (Niquet et al., 1994; Represa et al., 1995). As reported by Garzillo and Mello (2002), 60 days (chronic phase) after pilocarpine-induced SE, prominent astrocytes still could be seen in different brain areas. The activated microglia has been blamed as the source of the main inflammatory cytokines. De Simoni et al. (2000) described increased expression of mRNA for IL-1b, IL-6, iNOS, and tumor necrosis factor (TNF)a after seizures induced by electrical stimulation in the dorsal hippocampus. These factors remain increased 60 days after the insult. The glial scar, generally epileptogenic, is able to produces trophic factors, which will support the axonal sprouting and other anatomic substrates for maintenance of hyperexcitability (Represa et al., 1995). Another pathway that is involved in the inflammatory processes is linked to prostaglandin (PG) release. These eicosanoids are produced after the action of phospholipase A2 on phospholipids, the release of arachidonic acid, which could be done by action of glutamate on NMDA receptor (Pellerin and Wolf, 1991). In addition, Naffah-Mazzacoratti and colleagues (1995) showed an increased release of prostaglandin PGF2a during the acute phase; PGD2 during the acute, silent, and chronic period; and PGE2 only during the chronic phase of the epilepsy model induced by pilocarpine. According to Bazan (1989), in epileptic tissues, a backlog of these eicosanoids, which are released by neuronal and glial cells, occurs, increasing the inflammatory processes. During PG formation, free radicals are produced, increasing the inflammatory process. The free radicals are chemical entities produced during intermediary metabolism presenting an unpaired electron and for this reason show high reactivity. They are released during the mitochondrial transport chain, monoamine degradation, xanthine oxidase activity, and by the metabolism of arachidonic acid, which, when released in tissues, are capable of inducing membrane damage and cell death. Thus, during SE induced by lithium plus pilocarpine, hypermetabolism occurs—including increased glucose consumption, which results in abnormal respiratory chain and neuronal damage (Fernandes at al., 1999). Against free radicals, the tissues present enzymes such as superoxide dismutase (SOD) and glutathione peroxidase, which are able to remove the superoxide anion (O2-) or H2O2, considered potent oxidant agents. As reported by Bellissimo et al. (2001) rats presenting SE or spontaneous seizures showed decreased activity of SOD and increased levels of hydroperoxides (produces lipid peroxidation) in the hippocampus of animals submitted to the pilocarpine model of epilepsy. Because the brain is more vulnerable than other
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tissues, the decreased activity of SOD could be related to cell death and brain damage found in the hippocampus of these animals. To follow the spatial and temporal evolution of neuronal damage, cellular activation and stress response subsequent to lithium-pilocarpine seizures of various duration in adult rat. Motte et al. (1998) analyzed the expression of fos protein and glucose utilization as maskers of cellular activation, heat-shock protein (HSP72) immunoreactivity, and acid fuchsin staining as indicator of cell stress and injury. According to these investigators, regions with the highest expression of fos and largest metabolic activation were also highly stained with acid fuchsin and were most heavily damaged. Other compounds related to vessel dilatation, with consequence rupture of the blood-brain barrier, edema, pain, and inflammatory processes, are the kinins. These polypeptides are produced after proteolysis has limited the action of kallikreins on high- and low-molecular-weight kininogens. These short-lived peptides are rapidly degraded by kininases (Bhoola et al., 1992), originating active metabolites such as des-Arg9BK, des-Arg10, kallidin, and inactive products. The receptors are denominated B1 and B2, and both are coupled to G proteins. As reported, B2 receptor presents high affinity for bradykinin (BK) or Lys-BK (kallidin) and low affinity for the active metabolites des-Arg9BK and des-Arg10kallidin. In contrast, B1 receptor presents high affinity for des-Arg9BK and des-Arg10kallidin and less affinity for BK or Lys-BK (Menke et al., 1994; Regoli and Barabe, 1980). Kinin B1 receptor agonists led to inositol phosphate generation, promoting a transient rise in intracellular Ca2 + levels after phospholipase C activation (Liao and Homcy, 1993). Furthermore, stimulation of kinin B1 and B2 receptors induces tissue edema and phospholipase A2 activation, producing prostaglandins (Bhoola et al., 1992). Kinin B1 and B2 stimulation also activates MAPK (ERK1/ERK2) in cell culture, resulting in AP-1 translocation, modifying the immediate early gene expression (Naraba et al., 1999). Usually kinin B1 receptor is not expressed at a significant level under physiologic conditions in most tissues, but its expression is induced by injury or upon exposure in vivo or in vitro to proinflammatory mediators, such as lipopolysaccharide and cytokines (Marceau, 1995). Moreover, Ni and colleagues (1998) showed evidence that nuclear factor kB (NF-kB) is also involved in the dynamic regulation of human kinin B1 receptor gene expression during inflammatory processes. In contrast, kinin B2 receptor is constitutively and widely expressed in all nervous systems (Calixto et al., 2000) and has been found in the nucleus of neurons from hippocampus, hypothalamus, and cortex (Chen et al., 2000). Nevertheless, the real function of this receptor in neuronal nucleus is still unknown.
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Bregola et al. in 1999 showed that endogenous kinin B1 agonist Lys-des Arg9 BK increases the glutamate overflow in kindled rats slices (40%–50%) and, to a smaller extent (20%), in slices of kainate-treated animals, supporting the idea that kinin B1 receptor may play a role in TLE excitotoxicity. These investigators also suggest that the relationship between Lys-desArg9 BK and glutamate release is not a mere consequence of seizures, but it is associated with a condition of latent hyperexcitability found in epileptic tissues. In addition, Ongali et al. (2003) showed a significant decline of kinin B2 receptor binding sites, accompanied by an impressive increase of kinin B1 receptor binding site labeling in the brains of rats submitted to the kindling model of epilepsy. Studying the distribution of kinin B1 and B2 receptors and the expression of mRNA by real-time polymerase chain reaction (PCR) of these receptors during the development of the epilepsy model induced by pilocarpine, Argañaraz et al. (2004) found increased kinin B1 and B2 mRNA levels during the acute, silent, and chronic periods and changes in kinin B1 receptor distribution. In addition, the immunoreactivity against kinin B1 receptors was increased mainly during the silent period, where clusters of cells could be visualized, suggesting a local inflammation. The kinin B2 receptor immunoreactivity also showed augmentation but mainly during the acute and silent periods, supporting the hypothesis that both kinin receptors are related to TLE. Trying to understand the role of kinin B1 and B2 receptors in the physiopathology of TLE, we developed the epilepsy model induced by pilocarpine in B1 and B2 knockout mice (B1KO and B2KO, respectively); behavior parameters, cell death, and mossy-fiber sprouting were analyzed. B1KO mice showed increased latency for the first seizure, associated with a decreased frequency of spontaneous seizures (chronic phase) compared with their wild control mice. In addition, B1KO mice showed less cell death in all hippocampal formation associated to a minor grade of mossy-fiber sprouting compared with wild mice. Furthermore, B2KO mice presented minor duration of the silent period and an increased frequency of spontaneous seizures (chronic phase) compared with wild mice. B2KO and wild mice showed a similar pattern of cell death in the hippocampus, which was very intense compared with salinetreated animals. The mossy-fiber sprouting was also increased in B2KO mice compared with wild mice and saline-treated animals. Taken together, these data suggest a deleterious effect for B1 receptor and a protective effect for B2 receptor during the development of TLE. As reported previously, activation of several receptors, such as kinin receptors, glutamate receptors, P2 purinergic receptors, and others, have a strong relationship with calcium influx or calcium mobilization into the cells of the nervous system. Using the fura-2 method, Vianna et al. (2002) found that after adenosine triphosphatase (ATP)
stimulation, a discrete increase of intracellular Ca2+ occurred in normal hippocampal slices. However, slices from epileptic animals, submitted to the pilocarpine model, showed a biphasic response, indicating the presence of P2X7 receptors in calcium mobilization Quantification of P2X7 receptors showed an increased expression of 80% in the hippocampal formation in animals during the chronic phase, mainly in the dentate gyrus, suggesting a participation of these receptors in mossy-fiber sprouting.
Activity of ATPases Among the mechanisms involved in regulation the cytosolic calcium are the Ca2+ ATPases, whose function is to restore the normal level of this ion into the cell. These Ca2+ ATPases constitute a class of proteins that falls into two distinct groups, termed SERCAs and PMCA, depending on whether they are inserted into endoplasmic reticulum or into plasma membrane. SERCAs sequester calcium to sarcoplasmic or endoplasmic reticulum, and SERCA2b is found in several brain structures. PMCAs promote the extrusion of this ion from neural cells through plasma membrane. According to Funke et al. (2003), in the hippocampus of rats submitted to the pilocarpine model of epilepsy, the expression of SERCA2b as well as the PMCA enzymes is increased after 1 hour of SE, showing an attempt to control the tissue excitability during the early stages of the insult. The PMCA remained increased until the silent period, returning to control levels during the chronic phase. In contrast, regions vulnerable to cell death, such as CA1, CA3, and hilus, presented decreased expression of SERCA2b until the silent period, showing a deficit in the mechanisms related to calcium removal. The activity of the Na+K+ ATPase is also modified in the hippocampus of pilocarpine-treated animals. According to Fernandes et al. (1996), this enzyme has its activity reduced during the acute and silent period and has increased activity during the chronic phase, showing that the hippocampus of these animals also shows an ionic imbalance related to its maintained excitability.
Matrix Components Metalloproteases (MMPs) from matrix are proteolytic enzymes necessary to model the cell medium, recycling receptors, and other types of extracellular proteins. They are released as pro-enzymes, have the ion zinc as a cofactor, and are inhibited endogenously by tissue inhibitors of metalloproteases (TIMPs). An imbalance between MMPs and TIMPs has been related to brain damage. According to Junqueira et al. (2003) the activity of MMPs and the TIMPs is modified in the hippocampus of pilocarpine-treated animals, relating these proteins to plasticity after injury. Other matrix components are the glycosaminoglycans and proteoglycans,
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Chemical Alterations
increasing the excitability of the brain (Segal et al., 1988). This increased excitability probably occurs as a result of decreased activity of ATPases in the hippocampus, which could not repolarize the plasma membrane and does not promote the calcium extrusion (Fernandes et al., 1996; Funke et al., 2003). The high concentration of Ca2+ promotes the release of glutamate, inducing SE (Klan et al., 1999; Smolders et al., 1997). Glutamate, acting on AMPA/KA receptors, allows the entrance of Na+ and Ca2+ into the cell; as a consequence, the Mg++, which blockades the NMDA receptor, is removed, inducing activation of this receptor by glutamate and allowing entrance of more Ca2+ into the postsynaptic cell, which will induce excitotoxicity and cell death. Tissue excitability or SE increases the utilization rate of noradrenaline (NA) and serotonin (5HT), with a concomitant decrease in the utilization rate of DA (Cavalheiro et al., 1994). After docking to its own receptors, these monoamines are degraded by MAO and COMT, and during these processes free radicals can be formed. These free radicals are also freed during glucose metabolism and mitochondrial
which are also modified in the brain of epileptic animals (Naffah-Mazzacoratti, 1999). The receptor protein tyrosine phosphatase b (RPTPb), a proteoglycan, related to the sprouting of axons, has been associated with mossy-fiber sprouting (Perosa et al., 2002). The RPTPb, expressed only by astrocytes in control tissues, has its synthesis increased in pyramidal neurons, in the hippocampus during the acute and silent phases, showing that SE can modify gene expression in epileptic rats. By analyzing all these results, we can see that an insult can modify several signaling pathways in the central nervous system. Figure 2 summarizes the main findings, from pilocarpine injection until the occurrence of plastics events and cellular death. Pilocarpine may act on M1 and M2 muscarinic receptors. By activating M2, the adenylate cyclase is inhibited, decreasing the release of acetylcholine and decreasing the neuronal excitation (Smolders, 1997). On the other hand, binding to M1, the pilocarpine activates the phospholipase C, producing diacylglycerol (DG) and inositol triphosphate (IP3), resulting in alteration in a Ca2+ and K+ current and
M2 -
GABA Ach neurite outgrowth neurogenesis + ?
MAPK
AC
CS
+
HS
?
GAGs PG
trophic factors
MAPK protection/apoptosis
excitability ?
+
ATPase Na+,K+
?
GLU- SE
SERCA PMCA
Mg++
mossy fiber sprouting
RPTPb ?
+
+
protection/ apoptosis
+
M1 IP3, DG Ca++ ,K+
PILOCARPINE
-
PTyP ? Synaptic plasticity
AMPA
B2
NMDA
-
+ Mint1
trk Des-Arg9-Bk
+
B1 inflammation cell death necrosis ?
synaptophysin
GAP 43 SOD HPx
metabolites
DA ? FR
death
? AA
PTyP SOD
FR
HPx
prostaglandins NA
death necrosis, apoptosis
+
seizures
[Ca++]i, [Na+]i
synaptogenesis
BK
5HT protection
PGE2
PGD2 PGF2a protection? lesion?
FIGURE 2 Main biochemical pathways and physiologic consequences involved in the pilocarpine model.
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Chapter 35/The Pilocarpine Model of Seizures
transport chain, which is overactivated during SE. In addition, the superoxide dismutase (SOD) shows decreased activity during seizures, associated with an increased level of hydroperoxide in the hippocampus of epileptic animals (Bellissimo et al., 2001), showing tissue damage and lipid peroxidation. Glutamate on NMDA receptors promotes increased expression of GAP-43, which is linked to mossy-fiber sprouting and hippocampal plasticity (McNamara et al., 1995; Naffah Mazzacoratti et al., 1999). During SE, the expression of trophic factors such as NGF, BDNF, and FGF (Mùdo et al., 1996) increases in the hippocampus, propitiating MAPK and PTyP activation (Funke et al., 1998; Garrido et al., 1998), inducing modification in gene expression. MAPK also may have protector action (Berkeley et al., 2002), or it can be related to apoptosis process (Wang et al., 2003). The trophic factor receptors are also associated with PGs from the extracellular matrix. These PGs sometimes function as coreceptors for neurotrophins (Ruoslahti and Yamaguchi, 1991). In this context, the increased synthesis of chondroitin sulphate and RPTPb (Naffah Mazzacoratti et al., 1999), as found in the hippocampus of epileptic animals, can be related to neurite outgrowth or mossy-fiber sprouting (Fernaud-Espinosa et al., 1994; Peles et al., 1998). In addition, the RPTPb also present phosphatase activity, removing the phosphate group from tyrosine residues in PTyP, modified during SE (Funke et al., 1998). The SE or the excess of glutamate in tissue can activate routes that culminate in the release of kinins, and these polypeptides may act on kinin B1 and B2 receptors, which are overexpressed in the hippocampus of epileptic animals (Argañaraz et al., 2004). The kinin B2 receptor has a protector role during epileptogenesis, whereas B1 is deleterious (Argañaraz et al., 2004). Bradykinin (BK) as well as monoamines also induce PG release (Bazan et al., 1996). As reported by Naffah Mazzacoratti et al. (1995), the levels of PGE2, PGD2, and PGF2a increase in the hippocampus of epileptic rats. During PG synthesis free radical production also occurs, which could be visualized by SOD and HPx analyses (Bellissimo et al., 2001). In addition, BK stimulates the MAPK pathway and binds to neurotrophin receptors (Fleming and Busse, 1997), perhaps mediating the phosphorylation of proteins on tyrosine residue (PTyP), changing the gene expression and contributing to plasticity found in the epileptic phenomena.
CONCLUSION The pilocarpine model of epilepsy has generated a significant contribution for our current understanding of the underlying basis of a number of physiologic and pathologi-
cal events. No model adequately mimics all aspects of the human epilepsies. Spontaneous seizures are one of the hallmarks of epilepsy, and the pilocarpine model is among the best models for generating such seizures. The steady increase in the use of this model, as measured by the number of published papers (see Figure 1), is a clear reflection of its wide acceptance by the scientific community.
Acknowledgments Financial support from FAPESP, CNPq, CAPES, and PRONEX (Brazil) over the last 20 years has allowed a substantial portion of the experiments reported herein to be performed. We also appreciate the significant contribution made over the years to the development of the pilocarpine model by a vast group of technicians and students.
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36 Status Epilepticus: Electrical Stimulation Models ANDREY M. MAZARATI, KERRY W. THOMPSON, LUCIE SUCHOMELOVA, RAMAN SANKAR, YUKIYOSHI SHIRASAKA, JARI NISSINEN, ASLA PITKÄNEN, EDWARD BERTRAM, AND CLAUDE G. WASTERLAIN
develop therapies that are truly antiepileptogenic (Lothman et al., 1990; Shirasaka et al., 1994; Mazarati et al., 2002a,b; Brandt et al., 2003; Nissinen et al., 2003). This chapter is restricted to models of SE based on electrical stimulation of the brain, and space permits a detailed view of only a few of those that have been used most extensively. Because models are just tools that permit us to ask specific questions, we have to keep in mind that there is no such thing as a good (or a bad) model, only models that are good for a particular purpose. We will emphasize the practical, “cookbook” aspect of the models we describe, and only briefly tackle their assets and limitations. A number of reviews (Coulter et al., 2002; Loscher, 2002; Pitkänen 2002; Morimoto et al., 2004; Pitkänen and Kubova, 2004) provide many of the features that are not treated in-depth here.
INTRODUCTION Recent models of status epilepticus (SE) have begun to closely mimic the human illness. Gastaut’s dictum that “there are as many types of status as there are types of seizures” is reflected in the many models of SE available today. The tendency of SE to become self-perpetuating, and the fact that it is more than a series of severe seizures were recognized as early as the nineteenth century (Trousseau, 1868), and are prominent features of many current chemical and electrical stimulation models of SE. In a few of those models, usually done under anesthesia, seizure response is tightly coupled with the epileptogenic stimulus (Sloviter, 1987; Shirasaka and Wasterlain, 1994; Penix et al., 1994; Gruenthal, 1998). In awake, free-running animals, however, SE tends to become self-sustaining and to continue for hours after the epileptogenic stimulus is withdrawn. This is true regardless of the nature of the seizure trigger, which can be chemical (Buterbaugh et al., 1986; Morrissett, et al. 1987; Suchomelova, et al. 2003) or electrical (Lothman et al., 1989; Mazarati et al., 1998; Nissinen et al., 2000; Gorter et al., 2002). This independence of continuing seizures from their initial trigger makes self-sustaining status epilepticus (SSSE) a convenient model for studying basic pathophysiologic mechanisms (Inoue et al., 1992a,b; Mazarati et al., 1998b,c, 1999a,b, 2000b; Liu et al., 1999a,b). Understanding which changes are critical for the transition from stimulus-bound seizures to SSSE may help us to understand at what point SE becomes intractable and brain-damaging, and how to prevent these consequences. SE-induced epileptogenesis is a common and widespread phenomenon in experimental animals, and has become the main tool in our attempts to go beyond symptomatic treatment and to
Models of Seizures and Epilepsy
ELECTRICAL STIMULATION MODELS OF STATUS EPILEPTICUS The first model of electrical stimulation-induced SE derived from a serendipitous observation: Electroencephalographic (EEG) monitoring of rats that were paralyzed, ventilated with oxygen and kept in good metabolic balance, and received one electroconvulsive shock every minute for >25 minutes, showed that EEG seizures continued after stimulation stopped. The severity and duration of self-sustaining seizures correlated with the duration of stimulation (Wasterlain, 1972, 1974). After 100 stimulations, self-sustaining seizures continued until the animal died. Later, the kindling phenomenon (Goddard et al., 1969) was modified by Taber et al. (1977), de Campos and Cavalheiro (1980), and Milgram et al. (1985), who changed stimulation
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paradigms to trigger SE. Along the same vein, McIntyre et al. (1982, 1986) showed that 60 minutes of high-frequency stimulation of basolateral amygdala in kindled animals induced SSSE in about 60% of animals. Buterbaugh et al. (1986) showed that small amounts of pilocarpine would also induce SSSE in kindled animals. Buterbaugh (1986) and Morrisset et al. (1987) showed that, in chemical models of SE, seizures also become self-sustaining and independent from the initial trigger. Later studies showed that both continuous high- (Milgram et al., 1985) or low-frequency (Cain et al., 1992) stimulation of limbic structures can induce SSSE. Inoue et al. (1992a,b) reproduced SSSE in naïve rats by electrical stimulation of prepiriform cortex, a site with a low threshold for induction of SE. Handforth and Ackerman (1992, 1993) used continuous high-frequency stimulation of hippocampus or amygdala, and correlated metabolic (14C-deoxyglucose) mapping with seizure behavior. This approach was used by Lothman and colleagues (1989), to show that stimulation of the midventral hippocampus for 60 minutes with high-frequency trains with very short intertrain intervals, a protocol they called “continuous hippocampal stimulation” ’ (CHS), resulted in the development of SSE in many animals. Seizures were nonconvulsive or mildly convulsive and lasted for hours after the end of CHS. Metabolic activity was increased in many limbic structures (CA1, dentate gyrus (DG), presubiculum, and subiculum) (Bertram, 1993). These seizures lead to loss of GABAergic hippocampal inhibition, to hippocampal interictal spiking, and to delayed spontaneous recurrent seizures (SRS, Lothman et al. 1990, 1993). The role of the stimulation pathway used for induction of SSSE was investigated by Vicedomini and Nadler (1987, 1990). They showed that SSSE could be triggered by intermittent stimulation of any of several excitatory pathways. Repeated application of high-frequency trains to the perforant path, which induced afterdischarges in the DG, a key structure in the spread of hippocampal excitation and of seizure activity (Heinemann et al., 1992), induced selfsustaining seizure activity that lasted for many hours. SSSE developed in each animal that showed ten consecutive afterdischarges. Sloviter (1987, 1991) developed an elegant model in which stimulation of the perforant path under anesthesia resulted in localized and specific lesions, even if seizures were prevented, demonstrating that excessive synaptic activity was sufficient to fatally injure neurons. We describe the methods used to induce SE by perforant path stimulation, using a protocol derived from those of Vicedomini and Nadler (1987) and Sloviter (1987), as well as methods used to trigger SE by stimulating the hippocampus or the amygdala (Lothman et al., 1989, Mazarati et al., 1998, Nissinen et al., 2000, Gorter et al., 2003). Space limitations preclude discussion of the work done with many other models of SE.
Self-Sustaining Status Epilepticus Induced by Perforant Path Stimulation in Adult Rats Animals The anatomo-physiologic substrate of the model is an excitatory input delivered through the perforant path, the large glutamatergic projection from entorhinal cortex to DG. The procedure described applies to adult male Wistar rat, although in our hands, the protocol was successfully applied to male Sprague-Dawley rats as well. A similar protocol can be used in rat pups, and also in adult mice (see later in this chapter). Anesthesia Preparation of the animals is an important step to ensure a successful induction of SSSE, because it includes physiologic verification of proper placement of the stimulating electrode. Stereotaxic surgery is performed under general anesthesia. Recommended anesthetics include subcutaneous (s.c.) urethane (1.25 gm/kg), or inhalation anesthetics such as isoflurane or halothane. Ketamine is not recommended, because it induces seizure-like EEG activity, which can complicate interpretation of physiologic responses during electrode positioning. Electrode Implantation Surgery includes implantation of two electrodes: A twisted bipolar stimulating electrode is placed into the angular bundle of the perforant path, and a recording electrode is placed into the granule cell layer of the dorsal DG. For the stimulating electrode, the optimal bare wire diameter is 0.2 mm; the distance between the tips is 0.5 mm to ensure easier positioning. The tips of the electrode should be of equal length. The optimal distance between the two recording tips of the tripolar recording electrode is 0.5 mm. One tip should be 0.5 mm longer than the other. Because both electrodes are implanted simultaneously, they should be bent in such a fashion, that there is a 2-mm offset between the tip and the connector of the electrode (Figure 1A). A free untwisted uninsulated wire is used as ground. Both electrodes are placed in the electrode holder of a stereotaxic instrument. The stimulating electrode is connected to the electrical stimulator, and the recording electrode is connected to an oscilloscope. Three skull screws are placed before electrode implantation. One of the screws should be placed in the nasal bone for standard connection to the ground electrode. The coordinates for the stimulating electrode are 4.5 mm lateral from lambda, 0.5 mm rostral from lambdoid fissure, initial depth 3 mm. The electrode is positioned in such a way that the tips are in the coronal plane. The coordinates for the recording electrode are 4.16 mm caudal and 2.5 mm lateral
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Electrical Stimulation Models of Status Epilepticus
Stimulating electrode Side view Front view
Recording electrode Side view Front view
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FIGURE 1 Schematic drawing of electrodes (A), electrode positioning (B) and physiologic response (C) from the dentate gyrus. A. Recording and stimulating electrodes for perforant path stimulation (not for scale). B. Positioning of the electrodes. Note that the tips of the recording electrode are in the sagittal plane, and the longer tip is caudal with the reference to the shorter tip. The tips of the stimulating electrodes are in coronal plane. C. Excitatory postsynaptic potential per population spike complex from dentate granule cell layer in response to perforant path stimuli. Note that under anesthesia, the first population spike is substantially suppressed. POS, population spike; EPSP, excitatory postsynaptic potential.
from bregma, ipsilateral to the stimulating electrode, initial depth 3 mm. This corresponds to the dentate granule cell layer of rostral DG (Paxinos and Watson, 1986); however, the recording electrode can be placed in ventral DG if necessary. The electrode is positioned so that the tips are in the sagittal plane, and the longer tip is caudal with reference to the shorter tip (Figure 1B). The ground electrode is twisted around the skull screw. Testing the Electrodes On initial positioning of both electrodes, pairs of electrical stimuli are delivered through the stimulating electrode with the following parameters: 1 msec monophasic square wave, with frequency of 0.5 Hz (one every 2s). Higher frequency can affect the response. The goal is to position both electrodes so that stimulus delivered to the perforant path evokes excitatory postsynaptic potentials (EPSP) and population spikes of optimal size and shape from the DG (Figure 1C). General anesthetics inhibit the population spike, which, therefore often does not appear on the peak of EPSP in anesthetized animals, or is significantly suppressed. General
anesthetics, however, are known to induce paired pulse facilitation (i.e., if the second stimulus is delivered shortly after the first one, this evokes a population spike). It is therefore recommended to use pairs of stimuli, 40 msec apart. For the stimulation, voltage can be used (40 mV at the specified electrode parameters); however, to ensure standard conditions, it is recommended to use current; 1 mA is optimal. Once the initial response is obtained, both electrodes are slowly moved down until the EPSP-population spike complex appears (Figure 1C). To ensure successful SSSE induction, the population spike in response to the second stimulus should be at least 2 mV. With practice, it is easy to obtain population spikes of ≥4 mV. While the recording electrode is being moved down, the initial response is obtained from CA1 pyramidal cells, rather than from DG, and can be confused with the latter. The major difference between the two is the latency of the response. CA1 response is disynaptic and the time between the stimulation artefact and the peak of the population spike is 8 msec. Response from the DG is monosynaptic and its latency is 4 msec. The polarity of the two responses is
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opposite; therefore, 8 msec population spike obtained from the CA1 pyramidal cells is replaced with a 4-msec response of an opposite polarity from dentate granule cells. Keep in mind, however, that hypothermia leads to an increase of the response latency, therefore it is crucial that the core temperature be kept at 37° C. If the electrode goes beyond the granule cell layer, the polarity reverses again and the electrode should be pulled back. The maximal depth for the stimulating electrode is 4.5 mm, and for the recording electrode is 4 mm. If no EPSPpopulation spike complex is obtained at these depths, the chance for successful stimulation is low. Once the positions of both electrodes are optimized, we recommended waiting for another 10 to 15 minutes to make sure that the response is stable. Afterward, the electrodes are fixed to the skull by dental cement. The period between the surgery and SSSE induction should be at least 2 weeks. Attempts to induce SSSE earlier lead to a higher percentage of failures. Induction of SSSE SSSE is induced in free-running rats. The stimulating electrode is connected to the stimulator, and the recording electrode to the EEG acquisition system, using commercially available swivels.
After PPS
During PPS
A. 3 min. 2 Hz continuous
To induce SSSE, animals are stimulated for 30 minutes with the following parameters: 10-sec trains of 1-msec square wave monophasic stimuli at 20 Hz, delivered every minute (total, 30 trains). Simultaneously with the trains, 2 Hz continuous stimulation is delivered with otherwise same parameters. Our limited attempts to use only trains, without continuous stimulation, were not successful; in awake animals, 2-Hz stimulation may be needed to override DG inhibition. At the beginning of perforant path stimulation, the electrographic response from the DG is completely driven by electrical stimuli, therefore EEG pattern closely follows PPS (Figure 2A,B). After 10 to 15 minutes, stimulus-independent epileptiform potentials appear (i.e., those are not temporally related to electrical stimulation) (Figure 2C). By the end of the stimulation, animals show continuous stimulusindependent seizure activity. Initial behavioral responses are minimal and include motor arrest, facial myoclonus, or both (stage 1), especially, in response to trains. Gradually, seizure severity increases and animals display Racine stage 3–4–5 kindled-like seizures first during trains, and later independently of the trains (Racine, 1972). After PPS is stopped, seizures continue in a self-sustaining manner. Usually, if self-sustaining seizures last for 10 minutes, stimulation is successful. If seizures disappear by 10 minutes, this means that SSSE did not develop.
B. 4 min. 20 Hz train
C. 25 min. 2 Hz continuous. Seizure, stage 4
D. 1 min. Seizure, stage 4
E. 5 hrs. Spikes, stage 1
F. 8 hrs. Seizure, stage 4
G. 10 hrs. PEDs, stage 2
2 mV 1s FIGURE 2 Example of electrographic activity from the dentate granule cell layer, during (A–C) and after (D–F) 30 minutes of perforant path stimulation in the rat. Time after the beginning of stimulation (A–C) and after the end of the stimulation (D–F) is indicated over the tracings, along with the behavioral seizure score. PED-periodic epileptiform discharges.
Electrical Stimulation Models of Status Epilepticus
Two types of seizure responses occur. Electrographic seizures—episodes of high frequency (≥13 Hz) activity, usually accompanied by stage 3–4 seizures (Figure 2E). Spikes and short bursts occur between seizures and are usually accompanied by stage 1–2 seizures. Total duration of SSSE (i.e., the time between the end of PPS and the last seizure) is 20 to 24 hr, and animals spend 6 to 10 hours in EEG seizures. Periodic epileptiform discharges (PED) (rhythmic 1.5-Hz spikes) usually occur between 6 and 10 hr after PPS (Figure 2F), and represent a hallmark for brain damage. These numbers, of course, depend on the parameters and applications used for seizure quantification. To evaluate the effects of pharmacologic agents, the duration of stimulation can be shortened or prolonged. Normally, 7 minutes PPS does not lead to SSSE. If an agent is thought to be proconvulsant, its delivery can be combined with 7 minutes PPS, which might lead to the establishment of SSSE. In contrast, potentially anticonvulsant agents may not only prevent SSSE induced by 30 minutes PPS, but also by 60 minutes PPS. Stimulation >60 minutes is not recommended, because running seizures (bicuculline-like) can occur; such seizures are often lethal, and mean that SSSE is no longer limbic, but involved the brainstem. Despite prolonged and severe seizures, the survival rate is high (90% to 100%). Autoradiography studies using 2-deoxyglucose uptake showed bilateral involvement of hippocampi and other limbic areas. The long-term consequences of PPS-SSSE include neuronal injury to the hippocampus and other limbic areas, as well as to the thalamus and neocortex. The injury is bilateral, without differences between stimulated and contralateral sides. Animals show mossy fiber sprouting starting at 2 weeks after PPS, and spontaneous seizures starting at 3 weeks after PPS. Initial observations found that 80% of animals developed spontaneous seizures (17 of 21), but later studies showed that nearly all animals develop chronic epilepsy. Characteristics of Spontaneous Recurrent Seizures (Chronic Epilepsy) To describe the evolution of chronic epilepsy after SE induced by 30 minutes perforant path stimulation, we used video-EEG telemetry monitoring. Electrographic activity was analyzed off-line using Harmonie software (Stellate Systems, Montreal, Quebec, Canada), configured for automatic detection and saving of spikes and seizures. The settings for seizure detection were the following: amplitude threshold 2.7; seizure duration >2 sec, detection threshold 0; minimal frequency 3 Hz; coefficient of variation 65; short burst detection was turned off; length of EEG kept before and after each seizure was 1 minute. Amplitude threshold for spike detection was set at 4. Electrographic activity was recorded from DG electrodes either tethered, or wireless, using EEG transmitters from Data Science International,
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Arden Hills, MN, USA. Keep in mind that changing software settings can yield different results. Latent Period and Spontaneous Seizure Frequency Our initial observation included 21 animals, 17 of which developed spontaneous seizures. Time between PPS and first electrographic spontaneous seizure was 28 ± 2 days (minimum 20; maximum 32 days). Mean seizure count on day 1 was 6 ± 1 (from 1 to 18), and did not vary significantly for up to 1 year after SSSE. Each animal rapidly progressed to the plateau of seizure frequency, and maintained its stable seizure frequency. Twenty-four percent of animals (4 of 17) had relatively low seizure frequency, one to two seizures per day, and the rest showed four or more seizures per day. Our subsequent experiments revealed that nearly each animal that had experienced SSSE developed spontaneous seizures. Behavioral Pattern of the First Spontaneous Seizure In 5 of 17 animals, the first spontaneous seizure was a stage 1–2 limbic seizure (mean ± SEM seizure score was 1.4±0.2). In 8 rats, the first seizure was of stage 3–4 (mean seizure score 3.8 ± 0.16). The remaining 4 rats displayed stage 5 seizures at the onset of chronic epilepsy. The animals with initial stage 1–2 seizures progressed to overt limbic seizures after 14±3 mild seizures. In this subgroup of animals, the progression of behavioral seizure severity was statistically significant (score of the 20th seizure was 3.8 ± 0.7, p < 0.05 vs. 1st seizure). The animals with initial stage 3–4 seizures started displaying stage 5 seizure after 10 ± 2 stage 3–4 convulsions. No significant progression of seizure severity, however, was observed in this group of rats (score of the 20th seizure was 4.1 ± 0.3). The animals with initial full limbic seizures alternated between stage 5, stage 3, and stage 4 seizures; severity of the 20th seizure was 4.5 ± 0.5. Interictal spikes were observed starting from the 1st day after PPS. Frequency of spikes significantly varied (from 0.01 to 0.3 Hz over 4 hr sample epochs). Each animal, however, had a relatively stable spike frequency that did not change over time. Frequency of interictal spikes did not change after animals started showing spontaneous seizures. No correlation, however, was observed between interictal spike and spontaneous seizure frequency. Seizure origin Most of the interictal spikes in the animals, which were implanted with multiple electrodes, (n = 5) were equally generated from stimulated and contralateral ventral hippocampus and entorhinal cortex. Spontaneous seizures were preceded by a synchronous high-frequency oscillation (250 to 300 Hz) in the ventral hippocampi. The seizure itself
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originated from the stimulated or contralateral hippocampus with equal incidence, with fast involvement (1 to 2 s) of entorhinal cortex, and delayed involvement of other areas.
achieve the “ceiling” of mossy fiber sprouting earlier than animals with a low seizure frequency. Response to Antiepileptic Drugs
Neuropathology Neuronal injury was scattered throughout the hippocampus, with hilar interneurons, CA3 and CA1 pyramidal cells, and granule cells involved in order of decreasing severity. Other damaged regions included amygdala, entorhinal and pyriform cortices, thalamus, and to a lesser extent caudate and neocortex. Mossy fiber sprouting (Figure 3) preceded the occurrence of spontaneous seizures. Animals killed during the silent period, or several hours after the first spontaneous seizure (total n = 3) showed light, but clearly positive Timm staining in the inner molecular layer of DG (Figure 3A,B). No differences in Timm staining were observed between animals with high and low seizure frequency months after SSSE (Figure 3C,D). However, correlating the degree of Timm staining with seizure frequency in the animals euthanized between 1 day and 2 months after SSSE showed that the animals with higher seizure frequency
This model of SE develops time-dependent resistance to diazepam and phenytoin (Mazarati et al., 1998c). Felbamate and leveritacetam were relatively effective (Mazarati et al., 2000a, 2004). Blockers of N-methyl-d-aspartate (NMDA) receptors (MK-801, ketamine, or 5,7-Dichlorokynurenic acid) were highly effective in irreversibly stopping selfsustaining seizures, whereas NBQX, an AMPA receptor agonist, did not have a strong effect on seizures (Mazarati et al., 1999b). Applications and Limitations This model is very labor-intensive, but offers great reproducibility, and low mortality despite its prolonged seizures and the extensive brain damage that results. It is highly epileptogenic, with >80% of subjects developing recurrent spontaneous seizures (SRS).
A
B
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FIGURE 3 Timm staining was positive in the animals killed during the latent period, 20 days after self-sustaining status epilepticus (SSSE) (A), and 6 hours after the first spontaneous seizure (B). Timm staining in the animals euthanized 2-month after SSSE, after a total of 203 (C) and 42 (D) spontaneous seizures. (Reproduced from Mazarati et al., 2002a. Copyright Blackwell Publishing, 2002.) (See color insert.)
Electrical Stimulation Models of Status Epilepticus
The model is relatively easy to develop, but does not lend itself to mass-production. Physiologic monitoring of the population spike during electrode placement greatly improves the accuracy of placement compared with stereotaxic surgery alone, and is a major contributor to the excellent reproducibility of the model. It is somewhat time-consuming; furthermore, the number of animals in which SE can be induced simultaneously is limited to the number of available electrical stimulators. Limitations of the seizure detection software will be detailed in a future manuscript. Manual review of each seizure remains mandatory with currently available software.
Perforant Path Stimulation in P15-35 Rat Pups Similar perforant path stimulation protocols can be also used to induce SE in younger rats. This procedure induces hippocampal neuronal injury and alters physiologic properties of hippocampal neuronal circuits. At younger ages, however, PPS does not always lead to the development of self-sustaining seizures, thus making it necessary to apply prolonged stimulation (Thompson et al., 1998). This section focuses on the P15 (postnatal day 15) rats, which pose by far the most difficult challenge. Later ages (P20 to P35) require only minor adjustments from the technique used in adults. Perforant path stimulation in the 14- to 16-day-old rat has to be distinguished from SSSE induced by PPS in other developmental ages and from the adult. The maximal stimulation duration used in the awake animals at this age was 18 hr, and SSSE was never observed. The granule cell responses were entirely reliant on the repeated entorhinal stimulation, and they stopped as soon as the stimulation stopped. Older pups showed the progressive development of SSSE with increasing age (Sankar et al., 1999). Anesthesia Anesthesia is an important consideration with the 14- to 16-day-old group. Our studies (Thompson and Wasterlain, 2001), and those published recently (Iknomidou et al., 2000, 2001; Olney et al. 2002; Bittigau et al. 2002), have shown that anesthetics and NMDA antagonists (i.e., Ketamine and MK-801) can induce neuronal cell death at this age. We, therefore, used Metophane anesthesia during the surgical procedures. This also allowed paired-pulse testing and continuous stimulation within minutes after surgery. Special Instrumentation The animals typically weighed 25 to 35 g and this required the animals to be elevated into the plane of the ear
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bars. This was typically achieved with an empty Hamilton syringe box and overlaying heating pad but any stable raised plane would suffice. Additionally, because of the small size of the aural canal and the incomplete ossification at this developmental stage, standard rat ear bars were modified. Using the size and design of the David Kopf serrated mouse zygoma ear cups as a model, a plastic sleeve (a Rhodes electrode transport vial cut to size) was fitted over the tip of standard rat ear bars to expose 2 mm of the tip, and then fastened there by pouring and curing dental cement between the bar and the sleeve. This left the tips of the modified ear bars for entrance into the small interaural canal, and the flattened side walls of the attached sleeve to brace the pliant skull against movement. The dual arms of the stereotaxic frames need to be slightly modified to ensure adequate clearance as both arms are placed at the electrode target sites simultaneously. For this purpose, alligator clips were cemented to electrode clamps at the ends of the arms perpendicular to the axis of the arm. Electrode Placement The stimulating electrodes (Rhodes electrodes model SNEX .25mm separation) is placed into the perforant path at coordinates AP: -2.4, ML: 4.4, (Sherwood and Timiras, 1970) and the recording electrode (same model and vendor) is lowered into the facia dentate at coordinates AP: 2.0, ML: 1.5, (Sherwood and Timiras, 1970) (note that the nose bar is set at 0.0 for this atlas coordinate system). As with rats of other ages, we determined the final placement of both electrodes by the waveforms recorded at the dentate granule cell layer. Typically, starting with the stimulating electrode lowered ~3 mm, and the recording electrode lowered ~2 mm, slow, progressive downward descent of the recording electrode will produce granule cell spikes (using a stimulation intensity of 30 V for paired pulses delivered at 0.1 HZ and 50 msec apart). After the appropriate waveforms are established, further subtle manipulation of both electrode positions will optimize the response. The granule cell population spike was maximized for individual animals by increasing the voltage of the stimulation (30 to 40 V). General Surgical and Physiological Techniques These techniques are similar to those used in an adult rat. Implantation coordinates in 20- and 25-day-old rats are the following: stimulation electrode 4.4 mm lateral, 6.5 mm posterior to the bregma; recording electrode 1.8 mm lateral, 2.8 mm posterior to the bregma. The coordinates in 30- to 35day-old rats are stimulating electrode 4.5 mm lateral, 7.5 mm posterior to the bregma; recording electrode 2 mm lateral and 3.5 mm posterior to the bregma. Rapid ontogenic devel-
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opment is a significant limiting factor. Because of significant changes in seizure predisposition with the age and of the fast brain growth, postsurgical period is limited to 1 day. Duration of stimulation is longer—8 hours. Behavioral Manifestations and SSSE Both electrophysiologic and behavioral manifestations of SSSE in rat pups are less florid than in adult rats. It is not clear whether this is because animals are not allowed to recover completely from surgery or because of higher resistance of younger animals to seizures in this model. During PPS, the 20-day-old rats exhibit wet dog shakes with chewing and salivation. They also demonstrate periods of bilateral forelimb clonus and rare rearing behavior. In this age group, the seizures occasionally reach stage 5, but are between stages 3 and 4 much of the time. The 25-day-old animals exhibit rearing and falling much more commonly (stage 5), and a few animals also progressed to vocalizations and running (stage 6), and some of those progressed to tonus (stage 7) and death. At ages of 30 and 35 days, stage 6 and 7 was reached by about 15% of animals that survived. In this age group, approximately 40% of the animals die during the 8-hour stimulation period, and another 18% die during the following 3 days. Death is uncommon in 20- and 25-day-old rats (<5%) during and after the 8 hours of PPS. The proportion of animals that enter SSSE at the end of PPS is 0, 35%, 64%, 70%, and 88% at the ages of 15, 20, 25, 30, and 35 days, respectively. The epileptogenicity of SSSE in P15 rats is unknown. The initial group (Thompson et al., 1998) did not develop spontaneous recurrent seizures over 4 weeks of observation, but that duration may have been too short, and EEG monitoring was not carried out. Older animals show increasing epileptogenicity with advancing age (Sankar et al., 1999) and lithium-pilocarpine SE at P is epileptogenic (Sankar et al., 1999).
Perforant Path Stimulation Self-Sustaining Status Epilepticus in Mice The PPS protocol can be successfully applied to induce SSSE in mice and, therefore, is particularly useful for studying transgenic animals (Mazarati et al., 2000). Anesthesia and general implantation technique are similar to those in the rat. Coordinates for the stimulating electrode are 0.5 mm anterior and 2.5 mm lateral to lambda, 1.5 to 2.0 mm ventral from the brain surface; coordinates for the recording electrode are 2.0 mm posterior and 1.0 mm lateral to bregma, 1.5 to 2.0 mm ventral from brain surface (Paxinos and Franklin, 2001). Properties of evoked responses are similar to those in the rat. Perforant path stimulation parameters for SE induction are different from those used in the rat: train duration is 5 s,
frequency is 33 Hz, pulse duration 1 msec; frequency of continuous stimulation is 3 Hz. The longer duration of PPS needed to induce SSSE is 60 minutes in wild-type mice. In the mouse, SSSE does not last as long as in the rat: total SE duration is 5 to 6 hours with 30% to 50% of the time spent in seizures. This protocol was successfully used in C57Bl and 129OlaHsd mice. The parameters of SSSE were comparable in both strains.
Induction of Self-Sustaining Status Epilepticus by Amygdala Stimulation Animals Adult male Harlan Sprague–Dawley rats (275–350 g) are used (Nissinen et al., 2000). The rats are housed in individual cages in a controlled environment (constant temperature, 22 ± 1° C, humidity 50% to 60%, lights on 07:00 to 19:00 hours). Animals have free access to food and water. All animal procedures are conducted in accordance with the guidelines by the European Community Council Directives 86/609/EEC. Electrodes For amygdala stimulation, a bipolar electrode is made as follows: cut a 3-cm of bipolar electrode wire (Franco Corradi, Milano, Italy), diameter 0.127 mm with scissors. First, from another end of the wire, separate wires carefully by pressing the scalpel (size 11) between the tips of wires. After that, by pressing with fingers from the middle of the wire against a hard surface, separated wires were switched one to the left and the other to the right by a wooden toothpick. Insulation is removed with scalpel (size 10) at the distance of 3 mm from the tip of the wire. E363/0 Socket Contacts (Plastics One Inc., Roanoke, VA, USA) are soldered to the wires. In another end of the wire, the dorsoventral distance between the tips 0.4 to 0.5 mm was established after separating the wires carefully with scalpel (size 11) under the stereomicroscope. Beware of not destroying the insulation of neighboring wire. Electrode Implantation For amygdala stimulation, a bipolar electrode is implanted into the lateral nucleus of the left amygdala under deep sodium pentobarbital (58 mg/kg) and chloral hydrate (60 mg/ kg, i.p.) anesthesia. Coordinates of the lower electrode tip are 3.6 mm posterior to bregma, 5.0 mm lateral to bregma, and 6.5 mm ventral to the surface of the brain according to the rat brain atlas (Paxinos and Watson, 1986). Electrode is inserted to the arm of the Kopf stereotaxic apparatus (David Kopf Instruments [Tujunga, California, USA]), so that approximately 1.5 cm of wire comes out from the arm. It is easier to
Electrical Stimulation Models of Status Epilepticus
insert the electrode in a straight position, if the length of the electrode is adjusted correctly. For amygdala electrode, approximately 3-mm hole is burred into the skull. Dura is removed with tweezers and a hook made from the size 26 injection needle. Pia is cut, otherwise it may bend the electrode to go too laterally. Stimulation and Recording To record the spread of electrographic seizure activity to the contralateral cortex, a screw electrode (E363/20, Plastics One Inc., Roanoke, VA, USA) is inserted into the skull overlying the contralateral frontal cortex (coordinates: 3.0 mm anterior and 2.0 mm lateral to bregma). An anchor screw is used on the ipsilateral side (coordinates 3.0 mm anterior and 2.0 mm lateral to bregma). Two monopolar stainless steel screw-electrodes are fixed to the skull symmetrically over the cerebellum (coordinates 10.3 mm posterior and 2.0 mm lateral to bregma) to serve as ground and reference electrodes. All electrodes are secured with dental acrylate (Selectaplus CN, Dentsply DeTrey GmbH, Dreieich, Germany). Altogether, three cortical electrodes and one anchor screw are placed symmetrically over the skull. This confirms that the headset is tightly connected to the skull and the number of lost electrodes (headsets) is low. For cortical electrode and anchor screw, holes are burred with drill size 10 (Hager & Meisinger GmbH, Germany). Cortical electrodes and anchor screw are inserted first, followed by stimulation electrode. Order is important because screws are used as an anchor to secure the stimulation electrode with dental acrylate. After inserting the screws, contacts of electrodes are inserted into the six-channel pedestal (MS363, Plastics One Inc. Roanoke, VA, USA). Because there are only five contacts (two from stimulation and three from cortical electrodes), one hole in the pedestal is closed with Scotch tape not to allow dental acrylate to fill it. Otherwise, the cable cannot be connected to the animal later. After the contacts are inserted into the pedestal, they are connected to the holder that was made from the dust cap (303DCFT, Flat, Plastics One Inc., Roanoke, VA, USA), and then connected with injection needle (20 G). The injection needle is glued from its rigid luer-loc hub to the dust cap with dental acrylate. After inserting the contacts to the pedestal, carefully twist wires while pressing them down. This shortens the wires and reduces the amount of dental acrylate needed to secure the pedestal. Be sure that contacts do not make a short circuit by touching each other. Before adding dental acrylate, be sure that the skull surface is dry. Blow some air on it to make the skull dry. Add the dental acrylate first under the pedestal to secure the contacts. Place all wires and screws under the dental acrylate, but do not cover the skin with that. Two weeks after surgery, the baseline EEG for each rat is recorded for at least 15 minutes. Thereafter, SE is induced
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by stimulating the lateral nucleus of the left amygdala for 20 to 40 minutes. The stimulation consists of a 100-msec train of 1-msec biphasic square wave pulses [(0.5 msec upward (200 mA) and 0.5 msec downward (200 mA) (400 mA from peak to peak)] delivered at 60 Hz every 0.5 seconds using A300 Pulsemaster Stimulator (WPI, England) connected with two A360 Constant Current Stimulus Isolators (WPI, England) to which two animals are connected with a six-channel commutator (Plastics One Inc.) and six-channel shielded cables. The commutator allows animals to move freely during the stimulation. The stimulation electrode of the animal is connected to the switch in an external connection box. In the connection box, there is an input to the signal coming from stimulator and output to the amplifier. In one position, the switch allows the stimulation current to reach the animal; in the other, electrographic seizure activity can be measured. Development and duration of SE is monitored continuously via amygdala and cortical electrodes using the Nervus EEG Recording System (software version 2.4 or 3.1, Taugagreining, Iceland) connected with an M40 Amplifier (32channel amplifier, Taugagreining, Iceland). The behavior of the animal is recorded using SVT-S3000P Hitachi Time Lapse 168 VCR (Osaka, Japan). Panasonic WV-CL350 infrared light-sensitive video camera (Osaka, Japan) and infrared light (MUL, TIM Electrode Inc., Great Britain) or WFL2/15W IR-light (Panasonic, Japan) are positioned above the gages. A wide-angle lens permits videotaping of up to 8 to 10 animals simultaneously. Infrared light is used during nighttime to monitor animal behavior. Each rat is stimulated continuously for 20 minutes. Thereafter, the stimulation is interrupted, and the behavioral and electrographic seizure activity of the animal is observed for 1 to 2 minutes. If the animal does not meet the criterion of clonic SE (continuous electrographic epileptiform spiking and recurrent clonic seizures), stimulation is resumed and the behavior of the animal is checked again after 10 minutes. Once the criterion of SE is achieved, no further stimulation is given. The maximal stimulation time does not exceed 40 minutes.
Post-SSSE Care After the stimulation, animals have free access to powdered food pellets and Nutrisal Plus solution (Orion Pharma, Turku, Finland). Nutrisal Plus solution contains sodium chloride 2.3%, potassium chloride 3.4%, sodium bicarbonate 3.4%, and glucose 90.9%. If needed, Nutrisal Plus solution is given by gavage (2 mL per /injection). The stimulation electrode location is verified histologically at the end of the experiment, and only the animals with the stimulation electrode located correctly in the lateral nucleus of the amygdala are included in the study.
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Monitoring of Spontaneous Seizures
Diurnal Distribution of Seizures
Behavioral and electrographic monitoring of the appearance of spontaneous seizures was analyzed off-line using Nervus software (Nervus, Taugagreining, Iceland). EEG signals were recorded with sampling rate 200 Hz, high-pass filter 1 Hz and low-pass filter 100 Hz. Electrographic activity was recorded from amygdala and contralateral cortex for 6 months every other day (every other recording was included in analysis).
EEG analysis indicated that 57% of seizures occurred during the day (07:00 to 19:00 hours). Analysis of different seizure types separately indicated that 57% of partial and 56% of generalized seizures were detected during the day (07:00 to 19:00 hours). Interestingly, no difference was noted in the diurnal occurrence of seizures between animals with frequent and rare seizures. The peak seizure number appeared between 17:00 to 18:00 hours.
Latent Period and Spontaneous Seizure Frequency
Seizure Origin
Our initial observations included 15 stimulated animals, 13 of which developed spontaneous seizures during a 6month follow-up period. Recording of the animals was done every other day (every other recording was included in analysis). Latency and time between amygdala stimulation and first electrographic seizure was 33 ± 26 days (median 28 days, range 6 to 85 days). From these 13 animals, 4 (31%) had frequent seizures (seizure number varied from 697 to 1,317 seizures per animal) and 9 (69%) animals had rare seizures (1 to 107 seizures per animal). Latency to the first seizure differed between these two groups. In animals having frequent seizures, latency was 11 ± 4 days (median 10, range 6 to 16) compared with animals with rare seizures 48 ± 23 days (median 42, range 26 to 85). After 12 weeks, seizure frequency remained constant in both groups.
To detect independent seizure generation in hippocampus or amygdala, altogether 268 seizures in 7 animals (range of recorded seizures per animal varied from 3 to 80) were recorded via hilar and amygdala electrodes 3 to 5 months after induction of SE. Diffuse onset seizures arising from the amygdala and hippocampus simultaneously were the most typical. In the recordings available, however, only 20% of seizures were detected first in the amygdala and 9% in the hippocampus.
Behavioral Pattern of the Spontaneous Seizures The behavioral outcome of seizures changed during follow-ups. Calculated from all animals with seizures during the first 10 weeks, most seizures (79%) were generalized (on the Racine scale they were scored as stage 3–5 seizures). Thereafter, seizure type changed to more partial (Racine scale stage 0–2 seizures). Altogether, 77% of seizures were partial. During the first 10 weeks of recording, however, the proportion of generalized seizures was similar in animals with frequent and rare seizures (79% vs. 70%, respectively). Afterwards (weeks 11 to 26), seizures in animals with frequent seizures were partial (80% of all seizures). Only 4% of seizures in animals with rare seizures were partial. Seizure Duration Mean duration of individual seizures was 49 ± 24 sec (median 47, range 7 to 252). Mean duration of partial seizures was 44 ± 23 sec (median 42, range 7 to 232) and generalized seizures 61 ± 23 sec (median 59, range 16 to 252). Duration of generalized seizures did not change over time, however, duration of partial seizures became shorter during follow-up (p < 0.001 one-way ANOVA).
Neuropathology Histologic analysis indicated neuronal loss in the amygdala, hippocampus, and surrounding cortical areas. Also, reorganization of granule cell axons, mossy fiber sprouting, was detected. Altogether, 12 of 13 animals (92%) with seizures had damage in the amygdaloid complex, including the lateral nucleus, parvicellular division of the basal nucleus, the accessory basal nucleus, the ventral part of the central division of the medial nucleus, the anterior cortical nucleus, the posterior cortical nucleus, the amygdalohippocampal area, the periamygdaloid cortex, and the nucleus of the lateral olfactory tract. Interestingly, central nucleus of the amygdala was well preserved. Clear damage in hippocampus was detected in 4 of 13 (31%) of epileptic animals. The hilus and CA1 subfields were more often damaged than CA3. No clear granule cell loss was seen in any animal with seizures. Neuronal damage in subiculum was detected in 7 of 13 (54%) epileptic animals. Other damaged regions included entorhinal (in 92% of animals with seizures) and pyriform cortices. Mossy fiber sprouting was present in all (100%) animals with seizures. Great variability was seen in the density of mossy fiber sprouting between the animals and it appeared heavier temporally than septally. We did not observe difference between ipsilateral or contralateral sides.
Focal SE Induced by PP in Anesthetized Rats Status epilepticus can be induced by 24 hours of PPS delivered to anesthetized rats. In contrast to PPS-SSSE, no
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motor seizures occur, and the injury is confined to the hilus of the ipsilateral DG and to pyramidal cells in both hippocampi (90% ipsilateral, 10% contralateral). Essentially, no extrahippocampal lesions were seen. This model is useful for studying physiologic properties of dentate neuronal inhibitory and excitatory circuits. Sloviter (1987) has varied the method of stimulation to achieve specific goals, including an elegant demonstration that high-intensity neuronal stimulation can injure neurons without producing seizure activity. Anesthesia and Stimulation Surgery is performed under urethane anesthesia, as described in the section “Self-Sustaining Status Epilepticus Induces by Perforant Path Stimulation in Adult Rats”. PPS is delivered to the anesthetized rat placed in the stereotaxic frame, with the parameters described in the PPS-SSSE section, (10-sec trains of 1-msec square wave monophasic stimuli at 20 Hz, delivered every minute, together with continuous 2-Hz stimulation), but stimulation was continued for 24 hours. The animals were anesthetized throughout PPS. Maintaining an appropriate level of anesthesia is crucial: if the animal becomes insufficiently anesthetized, it may exhibit mild motor seizures, and more widespread and severe neuronal injury. Core temperature should be kept at 37° C thorough the procedure; this is needed both to ensure the animal’s survival and to sustain the physiologic response. We used two different methods of anesthesia, and found that the intravenous method achieved a steadier level of anesthesia than did the subcutaneous method. In the subcutaneous method, the first urethane injection (1.25 mg/ kg s.c.) was followed by two additional injections of 0.25 gm/kg s.c. 8 and 14 hours later, to ensure the maintenance of surgical anesthesia throughout the procedure (12). In the intravenous method (11) the rat is placed in an induction chamber; the anesthesia is induced by a gas anesthetic (5% isoflurane, or halothane in 1 litter per minute O2). Once anesthetized, the animal is injected with urethane intravenously through the tail vein (loading dose, 1,000 mg/kg in saline), and placed in a stereotaxic apparatus. Intravenous injection of urethane is continued until the end of experiments to maintain anesthesia, using an infusion pump, and the rate of infusion is changed according to the response to sensory stimulation. The maximal maintenance dose of urethane is 1.25 mg/kg over a 24-hour period, delivered in 50 mL of saline. After PPS, it takes several hours for the animal to recover consciousness. The major indicator of successful stimulation is a loss of paired-pulse inhibition. In normal awake animals, delivery of the second stimulus after either short (40 msec), or long (200 msec) interstimulus intervals suppresses the amplitude of the second response (Figure 4). Paired-pulse inhibition is observed both in response to low (0.1 to 0.5 Hz) and rela-
Before PPS
After PPS
40 ms, 0.5 Hz
200 ms, 0.5 Hz
40 ms, 2Hz
1 mV
FIGURE 4 Paired-pulse inhibition in the dentate granule cell layer of an awake rat. Pairs of stimuli were delivered to the perforant path of the rat before and 3 days after 24 hours of PPS, under urethane anesthesia. Interstimulus interval and stimulation frequency are indicated on the left. Note that in the naïve rat, the first population spike suppressed the second one under various conditions of stimulation. After focal status epilepticus, however, the second population spike was disinhibited. In addition, whereas in the naïve rat each stimulus evoked a single population spike, after focal status epilepticus, it evoked polyspikes.
tively high (2 Hz) stimulation frequencies (Figure 4). After 24 hours of PPS, the ratio of the second population spike to the first population spike increases. In addition, each stimulus elicits multiple population spikes instead of a single response (Figure 4). Throughout stimulation, it is difficult to assess paired-pulse inhibition because of frequent suppression of the first population spike. A sustained response to the second of the pair of stimuli, however, is a precondition for successful stimulation. If it is necessary to quantify the extent of the changes in the paired-pulse inhibition as a result of PPS-induced focal status epilepticus, then surgical implantation of the electrodes and PPS itself must be done on different days, because anesthesia alters paired-pulse inhibition. To obtain relevant responses as well, the test must be done in awake animals. Brain Damage and Epileptogenesis This model is very labor-intensive, and maintaining an even level of anesthesia is difficult. It does lead, however, to spontaneous recurrent seizures and to seizures triggered by handling in at least half the animals (Shirasaka and Wasterlain, 1994). Seizure-induced neuronal death is restricted to the hippocampus, involving the dentate hilus on the stimulated side, and CA1/CA3 pyramidal cells on both sides (approximately 90% ipsilateral, 10% contralateral). No extrahippocampal damage occurs, with the exception of a rare entorhinal cortex neuron (Wasterlain et al., 1996). Thus, it is histologically cleaner than any other model, to our knowledge, and could be useful for studying the role of local
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hippocampal circuits in epileptogenesis. Sloviter (1987) has also used this model to provide intense perforant path bombardment with single pulses, which caused injury and death of hilar neurons without using trains or seizure-like stimulation, supporting the view that intense synaptic activity is sufficient to cause neuronal injury.
Continuous Hippocampal Stimulation The CHS protocol is another variation for inducing SE in rats through intracerebral electrical stimulation. It has many similarities to the perforant path and amygdala stimulation models described in this chapter. We will focus on the aspects of the model that may be more unique. We will not repeat many of the descriptions of electrode implantation that are common to all of the models, unless critical differences in approach exist. More of the description of implantation techniques that we use for chronic recordings is contained in the chapter on EEG monitoring of rodents. Basic Features of Implantation and Stimulation Electrodes For stimulation twisted pair, Teflon-coated stainless steel electrodes are implanted in the mid ventral hippocampus (from bregma: AP -5.6 mm, ML 4.9 mm, DV -5.0 mm from dural touch point, bite bar -3.3 mm). The ventral hippocampus is a better site for inducing SE compared with the dorsal region because the success rate for inducing SE is much higher. Some investigators have attempted CHS using concentric bipolar electrodes, but had little to no success. Although no conclusive evidence exists, we suspect that the reason for the lowered success rate was that tip separation was insufficient to stimulate a large enough volume of tissue to induce SE. In twisting the wire during the fabrication of the electrode, do not over twist, because the mechanical forces of twisting can cause a break in the insulation and a short circuiting of the electrode with a consequent failure of the stimulation. Test for breaks and shorts with an Ohm meter before inserting the electrode. The tips of the twisted pair electrodes are cut with a pair of scissors at a 45-degree angle, so that one tip is slightly dorsal to the other, with the tips separated approximately 1 mm. Headsets The descriptions for the headsets was given in the earlier section of this chapter. One difference is our use of plastic connector strips that we can customize for each animal. Another is that we do not use skull screws as cortical recording electrodes. If we need cortical electrodes, we create a small burr hole in the skull and insert a single stainless steel wire, either in the cortex or on the surface of the cortex. In
general, the nature of the headset depends on the plan for the particular animal. If the plan is to monitor the animal continuously from the induction of SE to a predetermined endpoint several weeks or months following the induction of SE, a full, permanent headset is appropriate, with the number of electrodes determined by the study. On the other hand, if the plan is to induce SE, followed by maintaining the animal in the vivarium until needed for epilepsy-related studies weeks or months later, a temporary headset (euphemistically called a “pop top” in our laboratory) is all that is needed. In this scenario, the single twisted pair electrode is placed in the midventral hippocampus and held in place with two screws placed in the skull. They are held together by a small amount of dental acrylic. Stimulation can be performed within several days of the surgery, and the electrode assembly can be removed 1 or 2 days following the stimulation. The animals are anesthetized with an inhalation agent (e.g., isoflurane) and the assembly can be removed with a gentle upward traction with a set of forceps and the scalp is sutured back together. This approach is extremely useful for animals for which long-term recordings will be performed using a second, permanent implantation in several months when the animals have developed epilepsy. For many of these animals, especially the younger ones, maintaining a functional headset longer than a few months is difficult. If no need exists to have a headset in place, it is easier to maintain the rat for those months without one. Using the temporary headset is also valuable because it does not alter the skull landmarks (sutures), nor is there a large degree of scarring that would make second surgeries following lost full headsets so difficult. A second headset is often not necessary, if the sole purpose is to have rats with documented seizures. In this case, seizures can be documented by visual or video monitoring for the development of spontaneous seizures after an interval of at least several months. Stimulation Stimulation is standard. As originally described (Lothman et al., 1989), it is a 50-Hz, 1-msec (per phase) biphasic 400 : A (peak to peak) stimulus train lasting 10 seconds, delivered every 11 to 13 seconds (i.e., 10 seconds on, 1 to 3 seconds off) for 90 minutes. The actual duration of stimulation can vary, but in general 30 minutes is only occasionally successful in inducing true SE that over lasts the stimulation; if it has not started following 90 minutes, it is not likely that it will. The stimulus intensity was empirically chosen, but partly based on the rapid kindling model in which the stimulus is given every 5 minutes and the intensity had to be sufficiently above the baseline afterdischarge threshold to ensure an afterdischarge with every stimulation. 400 : A was chosen because it was sufficiently greater than the typical afterdischarge threshold (ADT) (to 10-second
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stimulation trains) of 60 to 120 : A. For the CHS model, a minimal stimulus intensity of twice the baseline ADT is used, so if the afterdischarge is 250 to 300 : A, a stimulus intensity of 500 to 600 : A is used. If the ADT is much higher, the chances of successfully inducing SE are reduced. It is important, therefore, to obtain an ADT before beginning CHS, first to assure that an afterdischarge can be obtained and second to be able to set an appropriate stimulus intensity. Status Epilepticus Following stimulation, it is worthwhile to monitor the EEG for the progression of the seizure activity, especially if intending to predict which animals are likely to develop epilepsy in the coming weeks or months. Treiman et al. (1990) described a pattern of EEG progression in human SE that had five distinct stages, and this evolution is also valid for most of the models of limbic SE, whether induced chemically or electrically. Animals that only have intermittent seizure activity or a brief period of continuous highfrequency activity immediately after stimulation rarely become epileptic or develop significant neuronal injury. On the other hand, the rats that over several hours following stimulation develop periodic discharges (PED) are likely to become epileptic (80% to 90%) (Bertram and Cornett, 1993; Prasad et al., 2002). Most rats that have high frequency activity at the end of stimulation progress to PED, but from time to time, some animals remit without full EEG evolution. On the other hand, some animals have minimal seizure activity at the end of stimulation that gradually progress to PED. The former do not become epileptic and the latter do. For this reason, it is helpful to monitor the animals over the 6 to 10 hours following stimulation to ascertain progression in EEG and to predict long-term outcome. Those animals that do not progress to PED are generally not kept, except to serve as control animals (effect of stimulation alone without SE or epilepsy). In general, these “stimulation failures” are no different physiologically or anatomically from naïve controls. Outcome and Evolution of Limbic Epilepsy Age, as has been suggested by many investigators, can have a significant influence on outcome. In this model, as with the other SE and post-SE limbic epilepsy models, there are three primary outcome measures: number of animals that enter SE, number of animals that survive SE during the first week following SE, and, finally, the percentage of SE survivors that become epileptic (and by implication have significant SE-associated neuronal loss). Although we have never kept formal records of the issue, a number of observations over the years have convinced us that age plays a significant role in these three outcomes. The informal survey results suggest that all ages enter into SE relatively easily,
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but the younger animals are more likely to survive the acute phase, but less likely to become epileptic. The older ones have a higher acute mortality (during CHS and in the week after), but those that survive are much more likely to become epileptic. Using these informal guidelines, we tend to select Sprague-Dawley rats with weights between 290 and 325 g for stimulation (implanted at weights around 250 to 275 g). It may be possible to rescue the animals during SE and, therefore, save for chronic use, the older animals with an injection of diazepam or phenobarbital about 2 hours after the cessation of stimulation. We have found that treatment around this time has little impact on the long-term outcome, but may sufficiently reduce the stress of the SE to allow for long-term survival. In the immediate recovery phase following SE, the animals’ behavior can be variable—from lethargic to hyperresponsive. Their oral consumption is reduced during this time, and some animals are sufficiently disoriented from the SE and presumed associated neuronal loss that they do not eat at all, and frequently die. This group is <5% of the total. We have attempted various approaches to assist animals during this period, such as giving them special foods (e.g., apple wedges) or fluid injections, but we have never been convinced that this additional care improved survival of these animals. As with the other models, the latent period between SE and the appearance of spontaneous seizures varies from animal to animal, and is difficult to predict from the course of SE, other than the appearance of PED in the later stages of SE. The presence of this pattern is strongly associated with the eventual development of epilepsy, and the absence of the pattern is almost universally predictive for not developing epilepsy (Bertram and Cornett, 1993). When we examined a cohort of animals for the evolution of their spontaneous seizures, we noted significant variability in the time course and ultimate severity of the epilepsy. The earliest spontaneous seizure occurred on the seventh day following SE (a few animals had one or two seizures in the first several days following SE, but then stopped and did not have further seizures for several weeks). The first recorded seizure was almost always nonmotor, and the first motor seizure appeared an average of a month after SE. Some animals, however, did not experience their first seizures for almost 3 months following SE. In general, animals that have not had a spontaneous seizure by 90 days following SE are unlikely to develop them later. Only rarely have we seen such an animal that had a seizure 6 or 9 months following SE. Rarely, an animal will have a seizure while being picked up. Many of these animals, when monitored later, fail to have a second one. Although the presence of PED during the late stages of SE is highly predictive of the development of epilepsy later, because a small percentage (10% to 20%) do not, it is good to confirm the presence of seizures if planning to study
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epileptic animals. Reimplantation is often not necessary, because the seizures are usually behaviorally obvious. Thus, seeing two seizures by chance observation is sufficient to confirm the diagnosis. For most animals, however, a period of monitoring is appropriate. In most cases, recording the animals for a week or two with VCR and reviewing the tapes in fast forward mode is a fairly efficient means to document the seizures. We rarely begin the monitoring before 60 days following SE, primarily because the yield will be relatively low. If one is interested in studying the changes leading up to spontaneous seizures, then continuous monitoring with the EEG is more appropriate to exclude any form of spontaneous seizure activity.
RELATIONSHIP TO HUMAN STATUS EPILEPTICUS All the models discussed produce kindled-like seizures, usually without tonic-clonic seizures, and without apnea or major changes in blood gases or acid-base balance. Imaging by 2-deoxyglucose autoradiography or c-fos expression shows predominant activation of the limbic system (Pereira et al., 1999). They are models of complex partial SE, rather than models of generalized convulsive (major motor) SE. They strikingly reproduce clinical phenomena such as the time-dependent development of resistance to benzodiazepines (Mazarati et al. 1998a) and, therefore, are probably good models for studying the pathophysiology of the human illness. Models involving local stimulation of perforant path, amygdala or ventral hippocampus have much in common with each other and with some chemical stimulation models (e.g., lithium-pilocarpine followed by atropine, leaving SSSE, Morrissett et al., 1987, Suchomelova et al., 2003). This might suggest that all these models access the same circuit that maintains SSSE, but it is clear that differences in pharmacologic responsiveness are seen from one model to another. Their circuits may have a lot in common, but they are certainly not identical. For example, SE can be initiated with pilocarpine in lithium-pretreated rats and, when seizures are fully developed, then the effect of pilocarpine blocked with atropine. This leaves SSSE that resembles SSSE induced by PPS. The latter, however, responds well to phenytoin or fosphenytoin; the former not at all (Suchomelova, personal communication). The age-dependence of many aspects of the PPS model suggests that circuit maturation plays a major role in the ontogeny of SSSE, and mimics the human situation where SE in infants is multifocal rather than generalized. Because SE is common in human infants and seizure-like activity is easily induced with high K or other stimuli in immature hippocampal slices, it has often been assumed that all aspects of SE are easily induced in the immature brain. The
inability of P15 rats to establish SSSE demonstrates that some types of SE require a minimal level of circuit maturation.
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References Inoue, K., Morimoto, K., Sato, K., Ishizu, H., Kawai, K., Yamada, N., and Otsuki, S. 1992a. A model of status epilepticus induced by intermittent electrical stimulation of the deep prepyriform cortex in rats. Japan J. Psychiatry Neurology 46: 361–367. Inoue, K., Morimoto, K., Sato, K., Yamada, N., and Otsuki, S. 1992b. Mechanisms in the development of limbic status epilepticus and hippocampal neuron loss: an experimental study in a model of status epilepticus induced by kindling-like electrical stimulation of the deep prepyriform cortex in rats. Acta Med Okayama 46: 129–139. Liu, H., Cao, Y., Basbaum, A.I., Mazarati, A.M., Sankar, R., and Wasterlain, C.G. 1999b. Resistance to excitotoxin-induced seizures and neuronal death in mice lacking the preprotachykinin A gene. Proc Natl Acad Sci U S A 96: 12096–12101. Liu, H., Mazarati, A.M., Katsumori, H., Sankar, R., and Wasterlain, C.G. 1999a. Substance P is expressed in hippocampal principal neurons during status epilepticus and plays a critical role in the maintenance of status epilepticus. Proc Natl Acad Sci USA 96: 5286–5291. Loscher, W. 2002. Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res 50: 105–123. Lothman, E.W., and Bertram, E.H. 1993. Epileptogenic effects of status epilepticus. Epilepsia 34(Suppl 1): S59–S70. Lothman, E.W., Bertram, E.H., and Bekenstein, J.W. 1989. Self-sustaining limbic status epilepticus induced by ‘continuous’ hippocampal stimulation: electrographic and behavioral characteristics. Epilepsy Res 3: 107–119. Lothman, E.W., Bertram, E.H., Kapur, J., and Stringer, J.L. 1990. Recurrent spontaneous hippocampal seizures in the rat as a chronic sequela to limbic status epilepticus. Epilepsy Res 6: 110–118. Mazarati, A.M., Baldwin, R.A., Sankar, R., and Wasterlain, C.G. 1998a. Time dependent decrease in the effectiveness of antiepileptic drugs during the course of self-sustaining status epilepticus. Brain Res 814: 179–185. Mazarati, A.M., Baldwin, R., Klitgaard, H., Matagne, A., and Wasterlain, C.G. 2004. Anticonvulsant effects of levetiracetam and levetiracetamdiazepam combinations in experimental status epilepticus. Epilepsy Res 58: 167–174. Mazarati, A.M., Baldwin, R.A., Sofia, R.D., and Wasterain, C.G. 2000a. Felbamate in experimental model of status epilepticus. Epilepsia 41: 123–127. Mazarati, A., Bragin, A., Baldwin, R., Shin, D., Wilson, C., Sankar, R., Naylor, D. et al. 2002a. Epileptogenesis after self-sustaining status epilepticus. Epilepsia 43(Suppl 5): 74–80. Mazarati, A.M., Hohmann, J.G., Bacon, A., Liu, H., Sankar, R., Steiner, R.A., Wynick, D. et al. 2000b. Modulation of hippocampal excitability and seizures by galanin. J Neurosci, 20: 6276–6281. Mazarati, A., Liu, H., Soomets, U., Sankar, R., Shin, D., Katsumori, H., Langel, U. et al. 1998b. Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. J. Neurosci. 18: 10070–10077. Mazarati, A.M., Liu, H., and Wasterlain, C.G. 1999a. Opioid peptide pharmacology and immunochemistry in an animal model of self-sustaining status epileptocus. Neuroscience 89: 167–173. Mazarati, A.M., Sofia, D., and Wasterlain, C.G. 2002b. Anticonvulsant and antiepileptogenic effects of fluorofelbamete in experimental status epilepticus. Seizure 11: 423–427. Mazarati, A.M., and Wasterlain, C.G. 1999b. NMDA receptors antagonists abolish the maintenance phase of self-sustaining status epilepticus. Neurosci. Lett 65: 187–190. Mazarati, A.M., Wasterlain, C.G., Sankar, R., and Shin, D. 1998c. Selfsustaining status epilepticus after brief electrical stimulation of the perforant path. Brain Res 801: 251–253. McIntyre, D.C., Nathanson, D., and Edson, N. 1982. A new model of partial status epilepticus based on kindling. Brain Res 250: 53–63.
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McIntyre, D.C., Stokes, K.A., and Edson, N. 1986. Status epilepticus following stimulation of a kindled hippocampal focus in intact and commissurotomized rats. Exp Neurol 94: 554–570. Milgram, N.W., Green, I., Liberman, M.K., and Petit, T.L. 1985. Establishment of status epilepticus by limbic system stimulation in previously unstimulated rats. Exp. Neurol 88: 253–264. Morimoto, K., Fahnestock, M., and Racine, R.J. 2004. Kindling and status epilepticus models of epilepsy: rewiring the brain. Progr Neurobio 73: 1–60. Morrisett, R.A., Jope, R.S., and Snead, O.C. 1987. Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats. Exp Neurol 97: 193–200. Nissinen, J., Halonen, T., Koivisto, E., and Pitkanen, A. 2000. A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res 38: 177–205. Nissinen, J., Large, C.H., Stratton, S.C., and Pitkanen, A. 2004. Effect of lamotrigine treatment on epileptogenesis: an experimental study in rat. Epilepsy Res. 58: 119–132. Olney, J.W., Wozniak, D.F., Jevtovic-Todorovic, V., Farber, N.B., Bittigau, P., and Ikonomidou, C. 2002. Drug-induced apoptotic neurodegeneration in the developing brain. Brain Pathol 12: 488–498. Paxinos, G., and Franklin, K.B.J. 2001. In The Mouse Brain in Stereotaxic Coordinates (2nd ed.). San Diego: Academic Press. Paxinos, G., and Watson, C. 1986. In The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press. Pereira de Vasconcelos, A., Mazarati, A.M., Wasterlain, C.G., and Nehlig, A. 1999. Self-sustaining status epilepticus after a brief electrical stimulation of the perforant path: a 2-deoxyglucose study. Brain Res 838: 110–118. Penix, L.R., and Wasterlain, C.G. 1994. Selective protection of neuropeptide-containing dentate hilar interneurons by non-NMDA receptor blockade in an animal model of status epilepticus. Brain Res 644: 19–24. Pitkanen, A. 2002. Drug-mediated neuroprotection and epileptogenesis. Neurology 59(Suppl. 5): S27–S33. Pitkanen, A. 2004. New pharmacotherapy for epilepsy. Drugs 7: 471–477. Pitkanen, A., and Kubova, H. 2004. Antiepileptic drugs in neuroprotection. Expert Opin Pharmacother. 5: 777–798. Prasad, A., Williamson, J.M., and Bertram, E.H. 2002. Phenobarbital and MK-801 but not phenytoin improve long-term outcome in status epilepticus. Ann Neurol 51: 175–181. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation. Electroenceph Clin Neurophysiol 32: 281–294. Sankar, R., Shin, D., Mazarati, A.M., Liu, H., and Wasterlain, C.G. 1999. Ontogeny of self-sustaining status epilepticus, Dev Neurosci 21: 345–351. Shirasaka, Y., and Wasterlain, C. 1994. Chronic epileptogenicity following focal status epilepticus epilepticus, Brain Res 655: 33–34. Sherwood, N.M., and Timiras, P.S. 1970. A Stereotaxic Atlas of the Developing Rat Brain. Berkeley: University of California Press. Sloviter, R.S. 1987. Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy. Science 235: 73–76. Sloviter, R.S. 1991. Permanently altered hippocampal structure, excitability and inhibition after experimental status epilepticus in the rat: the ‘dormant basket cell’ hypothesis and its possible relevance to temporal lobe epilepsy. Hippocampus 1: 41–46. Suchomelova, L., Baldwin, R.A., and Wasterlain, C.G. 2003. SE in the immature brain is not self-sustaining nor benzodiazepine-resistant. Neurology 60: A517–A518. Taber, K.H., McNamera, J.J., and Zornetzer, S.F. 1977. Status epilepticus: a new rodent model. Electroencephalogr Clin Neurophysiol 43: 707–724. Thompson, K., Holm, A.M., Schousboe, A., Popper, P., Micevych, P., and Wasterlain, C. 1998. Hippocampal stimulation produces neuronal death in the immature brain. Neuroscience 82: 337–348.
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Thompson, K.W., and Wasterlain, C.G. 2001. Urethane anesthesia produces selective damage in the piriform cortex of the developing brain. Dev Brain Res 130: 167–171. Treiman, D.M., Walton, N.Y., and Kendrick, C. 1990. A progressive sequence of electroencephalographic changes during generalized convulsive status epilepticus. Epilepsy Research 5: 49–60. Trousseau, A. 1868. Lectures on clinical medicine delivered at the Hotel Dieu, Paris. Volume 1: transl. P.V.Bazire, London: New Sydenham Society. VanLandingham, K.E., and Lothman, E.W. 1991. Self-sustaining limbic status epilepticus. I. Acute and chronic cerebral metabolic studies: limbic hypermetabolism and neocortical hypometabolism. Neurology 41(12): 1942–1949. Vicedomini, J.P., and Nadler, J.V. 1987. A model of status epilepticus based on electrical stimulation of hippocampal afferent pathways. Exp. Neurol 96: 681–691.
Vicedomini, J.P., and Nadler, J.V. 1990. Stimulation-induced status epilepticus: role of the hippocampal mossy fibers in the seizures and associated neuropathology. Brain Res 512: 70–74. Walker, M.C., White, H.S., and Sander, W.A.S. 2002. Disease modification in partial epilepsy. Brain 125: 1937–1950. Wasterlain, C.G. 1972. Breakdown in brain polysomes in status epilepticus. Brain Res 39: 278–284. Wasterlain, C.G. 1974. Mortality and morbidity from serial seizures. An experimental study. Epilepsia 15: 155–176. Wasterlain, C.G., Shirasaka, Y., Mazarati, A.M., and Spigelman, I. 1996. Chronic epilepsy with damage restricted to the hippocampus: possible mechanisms. Epilepsy Res 26: 255–265.
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FIGURE 25--3 A: GluR2 mRNA was significantly decreased in neocortex and hippocampus by 48 hours after hypoxia-induced seizures. B: In situ hybridization showed that GluR2 mRNA was significantly decreased in the pyramidal cell layers within 48 hours after hypoxia-induced seizures at P10. The most significant decrease was observed in CA1/CA2 pyramidal cells (arrows). C: GluR2 protein expression was significantly decreased within 96 hours after hypoxia-induced seizures. Western blots show that GluR2 protein expression was significantly decreased in hippocampus within 96 hours after hypoxia treatment. In contrast, GluR1 expression did not change significantly after hypoxiainduced seizures.
FIGURE 3 6 - 3 Timm staining was positive in the animals killed during the latent period, 20 days after selfsustaining status epilepticus (SSSE) (A), and 6 hours after the first spontaneous seizure (B). Timm staining in the animals euthanized 2-month after SSSE, after a total of 203 (C) and 42 (D) spontaneous seizures. (Reproduced from Mazarati et al., 2002a. Copyright Blackwell Publishing, 2002.)
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37 Posttraumatic Epilepsy Induced by Lateral Fluid-Percussion Brain Injury in Rats ASLA PITKÄNEN, IRINA KHARATISHVILI, JARI NISSINEN, AND TRACY K. MCINTOSH
with a GCS of 13 to 15 develop late posttraumatic seizures within 24 months after injury (Englander et al., 2003). Some TBI patients with a high risk of epilepsy present with penetrating head injury, from which up to 53% develop epilepsy (Salazar et al., 1985; for review, see Frey, 2003). Other risk factors for late posttraumatic seizures include early posttraumatic seizures (seizures that occur <1 week after TBI), skull fracture with dural penetration, biparietal contusions, acute intracerebral hematoma, subdural hematoma with surgical evacuation, multiple or bilateral contusions, multiple intracranial procedures, evidence of a >5-mm midline shift, and loss of consciousness for >24 hours (Annegers et al., 1998; Englander et al., 2003; Frey, 2003). Many of these factors reflect the severity of the initial injury and, therefore, their causal relationship to epileptogenesis is unclear. Contrary to previous assumptions regarding the contribution of hemosiderin to epileptogenesis, neither punctate, subarachnoid, nor intraventricular hemorrhages alter the probability of late posttraumatic seizures (Englander et al., 2003). Age is also a risk factor for TBI-induced epileptogenesis. About 10% of children with severe TBI (cf. 16% to 20% in adults) develop epilepsy (Annegers et al., 1980; Herman, 2002). The risk of late posttraumatic seizures in those >65 years of age is 2.5-fold compared with a younger population (Annegers et al., 1998; Frey, 2003). After the first late posttraumatic seizure (seizures that occur >1 week after TBI), 86% of patients develop a second seizure within 2 years (Haltiner et al., 1997). Almost 90% of patients have at least five seizures within 2 years of the first late seizure (Haltiner et al., 1997), and the latency to the first late seizure appears to depend on the severity of TBI. In a 2-year follow-up, 50% of those patients with multiple contusions who eventually developed epilepsy (25% of
Supported by the Academy of Finland, Sigrid Juselius Foundation, Finnish Cultural Foundation, and Paulo Foundation to AP and NIH NS08803 and NS40978 to TKM.
BACKGROUND Posttraumatic Epilepsy in Humans About 0.8% of the world population has epilepsy and, according to the World Health Organization, 50 million people worldwide have epilepsy at any one time (http://www.who.int/mediacentre/factsheets/fs165/en/). About 30% of all epilepsies are symptomatic and 30% are presumed symptomatic [previously called cryptogenic, Engel Jr. (2001)]. Traumatic brain injury (TBI) is estimated to cause 20% of all symptomatic epilepsies (Hauser et al., 1991). Seizures after TBI are often drug-refractory and, therefore, the need for medical care continues for years after epilepsy diagnosis (Herman, 2002). Thus, it is estimated that in the European Union (population 465 million) and USA (295 million), about 0.5 million individuals have posttraumatic epilepsy (PTE), which compromises their quality of life and well-being in addition to other functional impairments associated with TBI. The risk of TBI-induced epileptogenesis and epilepsy is believed to be related to the severity of TBI (Annegers et al., 1998; see Table 1 for classification of the severity of TBI). In the general population, the 30-year cumulative incidence of epilepsy is 2.1% for mild, 4.2% for moderate, and 16.7% for severe injuries (Annegers et al., 1998). According to a recent large multicenter study, 17% of TBI patients with a Glasgow Coma Score (GCS) of 3 to 8, 24% with a GCS of 9 to 12, and 8%
Models of Seizures and Epilepsy
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TABLE 1 Association of risk of epilepsy with the severity of traumatic brain injury (TBI) in humans and rats Human TBI Severity (% epilepsy)2 Mild
GCS (% epilepsy)3 13–15
(2%)
(8%)
Moderate
9–12
(4%) Severe
(> 17%)
Rat TBI induced by lateral FPI
1
(24%) 3–8
(17%)
Clinical findings2 (acute)
Severity4 (% epilepsy)5
One or more of the following: • absence of fracture • loss of consciousness or posttraumatic amnesia < 30 min
Pressure pulse < 1 atm (no data)
One or more of the following: • skull fracture • loss of consciousness or posttraumatic amnesia 30 min–24 h
Pressure pulse 1.5–2.5 atm (no data)
One or more of the following: • brain contusion • intracranial hematoma • loss of consciousness or posttraumatic amnesia >24 h
Pressure pulse 2.5–3.6 atm
Behavioral impairment6 (48 h)
• NeuroScore and spatial memory ~40–50% of deficit in controls • acute mortality ~20% • acute mortality >30%
(40%)
Estimation of the impairment in composite neuroscore and probe test of spatial memory and mortality in rats with TBI is based on analysis of data available from one of the authors lab (TKM). Abbreviations: GSC, Glasgow Coma Scale. 1 Teasdale and Jennett (1976). 2 Annegers et al. (1998). 3 Englander et al. (2003). 4 McIntosh et al. (1989). 5 present study. 6 McIntosh TK (laboratory files, unpublished).
total) did so in ~6 months. In cases with a single contusion, 50% of those patients who developed epilepsy (8% of total) did so within 10 months (Englander et al., 2003). Further, the second unprovoked seizure tended to appear faster in patients with a shorter latency to the first late seizure (Haltiner et al, 1997). Latency length, however, is not associated with seizure frequency (Haltiner et al., 1997). Studies that assessed seizures based on their behavioral appearance report that 67% to 79% of late seizures are (secondarily) generalized (Haltiner et al., 1997; Englander et al., 2003). A recent video-electroencephalography (EEG) monitoring study reported that only 24% of seizures in patients with PTE are secondarily generalized (Hudak et al., 2004). Previous studies report that 35% to 62% of patients with PTE manifest it as temporal lobe epilepsy (TLE) (Diaz-Arrastia et al., 2000; Hudak et al., 2004), and 53% of those patients with posttraumatic TLE have mesial temporal lobe sclerosis on magnetic resonance imaging (Hudak et al., 2004), which occurs bilaterally in some patients (Diaz-Arrastia et al., 2000). Histologic analysis of a few cases revealed hippocampal damage in surgically operated patients with TLE (Diaz-Arrastia et al., 2000). Although 35% of patients with PTE become seizure free with antiepileptic drug (AED) therapy, ~60% to 80% of patients continue to require polytherapy (Pohlman-Eden and Bruckmeir, 1997; Hudak et al., 2004). Finally, TBI can reactivate epilepsy that is in remission or even modify its course, resulting in drug refractori-
ness in a limited number of TBI survivors (Tai and Gross, 2004). Clinical trials with AED aimed at suppressing early seizures or the neurobiology of epileptogenesis failed to prevent or alleviate TBI-induced epileptogenesis (Temkin, 2001). Early administration of corticosteroids to relieve edema associated with trauma had no effect on epileptogenesis (Watson et al., 2004). Because TBI annually affects about 1.5 million Americans resulting in long-term disabilities in 80,000 to 90,000 people (Centers for Disease Control, Traumatic Brain Injury in the United States: A Report to Congress), it is a major challenge to identify patients with a poor outcome and to provide treatments that alleviate long-term somato-motor and cognitive impairments and prevent the development of epilepsy.
Animal Models of Posttraumatic Epilepsy Posttraumatic epilepsy was originally modeled by the application of metals (alumina, cobalt, iron) to the cortex in rats, cats, and nonhuman primates (Kopeloff et al., 1950; Dow et al., 1962; Willmore et al., 1972). One of the most successful attempts was the subpial application of FeCl2 or FeCl3 into the rat or cat sensorimotor cortex. Development of these models was based on observations that hemosiderin deposits are associated with a high risk of PTE. The cortical application of iron results in the appearance of focal
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Induction and Characterization of PTE Induced by Lateral FPI
onset partial seizures. In EEG, seizure duration was >20 seconds. Behavioral seizures persisted beyond 12 weeks in 94% of iron-injected rats (Willmore et al., 1972). Another early approach to model PTE was the subpial undercut model in rat, guinea pig, and cat (Prince and Tseng, 1993: Hoffman et al., 1994). Isolation of the sensorimotor cortex from the underlying white matter with minimal pial damage is achieved by advancing a bent needle under the pia with a micromanipulator. The needle is lowered into the white matter, and gray and white matter transection is accomplished by rotating the needle 180o (Graber and Prince, 2004). Lesions to immature rats (postnatal day 0 to 32) result in the appearance of epileptiform activity in in vitro cortical slices ~1 to 2 weeks after the lesion which persists for months (Prince and Tseng, 1993: Hoffman et al., 1994). Spontaneous seizures, however, have not been reported in these animals. The application of metallic cations to the cortex or lesioning of the brain reproduces few of the neurobiologic aspects of TBI in vivo. TBI results in a complex assembly of acute and delayed molecular, cellular, and network alterations, some of which are directly caused by trauma, whereas others are delayed and secondary to the initial physical impact. Secondary cascades of injury that presumably contribute to TBI-induced epileptogenesis include breakdown of the blood—brain barrier, edema formation, impairment of energy metabolism, changes in cerebral perfusion, ionic dyshomeostasis, activation of autodestructive neurochemicals and enzymes, generation of free radicals, induction of inflammatory cascades, and genomic changes (Laurer and McIntosh, 1999). These events might lead to acute and delayed cellular (neuronal, glial) death, gliosis, neurogenesis and gliogenesis, and axonal injury in both the traumatized cortex and in the hippocampus, thalamus, brainstem, and cerebellum. They can also contribute to modification of axonal and dendritic plasticity of surviving neurons, which might either enhance functional recovery, for example, by improving sensorimotor performance over time (Laurer and McIntosh, 1999), or is associated with functional impairment such as epileptogenesis. Moreover, the spectrum of alterations depends on the type and severity of TBI (Laurer and McIntosh, 1999). A hallmark of epilepsy is the occurrence of chronic, unprovoked spontaneous seizures. Several experimental models of TBI available mimic many of the clinical aspects of TBI in humans that could be adopted for the development of an in vivo PTE model (Laurer and McIntosh, 1999). So far, spontaneous seizures have been described only after the induction of experimental lateral fluid-percussion brain injury (FPI), which is currently the most widely used and investigated model for human closed head injury (Thompson et al., 2005). D’Ambrosio et al., (2004) recently induced rostral parasagittal FPI in juvenile male Sprague-Dawley rats at postnatal day (P) 30 to 32, which resulted in the
appearance of spontaneous generalized 7- to 9-Hz spikeand-wave discharges with a frontoparietal onset in 83% of animals in 1 month. A high percentage (92%) of rats developed ictal-like episodes in a 4-month follow-up period. According to the data shown, seizures were partial and the typical seizure duration was <10 seconds (D’Ambrosio et al., 2004; D’Ambrosio et al., 2005). Recently, D’Ambrosio et al. reported that the frequency of ictal-like episodes originating in the hippocampus increased over a 7-month follow-up period, suggesting that the ictal origin moved from the frontoparietal cortex to the temporal lobe (D’Ambrosio et al., 2005). Nissl and glial fibrillary acidic protein staining suggested that early pathologic changes were exclusively in the ipsilateral cortex around the lesion site without any hippocampal involvement (D’Ambrosio et al., 2004). In a chronic follow-up study, atrophy of the cortex and hippocampus was reported (D’Ambrosio et al., 2005). We describe a protocol that we use to induce PTE with lateral FPI in adult rats. In contrast to the results produced by rostral parasagittal FPI in juvenile rats (see above), ~43% to 50% of animals develop epilepsy by 11 months. Most of the spontaneous recurrent seizures are secondarily generalized, duration is about 100 seconds, and they occur one to two times every 2 weeks.
INDUCTION AND CHARACTERIZATION OF PTE INDUCED BY LATERAL FPI Animals Adult male Sprague-Dawley rats (n = 35, 310 to 350 g) were used. The rats were housed in individual cages in a controlled environment (constant temperature, 22 ± 1° C, humidity 50% to 60%, lights on 0700 to 1900 hours). Animals had free access to food and water. All animal procedures were approved by the Animal Care and Use Committee of the University of Kuopio and conducted in accordance with the guidelines set by the European Community Council Directives 86/609/EEC.
Lateral FPI Lateral FPI was induced as originally described (McIntosh et al., 1989). Animals were anesthetized with a single intraperitoneal injection (i.p., 6 ml/kg) of a mixture containing sodium pentobarbital (58 mg/kg), chloral hydrate (60 mg/kg), magnesium sulfate (127.2 mg/kg), propylene glycol (42.8%), and absolute ethanol (11.6%). After shaving the hair and wiping the skin with disinfectant, the head of the animal was fixed in a stereotaxic frame (lambda and bregma on the same horizontal level). A midline skin incision was made and the tissue was reflected to reveal the bregma, lambda, sagittal suture, and the lateral ridges. The
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Chapter 37/Posttraumatic Epilepsy Induced by Lateral Fluid-Percussion Brain Injury in Rats
anterior edge of the craniotomy was located—2.0 mm posterior to bregma and the lateral edge adjacent to the left lateral ridge. The center of the craniotomy was at -4.4. mm posterior and 3.6 mm lateral to bregma (Figure 1A). The bone flap was removed and the edges of the hole were cleaned. After implanting a stainless steel screw to the skull to support the dental cement (see below), a rigid plastic cap that was cut from a female Leur-Loc needle hub was placed into the hole over the intact dura. The cap was sealed to the skull with glue (3M VetbondRM) and allowed to dry. Thereafter, the plastic injury cap was fixed to the skull with dental acrylic. Proper sealing is critical to produce consistent injury severity. The injury cap was kept filled with 0.9% saline before connection to the injury device. After the dental cement had dried, the animals were connected to the fluid-percussion device via the Leur-loc fitting. Traumatic brain injury was produced with a fluidpercussion device (AmScien Instruments, Richmond, VA) consisting of a Plexiglas cylindrical reservoir, 63.5 cm long and 6.4 cm in diameter, filled with 25° C (room temperature) isotonic saline, and bound at one end by a cork-covered Plexiglas piston (Fig. 1B). The other end of the reservoir was fitted with a 5.7-cm long metal housing on which an extracranial pressure transducer (AmScien Instruments, model FP 302) was mounted and connected to a 3-cm tube that terminated with a male Leur-Loc fitting. At the time of injury, this tube was connected to the female injury cap of the rat. A metal pendulum was released from a predetermined angle to strike the piston. This produced a brief (21 to 23 ms) impact against the exposed dura. A transient pressure fluid pulse to the epidural surface produced a mechanical stress that was diffusely transmitted to a wide area of the brain resulting in little or no overt histologic damage at low levels of severity. It, therefore, produces features similar
to that of an acceleration impact injury, often resulting in a cerebral concussion. Pressure pulses were measured extracranially by a transducer and recorded on a storage oscilloscope. The force of the impact was adjusted to 2.9 to 3.3 atm to induce severe lateral FPI, which was assumed to maximize the likelihood of epileptogenesis based on previous data in humans showing that their injury severity positively correlates with the likelihood of PTE (Annegers et al., 1998). Animals were video-monitored (see following section) during the induction of lateral FPI to later quantify the behavioral seizure activity induced by injury. In addition, the duration of posttraumatic apnea was monitored. After injury, the dental cement and Leur-Loc fitting were removed, the incision sutured, and the animals allowed to recover. On recovery, animals were returned to their home cages. The weight of animals before impact and 1 week later did not differ between the controls and TBI group. Controls had identical anesthetic and surgical operations without the impact.
Mortality, Apnea, and Seizures Immediately after FPI The mortality rate in animals subjected to lateral FPI was 33%, which is consistent with results described previously by McIntosh et al. (1989). The duration of acute apnea immediately after the induction of TBI was defined as the time from injury to the moment when spontaneous breathing returned. Time in apnea varied greatly between the animals, from 10 to 65 seconds (mean 29 ± 18 seconds). Brief acute clonic seizures (10 to 15 seconds) were observed immediately after the induction of lateral FPI in 30% of rats with TBI. Only two of those animals that experienced immediate seizures developed epilepsy in long-term follow-up.
FIGURE 1 (A) A trephine (diameter 5 mm) is used to create a craniotomy that is centered between bregma and lambda. The lateral edge of the trephine is placed at the lateral ridge (open arrow). The grid lines on the left are placed 1 mm apart and show the location and size of the craniotomy (Paxinos and Watson, 1986). (B) Fluid-percussion apparatus. A pendulum that is set to a predetermined angle is released and hits the cork piston of the cylinder, which is filled with saline. While moving, the pendulum activates a photoelectric cell that triggers the transducer (placed immediately prior to the Leur-Loc connection) to read the pressure of the saline pulse that is also recorded on an oscilloscope. The pulse pressure is expressed in atmospheres (atm).
Induction and Characterization of PTE Induced by Lateral FPI
Video-EEG Monitoring of Behavioral and Electrographic Seizures Electrode Implantation One week after injury, the animals were anesthetized with an i.p. injection of the anesthesia cocktail described earlier and inserted into a stereotaxic frame (lambda and bregma at the same horizontal level). A depth electrode (Franco Corradi, Milano, Italy, ø 0.127 mm) was inserted into the ventral hippocampus ipsilateral to the trauma, placed at coordinates -6 mm caudal to the bregma, 4.6 mm lateral from the midline, and -7.0 mm ventral to surface of the skull according to the rat brain atlas of Paxinos and Watson (1986). Two cortical screw electrodes (E363/20 Plastics One Inc., Roanoke, VA) were placed over the parietal cortex, one rostral to the injury site and the other contralateral to the corresponding region. One stainless steel screw was inserted into the skull above the right frontal cortex as an anchor. Two stainless steel screw electrodes inserted into the skull bilaterally over the cerebellum served as indifferent and ground electrodes. All electrodes were connected to a plastic pedestal (Plastics One, Inc.) that was cemented onto the skull with dental acrylic (Selectaplus, Dentsply, DeTrey GmbH, Dreieich, Germany). Video-EEG Monitoring Monitoring was started 7 weeks after injury. Each animal was video-EEG monitored for 1 week (24 hours/day) every 6 weeks (Figure 2). At the end of the study, each animal was continuously monitored for 2 weeks. Details of the methodology of long-term video-EEG monitoring were described previously (Nissinen et al., 2000). Briefly, rats were placed singly in Plexiglas cages where they could move freely. Electrical brain activity was
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monitored using the Nervus EEG Recording System (Taugagreining, Iceland) connected with an ISO-1032 Amplifier (Braintronics, Netherlands), filtered (high-pass filter 1 Hz cut-off, low-pass 100 Hz). The behavior of the animals was recorded using a WV-BP312E Video Camera (Panasonic) that was positioned above the cages and connected with a SVT-S3000P Time Lapse 168 VCR (Sony) and a PVC-145E Video Monitor (Sony). A type 955 Infra Red Light (Videmech, Ltd., UK) was used at night to allow continuous video monitoring of the animals’ behavior. A wide-angle lens permitted simultaneous videotaping of up to eight animals. Analysis of Video-EEG Each EEG file was analyzed manually by browsing through the EEG recording on the computer screen. If an electrographic seizure was observed, behavioral severity was analyzed from the corresponding video-recording. An electrographic spontaneous seizure was defined as a high frequency (>5 Hz), high-amplitude discharge that lasted at least 5 seconds. The severity of behavioral seizures was scored according to a slightly modified Racine’s scale (Racine, 1972): score 0–electrographic seizure without any detectable motor manifestation; score 1—mouth and face clonus, head nodding; score 2—clonic jerks of one forelimb; score 3—bilateral forelimb clonus; score 4—forelimb clonus and rearing; score 5—forelimb clonus with rearing and falling. Seizures scored from 0 to 2 were considered partial, whereas seizures scored from 3 to 5 were considered secondarily generalized. In our study, animals with two or more seizures during the 11-month period of monitoring were considered epileptic. Animals that did not have any seizures during the follow-up were considered not epileptic.
FIGURE 2 Study design. An 11-month video-EEG (vEEG) monitoring follow-up period that started 7 to 9 weeks after induction of lateral fluid percussion injury (FPI). Each animal was continuously (24 hours/day) monitored for 7 consecutive days every 7 weeks at the time points indicated at the bottom of the striped boxes. At the end of the study, each animal was continuously monitored for 2 weeks. Thereafter, rats were perfused for histology.
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Histology Fixation The rats were perfused for histology 11 months after the induction of lateral FPI immediately after finishing videoEEG monitoring. The animals were deeply anesthetized and perfused according to the Timm fixation protocol described by Sloviter (1982). The brains were removed from the skull and postfixed in buffered 4% paraformaldehyde for 4 hours, and then cryoprotected in a solution containing 20% glycerol in 0.02 M potassium phosphate buffered saline (KPBS), pH 7.4, for 24 hours. The brains were then blocked, frozen in dry ice, and stored at -70° C until cut. The brains were sectioned in the coronal plane (30 mm, 1-in-5 series) with a sliding microtome. The sections were stored in a cryoprotectant tissue-collecting solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium phosphate buffer) at -20° C until processed. Adjacent series of sections were processed for Nissl and Timm stainings.
experimental animals developed epilepsy. The number of epileptic animals increased steadily throughout the monitoring period and constituted 6% (1/18 rats) in the first monitoring at 9 weeks, 11% (2/18 rats) in the second monitoring at 17 weeks, 22% (4/18 rats) in the third monitoring at 24 weeks, 39% (7/18 rats) in the fourth monitoring at 32 weeks, and 50% (9/18 rat) in the fifth monitoring at 42 weeks after TBI. In our second series of animals with a 1-year followup, 43% of the rats developed epilepsy after lateral FPI (data not shown).
Latency Period According to our first and second (data not shown) experiments, the latency period varied greatly between individual animals and ranged from 7 weeks to 11 months using the video-EEG monitoring paradigm described above (Figure 2). Our preliminary estimate is that 50% of animals that will develop epilepsy express spontaneous seizures within 7 to 8 months after induction of FPI.
Nissl Staining The first series of sections was stained for thionin to identify the cytoarchitectonic boundaries and the distribution and severity of neuronal damage. The severity of neuronal damage in different subfields of the hippocampus (CA1, CA3, hilus) was scored semiquantitatively as follows: Score 0—no damage; 1—less than 10% neuronal loss; 2— between 11 and 50% neuronal loss; and 3—greater than or equal to 50% neuronal loss. To assess the total hippocampal damage, we also calculated the sum score (sum of damage scores in the hilus, CA3, and CA1). Timm Staining Mossy fiber sprouting was analyzed from Timm-stained sections at the septal end, including coronal sections between levels—2.3 and—6.0 mm posterior to bregma (Paxinos and Watson, 1986). The density of sprouting was semiquantitatively assessed according to Cavazos et al., (1991): Score 0—no granules; 1—sparse granules in the supragranular region and in the inner molecular layer; 2—granules evenly distributed throughout the supragranular region and the inner molecular layer; 3—almost a continuous band of granules in the supragranular region and inner molecular layer; 4—continuous band of granules in the supragranular region and in the inner molecular layer; 5—confluent and dense laminar band of granules that covers most of the inner molecular layer, in addition to the supragranular region.
CHARACTERISTICS OF PTE Occurrence of Epilepsy Chronic video-EEG monitoring was performed for 11 months after injury. By the end of the study, 50% of the
Behavioral and Electrographic Characteristics of Spontaneous Seizures Seizure Type Mean behavioral seizure score was 3.3 ± 1.4 (range 1 to 5; median 4). The mean percentage of secondary generalized seizures of all seizures was 78 ± 37% (range 20 to 100; median 100). There was a tendency towards worsening in the mean behavioral seizure score from 3.2 to 4.1 along the course of the follow-up period. The electrographic seizure activity was detected first by the depth electrode located in the ventral hippocampus ipsilateral to the injury (Figure 3). From there, it rapidly spread to the contralateral cortex. The ictal pattern was always represented by repetitive, rhythmic spike-and-wave or polyspikeslow wave complexes, changing in shape, amplitude, and frequency throughout the seizure. Subclinical electrographic seizure activity without behavioral manifestations was detected initially in three animals with PTE that then developed score 5 behavioral seizures in subsequent recordings. Seizure Frequency Mean seizure frequency in animals with PTE was 0, 3 ± 0, 3 seizures per day (range 0.04 to 0.4 seizures/day; median 0.15 seizures/day). There was a tendency towards an increase in the mean seizure frequency from 0.29 seizures/day to 0.86 seizures/day along the course of the follow-up. Seizure Duration Mean seizure duration in animals that developed PTE was 104 ± 56 seconds (range 29 to 196 seconds; median 85
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Characteristics of PTE
left HC
contralateral Cx
left HC
contralateral Cx
5 seconds
FIGURE 3 A typical example of an electroencephalographic (EEG) recording from an animal with secondarily generalized seizures. Electrographic seizure originated in the hippocampus (HC) and spread to the contralateral cortex (Cx). Electrographic seizure lasting 1 minutes and 44 seconds was associated with a stage 5 behavioral seizure [rearing and falling, according to Racine, (1972)]. Max. amplitude 1.5 mV.
seconds). Once registered, seizure duration remained essentially stable for each epileptic animal throughout the five monitoring sessions. Diurnal Occurrence of Seizures Epileptic seizures were observed both during daytime and nighttime monitoring. Seizures tended to occur more often during lights-off period (55% of seizures occurred between 1900 and 0700 hours). Interictal Activity Epileptiform interictal activity was usually intermittent, represented by spike-wave paroxysmal discharges in the hippocampus ipsilateral to trauma. It was observed only in those animals that also had seizures on the EEG. Remission No remission was observed in any epileptic animal during the 11-month monitoring period.
Hippocampal Cell Loss Analysis of coronal thionin-stained sections from animals sacrificed 11 months after lateral FPI indicated substantial neuronal loss both ipsilaterally (total damage score 2.68) and contralaterally (1.83) compared with controls (p < 0.01; Figure 4A, B, D). Damage was more severe ipsilaterally than contralaterally (p < 0.05).
Mossy Fiber Sprouting An adjacent series of sections was stained using the Timm method to detect the severity of mossy fiber sprouting in the dentate gyrus. Analysis indicated an increase in mossy fiber sprouting in the septal hippocampus ipsilateral to the trauma compared to that in control animals (p < 0.001; Fig. 4C, E). Sprouting was denser in epileptic animals compared to those without epilepsy (p < 0.01). Further, sprouting was denser ipsilaterally than contralaterally (p < 0.05).
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A HC
Th
B
Control g
mol
C
Control
CA3c
CA3c
H
H Nissl
D
g
o m i
Timm PTE
E
PTE
FIGURE 4 Digitized bright-field photomicrographs showing damage in the brain of rats with posttraumatic epilepsy (PTE) as a consequence of lateral fluid-percussion injury (FPI). (A) A thionin-stained coronal section from the brain of an animal that had lateral FPI 8 months earlier and had spontaneous seizures. Notice the lesion in the left primary somatosensory and auditory cortices (between open arrows, according to Paxinos and Watson, 1986). The hippocampus (HC) and thalamus (Th) show substantial atrophy compared with the contralateral side. (B) A thionin-stained section from the septal end of the hippocampus in a control rat. (C) A Timm-stained section of the septal hippocampus in a control rat. (C) A thionin-stained section from a rat with PTE that was killed 11 months after lateral FPI. Note the loss of hilar cells. (D) A Timm-stained section from a rat with posttraumatic epilepsy (PTE). Note abnormal dense labeling of mossy fibers in the inner molecular layer of the dentate gyrus (open arrows). g, granule cell layer; H, hilus; i, inner molecular layer; m, midmolecular layer; mol, molecular layer; o, outer molecular layer. Scale bars: panel A, 1 mm; panels B-E, 100 mm.
Lateral Fluid-Percussion Induced Epilepsy: What Does It Model?
LATERAL FLUID-PERCUSSION INDUCED EPILEPSY: WHAT DOES IT MODEL? Lateral FPI Lateral FPI is currently the most widely used animal model of human TBI (Thompson et al., 2005). It produces several focal and diffuse characteristics of moderate to severe closed head injury in humans, including focal contusion, blood—brain barrier disruption, altered cerebral metabolism, altered blood cerebral flow, subdural hematoma, intraparenchymal and subarachnoid hemorrhage, local and remote axonal injury, progressive neuronal loss, altered electrical activity (acute seizures, alterations in evoked potentials), and acute and chronic behavioral abnormalities (Thompson et al., 2005). Association Between the Severity of TBI and the Development of PTE In the present study, the acute mortality of the animals subjected to lateral FPI was 30% to 40%, indicative of animals experiencing severe TBI (McIntosh et al., 1989). Approximately 43% to 50% of the rats with severe injury developed epilepsy, comparable with epidemiologic data available from patients with severe TBI under civilian or military circumstances (see section Background). Whether milder damage results in epileptogenesis remains to be studied. Latency Period The latency period typically lasted several months before the first detection of spontaneous seizures. We monitored the animals continuously for 1 week every 7 weeks, therefore it is possible that we missed some seizures. Data from our second series of animals that included a more intensive monitoring (data not shown), however, support the data shown here. Characteristics of Late Seizures Several previous studies reported hyperexcitability and lowered seizure threshold for convulsants in animals with moderate to severe lateral FPI (Lowenstein et al., 1992; Coulter et al., 1996; Toth et al., 1997; D’Ambrosio et al., 1999; Golarai et al., 2001; Santhakumar et al., 2001), but no clear spontaneous seizures. In the present study, 43% to 50% of rats with severe TBI developed epilepsy and the mean duration of seizures was 10 seconds. Further, based on video-EEG analysis, most late spontaneous seizures in rats with PTE were secondarily generalized. Information about the seizure characteristics in PTE in humans is compromised by the fact that typically patients are on antiepileptic medication. Haltiner et al. (1997) and
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Englander et al. (2003) reported that 67% to 79% of patients with PTE had secondarily generalized seizures. A recent video-EEG study by Hudak et al. (2004), however, indicated that only 24% of patients with PTE had generalized seizures (primary or secondary generalization), suggesting that the occurrence of partial seizures might have been underestimated in previous studies. Hippocampal Pathology Rats with PTE had neuronal damage in several hippocampal subfields, including the hilus, CA3, and CA1. Previous histologic studies demonstrated neuronal loss in the CA4 (includes part of the CA3 and hilus), CA3, and CA1, whereas the CA2 was preserved in the hippocampus of TBI patients with or without epilepsy (Diaz-Arrastia et al., 2000; Maxwell et al., 2003). Further, based on magnetic resonance imaging (MRI), hippocampal atrophy is often bilateral in human PTE (Diaz-Arrastia et al., 2000). As we show here, rats with PTE typically have bilateral damage, even though the neuronal loss was more substantial ipsilateral to the trauma, consistent with previous reports in rats with lateral FPI (Lowenstein et al., 1992; Smith et al., 1997). Immunohistochemical studies demonstrated that subpopulations of hilar inhibitory neurons contributing to perisomatic (parvalbumin, cholecystokinin) and dendritic (somatostatin) inhibition are lost after FPI (Lowenstein et al., 1992; Coulter et al., 1996; Toth et al., 1997; Golarai et al., 2001). In addition, approximately 60% of excitatory mossy cells that innervate inhibitory neurons are lost, which might also contribute to the increased excitability of the dentate gyrus after FPI (Toth et al., 1997). Contrary to these observations, Reeves et al. (1997) reported increased g-aminobutyric acid (GABA) immunoreactivity in the granule cell layer and inner molecular layer 2 and 15 days after FPI, which was associated with increased inhibition in the dentate gyrus. Substantial bilateral mossy fiber sprouting occurred after FPI, which as with the neuronal damage, was more severe ipsilateral to TBI. TrkB-ERK1/2-CREB/Elk-1 signaling pathways associated with axonal plasticity become activated within 24 hours after FPI (Hu et al., 2004). Golarai et al. (2001) observed bilateral mossy fiber sprouting in a weightdrop model when brains were analyzed 2 to 15 weeks after TBI. No systematic studies have been done on the development of mossy fiber sprouting after TBI in humans. Other Pathologies Data available indicate that in addition to acute and delayed neuronal death and axonal sprouting, other pathologies have a role in structural reorganization and functional impairment and recovery after TBI, and consequently, can contribute to posttraumatic epileptogenesis. Immunohistochemical studies demonstrated increased numbers of
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activated astrocytes during the first weeks following lateral FPI in rats at the cortical injury site and the hippocampus (Hill et al., 1996). Analysis of hippocampal tissue using stereologic cell counting, however, could not demonstrate any differences in the total number of astrocytes compared to controls 2 weeks after lateral FPI (Grady et al., 2003). Further, by using gold sublimate staining, Hill-Felberg et al. (1999) demonstrated an approximate 40% loss of the total astrocyte population in the ipsilateral hippocampus occurred during the first week after lateral FPI. Inflammatory neutrophil and macrophage recruitment occurs in the lateral FPI model within 3 days after injury in regions with neuronal loss and blood-brain—barrier disruption, including the hippocampus (Soares et al., 1992). Using stereology, Grady et al. (2003) demonstrated that the number of microglial cells continued to be increased at 14 days after injury in a different CA subfields of the hippocampus as well as in the hilus of the dentate gyrus in rats with lateral FPI. No differences were seen in the total number of oligodendrocytes (Grady et al., 2003). In addition to neuronal and glial cell death, TBI can induce cell proliferation in the traumatized cortex, dentate gyrus, and subventricular zone in rats that can last for up to 1 year (Dash et al., 2001; Braun et al., 2002; Chirumamilla et al., 2002; Chen et al., 2003; Rice et al., 2003). At 48 hours after lateral FPI, proliferating cells in the hippocampus express markers of immature astrocytes as well as of proliferating microglia and macrophages (Chirumamilla et al., 2002). In a rat cortical impact injury model, most (65%) of proliferating cells in the dentate gyrus gain the phenotype of mature granule cells in approximately 1 month, whereas only 5% of newly born cells express the astrocytic marker glial fibrillary acidic protein (Dash et al., 2001). Interestingly, when assessed 10 days after cortical impact injury, neurogenesis also occurs in the contralateral hippocampus, although to a lesser extent than ipsilaterally (Dash et al., 2001). As the present and previous studies show, axon sprouting of remaining neurons is most prominent several weeks or months after lateral FPI. Several types of TBI, including lateral FPI, can also cause axonal injury, which is presumably one of the major factors contributing to poor sensorimotor and cognitive outcome. Histologic studies using markers of damaged axons (e.g., amyloid precursor proteins) reveal that axonal injury continues for weeks to months after impact in the major fiber pathways as well as the thalamus and hippocampus, both in rats and humans, and, therefore, occurs in parallel with axonal sprouting (Roberts et al., 1991; Graham et al., 1995; Pierce et al., 1996). In addition to axonal alterations, recent evidence indicates that the dendrites also undergo remodeling during the first month after injury in rats with FPI at postnatal days 19 to 20 (Ip et al., 2002). Dendritic density was increased in
regions remote to the primary injury site (i.e., in the contralateral parietal cortex and ipsilateral and contralateral occipital cortex). The PTE induced by lateral FPI in rats has clinical and pathologic characteristics similar to those of PTE in humans. The presence of an initial insult (TBI), latency period, and spontaneous seizures demonstrates that the model has all of the components of the epileptic process in humans that eventually results in symptomatic epilepsy.
LIMITATIONS Technically, FPI is easy to induce and highly reproducible. Recent studies, however, indicate that small differences in the location of the FPI in the cortex result in substantial variability in the distribution of structural damage and associated functional impairment (Vink et al., 2001; Floyd et al., 2002). For example, bilateral hippocampal damage is more likely if the craniotomy is located medially rather than laterally (Vink et al., 2001). Lesion location is an important variable to consider when data from different laboratories are compared, as is the present one, and the recent observations by D’Ambrosio and colleagues (2004). Severe injury used to induce epileptogenesis results in 30% to 40% mortality after FPI. This increases the number of animals required to achieve statistical power in the final analysis. Further, because the development of epilepsy occurs in 43% to 50% of severely brain-injured rats in a 1year follow-up, a larger number of animals is required for studies investigating the effect of novel antiepileptogenic or disease modifying compounds after TBI. One of the major challenges in working with FPI-induced PTE relates to the long follow-up (6 to 12 months) of animals that is required to detect spontaneous seizures. Low seizure frequency makes it difficult to detect seizures without long-term video-EEG monitoring, which is costly and labor-intensive. Despite some practical issues, the epileptogenesis and epilepsy induced by lateral FPI provides a model that reproducibly replicates the entire epileptogenic process in humans after TBI and, therefore, provides a promising tool to investigate the mechanisms of epileptogenesis, and novel therapeutic targets for its prevention.
FUTURE CHALLENGES Several important issues relevant to human posttraumatic epileptogenesis remain to be modeled and studied. These include the age-dependence of TBI-induced PTE, effect of genetic background on posttraumatic epileptogenesis, effect of location, severity, and type of injury on the duration of latency period and severity of epilepsy, detailed analysis of
References
the distribution and severity of molecular alterations and cellular pathologies, interanimal variability, and assessment of sensorimotor and memory dysfunction and its association with epilepsy. Currently, we also do not have any data on the response of late recurrent seizures to AED or the development of drug refractoriness in rats with PTE. Another important area of future research should be to identify surrogate markers that would predict epileptogenesis. Further, the usefulness of lateral FPI-induced PTE as a tool to study novel focal therapies (e.g., gene therapy, cell transplantations, or neurostimulation) in the treatment of epileptogenesis or epilepsy needs to be investigated. Data already collected on the effects of different treatments on somatosensory and cognitive recovery after FPI provide a fascinating starting point for studies investigating whether these treatments would also lead to favorable antiepileptogenic or disease modifying effects.
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Dow, R.S., Fernandez-Guadriola, A., Manni, E. 1962. The production of cobalt experimental epilepsy in the rat. Electroencephalogr Clin Neurophysiol. 14: 399–407. Engel J. Jr. 2001. International League Against Epilepsy (ILAE). A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classification and Terminology. Epilepsia 42: 796–803. Englander, J.E., Bushnik, T., Duong, T.T., Cifu, D.X., Zafonte, R., Wright, J., Hughes, R. et al. 2003. Analyzing risk factors for late posttraumatic seizures: a prospective, multicenter investigation. Arch Phys Med Rehabil 84: 365–373. Floyd, C.L., Golden, K.M., Black, R.T., Hamm, R.J., Lyeth, B.G. 2002. Cranioectomy position affects Morris water maze performance and hippocampal cell loss after parasagittal fluid percussion. J Neurotrauma 19: 303–316. Frey, L.C. 2003. Epidemiology of posttraumatic epilepsy: A critical review. Epilepsia 44(Suppl. 10): 11–17. Golarai, G., Greenwood, A.C., Feeney, D.M., Connor, J.A. 2001. Physiological and structural evidence for hippocampal involvement in persistent seizure suspectibility after traumatic brain injury. J Neurosci 21: 8523–8537. Graber, K.D., Prince, D.A. 2004. A critical period for prevention of posttraumatic neocortical hyperexcitability in rats. Ann Neurol 55: 860–870. Grady, M.S., Charleston, J.S., Maris, D., Witgen, B.M., Lifshitz, J. 2003. Neuronal and glial cell number in the hippocampus after experimental traumatic brain injury: analysis by stereological estimation. J Neurotrauma 20: 929–941. Graham, D.I., Gentlemen, S.M., Lynch, A., Roberts, G.W. 1995. Distribution of beta-amyloid protein in the brain following severe head injury. Neuropathol Appl Neurobiol 21: 27–34. Haltiner, A.M., Temkin, N.R., Dikmen, S.S. 1997. Risk of seizure recurrence after the first late posttraumatic seizure. Arch Phys Med Rehabil 78: 835–840. Hauser, W.A., Annegers, J.F., Kurland, L.T. 1991. Prevalence of epilepsy in Rochester, Minnesota: 1940–1980. Epilepsia 32: 429–445. Herman, S.T. 2002. Epilepsy after brain insult. Neurology 59: 21–26. Hill, S.J., Barbarese, E., McIntosh, T.K. 1996. Regional heterogeneity in the response of astrocytes following traumatic brain injury in the adult rat. J Neuropathol Exp Neurol 55: 1221–1229. Hill-Felberg, S.J., McIntosh, T.K., Olivier, D.L., Raghupathi, R., Barbarese, E. 1999. Concurrent loss and proliferation of astrocytes following lateral fluid percussion brain injury in the adult rat. J Neurosci Res 57: 271–279. Hoffman, S.N., Salin, P.A., Prince, D.A. 1994. Chronic neocortical epileptogenesis in vitro. J Neurophysiol 71: 1762–1773. Hu, B., Bramlett, H., Sick, T.J., Alonso, O.F., Chen, S., Dietrich, W.D. 2004. Changes in TrkB-ERK1/2-CREB/Elk-1 pathways in hippocampal mossy fiber organization after traumatic brain injury. J Cereb Blood Flow Metab 24: 934–943. Hudak, A.M., Trivedi, K., Harper, C., Booker, K., Caesar, R.R., Agostini, M., Van Ness, P.C. et al. 2004. Evaluation of seizure-like episodes in survivors of moderate and severe traumatic brain injury. J Head Trauma Rehabil 19: 290–295. Ip, E.Y.-Y., Giza, C.C., Griesbach, G.S., Hovda, D.A. 2002. Effects of enriched environment and fluid percussion injury on dendritic arborization within the cerebral cortex of the developing rat. J Neurotrauma 19: 573–585. Kopeloff, N., Whittier, J.R., Pacella, B.L., Kopeloff, L.M. 1950. The epileptogenic effect of subcortical alumina cream in the rhesus monkey. Electroencephalogr Clin Neurophysiol 12: 163–168. Laurer, H.L., and McIntosh, T.K. 1999. Experimental models of brain trauma. Curr Opinion Neurol 12: 715–721. Lowenstein, D.H., Thomas, M.J., Smith, D.H., McIntosh, T.K. 1992. Selective vulnerability of dentate hilar neurons following traumatic brain
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injury: a potential mechanistic link between head trauma and disorders of the hippocampus. J Neurosci 12: 4846–4853. Maxwell, W.L., Dhillon, K., Harper, L., Espin, J., McIntosh, T.K., Smith, D.H., Graham, D.I. 2003. There is differential loss of pyramidal cells from the human hippocampus with survival after blunt head injury. J Neuropathol Exp Ther 62: 272–279. McIntosh, T., K., Vink, R., Noble, L., Yamakami, I., Fernyak, S., Soares, H., Faden, A.L. 1989. Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model. Neuroscience 28: 233–244. Nissinen, J., Halonen, T., Koivisto, E., Pitkänen, A. 2000. A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res 38: 177–205. Paxinos, G., and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates. New York: Academic Press. Pierce, J.E.S., Trojanowski, J.Q., Graham, D.I., Smith, D.G., McIntosh, T.K. 1996. Immunohistochemical characterization of alterations in the distribution of amyloid precursor proteins and beta-amyloid peptide after experimental brain injury in the rat. J Neurosci 16: 1083–1090. Pohlmann-Eden, B., and Bruckmeir, J. 1997. Predictors and dynamics of posttraumatic epilepsy. Acta Neurol Scand 95: 257–262. Prince, D.A., and Tseng, G.F. 1993. Epileptogenesis in chronically injured cortex: in vitro studies. J Neurophysiol 69: 1276–1291. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizures. Electroencephalogr Clin Neurophysiol 32: 281–294. Reeves, T.M., Lyeth, B.G., Phillips, L.L., Hamm, R.J., Povlishock, J.T. 1997. The effects of traumatic brain injury on inhibition in the hippocampus and dentate gyrus. Brain Res 757: 119–132. Rice, A.C., Khaldi, A., Harvey, H.B., Salman, N.J., White, F., Fillmore, H., Bullock, M.R. 2003. Proliferation and neuronal differentiation of mitotically active cells following traumatic brain injury. Exp Neurol 183: 406–417. Roberts, G.W., Gentleman, S.M., Lynch, A., Graham, D.I. 1991. BetaA4 amyloid protein deposition in brain after head trauma. Lancet 338: 1422–1423. Salazar, A.M., Jabbari, B., Vance, S.C., Grafman, J., Amin, D., Dillon, J.D. 1985. Epilepsy after penetrating head injury. I. Clinical correlates: a report of the Vietnam Head Injury Study. Neurology 35: 1406–1414.
Santhakumar, V., Ratzliff, A.D.H., Jeng, J., Toth, Z., Soltesz, I. 2001. Longterm hyperexcitability in the hippocampus after experimental head trauma. Ann Neurol 50: 708–717. Sloviter, R.S. 1982. A simplified Timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of the rat brain. Brain Res Bull 8: 771–774. Smith, D.H., Chen, X.-H., Pierce, J.E., Wolf, J.A., Trojanowski, J.Q., Graham, D.I., McIntosh, T.K. 1997. Progressive atrophy and neuron death for one year following brain trauma in the rat. J Neurotrauma 14: 715–727. Soares, H.D., Thomas, M., Cloherty, K., McIntosh, T.K. 1992. Development of prolonged focal cerebral edema and regional changes following experimental brain injury in the rat. J Neurochem 58: 1845–1852. Tai, P.C., and Gross, D.W. 2004. Exacerbation of pre-existing epilepsy by mild head injury: a five patient series. Can J Neurol Sci 31: 394– 397. Teasdale, G., and Jennett, B. 1976. Assessment and prognosis of coma after head injury. Acta Neurochirurgica 34: 45–55. Temkin, N.R. 2001. Antiepileptogenesis and seizure prevention trials with antiepileptic drugs: meta-analysis of controlled trials. Epilepsia 42: 515–524. Thompson, H.J., Lifshitz, J., Marklund, N., Grady, M.S., Graham, D.I., Hovda, D.A., McIntosh, T.K. 2005. Lateral fluid percussion brain injury: a 15-year review and evaluation. J Neurotrauma 2005, 22(1): 42–75. Toth, Z., Hollrigel, G.S., Gorcs, T., Soltesz, I. 1997. Instantaneous perturbation of dentate interneuronal networks by a pressure wave-transient delivered to the neocortex. J Neurosci 17: 8106–8117. Vink, R., Mullins, P.G.M., Temple, M.D., Bao, W., Faden, A.I. 2001. Small shifts in craniotomy position in the lateral fluid percussion injury model are associated with differential lesion development. J Neurotrauma 18: 839–847. Watson, N.F., Barber, J.K., Doherty, M.J., Miller, J.W., Temkin, N.R. 2004. Does glucocorticoid administration prevent late seizures after head injury? Epilepsia 45: 690–694. Willmore, L.J., Sypert, G.W., Munson, J.V., Hurd, R.W. 1972. Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science 200: 1501–1503.
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become progressively more hyperexcitable (Grafstein and Sastry, 1957; Sharpless and Halpern, 1962), and spontaneous interictal discharges can be recorded for at least 1 year (Echlin and Battista, 1963), with spread to adjacent and contralateral cortical areas (Echlin and Battista, 1961). One brief report indicates that ictal episodes could be evoked in partially isolated cortex of monkey by peripheral nerve stimulation (Echlin and Battista, 1961). The duration of paroxysmal afterdischarges evoked in isolations is greatly prolonged compared with naïve cortex (Grafstein and Sastry, 1957; Echlin, 1959; Sharpless and Halpern, 1962).
GENERAL DESCRIPTION OF MODEL Model of Traumatic Neocortical Injury The importance of epilepsy occurring after a traumatic cortical injury is emphasized by its high incidence after penetrating brain wounds, 53% according to Salazar and colleagues (1985). Frequently a long latent period exists between injury and clinical manifestations of epilepsy that may provide an opportunity for therapeutic intervention (Salazar et al., 1985; Annegers et al., 1998; Graber and Prince, 1999, 2004). Partially isolated and undercut slabs of neocortex with intact pial circulation (“isolations” or “undercuts” below) are an established in vivo and in vitro model for development of chronic post-traumatic hyperexcitability and epileptogenesis (Echlin and McDonald, 1954; Grafstein and Sastry, 1957; Purpura and Housepian, 1961; Echlin and Battista, 1961, 1962, 1963; Sharpless and Halpern, 1962; Prince and Tseng, 1993; Hoffman et al., 1994; Halpern, 1972). A number of early studies were focused on electroencephalographic (EEG) and neuronal activities in acutely isolated cortex, according to Burns (1954) and others). This chapter is limited to aspects of the chronic isolation that make this model suitable for exploring the mechanisms underlying delayed cortical epileptogenesis after injury.
In Vitro Studies A latent period allows study of alterations of anatomy and physiology before, during, and after onset of epileptogenesis and the model, therefore, may be ideal for experiments focused on underlying cellular mechanisms and antiepileptogenic strategies. In recent years, the model has not been used extensively for in vivo studies, and the incidence and characteristics of behavioral seizures in lesioned animals have not been well characterized. Generalized motor seizures are not a feature of the partial cortical isolation model in rats, although more subtle behavioral seizures and associated electrographic ictal discharges have been recorded (see Figure 2). This may result from the relative isolation of the epileptogenic cortex from subcortical and adjacent cortical structures and the absence of significant damage to remote structures such as hippocampus. Chronic neocortical isolations, however, are particularly well-suited for detailed in vitro studies of hyperexcitability and epileptogenesis, as neocortical slices made through areas of the
Epileptogenesis after a Latent Period First studied chronically in cats and monkeys, partially isolated cortical islands in vivo tend to be quiescent electrophysiologically, but after a 2- to 3-week latent period they
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Coronal Slice FIGURE 1 Schematic of undercutting methods. A: View of rodent neocortex and underlying white matter in the parasagittal plane. 1: Fine gauge needle, bent 90 degrees from the tip, is inserted at a near-tangential angle through dura and beneath the pia into superficial cortex. 2: Shaft is aligned normal to pial surface, raised slightly through layer I (not shown) sparing pial vasculature and then depressed through all cortical layers to underlying white matter, creating a transcortical cut (gray shading in 3). 3: Needle is rotated laterally 180 degrees to create a white matter undercut (dark line in 4, 5). 4: Needle is raised to extend the transcortical cut (gray shading). 5: Needle is removed through the point of insertion. B: Schematic of different types of cortical and white matter transactions. Bilateral lesions shown only for illustrative purposes. 1: Steps in A used to create a longer transcortical cut (dark line; small dark circle represents point of needle insertion) and underlying white matter undercut (gray shaded semicircle). 2: Associated lateral transcortical cut (dark line) placed without needle rotation can be used to create a more complete isolation (“U”-shaped in coronal brain slices, C 3: Rotation of the depressed needle approximately 90 to 135 degrees can be used to create smaller lesion with two adjacent transcortical cuts. 4: Rostral (or caudal, not shown) transcortical cuts can also be used for more extensive isolation. C: Typical recording of field potentials from two electrodes spaced 1.5 mm apart in layer V demonstrates transcortical propagation of evoked interictal discharges. Dot: stimulus at white matter—layer VI junction. Actual slice contained only unilateral lesion.
partially isolated cortex in rats and guinea pigs reliably generate evoked and occasional spontaneous polyphasic, prolonged epileptiform discharges after a latency of about 10 days after injury (Prince and Tseng, 1993; Hoffmann et al., 1994, Graber and Prince, 1999), and for at least 2 years after the initial lesion.
Several anatomic abnormalities result from the direct cortical trauma inherent in the isolation procedure. The principal descending axons of pyramidal cells in layers V and VI are severed by the undercutting lesion, leaving a population of surviving axotomized projection cells whose anatomical and electrophysiologic properties can be studied. At the same time, neurons in all layers lose intracortical and subcortical inputs to varying degrees, so that deafferentation becomes a prominent feature in this model. Subcortical ascending neurotransmitter systems (e.g., for acetylcholine, norepinephrine, and serotonin) must also be affected. The extensive intracortical axonal arbors of both pyramidal cells and interneurons near the intracortical lesions are likely injured to varying degrees. Most intracortical synapses are from axons intrinsic to the cortex (Gruner et al., 1974; Douglas and Martin, 1991, 2004), rather than ascending pathways, making it likely that the transcortical cut induces much greater deafferentation than the undercutting white matter lesion. Partial dendrotomy also must affect both pyramidal cells and interneurons located adjacent to the transcortical cuts. Despite this, the partially isolated cortical island maintains its laminar appearance. Thus, the isolated cortex is a model of chronic focal neocortical injury in which most neurons survive, albeit under abnormal anatomic and physiologic conditions. Studies of neocortical isolations have yielded information about injury-induced alterations in neuronal intrinsic properties and cortical circuitry that may be relevant to mechanisms underlying human posttraumatic epileptogenesis and possibly other aspects of symptomatic focal (localization-related) epilepsy syndromes. Both acutely and chronically isolated cortical islands have also been used to assess various aspects of cortical function (Goldring et al., 1961; Chow, 1964; Suzuki and Ochs, 1964), and the capacity of cortical networks to generate rhythmic activity or epileptiform discharges in the absence of subcortical influences (Prince, 1965). Other experiments have focused on alterations in amino acids and transmitter-related enzymes in isolations (Duncan et al., 1968; Green et al., 1970a,b, 1973a,b; Koyama and Jasper, 1976), as well as responses to convulsant drugs and transmitters (Wright et al., 1954; Echlin and Battista, 1962; Krnjevic et al., 1970; Spehlmann et al., 1970, 1971; Reiffenstein and Triggle, 1972; Farrance and Halpern, 1975).
Focal Injury Partial cortical isolations represent a relatively focal injury, compared with the more widespread brain regions that may be damaged and involved in epileptogenesis in animals surviving status epilepticus, or other more diffuse
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2 3 4 5 6 7 8 FIGURE 2 Ictal episode in an unanesthetized rat with implanted electrodes. Digital Video-EEG recording of a brief ictal discharge arising in the posterior aspect of the cortical partial isolation in left cortex. A: Electroencephalogram (EEG) shows ~10-Hz repetitive sharp waveforms with phase reversal at the second most caudal electrode (between bipolar channels 3 and 4). B,C: Accompanying behavioral changes with this and other discharges were subtle, consisting of freezing (a,d), frequently slight raising of the head (b), followed by head drop and resumption of normal motor activity (c,e) abruptly with cessation of the discharges. Some discharges spread focally to contralateral hemisphere (C), but no generalized convulsive seizures were recorded. From K. Graber and D. A. Prince, unpublished data.
models. The hippocampus is presumably unaffected, compared with the alterations produced in this structure by trauma induced with lateral fluid percussion where a pressure wave induces injury deep and remote to the point of impact (Lowenstein et al., 1992; Coulter et al., 1996; Toth et al., 1997; Santhakumar et al., 2000, 2001; D’Ambrosio et al., 2004, 2005). The lesions producing partial neocortical isolations, however, sever reciprocal
thalamic and callosal connections, and those from more remote subcortical structures (e.g., ascending transmitter systems originating in the brainstem), likely leading to deafferentation and retrograde neuronal changes in neurons at these sites and loss of extrinsic modulation of cortical excitability. Thus, the effects of a “focal” cortical injury in this and other models likely extend beyond the site of direct trauma.
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“Naturally Occurring” Injury In contrast to other chronic models of focal neocortical epileptogenesis produced by local injections of iron salts and blood (Willmore et al., 1978; Lange et al., 1980; Hammond et al., 1980; Ueda et al., 1998; see Willmore this volume for review), cobalt and alumina (Dow et al., 1962; see Ward, 1972 and Ribak, Chapter 14, for review), tetanus toxin (see Chapter 33), and so forth, the direct neocortical trauma in the undercut model may more closely resemble aspects of penetrating cortical injuries in humans that carry a high risk for development of late posttraumatic seizures (Salazar et al., 1985).
Anesthesia For ~P30 (80 to 120 g) rats, administration of ketaminexylazine (40–4 mg/kg intraperitoneally) will allow 45 to 60 minutes of adequate anesthesia. The entire procedure requires ~30 minutes in experienced hands and animals typically recover within an additional 30 to 60 minutes. Similar dosages can be used in guinea pigs. Higher dosages may be necessary for older rats and lower dosages for ~2- to 3week-old animals. Inhalation anesthetics (e.g., Metofane) may be more optimal in younger animals and hypothermia is effective in neonatal rats
METHODS OF GENERATION The chronic neocortical isolation model was initially used in cats or monkeys to assess electrophysiologic properties of the injured cortex in vivo (Grafstein and Sastry, 1957; Sharpless and Halpern, 1962; Echlin and Battista, 1963; Prince, 1965; Halpern, 1972). The lesion was usually produced by inserting a bent spatula through a cortical incision to section underlying white matter and then severing connections to surrounding cortex with a probe inserted through the same incision (Halpern, 1972). After recovery from anesthesia, animals could be studied chronically with implanted electrodes (e.g., Sharpless and Halpern, 1962), or reanesthetized at a later time for electrocorticography (Echlin, 1959) or microelectrode studies in the isolated island (Krnjevic et al., 1970). More than a decade ago, this model was adapted for use in rodents, and it was found that hyperexcitability and epileptiform activities persisted in slices in vitro cut through the partially isolated cortex of rats and guinea pigs (Prince and Tseng, 1993; Hoffman et al., 1994; Salin et al., 1995). This has allowed more detailed neurophysiologic and anatomic studies of the chronically injured, epileptogenic cortex.
Surgical Techniques Animal Selection Most recent experiments have involved partial isolations in male, Sprague-Dawley rats, 1 to 5 weeks of age (Prince and Tseng, 1993; Hoffman et al., 1994; Salin et al., 1995; Graber and Prince, 1999, 2004; Kharazia and Prince, 2001; Li and Prince 2002; Li et al., 2005). Other rat strains have not been investigated. We have also made partial cortical isolations in albino guinea pigs and mice (C57/BL6) and found that epileptiform activity occurs in slices in vitro from isolations in these animals (Prince and Tseng, 1993; Graber and Prince, unpublished observations).
Animal Preparation Once the animal is anesthetized, the scalp is shaved with an electric clipper, skin cleansed with betadyne and alcohol, and animals placed in a stereotaxic frame. We are currently using either a locally fabricated head holder for the mouse or rat pup adapter of a stereotaxic frame for small animals (David Kopf, Tujunga, CA). Ear bars are coated with 5% lidocaine ointment and artificial tear lubricant is placed over the eyes to prevent postoperative conjunctivitis. Bone Window Under an operating surgical microscope, the scalp is incised longitudinally and retracted, the skull exposed and the periosteum removed with a bone scraper. A rectangular bone window slightly larger than the intended lesion is removed over one hemisphere, using an electric drill (Micromotor, Foredom Tools, Bethel, CT) and diamond cutting burr (~1.2 mm head diameter; e.g., George Tiemann and Company, Hauppauge, NY) and avoiding damage to the underlying dura, pial vessels, and cortical surface. The skull should be kept moist with sterile saline to facilitate cutting the bone and to avoid significant heating during drilling. By gradually increasing the depth of grooves made at the margins of the bone window, unintentional dural penetration and cortical injury can be avoided. When only a thin layer of the inner table remains, the loosened bone can be easily removed with curved forceps by lifting the medial edge and reflecting it laterally. It is sometimes necessary to gently loosen adhesions between the dura and bone flap along the coronal suture and the lateral—inferior margin of the bone window by sliding a forceps or small dental probe along the inside of bone flap. It is important to place the medial edge of the bony opening ≥1 mm from the midline to avoid damage to the superior sagittal sinus. Typically, a rectangular bone window measuring at least ~4 ¥ 8 mm, with the
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longer dimension parallel to the sagittal suture, is made when rats ≥P21 are used, but smaller openings are required in rat pups or mice. Depending on the site of the bone window, almost any cortical region can be the chosen for the isolation using the surface coordinates in the atlas of Zilles (1985). For sensory-motor isolations, the bone window is centered over the coronal suture. Lesion Placement A 30-gauge needle or fine wire, bent to 90 degrees 2 to 3 mm from the tip, is used to make the lesion. A mark is placed along the vertical needle shaft ~2 mm from the bend, in line with the direction of the needle point so that the surgeon can gauge the depth of the transcortical cut and the location of the needle tip within the brain (Figure 1.A.1.). Being careful to avoid large pial vessels, under microscopic control, the needle is gently inserted through the dura and under the pia, typically in the parasagittal plane, and straightened so that the bent end is visualized just beneath and parallel to the pial surface (Figure1A2.). The needle is then pushed down through the cortex for 2 mm (full cortical thickness; 1 to 1.25 mm in mice or young rats) to make a transcortical cut, rotated 180 degrees (away from midline) to undercut the cortex, raised through cortex until it is visualized just beneath the pial surface to make an extension of the transcortical cut, and carefully removed through the point of insertion (Figure 1A3–5., Figure 1B1). Alternatively, rotation of 90 to 135 degrees can be used to make smaller partial isolations bordered by two transcortical cuts (Figure 1B3). The needle can be reinserted, depressed, raised, and removed without rotation along other edges of the desired lesion to make the isolation more complete; for example, 2 to 3 mm lateral in the parasagittal plane (Figure 1B2), or in the coronal plane 2 to 3 mm anterior or posterior to the initial point of insertion (Figure 1B4). In our experience, attempts to make more complete isolations (e.g., surrounded by four transcortical cuts) can lead to infarction. We typically make two transcortical cuts and a white matter undercut as above, followed by a more lateral transcortical cut. Even a single transcortical cut, however, can result in hyperexcitability in slices, adjacent to the lesion (Hoffman et al., 1994). Lesions can be placed free hand or by mounting the needle in a hinged holder mounted on a micromanipulator. The latter approach tends to make more consistent lesions, and is particularly desirable in rat pups and mice where small movements of the needle caused by physiologic tremors can produce significant damage and bleeding. The holder must allow horizontal and rotational movement; the needle is positioned tangential to the cortex; for example, 18 degrees off horizontal at 2 mm posterior to the bregma, 2 mm lateral to midline, with the straight shaft of the needle aimed at the intended point of insertion. The holder is then
moved slightly forward and needle pulled back on the hinge to allow an angled approach into the cortex. Once inserted, the holder is moved back into the original position, initially raised to transect superficial layers, and then depressed, rotated, and so forth. If desired, a thin piece of drug-eluting polymer (Elvax 40W®) can be inserted subdurally over lesioned cortices for studies of antiepileptogenesis (Graber and Prince, 1999; 2004). A sterile piece of Saran Wrap® is placed over the bone window and the scalp sutured or glued with Vetbond TM Tissue Adhesive, (3M, St. Paul, MN). With experience, >90% of attempted isolations can be completed successfully with only minimal bleeding from subpial vessels, or bridging veins that can be easily controlled with Gelfoam®. Should significant cortical swelling or bleeding occur at surgery, it is best to kill the animal because of the likelihood that the isolation will be the site of infarction. Electrode Implantation For protocols involving implanted electrodes, lesions can be placed through narrow oblong skull openings instead of through a bone window, allowing screws to be implanted in the bone over the isolation at the initial surgery. This approach can be difficult in animals >P30 because the thicker skull makes it difficult to visualize and avoid pial vessels. Alternatively, electrodes can be implanted at a second surgery 2 to 4 weeks after the initial lesion, when regrowth and remineralization of bone over the previous bone window is adequate to allow screw electrode placement. Animal Recovery Warming with a heating blanket or heat bulb will aid in recovery and prevent hypothermia. A postoperative analgesic is administered subcutaneously (s.c.) (0.02 to 0.03 mg/kg buprenorphine) as animals are beginning to awaken from anesthesia and later at intervals, if necessary. Animals typically resume normal behavior within in a few hours of the procedure. If the procedure and recovery are prolonged, saline at the rate of 3 ml/100 g can be given s.c. over the back, with not >3 ml at any one site.
In Vitro Recording Neocortical Slices In vitro neocortical slices can be prepared and studied electrophysiologically at any time after the partial isolation is prepared, although the slice procedure is more difficult during the first few days after the lesion because the isolated area tends to separate from the rest of the slice, and some edema may be present. Hyperexcitability is reliably present in slices as early as 10 to 14 days following the isolation
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(Hoffman et al., 1994). Animals are anesthetized intraperitoneally (i.p.) with pentobarbital (2 50 mg/kg), decapitated, and the brain removed with careful attention to the area of previous surgery to avoid tearing or distortion if adhesions are present. The isolation site can be easily seen and slices cut through it and the adjacent and homotypic contralateral cortex using standard techniques (Prince and Tseng, 1993; Hoffman et al. 1994; see also Chapters 6 and 7). Homotypic neocortex contralateral to the lesion can serve as a reasonable control for some studies; however, some neurons in this cortex are likely partially deafferented and callosal cells axotomized by the lesion, making it important to also use slices from homotypic cortex of nai’ve littermates as controls in at least some experiments. More care is necessary to prepare viable slices from mature animals with cortical isolations than is the case in naive or younger animals; delays during the cutting procedure, or inadvertent trauma (e.g., stretching) must be avoided. A cutting solution in which NaCl is replaced by sucrose is used routinely (Aghajanian and Rasmussen, 1989; Fukuda and Prince, 1992), as it appears to improve slice health. Although optimal thickness has not been systematically studied in undercut
slices, 400 pm thick slices are thin enough to remain “healthy” at least 6 hours within interface recording chambers perfused with oxygenated artificial cerebral spinal fluid (ACSF), after at least 1 hour of prior incubation in normal ACSF. Field Potential Recording Slices from undercuts retain sufficient connectivity to reliably generate normal and epileptiform field potentials. Stimuli are usually applied in deep neocortical layer VI at the white matter border, however, “normal” short latency field potentials and interictal epileptiform events can be evoked and recorded in any lamina of the same cortical column (Prince and Tseng, 1993; Hoffman, et al. 1994). Epileptiform events, which are typically evoked near threshold for the short latency field potential, consist of prolonged polyphasic discharges and a baseline voltage shift in DC recordings (Figure l.c., Figure 3.A.,B.). Evoked discharges are followed by a variable refractory period (~10 seconds) and spontaneous epileptiform field potentials are also occasionally present in slices containing chronic isolations. Hyperexcitability is not typically evident in “healthy,” well-maintained slices, with
A i-f---~------m C
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FIGURE 3 Interictal and ictal epileptiform discharges from chronic partial cortical isolation in rat, recorded in virro. A: Average of 10 evoked interictal epileptiform held responses recorded on-column in layers III and V. Onset latency to epileptiform potential peak (dashed vertical lines) was -1Omsec shorter in layer V than in layer III. Stimuli (dots) applied to layer VI-white matter border at 0.1 Hz (1.5 x threshold). B: Same slice and recording sites as in A, except that stimulus applied just beneath the pial surface. Latency to peak of evoked epileptiform event was again shorter in layer V than in layer III. C: Top trace: Current clamp (sharp electrode) recording from biocytin-identified layer III pyramidal cell during a brief evoked “ictal” lasting a number of seconds. Bottom trace: Simultaneous field recording nearby in layer 111. D: Section from a chronic cortical isolation -4 weeks after lesion. Qxn UYYOWS mark tranacortical cuts and undercut. Filled WYOW points to layer V pyramidal cell filled with biocytin. (Modified from Hoffman, S.N., Salin, P.A., and Prince, D.A. 1994. Chronic neocortical epileptogenesi? in vitro. J Neuroph~siol 71: 1762-1773, with permission.)
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Methods of Generation
supramaximal stimulation. The capacity to generate short latency field potentials with an amplitude of at least 1 mV is a general measure of slice health (Connors et al., 1988). Cellular Recording Intracellular activities from neurons in partial isolations during interictal events were initially recorded with sharp microelectrodes in an interface chamber (Prince and Tseng, 1993; Figure 4.A,B). Patch clamp recordings from neurons in slices containing isolations can be obtained using either the “blind” slice-patch technique (Blanton et al., 1989; Salin et al., 1995; Figure 4.C,D). or from cells visualized with a compound microscope equipped with differential interference contrast (DIC) optics and infrared illumination (Edwards, et al., 1989; Li and Prince, 2002). The yield of
high-quality, whole-cell patch clamp recordings is lower in lesioned slices from relatively mature animals using either of these approaches. Direct visualization of cells with a water immersion lens requires using thinner slices (~250 to 350 mm) and a recording chamber in which slices are submerged. Such slices tend to be less viable over time than those maintained in an interface chamber, a limitation that can be partially offset by perfusing them at a higher rate (>2 ml/minute) and cooler temperature (~33° C to 35° C versus 37° C in interface chambers). Polysynaptic activities, such as those required to generate epileptiform events, are depressed at cool temperatures (~<30° C). Field potentials are more difficult to obtain in submerged slices; however, with high amplification and the use of electrodes suitable for multiunit recordings, population events can be recorded that reflect interictal epileptiform activities.
C
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FIGURE 4 Recordings from layer V pyramidal neurons and extracellular field potentials in partially isolated cortex during evoked and spontaneous interictal epileptiform events. A,B: Sharp microelectrode current clamp recording of evoked (A) and spontaneous (B) interictal discharges in an intrinsically bursting neuron (upper traces) and simultaneously recorded field potentials (lower traces). Dots under traces of A: pair of extracellular stimuli delivered on column in layer VI. Polysynaptic activities in this cell mirror extracellular discharges (“interictal spikes”) in the population. (From Prince, D.A., and Tseng, G.F. 1993. Epileptogenesis in chronically injured cortex: in vitro studies. J Neurophysiol 69: 1276–1291, 1993, with permission.) C,D: “Blind” slice-patch recordings of whole cell currents from a neuron in another partial isolation, and simultaneous extracellular fields. C: When membrane potential is held near the calculated reversal potential for IPSC (Vh = -50 mV), field potential polyphasic activity (Field) is associated with a summed polyphasic inward (excitatory) currents (EPSCs) lasting several hundred milliseconds. D: Summed polyphasic outward (inhibitory) currents (IPSCs) coincident with the epileptiform field potential are revealed when the membrane potential is held near the EPSC reversal potential (Vh = 0 mV). Background spontaneous EPSCs and IPSCs are present in C and D, respectively. Cs+ based intracellular solution. (Modified from Salin, P., Tseng, G.F., Hoffman, S., Parada, I., and Prince, D.A. 1995. Axonal sprouting in layer V pyramidal neurons of chronically injured cortex. J Neurosci 15: 8234–8245., with permission.)
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CHARACTERISTICS/DEFINING FEATURES Behavioral Features Limited data are available with respect to behavioral seizures in this model. As mentioned above, generalized motor seizures have not been seen in the lesioned animals on casual observation. Focal spontaneous epileptiform activities have been recorded during electrocorticography from chronic isolations in unanesthetized monkeys (Echlin and Battista, 1961; Echlin, 1959) and as well as in chronically implanted unanesthetized cats (Sharpless and Halpern, 1962). Long-term video-EEG recordings, however, have been done in our laboratory in only a few rats. Continuous video-EEG recordings for 3 days from the time of injury showed only focal slowing without clear interictal discharges or behavioral seizures (Graber and Prince, 2004). Behavioral and associated electrographic seizure activity was seen in an animal with a sensory—motor cortical isolation studied several months after the isolation was placed. Video-EEG monitoring showed focal ictal episodes lasting many seconds, beginning in the area of the undercut and at times spreading to contralateral cortex, as shown in Figure 2. The accompanying behavioral changes were subtle, consisting of sudden freezing, followed by head drop and abrupt resumption of normal motor activity with cessation of the discharge. Previous studies in monkey have documented the occurrence of ictal episodes in vivo (Echlin and Battista, 1961).
Electrophysiologic Features Characteristics of the Epileptogenic Lesion In vitro field potential recordings A number of variables affect the incidence of epileptogenesis in this model. These factors have been systematically assessed in rat and guinea pig lesioned slices (Hoffman et al., 1994). Analysis has shown that the transcortical, but not the undercutting lesion is the critical one leading to hyperexcitability. Slices containing transcortical cuts alone generate interictal events, whereas those with undercutting white matter lesions alone rarely do so. The combination of these two lesions, however, yields slices that are more excitable than those with transcortical cuts alone and generate occasional ictal episodes (Figure 3C). In a typical coronal slice containing two transcortical cuts and a contiguous white matter lesion, the area from which epileptiform activity can be most effectively evoked is usually within 2 mm of the transcortical cut. Both short latency “normal” field potentials and epileptiform events are typically attenuated <0.5 mm from a transcortical cut, presumably because of immediately adjacent gliosis and neuronal loss. The interictal discharges propagate variable distances
across the slice (Figure 1C); however, the propagation has characteristics that are different from those in unlesioned slices treated with convulsant drugs (Hoffman et al., 1994, and Chagnac-Amitai and Connors, 1989). Another variable affecting the incidence of epileptogenesis in slices is the latency between the initial lesion and the slice experiment. Abnormal evoked activity is rare if this interval is < 7 days, but regularly seen at latencies of at least 15 days (5% and >80% of slices, respectively, in Hoffman et al., 1994; and Graber and Prince, 1999). Slices from animals lesioned during cortical development (
30 days). Epileptiform discharges can be recorded from any cortical lamina within the lesion, but simultaneous recordings in different laminae (Figure 3.A.,B.) and current source density analyses show that interictal epileptiform events originate in layer V (Hoffman et al., 1994). The stimulus threshold for evoking an interictal discharge is also lowest in layer V, a lamina that also has the lowest threshold for initiating interictal events in unlesioned slices exposed to convulsant drugs (Connors, 1984). A careful survey of slices in an interface chamber perfused with ACSF containing 5 mM potassium showed that polyphasic, all-or-none, variable latency epileptiform abnormalities could be evoked in at least some slices from almost every animal with a successful partial cortical isolation (Hoffman et al., 1994; Graber and Prince, 1999). As mentioned, the percentage of slices showing interictal epileptiform events varies, depending on the nature of the lesion and the latency from injury to the slice experiment (Hoffman et al., 1994). By contrast, similar polyphasic discharges are rare in healthy sensorimotor cortical slices from naïve, mature (>P21) animals under the same recording conditions (Luhmann and Prince, 1990, 1991). Spontaneously occurring epileptiform events also are present in some undercut slices (Hoffman et al., 1994; Graber and Prince, 1999). Robust epileptiform discharges lasting from hundreds of milliseconds to seconds can occasionally be evoked by single stimuli ~0.5 to 2 mm adjacent to transcortical lesions, but not outside the lesioned area (Figure 3). Epileptiform discharges are evoked near threshold for the normal short-latency field response but typically are not elicited with intense stimuli, hypothetically because of enhanced recruitment of inhibitory neurons. In contrast to recordings from unlesioned neocortex acutely exposed to convulsant drugs in vivo (Matsumoto and Ajmone Marsan, 1964; Prince, 1968, 1971) and in vitro (Gutnick et al., 1982; Galvan et al., 1982), the epileptiform events in this model usually are not as stereotyped in amplitude, duration, or waveform, and may be followed by periods of several seconds when only normal short latency field potentials can be evoked. For these reasons, an optimal survey protocol
Characteristics/Defining Features
for identifying epileptogenic slices involves application of 0.1 Hz submaximal intensity stimuli delivered through an electrode on cortical column with the recording electrode. Cellular Electrophysiology Although some studies are available in which cellular activities in acute or chronic partially isolated cortex have been examined in vivo (Burns et al., 1979; Krnjevic et al., 1969, 1970), data relevant to the cellular mechanisms underlying hyperexcitability in chronic partial cortical isolations have come predominantly from neocortical slices maintained in vitro. The timing of the onset of abnormal hyperexcitability at the cellular level in chronically isolated islands prepared as described above has not been clearly delineated. Results of recent experiments on acute partial isolations in ketamine-xylazine-anesthetized cats studied in vivo suggest that epileptiform activities can be generated within hours of the injury in cortex bordering the isolation (Topolnik et al., 2003a,b). Other experiments performed on naïve neocortical slices in vitro, where possible effects of anesthetic agents cannot be involved, have shown that subgranular layers become acutely epileptogenic when separated from overlying cortex (Yang and Benardo, 1997, 1998). The mechanisms for development of epileptiform activity in these acute injury experiments are likely to be different from those in chronically injured cortex; however, results raise the possibility that hyperexcitability within neuronal aggregates might be present early on in the chronic partial isolation model, before evoked epileptiform events can be reliably detected in slice experiments at ~10 to 14 days (but see Graber and Prince, 2004). Acute seizure activity is a well-known consequence of brain trauma and is a risk factor for development of late posttraumatic seizures, although occurrence does not usually presage epilepsy (Annegers et al., 1998). Intracellular recordings with “sharp” microelectrodes in slices containing partially isolated cortex (Prince and Tseng, 1993; Figure 3C, Figure 4A,B) and those recorded with the whole cell configuration of the patch clamp (Figure 4C,D) have similar features. Use of an interface chamber for either sharp electrode or blind slice-patch recordings has the advantage that it is easier to record field potentials from multiple sites along with the intracellular activity. Use of a submersion chamber and compound microscope with a water immersion lens allows direct visualization of neuronal somata and processes within normal cortex or cortical isolations and facilitates acquisition of high quality recordings (Edwards, 1989; Li and Prince, 2002; Li et al., 2005). Slice health is more limited in submerged chambers and the yield of high-quality, whole-cell patch clamp recordings from injured tissue of >P21 rats is significantly lower than from naïve slices of younger animals.
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Results have shown that field potential epileptiform events are associated with complex polyphasic synaptic potentials containing both excitatory and inhibitory components (Prince and Tseng, 1993; Salin et al., 1995; Figure 4C,D). Variations in the form and amplitude of field potentials and intracellular events, as well as in the propagation of discharges and the synchrony between field potentials and neuronal activities, suggest that the epileptiform neuronal aggregate is more dispersed and less uniformly affected than is the case in acute epileptogenesis induced by convulsant drugs. The stability of neuronal recordings in the in vitro slice preparation allows quantitative assessment of neuronal membrane and synaptic properties. Abnormalities of intrinsic membrane properties, including increased input resistance, prolonged membrane time constant, and a steeper relationship between depolarization and firing frequency, make layer V pyramidal neurons in the isolation more excitable (Prince and Tseng, 1993). Synaptic activities in these neurons also are altered in a manner favoring increased excitability, with an increase in the strength and frequency of excitatory currents, whereas the frequency of inhibitory events is decreased (Li and Prince, 2002). Recent results also suggest that functional abnormalities exist in the presynaptic terminals of injured layer V pyramidal cells consistent with an increased probability of transmitter release (Li et al., 2005). Results of recent experiments (Jin et al., 2005) also show that these cells have a decreased capacity to maintain normal Cl- gradients in the face of intense activity—an abnormality that can result from down-regulation of the Cl-/K+ transporter, KCC2 (Prince et al., 2000), and one that would make GABAergic inhibition less effective during epileptiform activity.
Anatomic and Pathologic Features A variety of experimental approaches have been used to assess the anatomic alterations induced within the chronic neocortical isolation. Significant loss of cortical thickness occurs in the undercut area that affects infragranular layers, accompanied by a decrease in the number and somatic size of layer V pyramidal neurons (Prince and Tseng, 1993; Hoffman et al., 1994; Grüner et al., 1974). Analysis of biocytin-filled layer V pyramidal cells (Figure 5C,D) showed that the axonal arbors of these injured neurons are grossly altered with increased length, numbers of branches, and presumed boutons. Increased recurrent excitatory connectivity shown in these experiments may serve as one functional substrate for hyperexcitability and epileptogenesis within the injured cortex (Salin et al., 1995). Results of earlier anatomic studies (e.g. Grüner et al., 1974) also indicated significant changes in connectivity within the isolated cortex (Salin et al., 1995).
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B
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somadendrites axonal arbor IV V
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FIGURE 5 Anatomic plasticity in layer V neurons of chronic partial neocortical isolations. A: Photomicrograph of a coronal section of adult rat brain reacted with 68 kd neurofilament antibody and diaminobenzidine. Arrows and dashed lines mark the approximate extent of the isolation. Increased immunoreactivity, most intense in infragranular layers, is present in the isolation and adjacent cortex medial to parasagittal cut. Lesion made at P21, ~4 weeks before perfusion. B: Pyramidal neurons (double arrowheads) and interneurons (arrow) within layer V of the partial isolation of A are intensely immunoreactive. Hoffman optics. A,B from I. Parada and D. A. Prince, unpublished data. C,D: Camera lucida tracings from biocytin-filled layer V pyramidal neurons. Examples of a neuron from partially isolated neocortex (C) and control neocortex (D) graphically demonstrate marked differences in axonal arborizations. Axonal arborization shown to right of each neuron and tracings of the soma-dendritic tree of the same neuron to left. Arrow heads mark positions of somata. IV/V marks the boundary between neocortical layers IV and V. PIA: pial surface. (From Salin, P., Tseng, G.F., Hoffman, S., Parada, I., and Prince, D.A. 1995. Axonal sprouting in layer V pyramidal neurons of chronically injured cortex. J Neurosci 15: 8234–8245., with permission.) (See color insert.)
Immunocytochemical results are also consistent with substantial reorganization induced by injury. Neurofilament immunoreactivity (IR) is increased within the neuropil of the isolation and is prominent in both pyramidal cells and interneurons for weeks after the lesion (Figure 5A,B), and c-fos IR is increased as well, likely an important early step in the significant molecular alterations known to occur after injury or epileptiform activity itself (Dragunow et al., 1990; Ernfors et al., 1991; Tetzlaff et al., 1991; Graber et al., 1998, 2003). A variety of cellular markers have been used to assess the anatomic state of GABAergic interneurons in the partially isolated cortex. Immunocytochemical labeling for calbindin, GABA, glutamic acid decarboxylase (GAD) and neuropeptide Y has not shown any obvious reduction in numbers of immunoreactive interneurons (I. Parada and D.A. Prince, in preparation). Counts of parvalbumin posi-
tive interneurons in the injured cortex likewise show no reductions in labeled cells/mm2 (Graber et al., 1999). Recent results in other experiments on biocytin-filled fast-spiking interneurons in the partially isolated cortex indicate, however, that axons of these cells are abnormal in that they possess fewer presumed boutons containing vesicular GABA transporter (VGAT1) that are juxtaposed to postsynaptic gephyrin clusters (I. Parada, J. Li, K. Fu, F. Shen, A. Bacci, and D.A. Prince, unpublished observations). Electron microscopic observations also suggest that fewer symmetric synapses occur on layer V pyramidal cell somata in the injured cortex (J. Wenzel and P.A. Schwartzkroin, personal communication). These anatomic findings fit with electrophysiologic data showing that the frequency of miniature IPSC is decreased in epileptogenic partially isolated cortex (Li and Prince, 2002).
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Advantages and Limitations of the Model
Studies of Antiepileptogenesis Electrophysiologic studies reveal that it is possible to block the development of epileptogenesis in the partially isolated cortex pharmacologically (Graber and Prince, 1999; 2004). In these experiments, tetrodotoxin (TTX), a potent blocker of voltage-dependent sodium channels, was embedded in a slow-release resin (Elvax 40W®; Chiaia et al., 1992; Graber and Prince, 1999) and placed subdurally over the lesioned area in vivo immediately following injury. Slices were cut through the partially isolated, TTX-treated cortex after the ~2-week latent period for epileptogenesis and assessed in vitro. It was found that the presence of TTX prevented subsequent development of hyperexcitability in these slices. Additional studies indicate a critical period for epileptogenesis in this model (Graber and Prince, 2004). TTX-treatment starting at time of injury and continuing for a minimum of 3 days is effective prophylaxis against subsequent development of hyperexcitability after the latent period; treatment of shorter duration is ineffective. Effective treatment can be delayed following injury, but no longer than 3 days after the lesion is placed. Data from these studies suggest that the seemingly physiologically quiescent latent period, before expression of hyperexcitability in slices, can be divided in to a critical period for epileptogenesis followed by a period when intervention is ineffective. An apparent link exists between ongoing electrical activity after the injury and subsequent development of hyperexcitability. The cascade of cellular and molecular changes resulting in epileptogenesis thus appears to start soon after trauma, but remains somewhat plastic for ~3 days; intervention during this critical period either prevents pathologic changes resulting in hyperexcitability or results in compensatory changes. This novel finding provides a useful tool for further delineating alterations that are critical to epileptogenesis, so that more practical approaches to prophylaxis might be developed in the future. It is likely that not all postinjury anatomic and physiologic changes present in undercut neocortex are responsible for epileptogenesis. Some may be coincidental, whereas others might represent failed potentially antiepileptogenic alterations. By comparing like-injured cortices that differ in hyperexcitability (i.e., TTX-treated and untreated), it may be possible to find differences in anatomic, electrophysiologic, or molecular variables that are most relevant to occurrence of epileptogenesis. Studies are in progress using Affymetrix GeneChip microarrays (Affymetrix, Santa Clara, CA) to assess differences in gene expression between partially isolated, TTX-treated nonepileptogenic cortex, and TTXuntreated epileptogenic cortex with similar lesions. This comparison has revealed significantly less differential gene expression between injured, nonepileptogenic and injured, epileptogenic cortices than between injured epileptogenic and naïve cortices (Graber et al., 2002, 2003).
ADVANTAGES AND LIMITATIONS OF THE MODEL Limitations The most significant limitations in any study of a model of epileptogenesis lie in potential differences in pathophysiology between humans and animals, and that human epilepsy is a heterogeneous set of syndromes with presumably differing epileptogenic pathologies. Epileptogenesis is undoubtedly a multifactorial process even in the “simplest” cases (e.g., single gene defects), making it important to know whether the particular abnormality uncovered by a given investigator’s approach is a critical one (a problem that might be termed “the blind man and the elephant” syndrome). Partially isolated neocortex may not necessarily reproduce all aspects of penetrating traumatic brain injury in humans. For example, the technique used produces a relatively limited cortical lesion and likely does not induce simultaneous injury to remote structures, as occurs from blunt trauma and the resulting pressure wave in the lateral fluid percussion model (Lowenstein et al., 1992; Coulter et al., 1996; Santhakumar et al., 2001; Kharatishvili et al., 2003; D’Ambrosio et al., 2004, 2005; see Chapter 37 for review). The lack of widespread brain injury in the cortical partial isolation model is advantageous in some respects; a more localized lesion may limit variables and increase the possibility that epileptiform activity is actually originating in the circuit of interest. Differences may occur in mechanisms underlying partial epilepsy produced by different types of lesions, making it important to compare results from experiments in various models. This approach may also serve to highlight the importance of basic epileptogenic mechanisms that are common to different types of cortical injury (e.g., axonal sprouting and establishment of new and excessive recurrent excitatory circuitry, disinhibition). It is well known that even severe cortical injuries may not induce epilepsy in some patients (Salazar et al., 1985; Annegers et al., 1998), but the reasons for occurrence of epileptogenesis in some brains, but not others, are not known. Not all slices from partially isolated cortex generate epileptiform activities (Graber and Prince, 1999 Hoffman et al., 1994). These differences, which may be related to quantitative (e.g., the amount of new recurrent circuitry) or qualitative (e.g., genetic variations; Andermann, 1969; Schauwecker et al., 2000) factors, emphasize the importance of examining injured, but nonepileptogenic tissue, as well as cortex from naïve controls wherever possible. It should also be emphasized that the link between the occurrence of epileptiform activities in brain slices and development of behavioral seizures in vivo is intuitive, but presumptive and unproved. Attempts to establish such a link might involve correlations between the in vitro and in vivo findings, such
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as the occurrence of epileptiform events from a cortical region from which seizures are initiated in vivo, and subsequent recordings of similar events in vitro from slices through the area. This has been possible in slice experiments from human epileptogenic cortex and hippocampus (Wong and Prince, 1981; Mattia et al., 1995; Avoli et al., 1999; D’Antuono et al., 2004; Schwartzkroin, 1986; see Chapter 8 for review). The properties of epileptiform discharges recorded in vitro from slices of partially isolated cortex (Tseng and Prince, 1993; Hoffman et al., 1994; Graber and Prince, 1999) are similar to in vivo epileptiform abnormalities, and the hyperexcitable cortical networks demonstrated in brain slices would certainly have important physiologic and behavioral consequences, were they to be embedded in the cortex of a behaving animal.
epileptogenic changes persist for the life of the animal (Hoffman et al., 1994; our unpublished observations).
Utility and Ease of Development The model is useful for examination of critical changes occurring during the latent period between injury and development of hyperexcitability (Graber and Prince, 1999; 2004; Graber et al., 2002, 2003). The persistence of epileptiform abnormalities in vitro allows for detailed physiologic and anatomic studies as well as electrophysiologic examination of the cellular mechanisms of drug action in epileptogenic tissue. Although lesion placement takes only ~1/2 hour in experienced hands, the model is probably less well suited for experiments in which a high “throughput” is required (e.g., drug screening).
Reliability The model is reliable, reproducible, and may better mimic “naturally occurring” human pathology than chemically or electrically induced lesions. The partial cortical isolation lesion itself involves actual penetrating injury, axotomy, deafferentation, mild hemorrhage (along needle tracts), and limited blunt trauma because of some stretching of the neocortex as the relatively blunt needle shaft moves through it. High incidence of rodent survival is another advantage of the undercut model; the placement of lesions is well tolerated and mortality is usually related to anesthesia. Overall, the occurrence of evocable epileptogenic activity in chronically injured brain slices, prepared as detailed above, is present in ~80% of slices from ~93% to 100% of lesioned animals (Hoffman et al., 1994; Graber and Prince, 1999). The detection of hyperexcitability is increased by the following measures: (1) use of otherwise healthy slices in which short latency field potentials of at least 1 mV can be evoked; (2) appropriate stimulus intensity near threshold for the normal short latency field response and a relatively low rate of stimulation (£0.1 Hz); intense or more frequent stimuli tend to block occurrence of epileptiform discharges (Prince and Tseng, 1993); (3) exploration of multiple stimulus and recording sites within a given slice, as epileptiform discharges are not present uniformly across the partially isolated cortex within a slice (Graber and Prince, 1999); (4) use of ACSF containing ~5 mM [K+].
Timing of Lesions Lesions made before 1 week of age have not been studied electrophysiologically; however, partial isolations placed as early as P7 can result in hyperexcitable slices after a latent period, a finding that may allow future studies of differences in mechanisms underlying posttraumatic epileptogenesis in immature versus mature neocortex. Hyperexcitability persists in slices obtained >2 years after injury, suggesting that
Future Development As noted, a potential disadvantage of the partial cortical isolation model is that studies of incidence of spontaneous electrographic and behavioral seizures in vivo are incomplete. It is known that epileptiform activity can be reliably evoked in vivo in cortical isolations of cats and monkeys (Grafstein and Sastry, 1957; Sharpless and Halpern, 1962; Echlin and Battista, 1963; Prince, 1965; Halpern, 1972). In implanted rats, electrographic ictal episodes originating from partially isolated sensory-motor neocortex are accompanied by subtle behavioral seizures that might go undetected without simultaneous EEG-video monitoring (K. Graber and D.A. Prince, unpublished observations). Work along these lines to determine the incidence, latency to onset, and frequency of seizures in vivo, as well as characterization of effects of anticonvulsant drugs, would further strengthen the relevance of the partial cortical isolation model to posttraumatic epilepsy (PTE). Currently, a number of unsettled issues remain with respect to epileptogenesis in this model. Although mounting anatomic evidence exists for synaptic reorganization of chronically undercut neocortex (see Section IV.C.), the precise underlying basis for decreased inhibition and increased excitation onto layer V pyramidal neurons (Li and Prince, 2002; see section on Cellular Electrophysiology) remains uncertain. Detailed properties of GABAergic and glutamatergic neurons and their receptors; intrinsic membrane conductances; transporters; functions of reactive astrocytes; and numerous other regulatory mechanisms that affect cortical network excitability in the chronically injured tissue, remain to be explored. Although pathophysiologic alterations in such variables are likely substrates for hyperexcitability in the model, additional studies are necessary to delineate which combination of alterations is critical and sufficient for epileptogenesis. Given the range of head injuries that can be encountered in clinical practice and their varying consequences in terms
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Insights into Human Disorders
of epileptogenesis (Annegers et al., 1998), results of experiments in newer models that might combine aspects of both percussive and penetrating injury, as well as use of genetically susceptible and resistant strains, will be of considerable interest.
INSIGHTS INTO HUMAN DISORDERS Similarity to Human Pathology One issue with respect to this model is how closely it might mimic human pathology in epileptogenic lesions. All aspects of traumatic brain injury in humans are clearly not reproduced by the partial cortical isolation. It does, however, produce a significant focal penetrating cortical injury, in some respects similar to the penetrating lesions that carry the highest risk of development of PTE in patients (Salazar et al., 1985; Annegers et al., 1998). It is also of interest that significant cavitary white matter lesions just beneath the cortex can be a pathologic feature at the site of fluid percussion injury, causing partial isolation of the traumatized cortex (Feeney et al., 1981). The partial isolation model may also have implications for iatrogenic (i.e., surgical) trauma. Seizures were reportedly an undesired consequence of “frontal lobe undercutting” performed in patients for medically intractable psychiatric conditions several decades ago (Scoville, 1960). Epileptogenesis from neurosurgically induced cortical transection may be one of several factors responsible for only ~50% complete seizure control following resection of medically intractable epileptic neocortical foci (Engel et al., 2003). Isolated islands of neocortical gray matter, with neuropathologic evidence of substantial reorganization, are also present in postmortem specimens from epileptic children who developed extensive underlying white matter lesions as infants (Marin-Padilla, 1997), and seizures can also be a clinical feature of some neuropathologic processes with prominent white matter lesions (Williams et al., 1979). Thus, the model may have broader implications for epileptogenic alterations in cortical neurons and circuits following axotomy and deafferentation resulting from ischemic or other nontraumatic insults.
Epileptic Neurons and Circuits Although pathophysiologic mechanisms underlying human PTE are unknown, numerous findings in this model may have potentially important implications. Decreases in somatic size, increases in input resistance, decreases in potassium conductances that normally control the frequency of action potentials, and prolonged membrane time constants in chronically injured pyramidal neurons (see section on Cellular Electrophysiology; Prince and Tseng, 1993) would render them more responsive to excitatory dendritic inputs and increase their input or output function. Changes
in these intrinsic neuronal properties, coupled with excitatory axonal sprouting and formation of new recurrent excitatory synapses, even in the absence of other alterations, might be sufficient to result in hyperexcitability in cortical circuits and an epileptogenic brain. Some of these alterations have been noted in humans; for example, Ramon y Cajal described axonal sprouting of injured neocortical neurons decades ago (1928), suggesting a resultant increase of activity within the neuronal circuit. Given the multiple roles of GABAergic inhibition in (1) controlling burst generation and underlying calcium conductances known to be activated during epileptiform discharges in hippocampus (Wong and Prince, 1979) and neocortex (Kim et al., 1995); (2) suppressing activation of N-methyld-aspartate (NMDA) receptors that are also important in generating epileptiform activity (Hwa and Avoli, 1989); (3) direct control of action potential output of pyramidal cells (Somogyi et al., 1985; Freund, 2003; Tamas and Szabadics, 2004); (4) shunting of excitatory inputs to dendrites (Anderson et al., 1980; Qian and Sejnowski, 1990); and (5) generation of synchronized rhythms in cortical networks, it is obvious that alterations in inhibitory systems within injured cortex will have important effects on regulation of cell and network excitability. Results of additional experiments are required to further assess inhibitory electrogenesis in the partially isolated cortex and to interpret the functional effects of the decreases in anatomic and electrophysiologic indices of inhibition found to date (see section on Cellular Electrophysiology) and their role in epileptogenesis.
Strategies for Antipileptogenesis Studies in this model demonstrate the “proof of concept” that prevention of epilepsy following traumatic injury may be possible. Clinical trials to date in patients suffering traumatic brain injury and treated with conventional antiepileptic agents prophylactically, however, have not been successful in preventing epilepsy (Temkin, 2003). Although agents investigated have efficacy in preventing seizures from occurring while being administered, the incidence of PTE after drug withdrawal remains unchanged. It has been suggested that one possible reason for this failure is that agents have not been given quickly enough after injury (Benardo, 2003). In an acute model of epileptiform activity following placement of a cut between supra- and infragranular layers, valproic acid prevents development of epileptiform activity in the infragranular segment, only if given within 20 minutes of the injury (Yang and Benardo, 2000). In most human studies, medications have been given within 24 hours of the brain trauma, well within the critical period for prevention in the undercut model. Because the latent period in posttraumatic clinical epilepsy can be so much longer than that for occurrence of epileptiform activity in acutely or chronically injured cortex of models studied in
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vivo (Sharpless and Halpern, 1962; Topolnik et al., 2003a,b; D’Ambrosio et al., 2005) or in brain slices from chronic partial isolations (Tseng and Prince, 1993; Hoffman et al., 1995; Graber and Prince, 1999, 2004), it is likely that at least some of the presumably multiple underlying pathogenic mechanisms are different. Effective prophylactic strategies, therefore, may have to be varied, depending on the pathophysiologic nature of the lesion. One importance difference between the actions of antiepileptic drugs that have failed to block human epileptogenesis and that of focal TTX used successfully in the partial isolation model is obviously the degree of suppression of cortical activities. For example, phenytoin, which is known to affect sodium channels at therapeutic serum levels, only limits the frequency and duration of action potential firing during depolarization, whereas even submicromolar concentrations of TTX completely block impulse generation. It is obviously not possible to use agents with such potent global effects in humans, and additional experiments are required to discover the precise molecular links between activity and development of epileptiform activity so that more targeted prophylactic strategies can be developed. Is abnormal activity early in the latent period required for epileptogenesis in the partially isolated cortex, or are other factors involved (e.g., the injury-induced changes known to be present in the model) (Prince and Tseng, 1993; Salin et al., 1995, Li and Prince, 2002; Li et al., 2004) and in epileptic human neocortex (Marco and DeFilepe, 1997; Graber and Prince, 1999 and 2004)? Although it has been previously suggested that PTE might result from kindling-like a phenomenon (Silver et al., 1991 and others), data from videoEEG monitoring of implanted undercut rats during the 3-day postinjury critical period has shown that neither seizures nor epileptiform activity are present, although hyperexcitability occurs in slices following the latent period in the same animals (Graber and Prince, 2004). These data differ from those in ketamine-anesthetized cats where recordings obtained acutely after partial isolations contain epileptiform activity (Topolnik et al., 2003a,b). Anesthesia or species differences might be a factor in this discrepancy. Whether suppression of epileptogenesis following human brain trauma is possible without adversely affecting optimal recovery and rehabilitation (Hernandez, 1997) remains uncertain, as it may be that similar processes are involved (e.g., axonal sprouting and establishment of new synaptic connectivity). Of the myriad of molecular and cellular changes resulting from physical brain trauma (McIntosh et al., 1998), individual ones certainly could be pro- or antiepileptogenic or unrelated to epileptogenesis or recovery. Studies utilizing chronic TTX-treatment point to a welldefined critical period for prevention by suppressing cortical activity in the partial isolation model; however, other relative critical periods might exist during which more focused interventions targeted to specific pathophysiologic events
could be possible. Additional studies utilizing the partial cortical isolation model may be helpful for understanding which postinjury changes are critical and sufficient for posttraumatic epileptogenesis, and provide a better theoretical background for clinical studies leading to successful prophylaxis.
Acknowledgments Portions of the work presented here were supported by NIH grant NS06477, NS12151, NS07280, K08 NS02167 and the Morris and Pimley Research Funds. We thank Isabel Parada for her invaluable assistance in the course of this work.
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FIGURE 3 8 - 5 Anatomic plasticity in layer V neurons of chronic partial neocortical isolations. A: Photomicrograph of a coronal section of adult rat brain reacted with 68 kd neurofilament antibody and diaminobenzidine. Arrows and dashed lines mark the approximate extent of the isolation. Increased immunoreactivity, most intense in infragranular layers, is present in the isolation and adjacent cortex medial to parasagittal cut. Lesion made at P21, ~4 weeks before perfusion. B: Pyramidal neurons (double arrowheads) and interneurons (arrow) within layer V of the partial isolation of A are intensely immunoreactive. Hoffman optics. A,B from I. Parada and D. A. Prince, unpublished data. C,D: Camera lucida tracings from biocytin-filled layer V pyramidal neurons. Examples of a neuron from partially isolated neocortex (C) and control neocortex (D) graphically demonstrate marked differences in axonal arborizations. Axonal arborization shown to right of each neuron and tracings of the soma-dendritic tree of the same neuron to left. Arrow heads mark positions of somata. IV/V marks the boundary between neocortical layers IV and V. PIA: pial surface. (From Salin, R, Tseng, G.F., Hoffman, S., Parada, I., and Prince, D.A. 1995. Axonal sprouting in layer V pyramidal neurons of chronically injured cortex. J Neurosci lg: 8234-8245., with permission.)
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39 Head Trauma: Hemorrhage-Iron Deposition YUTO UEDA, MD, PHD, WILLIAM J. TRIGGS, MD, AND L. JAMES WILLMORE, MD
methods as electrical stimulation (McNamara, 1986; Sutula, et al., 1986; Goddard, 1967; Goddard, et al., 1969) or by chemical injections (Fisher, 1989; Piredda, et al., 1986; Ueda, et al., 2000). Although most paradigms cause animals to have stimulus-dependent seizures, some manipulations do induce spontaneous kindled behaviors, albeit in a small percentage of animals (Pinel and Rovner, 1978b; Pinel and Rovner, 1978a; Hiyoshi, et al., 1993; Milgram, et al., 1995; Mathern, et al., 1997). Injection of microliter quantities of ferrous or ferric cations into isocortex does cause development of epileptiform discharges and focal behavioral seizures (Willmore, et al., 1978b; Willmore, et al., 1978a; Reid, et al., 1979). These effects become spontaneous and chronic (Engstrom, et al., 2001), lasting up to 1 year in 37% of animals (Moriwaki, et al., 1990). Initiation of free radical reactions and lipid peroxidation at the injection site are thought to be critical to iron-induced epileptogenesis (Willmore and Rubin, 1982; Willmore, et al., 1983b; Triggs and Willmore, 1984; Willmore and Triggs, 1991). In our model, injection of the component of blood in the form of an aqueous solution of ferric chloride into rat amygdala produced spontaneous, chronic kindled behavior (Csernansky, et al., 1983; Ueda, et al., 1998).
RATIONALE FOR THE MODEL Traumatic contusion and intracerebral hemorrhage result in focal encephalomalacia, hemosiderin deposition, and, in some patients, chronic and recurrent seizures or posttraumatic epilepsy (Faught, et al., 1989; Willmore, 1990). Severe head trauma in humans results in a cascade of changes that include shearing injury, contusion with accumulation of an admixture of necrotic brain, edema, and hemorrhage (Willmore, 1990). During the latency that reflects the process of epileptogenesis, isolation of regions of neocortex, synaptic reorganization, and altered balance between excitation and inhibition are known to occur. Infarction and cerebral contusion have, in common, the extravasation of red blood cells followed by liberation of iron from hemoglobin and hemosiderin deposition within the neuropil. (Payan, et al., 1970) Iron compounds in biological systems are critical to the process of oxygen transport, for electron transfer reactions and as enzyme cofactors. Iron, however, poses hazards because of two stable oxidation states and the redox properties of iron (Aisen, 1977). Oxidation of ferrous iron to ferric yields an insoluble hydroxide complex. Autoxidation of iron in an aqueous solution such as a biological fluid initiates a series of one-electron transfer reactions that yield free radical intermediates (Willmore, et al., 1983a; Mori, et al., 1992). Indeed, addition of inorganic iron or heme to membrane preparations from subcellular organelles causes formation of superoxide radicals and production of lipid peroxidation (Willmore and Triggs, 1991) with accumulation of malonaldehyde (Willmore, et al., 1983b; Triggs and Willmore, 1984). Manipulation of amygdalar nuclear complexes changes rodent behaviors by kindling that can be induced with such
Models of Seizures and Epilepsy
METHODS OF GENERATION We use Sprague-Dawley rats weighing 200 to 280 g. The appropriate animal use committees review and approve all of our experiments. Surgical procedures are performed with pentobarbital sodium anesthesia injected intraperitoneally (i.p.) (37.5 mg/kg). We prepare animals for parallel observations of
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either behavior or for both behavior and electrophysiologic assessment. Control for the experimental injectate is saline (0.9%) with pH adjusted to equal the acidity of the iron salts solution. We do not adjust pH of either ferrous or ferric chloride because complexes form and cations will precipitate from solution. Preparation of the physiology group begins with stereotaxic placement of a 22-gauge guide canula with the lumen occluded with a stylet and the end positioned just above the left amygdalar body. The canula, held in place on the calvarium with dental acrylic, is prepared with a bare-tipped insulated wire affixed to the lip of the guide canula orifice. After positioning the guide canula, the stylet is removed and replaced with a fused silica tube that is attached to a microinjection pump (CMA Type 100) and positioned into the left amygdaloid body. Animals are then injected with a total of 1.5 ml of 100 mM FeCl3 over 1.5 minutes, or with an equal volume of 0.9% NaCl with the pH adjusted to 2.2 to equal that of the ferric chloride solution. During injection, and for the following 30 minutes, all of our animals undergo continuous electroencephalographic (EEG) recording (Grass Model 79). The canula is removed at the end of the injection and the animals are allowed to recover. We have used video recording in a dimly lit room for 2 hours on selected days between 8 and 10 am. Behaviors are scored by the criteria of Racine (Racine, 1972). Blinding is important and the observer scoring animals should not know the nature of the injectate. For chronic recording from freely moving animals, we add three additional electrodes. Teflon-coated stainless steel electrodes with <0.25-mm bare tips are positioned into the basolateral nucleus of the right amygdaloid body and into both dorsal hippocampi. Stainless steel screws are positioned in the occipital bone to provide reference and ground contacts. All electrodes are attached to wires with pin adapters and then affixed to the skull with dental acrylic. After 5 days of recovery, the stylet is removed from the guide canula and is replaced with an injection canula consisting of a 24-gauge guide wire, the tip of which is a 0.5-mm length of fused silica (0.075 mm inside diameter, 0.15 mm outside diameter). These freely moving animals are then injected with an aqueous solution of 1.5 ml of 100 mM ferric chloride (FeCl3, pH 2.2) injected at a rate of 1ml per minute using a microinfusion pump (CMA Type 100). Following injection, EEG recording and behavioral observations can be obtained daily for the duration of the experiment. Animals prepared for neurochemical assessment or for molecular biological experiments are injected, but chronic recording electrodes are not placed within the depths of the brain. Behaviors on the Racine scale are recorded for each animal during scheduled observations. In our experiments, we use stereotaxic coordinates determined with the rat brain atlas of Paxinos and Watson (1986). The incisor bar is set 3.3 mm below the intraaural line. Stereotaxic coordinate for
the amygdaloid body are 3.3 mm posterior and 5.0 mm bilateral to the bregma, and 8.5 mm below the surface of the skull. Coordinate for the dorsal hippocampus are 3.3 mm posterior and 1.5 mm bilateral to the bregma, and 3.4 mm below the surface of the skull.
CHARACTERISTICS, DEFINING FEATURES, AND RESULTS Acute injection of FeCl3 into rat amygdala causes a gradual increase in spike discharges over 20 minutes, followed by quiescence of EEG patterns by the end of 30 minutes of recording (Ueda, et al., 1998). Animals injected with acidified saline have occasional isolated spike discharges. Of animals injected into the left amygdaloid body with FeCl3, up to 92% develop spontaneous seizures of stage 4 or stage 5 (Racine) by the fifth day after injection. None of the acidified saline injected control rats have either sustained EEG discharges or behavioral seizures. We have prepared parallel groups of animals with either varied recording systems or no intracranial electrodes to allow neurochemical experiments using microdialysis (Ueda and Tsuru, 1995; Engstrom, et al., 2001) or molecular biological experiments (Ueda, et al., 2001; Ueda and Willmore, 2000c; Doi, et al., 2000). Among those animals prepared for chronic recording, all of those injected with FeCl3 in our initial experiment developed spontaneous behavioral seizures and epileptiform discharges from the depth electrodes. Epileptiform patterns are recorded from the ipsilateral amygdaloid body within 5 to 10 minutes after injection of FeCl3. Over the following days, mainly ipsilateral epileptiform discharges will be recorded from the ipsilateral FeCl3-injected amygdala. During the period of 1 to 3 days, focal spike discharges are much more abundant from the ipsilateral amygdaloid body. Within 5 to 7 days after injection, isolated spikes from both hippocampi and contralateral amygdaloid body are readily observed. From 7 days after injection, spontaneous rearing and limb clonus seizure (Racine stage 4 to 5) with rhythmic epileptiform discharges can be observed (Figure 1). Both the electrographic patterns and the behavioral seizures are observed until we terminated our experiments at 30 days after injection. Saline-injected animals remain without spontaneous seizures or epileptiform abnormalities on the EEG recordings.
HISTOPATHOLOGY Timm’s stain of the hippocampus in sequences up to 30 days following injection in rats with Racine stage 4 seizures show mossy fiber sprouting (Figure 2). Timm’s staining followed the protocol of Mathern et al. (1994; 1995). In addi-
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Histopathology
FIGURE 1 Chronic electrographic recordings obtained from an animal implanted in the locations noted with Tefloncoated steel electrodes: LA in the left amygdala, RA in the right amygdala. Hippocampal recordings were LH for the left hippocampus and RH for the right hippocampus. Spontaneous electrographic and behavioral seizure recorded during routine scheduled observation and recording of a rat injected with 1.5 ml of 100 mM ferric chloride into the left amygdalar nuclear region 15 days before this recording. Rhythmic epileptiform discharges appear from the amydgalar regions and rapidly propagate to hippocampal electrodes. Behavioral stages according to the scheme of Racine (Racine, 1972) are noted as S1 through S 4. Reprinted from Ueda et al. (1998) with permission of Elsevier.
tion, with Berlin blue and with combined H and E staining we find loss of cells in the ipsilateral and contralateral hippocampi in the CA1 and 4 regions and in the dentate hilus.
Genetic and Molecular Changes Given that repetitive microinjection of glutamate into the amygdala causes Racine behaviors (Croucher and Bradform, 1989), we examined the regulation of neurotransmitter transporters by measuring mRNA and proteins responsible for glutamate and g-aminobutyric acid (GABA) removal. We created a colony of animals with recurrent seizures induced by ferric chloride injection into the amygdala and killed animals from 1 hour to 30 days after injection. We extracted crude synaptic membrane fractions from the hippocampus and used immunoblotting and quantitative densitometry to measure the regulation of the high-affinity glutamate transporters GLAST, GLT-1 and EAAC-1 (Doi et al., 2000). As shown in Figure 3, by 30 days following iron injection into the amygdala, the production of glial
glutamate transporter GLAST is down-regulated in both hippocampi. In our opinion, the collapse in the ability of brain regions to remove synaptic glutamate is fundamental to the process of posttraumatic epilepsy as modeled in these animals. Of interest, GABA transporters, particularly GAT3, are up-regulated in a pattern that in bidirectional function suggests compensatory responses within the inhibitory system (Ueda and Willmore, 2000a).
Effects of Drugs Transporter proteins GAT-1 and GAT-3 remove GABA from synaptic regions. To assess the molecular effects of the antiepileptic drug valproate, albino rats with chronic, spontaneous, recurrent seizures induced by amygdalar injection of FeCl3 were treated for 14 days with either valproic acid or with saline as an injection control (Ueda and Willmore, 2000b). Regions of the hippocampus were assayed for glutamate and GABA transporters by western blot. Although epileptogenesis is correlated with the down-regulation of
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FIGURE 3 Sequential changes in the levels of the glial glutamate transporter GLAST measured in tissue taken from the hippocampus from the time of injection of with 1.5 ml of 100 mM ferric chloride into the left amygdalar nuclear region from baseline up to 30 days after injection. Note marked up-regulation of GLAST within 5 days of injection, whereas at 15 and 30 days, when animals have developed chronic seizures, down-regulation is seen of the production of GLAST. Modified and reprinted from Ueda and Willmore (2000c) with permission of Elsevier.
FIGURE 2 Representative Timm silver sulfide staining demonstrating the presence of zinc-containing mossy fibers in the dentate gyrus (A, square area, and CA3 circle). Rats were killed 15 days after a single injection into the left amygdalar region with 1.5 ml of 100 mM ferric chloride. Animals show Racine (Racine, 1972) stage 4 behaviors. Note the mossy fiber sprouting (B) both in the suprapyramidal (closed arrows) and infrapyramidal blades (open arrows). (C) Arrowheads mark sprouting of mossy fibers in the CA3. (See color insert.)
GLAST and up-regulation of EAAC-1, valproate caused an increase in the quantity of GLAST protein measured in the hippocampus. Further, valproate treatment decreased GLT1 in both control and experimental animals in both hippocampi. Although GABA transporters GAT-1 and GAT-3 in the hippocampus were up-regulated by FeCl3 injection
into the amygdala, valproate caused the down-regulation of these GABA transporters in both control and experimental animals (Ueda and Willmore, 2000b). Thus, valproate appears to have unique mechanisms of action; specifically, it affects the removal of glutamate by up-regulating GLAST and decreasing GABA transport, which could result in increased tissue concentrations of GABA (Hassel, et al., 2001). Zonisamide is a 1,2-benzisoxazole drug that contains a sulfonamide side chain. Albino rats with chronic, spontaneous recurrent seizures induced by the amygdalar injection of FeCl3 (Ueda, et al., 1998) were treated for 14 days with zonisamide. Zonisamide has unique effects with increase in the quantity of EAAC-1 protein in hippocampus and cortex and down-regulation of the GABA transporters, GAT-1. These changes occurred in both experimental and zonisamide treated control animals (Ueda et al., 2003). These data demonstrate that zonisamide has molecular effects in that up-regulation of EAAC-1 and decreased production of GABA transporters will increases tissue and synaptic concentrations of GABA.
Insights Into Human Disorders Epilepsy complicates head trauma in approximately 7% of injured civilians (Annegers, et al., 1980) and up to 34%
References
of those injured in combat (Meirowsky, 1982). Development of epilepsy appears to correlate with the severity of brain injury. Caveness (1979) defined a spectrum of severity of injury that showed those with dural penetration, neurologic deficits, and intracerebral hemorrhage were at greatest risk. Salazar et al. evaluated patients with posttraumatic epilepsy following head injury during the Viet Nam war. Factors correlating with greatest risk were retained metal fragments, moderate volume of brain tissue loss, and the presence of an intracranial hematoma (Salazar et al., 1985). In common with epilepsy following hemorrhagic vascular disease (Faught et al., 1989), extravasation of blood in contact with neuropil, usually in the context of contusion of cortical tissue, appears to be a consistent occurrence in patients with posttraumatic epilepsy (Richardson and Dodge, 1954). Injection of iron salts or heme-containing blood products into rat isocortex results in focal cerebral edema, cavitary necrosis, and gliosis, all changes found in cerebral tissue foci of seizures from humans with posttraumatic epilepsy. These profound histopathologic changes caused by iron injection into the isocortex can be prevented by pretreatment of animals with a-tocopherol and selenium (Rubin and Willmore, 1980; Triggs and Willmore, 1994). Protection against the histopathologic damage of iron injection has been observed in iron injection into feline spinal cord (Anderson and Means, 1983) and after compression-induced edema in rat brain (Yoshida et al., 1983). Because antioxidants prevent both acute epileptiform responses and lipid peroxidation following stereotactic injection of ferrous chloride into rat hippocampus, it would appear that iron-induced lipid peroxidation has a critical role in the development of posttraumatic epilepsy.
References Aisen, P. 1977. Some physicochemical aspects of iron metabolism. Ciba Foundation Symposium. pp. 1–14. New York: Elsevier. Anderson, D.K., and Means, E.D. 1983. Lipid peroxidation in spinal cord. FeCl2 induction and protection with antioxidants. Neurochem.Pathol 1: 249–264. Annegers, J.F., Grabow, J.D., Grover, R.V., Laws, E.R., Elveback, L.R., and Kurland, L.T. 1980. Seizures after head trauma: a population study. Neurology 30: 683–689. Caveness, W.F., Meirowsky, A.M., Rish, B.L., Mohr, J.P., Kistler, J.P., Dillon, J.D., and Weiss, G.H. 1979. The nature of posttraumatic epilepsy. J Neurosurg 50: 545–553. Croucher, M.J., and Bradform, H.F. 1989. Kindling of full limbic seizures by repeated microinjection of excitatory amino acids into the rat amygdala. Brain Res 501: 58–65. Csernansky, J.G., Holman, C.A., Bonnet, K.A., Grabowsky, K., King, R., and Hollister, L.E. 1983. Dopaminergic supersensitivity at distant sites following induced epileptic foci. Life Sci 32: 385–390. Doi, T., Ueda, Y., Tokumaru, J., Mitsuyama, Y., and Willmore, L.J. 2000. Sequential changes in glutamate transporter mRNA during Fe+++ induced epileptogenesis. Mol Brain Res 75: 105–112. Engstrom, E.R., Hillered, L., Flink, R., Kihlstrom, L., Lindquist, C., Nie, J.X., Olsson, Y. et al. 2001. Extracellular amino acid levels measured
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with intracerebral microdialysis in the model of posttraumatic epilepsy induced by intracortical iron injection. Epilepsy Res 43: 125–144 Faught, E., Peters, D., Bartolucci, A., Moore, L., and Miller, P.C. 1989. Seizures after primary intracerebral hemorrhage. Neurology 39: 1089–1093 Fisher, R.S. 1989. Animal models of the epilepsies. Brain Res Brain Res Rev 14: 245–278. Goddard, G.V. 1967. Development of epileptic seizures through brain stimulation at low intensity. Nature 214: 1020–1023. Goddard, G.V., Mcintyre, D.C., and Leech, C.K. 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 25: 295–330. Hassel, B., Iversen, E.G., Gjerstad, L., and Tauboll, E. 2001. Up-regulation of hippocampal glutamate transport during chronic treatment with sodium valproate. J Neurochem 77: 1285–1292. Hiyoshi, T., Seino, M., Kakagawa, N., Higashi, T., Yagi, K., and Wada, J.A. 1993. Evidence of secondary epileptogenesis in amygdaloid overkindled cats: electroclinical documentation of spontaneous seizures. Epilepsia 34: 408–415. Mathern, G.W., Bertram, E.H. III, Babb, T.L., Pretorius, J.K., Kuhlman, P.A., Spradlin, S., and Mendoza, D. 1997. In contrast to kindled seizures, the frequency of spontaneous epilepsy in the limbic status model correlates with greater aberrant fascia dentata excitatory and inhibitory axon sprouting, and increased staining for n-methyl-daspartate, AMPA and GABAa receptors. Neuroscience 77: 1003– 1019. Mathern, G.W., Leite, J.P., Pretorius, J.K., Quinn, B., Peacock, W.J., and Babb, T.L. 1994. Children with severe epilepsy: evidence of hippocampal neuron losses and aberrant mossy fiber sprouting during postnatal granule cell migration and differentiation. Brain Res Dev Brain Res 78: 70–80. Mathern, G.W., Pretorius, J.K., and Babb, T.L. 1995. Quantified patterns of mossy fiber sprouting and neuron densities in hippocampal and lesional seizures. J Neurosurg 82: 211–219. McNamara, J. 1986. Kindling model of epilepsy. Adv Neurol 44: 303–318. Meirowsky, A.M. 1982. Notes on posttraumatic epilepsy in missile wounds of the brain. Mil Med 147: 632–634. Milgram, N.W., Michael, M., Cammisuli, S., Head, E., Ferbinteanu, J., Reid, C., Murphy, M.P. et al. 1995. Development of spontaneous seizures over extended electrical kindling. II. Persistence of dentate inhibitory suppression. Brain Res 670: 112–120. Mori, A., Hiramatsu, M., and Yokoi, I. 1992. Posttraumatic epilepsy, free radicals and antioxidant therapy. In Free Radicals in the Brain Aging, Neurological and Mental Disorders. Ed. L. Paker, L. Prilipko, and Y. Christen, pp. 109–122. Berlin: Springer-Verlag. Moriwaki, A., Hattori, Y., Nishida, N., and Hori, Y. 1990. Electrocorticographic characterization of chronic iron-induced epilepsy in rats. Neurosci Lett 110: 72–76. Paxinos, G., and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press. Payan, H., Toga, M., and Berard-Badier, M. 1970. The pathology of posttraumatic epilepsies. Epilepsia 11: 81–94. Pinel, J.P.J., and Rovner, L.I. 1978a. Electrode placement and kindlinginduced experimental epilepsy. Exp Neurol 58: 335–346. Pinel, J.P.J., and Rovner, L.I. 1978b. Experimental epileptogenesis: kindling-induced epilepsy in rats. Exp Neurol 58: 190–202. Piredda, S., Yonekawa, W., Whittingham, T.S., and Kupferberg, H.J. 1986. Enhanced bursting activity in the CA3 region of the mouse hippocampal slice without long-term potentiation in the dentate gyrus after systemic pentylenetetrazole kindling. Exp Neurol 94: 659–669. Racine, R. 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroencephalogr Clin Neurophysiol 32: 281–294. Reid, S.A., Sypert, G.W., Boggs, W.M., and Willmore, L.J. 1979. Histopathology of the ferric-induced chronic epileptic focus in cat: A golgi study. Exp Neurol 66: 205–219.
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Richardson, E.P., and Dodge, P.R. 1954. Epilepsy in cerebrovascular disease. Epilepsia 3 (series 3): 49–74. Rubin, J.J., and Willmore, L.J. 1980. Prevention of iron-induced epileptiform discharges in rats by treatment with antiperoxidants. Exp Neurol 67: 472–480. Salazar, A.M., Jabbari, B., Vance, S.C., Grafman, J., Amin, D., and Dillon, J.D. 1985. Epilepsy after penetrating head injury. I. Clinical correlates: A report of the Vietnam Head Injury Study. Neurology 35: 1406– 1414. Sutula, T., Harrison, C., and Steward, O. 1986. Chronic epileptogenesis induced by kindling of the entorhinal cortex: the role of the dentate gyrus. Brain Res 385: 291–299. Triggs, W.J., and Willmore, L.J. 1984. In vitro lipid peroxidation in rat brain following intracortical Fe++ injection. J. Neurochem 42: 976–980. Triggs, W.J., and Willmore, L.J. 1994. Effect of [dl]-a-tocopherol on FeCl2-induced lipid peroxidation in rat amygdala. Neurosci Lett 180: 33–36. Ueda, Y., Doi, T., Tokumaru, J., Mitsuyama, Y., and Willmore, L.J. 2000. Kindling phenomena induced by the drepeated short-term high potassium stimuli in the venteral hippocamus of rats: on-line monitoring of extracellular glutamate overflow. Exp Brain Res 135: 199–203. Ueda, Y., Doi, T., Tokumaru, J., and Willmore, L.J. 2003. Effect of Zonisamide on molecular regulation of glutamate and GABA transporter proteins during epileptogenesis in rats with hippocampal seizures. Mol Brain Res 116: 1–6. Ueda, Y., Doi, T., Tokumaru, J., Yokoyama, H., Nakajima, A., Mitsuyama, U., Ohya-Nashiguchi, H. et al. 2001. Collapse of extracellular glutamate regulation during epileptogenesis: Down-regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by dainic acid. Exp Brain Res 135: 199–203. Ueda, Y., and Tsuru, N. 1995. Simultaneous monitoring of the seizurerelated changes in extracellular glutamate and gamma aminobutyric acid concentration in bilateral hippocampi following development of amygdaloid kindling. Epilepsy Res 20: 213–219.
Ueda, Y., and Willmore, L.J. 2000a. Hippocampal gamma-aminobutyric acid transporter alterations following focal epileptogenesis induced in rat amygdala. Brain Res Bull 52: 357–361. Ueda, Y., and Willmore, L.J. 2000b. Molecular regulation of glutamate and GABA transporter proteins by valproic acid in rat hippocampus during epileptogenesis. Exp Brain Res 133: 334–339. Ueda, Y., and Willmore, L.J. 2000c. Sequential changes in glutamate transporter protein levels during Fe+++ induced epileptogenesis. Epilepsy Res 39: 201–219. Ueda, Y., Willmore, L.J., and Triggs, W.J. 1998. Amygdalar injection of FeCl3 causes spontaneous recurrent seizures. Exp Neurol 153: 123–127. Willmore, L.J. 1990. Posttraumatic epilepsy: cellular mechanisms and implications for treatment. Epilepsia 31 (Suppl. 3): S67–S73. Willmore, L.J., Hiramatsu, M., Kochi, H., and Mori, A. 1983a. Formation of superoxide radicals after FeCl2 injection into rat isocortex. Brain Res 277: 393–396. Willmore, L.J., Hiramatsu, M., Kochi, H., and Mori, A. 1983b. Formation of superoxide radicals, lipid peroxides and edema after FeCl3 injection into rat isocortex. Brain Res 277: 393–396. Willmore, L.J., and Rubin, J.J. 1982. Formation of malonylaldehyde and focal brain edema induced by subpial injection of FeCl2 into rat isocortex. Brain Res 246: 113–119. Willmore, L.J., Sypert, G.W., and Munson, J.B. 1978a. Recurrent seizures induced by cortical iron injection: a model of post-traumatic epilepsy. Ann Neurol 4: 329–336. Willmore, L.J., Sypert, G.W., Munson, J.B., and Hurd, R.W. 1978b. Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science 200: 1501–1503. Willmore, L.J., and Triggs, W.J. 1991. Iron-induced lipid peroxidation and brain injury responses. Int. J. Dev. Neuroscience. 9: 175–180. Yoshida, S., Busto, R., Ginsberg, M.D., Abe, K., Martinez, E., Watson, D.B., and Scheinberg, P. 1983. Compression-induced brain edema: modification by prior depletion and supplementation of vitamin E. Eur Neurol 33: 166–172.
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FIGURE 3 9 - 2 Representative Timm silver sulfide staining demonstrating the presence of zinc-containing mossy fibers in the dentate gyms (A, square area, and CA3 circle). Rats were killed 15 days after a single injection into the left amygdalar region with 1.5 btl of 100mM ferric chloride. Animals show Racine (Racine, 1972) stage 4 behaviors. Note the mossy fiber sprouting (B) both in the suprapyramidal (closed arrows) and infrapyramidal blades (open arrows). (C) Arrowheads mark sprouting of mossy fibers in the CA3.
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FIGURE 4 0 - 8 Photothrombosis. (A) Placement of an argon laser beam on the exposed skull of a rat overlying the left sensorimotor cortex. (B) 2,3,5-triphenyltetrazolium chloride (TTC)-stained coronal slice of brain performed 24 hours after lesioning of a 20-month-old animal.
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40 Stroke KEVIN M. KELLY
mic spike wave discharges in cortex ipsilateral to the occlusion during both ischemic and reperfusion periods in animals studied for 24 hours to 1 week after lesioning (Lu et al., 2001; Williams et al., 2001), and in both ipsilateral and bilateral electrographic seizures when studied up to 3 days after lesioning (Hartings et al., 2003; Williams et al., 2004). Although these findings are associated with the acute phase of injury following arterial occlusion, they suggest the potential for the development of epileptogenicity in the periinfarct area. It is indeterminate, however, whether these animals would have become epileptic, because these studies were terminated shortly after lesioning. Long-term monitoring studies of young animals that underwent MCAO did not provide evidence of seizure activity with monitoring periods extending up to 1-year after lesioning (Karhunen et al., 2003). Because the standard model of MCAO in rats frequently involves injury to the hippocampus, unlike MCAO in humans (Del Zoppo, 1998, Kelly 2002), our laboratory chose to use the technique of transient (3 hour) unilateral (left-sided) MCA and common carotid artery occlusion (CCAO), which results in moderately large cortical infarcts (~100 mm3) of relatively reproducible size and location, while sparing injury to the hippocampus (Aronowski et al; 1994, 1997). Our initial studies using this model were designed to study young animals with extended periods of monitoring. We performed MCA/CCAO and sham operations in 2.5-month-old male Long Evans rats, followed by intermittent video-EEG monitoring for 6 months. These studies failed to demonstrate poststroke epilepsy in any animal (Kelly et al., submitted). Because of this negative result, our recent studies have been designed to determine whether animal age might be a critical variable
MIDDLE CEREBRAL ARTERY OCCLUSION General Description of Model Poststroke seizures and epilepsy have been described in numerous clinical and population studies. In general, poststroke epileptic seizures are considered focal in onset, arising within the “ischemic penumbra,” the area of compromised tissue surrounding the infarct (Loiseau, 1997). The pathophysiologic events of injured brain that establish poststroke epileptogenesis are not well understood, however, primarily because no standard animal model of poststroke epilepsy exists (Kelly, 2002, 2004a). Although arterial occlusion models of brain infarction are used in hundreds of laboratories to study the pathophysiology of stroke, neuroprotection, and functional outcome after stroke, typically, no description of seizure activity in lesioned animals exists. The lack of observed seizure activity in lesioned animals may be associated with relatively brief periods of observation from the time of infarction to animal sacrifice (hours, days), young age, nonconvulsive seizures, and no electroencephalographic (EEG) monitoring. All of these factors have likely contributed to a relatively modest effort of extending arterial occlusion models of stroke to the study of poststroke epileptogenesis. Middle cerebral artery occlusion (MCAO) is the standard technique of vascular occlusion used in models of cerebral infarction. Similar to human MCAO, experimental MCAO produces a large cortical and subcortical infarct ipsilateral to the occlusion. As described above, little work has been performed extending the use of MCAO as a potential model of poststroke seizures and epilepsy. In studies performed shortly after lesioning, however, MCAO resulted in rhyth-
Models of Seizures and Epilepsy
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in generating epileptogenesis after lesioning with MCA/ CCAO.
What Does It Model? The MCA/CCAO procedure is intended to model poststroke neocortical epilepsy characterized by focal seizures with elementary clonic or inhibitory motor signs, with or without secondary generalization (Engel, 2001).
Methods of Generation Animal Issues Our laboratory has conducted several studies of aged rats (Kharlamov et al., 2000; Kelly et al., 2001a; 2001b; 2003) and chose to use Fischer 344 (F344) rats because of their longevity and availability; aged animals of other common strains (Long Evans, Sprague-Dawley, Wistar) are not currently available for purchase. Studies were planned with 4and 20-month-old male F344 animals in accordance with general principles of animal use for gerontologic research (Miller and Nadon, 2000). Four-month-old animals are considered more reliably “young adult” than animals that are 2 to 3 months old, and thereby reflect a conservative strategy for experimental comparisons with an aged adult cohort. Twenty-month-old F344 animals are appropriately considered “aged” (Kharlamov et al., 2000) and, based on our experience with the MCA/CCAO model and with 24- and 30-month-old animals in the photothrombosis model (see below; Kelly et al., 2001a), we hypothesized that most 20-month-old F344 rats would demonstrate problem-free survival after lesioning, allowing extended periods of video-EEG monitoring. Pilot studies using transient unilateral MCA/CCAO in 4- and 20-month-old lesioned and sham-operated F344 animals have been conducted with video-EEG monitoring for 2 months after lesioning to assess the effects of age on animal survival, potential evidence of seizures or epilepsy, and lesion-induced changes in brain anatomy. To date, these studies have demonstrated reasonable animal survival, evidence of convulsive seizures that differ by age, and infarcts restricted to the cerebral cortex. Data analysis of these studies is ongoing at this time. Procedures Transient Unilateral MCA/CCAO Transient focal ischemia is induced by occlusion of the left MCA and left CCA as described by Aronowski et al. (1994, 1997), with minor modifications. All animal lesioning in our studies has been performed by Dr. Jaroslaw Aronowski at the University of Texas—Houston. Briefly, animals are fasted overnight with free access to water and anesthetized with an intraperitoneal (i.p.) injection of chloral
hydrate (0.45 g/kg in 1 ml of saline). The femoral artery is cannulated to measure blood pressure. Temperature is monitored from the right temporalis muscle and maintained at 37.0° C + 0.4° C during ischemia and the first hour of reperfusion by a feed-forward temperature controller (YSI Model 72, Yellow Springs, OH), heating lamp, and warming blanket. The CCA is isolated through a midline incision and tagged with a suture. An incision is made through the left temporalis muscle perpendicular to a line drawn between the external auditory canal and the lateral canthus of the left eye. Under direct visualization with the surgical microscope, a rectangular burr hole 1 ¥ 3 mm is drilled 2 to 3 mm rostral to the fusion of the zygomatic arch and the squamosal bone to expose the left MCA rostral to the rhinal fissure, and a 1mm round burr hole is made 4 mm dorsal to the MCA exposure to allow measurement of cerebral perfusion (CP). The beveled edge of a 23-gauge hypodermic needle is used to pierce and open the dura along the entire length of the rectangular burr hole. A 0.005-inch diameter stainless steel wire (Small Parts Inc, Miami, FL) is placed underneath the left MCA rostral to the rhinal fissure, proximal to the major bifurcation of the MCA, and distal to the lenticulostriate arteries. The artery is lifted and the wire is rotated clockwise. The left CCA is then occluded using atraumatic Heifetz aneurysm clips. After 3 hours, reperfusion is established by first removing the aneurysm clips from the CCA, and then rotating the wire counterclockwise and removing it from beneath the MCA. Interruption of flow through the MCA is inspected under the microscope and verified by CP measurements using a laser Doppler flowmeter (LDF; model BPM2, Vasamedics Inc, St Paul, MN). Only those animals that display reduction of CP following ischemia to <15% of the preischemic value are included in the study. The same surgical procedure is performed on sham-operated animals, except that the MCA and CCA are not occluded. Following adequate postoperative stabilization, behavioral deficit testing is performed and scored (Aronowski et al., 1995). Skull Screw Electrodes Screws for EEG recordings can be placed in the skull of an animal 1 week after MCA/CCAO; the procedure is the same for lesioned and sham-operated animals. Animals are anesthetized with injections of a 9 : 1 mixture of ketamine and xylazine. After the loss of the tail pinch reflex, animals are positioned in a stereotaxic frame, ophthalmic ointment is applied over the eyes, and a midline incision is made along the scalp, which is reflected bilaterally. Partial burr holes are made in the skull for six recording and two anchoring stainless steel screws (MX-080-2; Small Parts Inc., Miami Lakes, FL). Three recording screws are placed on each side of the skull to sample neocortical activities symmetrically (Figure 1). Odd numbered electrodes represent parasagittal placements over the left (lesioned) hemisphere and even numbered electrodes represent placements over the right
Middle Cerebral Artery Occlusion
Electrode Anchoring FIGURE 1 Positions of screws in the rat skull. Electrodes are designated to indicate relative positions over the cerebral cortex.
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(Advanced Technology Video, Inc., Redmond, WA) are used to monitor animal behavior. Digital video files are obtained for all animals (Diva, Stellate Systems) and recorded directly to high capacity (120 GB) hard disk drives using a removable hard-drive bay. The 32-channel Stellate monitoring system is used to monitor four animals concomitantly (eight channels per animal). This system includes a hardwired common reference that is arbitrarily linked to each animal’s F4 electrode. This method, however, results in unavoidably contaminated electrode pairs that include the F4 electrode, although gross signal distortion is generally inapparent in these derivations. EEG Data Analysis
hemisphere; the rostrocaudal placement is designated as F = frontal, C = central, and P = parietal. Two screws are placed laterally for anchoring the headset. An exposed end of an insulated copper wire is wrapped tightly around each recording screw while the other end of the wire is crimped into a six-conductor (RJ12) modular plug and secured to the skull surface with dental acrylic. The skin around the headset is sutured and the animal is allowed to recover from anesthesia. Bipolar EEG recordings are initiated 1 week after electrode placement to allow complete recovery from anesthesia and the local effects of surgery, and to ensure adequate hydration, nutrition, and vitality.
Monitoring Ease of Development Digital Video-EEG Recordings The digital video-EEG recording techniques used for this model have been expanded from previously published methods (Kharlamov et al, 2003) and have proved to be relatively easy to implement. Individual animal recording chambers (Dragonfly Inc., Ridgeley, WV) are equipped with a custom-designed swivel commutator and provide a low noise recording environment, full freedom of animal movement, and excellent visualization of animal behavior. Headset electrodes are connected to a 32-channel Stellate Systems video-EEG monitoring system. Recordings include active wakefulness, passive wakefulness, and sleep for all animals during daytime and nighttime hours. Individual animals are recorded with a standard montage of eight EEG channels that sample brain activity from both hemispheres using different bipolar electrode derivations. EEG signals are obtained without filtering but, for display purposes, typically are filtered at 1 and 70 Hz, and with a 60-Hz notch filter (Harmonie, Stellate Systems, Montreal). Closed-circuit television cameras connected to a video splitter unit
Qualitative EEG analysis is performed off-line by a complete visual examination of the records with special attention paid to intermittent focal and generalized activities as determined by changes in waveform morphology, amplitude, and frequency. Available event detection software (SensA, Stellate Systems; Reveal, Persyst) is not designed to be sufficiently discriminating for all ictal activities and, therefore, is used in a limited fashion for “first pass” detection of grade 4 to 5 seizure activity (Racine, 1972). Standard terms for frequency ranges are used: delta (0.1–3.9 Hz); theta (4.0–7.9 Hz); alpha (8.0–12.9 Hz); and beta (13.0–30 Hz; Fisch, 1999). EEG nonictal activities are distinguished from ictal activities based on waveform morphology and frequency and the associated absence or presence of behavioral change, respectively. Reliability To maximize the yield of video-EEG recordings during periods of epileptogenesis that may be £2 months, recording protocols are standardized for frequent video-EEG monitoring during the first 2 months after lesioning. This is done recognizing the potential variability of epileptogenesis within and between animal cohorts, not unlike the variability that is observed clinically. Because available resources prohibit continuous (around-the-clock, every day) videoEEG recordings for all animals, both lesioned and shamoperated animals undergo periodic monitoring for shorter periods of time. At the start of each day, four animals begin monitoring coinciding with their light (inactive) cycle and are recorded simultaneously for 6 hours, after which they are returned to the vivarium. At the end of each day, a different set of four animals are hooked up to coincide with the beginning of their 12-hour dark (active) cycle and recorded for 6 hours. These animals are returned to the vivarium the next morning. We recognize that monitoring two sets of animals every day for 6 hour each requires more labor than monitoring just one set for 12 hour; however, aged animals experience less stress and are better able to
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tolerate limited periods of monitoring and minimized disruption of their routines. In this way, equal periods of relative activity and inactivity are sampled from all animals. During daytime monitoring periods and the 3 days per week (including weekend days) that monitoring does not take place, off-line analysis of video-EEG records is performed. We feel that this monitoring protocol reliably samples and identifies ictal activities as they evolve over the 2 months following lesioning. Chronic recordings lasting several months or longer can be executed to enhance the capture of ictal activities and to provide additional information on the evolution of the epileptic state. Occurrence of Spontaneous Seizures Seizures occur spontaneously in this model, including hemibody clonic contractions and grade 3 to 5 convulsions (discussed later). Remission Although the MCA/CCAO technique is used to generate a model of chronic epilepsy, no information is currently available regarding the possibility of the condition undergoing remission.
Characteristics and Defining Features Behavioral Features All animals (sham-operated and lesioned) variably demonstrated spontaneous generalized 7- to 9-Hz spike-wave discharges (SWD) associated with behavioral absence seizures, a frequent finding in common laboratory rat strains (Kelly, 2004b), and will not be described further.
Seizure Severity Vertical Jumps Review of video-EEG records revealed surprising evidence of a stereotypic convulsion that occurred in all (nine lesioned and nine sham-operated) 4- and 20-month-old animals, suggesting that the event was strain-specific, and age- and lesion-independent. The convulsion was an unexpected, abrupt, solitary vertical jump that occurred during sleep (Figure 2). These convulsive jumps could be severe, at times resulting in the animal hitting the top of the recording chamber. Immediately after the jump, the animal would be awake and motionless for a brief period after which normal behavior resumed, if somewhat slowly. EEG typically showed little to no evidence of significant change before or after the convulsion; brief (2 seconds) parietally dominant rhythmic theta (5 Hz) activity was seen following a severe vertical jump in a 20-month-old lesioned animal (data not shown), more likely reflecting a changed state of arousal rather than termination of an ictal discharge. Hemibody Clonic Contractions One of four (25%) lesioned 4-month-old animals demonstrated a single severe hemibody clonic contraction that involved the left forelimb and hindlimb lasting 6 seconds (Figure 3), after which the animal resumed normal behavior. The EEG showed no clear evidence of significant change before or after the event; the apparent asymmetry seen in the EEG in Figure 3 is caused by movement artifact that is most evident in the F4 electrode, which is contaminated as a linked common reference. Bilateral Forelimb Clonus Two of five (40%) lesioned 20-month-old animals demonstrated recurrent episodes of forelimb clonus (grade 3;
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FIGURE 2 Middle cerebral artery–common carotid artery occlusion (MCA/CCAO). Solitary vertical jump of a 4month-old sham-operated animal 2 months after surgery. Cursors indicate the time of each picture. The high amplitude irregular activity seen during and after the convulsion is movement artifact.
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FIGURE 3 Middle cerebral artery–common carotid artery occlusion (MCA/CCAO). Left hemibody clonic convulsion of a 4-month-old animal 1.5 months after lesioning. The apparent asymmetry seen in the electroencephalogram (EEG) is caused by movement artifact that is most evident in the F4 electrode (see text for a description of common reference electrodes).
Figure 4). The EEG typically showed mild attenuation of baseline voltages but otherwise little electrographic change was apparent during these episodes, which lasted 5 to 20 seconds. Subdural electrode recordings may be required to detect these ictal discharges. Normal behavior resumed shortly after these events. Continuous Running, Jumping, Rearing, and Falling Three of five (60%) lesioned 20-month-old animals (different animals than those with bilateral forelimb clonus) demonstrated recurrent convulsive episodes of continuous running, jumping, rearing, and falling (grade 4 to 5; Figures 5 and 6). This convulsive activity occurred in an animal with a right cerebral tumor (Figure 5), and was associated with sudden death in a different, otherwise healthy-appearing animal (Figure 6). EEG recordings typically did not lateral-
ize, but could show right parietal emphasis of bilateral rhythmic activities before convulsive activity (Figure 5, top panel), as well as clear evolution of the discharge pattern (Figure 5, middle and bottom panels). In summary, all lesioned and sham-operated F344 animals demonstrated intermittent solitary convulsive jumps arising out of sleep that appeared to be specific to the F344 strain and not to cohort age or lesioning. One lesioned 4-month-old animal demonstrated a solitary left hemibody clonic contraction. No sham-operated 4-monthold animal demonstrated seizure activity. All of the lesioned 20-month-old animals demonstrated epileptic seizures characterized by either recurrent episodes of grade 3 or grade 4 to 5 convulsive seizures. One of six sham-operated 20-month-old animals demonstrated grade 4 convulsive seizures.
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FIGURE 5 Middle cerebral artery–common carotid artery occlusion (MCA/CCAO). Initiation of bilateral rhythmic discharge (top) followed by continuous clonic phase discharge (middle), and termination of ictal discharge (bottom) recorded in a 20-month-old animal 1.5 months after lesioning. The entire seizure lasted approximately 1 minute.
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FIGURE 6 Middle cerebral artery–common carotid artery occlusion (MCA/CCAO). Electroencephalogram (EEG) record representing continuous convulsive activity (Racine stage 5) before the sudden death (flatline EEG) of a 20month-old animal 1 month after lesioning.
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Forebrain vs. Hindbrain Seizures Although the functional anatomy of recorded seizures has yet to be elaborated in this model, we believe that seizures may originate in forebrain neocortex. Electrographic and EEG Features Duration of Latency Period The latency to the first seizure following MCA/CCAO has yet to be determined in a comprehensive fashion; however, a conservative estimate of latency derived from our pilot studies suggests that seizures can occur within 2 to 4 weeks of lesioning.
however, preliminary estimates of gross infarct volume in these brains suggest reproducible cortical lesions that are larger in 20-month-old animals (~100 mm3) compared with those of 4-month-old animals (~60 mm3). These studies form the basis of more detailed evaluations to determine infarct volume, the extent and variability of neuronal injury, and whether identifiable brain injury is associated with epileptogenesis following MCA/CCAO in animal cohorts of different ages. Reactive Gliosis Some degree of gliosis occurs in penumbral tissue flanking the infarct core. In contrast, tissue within the infarct core is resorbed completely.
Seizure Duration
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Seizure duration was as brief as 6 seconds in the hemibody clonic contractions (Figure 3) and between 5 and 20 seconds for episodes of forelimb clonus (Figure 4). Grade 4 seizures would typically last for ~1 minute (Figures 5 and 6).
Previous study of the MCAO model did not reveal aberrant mossy fiber sprouting in either the supragranular region or inner molecular layer of the dentate gyrus (Karhunen et al., 2003). Similar studies in the MCA/CCAO model have yet to be performed. No other measures of neuronal plasticity apropos epileptogenesis have been assessed in the MCA/CCAO model.
Frequency Estimates of seizure frequency are necessarily guarded based on our pilot studies. Counts of seizures in 20-monthold F344 animals suggest a frequency of approximately one to two grade-4 seizures per week. Neuropathology Cell Loss Microtubule associated protein-2 (MAP-2) immunostaining of coronal brain sections performed in our pilot studies of MCA/CCAO in 4- and 20-month-old F344 rats 2 months after lesioning demonstrated infarction of a relatively restricted area of cortex (Figure 7) that spared both basal ganglia and hippocampus. The brain slices shown in Figure 7 were obtained from animals with identical behavioral deficit scores determined immediately after lesioning (Aronowski et al., 1995). Detailed morphometry of the infarcts produced in this study is incomplete at present;
Biochemical and Biophysical Changes Techniques of transient arterial occlusion that result in infarcted tissue do so largely because of “reperfusion injury” (Yang and Betz, 1994; Aronowski et al., 1997; Bright et al., 2004), which is likely mediated by numerous biochemical processes (e.g., excitotoxicity, free radical formation, apoptosis) and biophysical factors (e.g., blood–brain barrier breakdown, edema, altered cellular membrane permeability; Lipton, 1999). Although many of the processes and factors underlying reperfusion injury would appear to be candidate mechanisms or contributors to poststroke epileptogenesis, the potential relationship of reperfusion injury to epileptogenesis in the MCA/CCAO model is not known. Response to Antiepileptic Drugs and Usefulness in Screening Drugs Use of the permanent (no reperfusion) MCAO model has been used to test the effects of antiepileptic drugs (AED) on the nonconvulsive seizures that are induced shortly after lesioning (Williams et al., 2004). AED testing has not been performed in the MCA/CCAO model either before or after the establishment of epileptic seizures.
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FIGURE 7 Middle cerebral artery–common carotid artery occlusion (MCA/CCAO). MAP-2 immunostained coronal sections of a 4-monthold (A) and a 20-month-old (B) animal 2 months after lesioning. Arrows demarcate a smaller infarct in A compared with B. No infarct was noted in the basal ganglia.
Limitations How Easy To Develop In general, application of the MCA/CCAO model to the study of epileptogenesis was relatively easy to develop
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because of two main factors: the expertise of Dr. Aronowski’s stroke research laboratory in animal lesioning and our previous experience with video-EEG monitoring using the photothrombosis model (Kharlamov et al., 2003). The MCA/CCAO procedure, however, is both invasive and technically demanding. Because the transient unilateral MCA/CCAO procedure has had considerable study and refinement over the last 10 years, variability in lesion size and distribution has been minimized as has animal morbidity and mortality. Without these attributes, vascular occlusion models can prove to be less than ideal for the study of epileptogenesis and the epileptic state, especially when extended survival of animals is required. Mortality In our pilot study, F344 animals were procured at 4 and 20 months of age (n = 24): 12 animals (4 months, n = 6; 20 months, n = 6) were lesioned by transient (3 hours) unilateral (left-sided) MCA/CCAO; 12 animals underwent sham operation (4 months, n = 6; 20 months, n = 6). Overall animal survival was 18 of 24 (75%): one 20-month-old animal died during lesioning from the effects of an obstructive pituitary mass; two 4-month-old animals died during our initial attempts to implant screw electrodes from anesthesia effects; and three 4-month-old animals died during summer transit from Houston to Pittsburgh from presumed hyperthermia (dead on arrival). Of the surviving animals, nine lesioned (4 months, n = 4; 20 months, n = 5) and nine sham-operated (4 months, n = 3; 20 months, n = 6) were used to obtain intermittent digital video-EEG recordings for up to 2 months following surgery. Two 20-month-old lesioned animals died 5 to 6 weeks after lesioning (one animal was sickly, the other died during seizure activity); one 20-month-old sham-operated animal died 7 weeks after surgery from abdominal surgical wound dehiscence. Importantly, animal survival improved steadily as we attained greater experience with aged F344 animals used in the MCA/CCAO model. Reproducibility Lesion generation in the MCA/CCAO model is restricted to ipsilateral neocortex and has proved to be reproducible (Aronowski et al., 1994, 1997). Because this model has been used only recently in the study of poststroke epileptogenesis and epilepsy, limited information is available regarding the reproducibility of specific seizure types and the evolution of the epileptic state. Age-related As noted, MCA/CCAO did not result in epileptic seizures in 2.5-month-old Long Evans rats monitored for 6 months
after lesioning (Kelly et al., submitted) or in 4-month-old F344 rats monitored for 2 months after lesioning. Similarly, MCAO did not result in epileptic seizures in adult SpragueDawley rats monitored up to 1 year after lesioning (Karhunen et al., 2003). To date, we have observed epileptic (recurrent unprovoked) seizures in the MCA/CCAO model only in 20-month-old F344 animals. These findings, however, are preliminary and it remains indeterminate whether the differences in convulsive seizures observed in the pilot studies were time-dependent rather than clearly age-related events (i.e., it will be necessary to determine whether longer periods of monitoring can result in seizures in 4-month-old animals similar to those demonstrated in the 20-month-old cohort). An important corollary of this issue is whether MCA/CCAO results in age-related differences in infarct volume that might correlate with the differences in seizure expression observed in the two age cohorts; MCA/CCAO may result in larger lesions in 20-month-old animals compared with 4-month-old animals, similar to the findings of other studies using aged animals and MCAO without CCAO (Futrell et al., 1991; Davis et al., 1995; Sutherland et al., 1996; Wang et al., 2003). Therefore, precise morphometry of the infarct core and comprehensive histologic studies of cortical and subcortical areas are required to determine potential age-related differences in lesion formation. Need for Future Development Use of arterial occlusion models for the study of poststroke epileptogenesis and the epileptic state has just begun and much work is needed to advance the area. Currently, it is not clear what elements of these models are necessary and sufficient for them to work and to be reliable. Basic experimental issues such as age of animal, MCAO with or without CCAO, transient versus permanent occlusion, and type of recording for seizure detection (depth, epidural, skull) still require much exploration and refinement.
Insights Into Human Disorders Underlying Mechanisms The mechanisms that generate epileptogenesis following stroke are not known. Use of an arterial occlusion model of stroke serves the immediate goal of establishing a physiologically relevant model of poststroke epilepsy in humans that can enable the exploration and discovery of those mechanisms essential for epileptogenesis. Future work with arterial occlusion models will elaborate the functional anatomy of seizure generation and thereby provide a better understanding of the pathophysiology of stroke-related brain injury and its ability to promote epileptogenesis and the epileptic state.
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Usefulness for Treatment The long-term goal of these models is to determine critical steps or pathways of poststroke epileptogenesis so that potential targets can be identified for antiepileptogenic strategies. In this way, we hope to shift the focus of therapeutic approaches from controlling seizures following stroke to preventing or limiting the occurrence of poststroke epilepsy.
PHOTOTHROMBOSIS General Description of Model An alternative to vascular occlusion models of stroke is the model of cortical photothrombosis, which induces cortical infarcts with reproducible area, depth, and location (Watson et al., 1985; Dietrich et al., 1987; Clissold et al., 1991; Kharlamov et al., 1994, 1996; Schiene et al., 1996; DeRyck, 1997). The photothrombosis model is based on a class of light-sensitive molecules and the reactions they undergo following photic stimulation. One such molecule is rose bengal (disodium 4,5,6,7-tetrachloro-2¢,4¢, 5¢,7¢tetraiodo-fluorescein; Sigma, St. Louis, MO), a derivative of fluorescein, which can be injected directly into the circulation. Because of hepatic metabolism, rose bengal is removed from the blood quickly, resulting in a half-life of ~2 minutes for doses up to ~40 mg/kg of the animal’s body weight. At higher doses, the half-life increases because of saturation of hepatic enzymes. Molecules of rose bengal that are not metabolized bind to proteins, including those of the intimal lining of blood vessels. These molecules become excited following absorption of photons (peak absorption, 562 nm), typically activated in brain vasculature in the photothrombosis model by an external light beam that is focused on and penetrates the animal’s translucent skull and underlying cerebral cortex. This results in the transition of the molecules from the ground state to a triplet state. Direct reaction of the triplet state molecules with molecular oxygen creates an excited singlet state species of molecular oxygen. Singlet oxygen is not a free radical, but it can directly peroxidize certain cell membrane constituents such as unsaturated fatty acids and proteins, especially proteins containing histidine, methionine, cysteine, and tryptophan. Small cortical vessels (diameter <50 mm) exposed to singlet oxygen attack are easily occluded by platelet agglutinates concentrated mainly at the pial surface; fibrin is essentially absent. The platelet response is apparently stimulated by endothelial discontinuities such as luminal surface microruptures. These defects facilitate vasogenic edema seen as perivascular swelling leading to parenchymal displacement and luminal distortion, and greatly increased content of tissue water. Vasogenic edema is sufficiently severe to occlude the deeper cortical vasculature by mechanical compression, thereby enhancing
the depth and volume of the infarction. The typical infarct is a sharply circumscribed, wedge-shaped necrotic lesion occupying the entire depth of the cortex (Dietrich et al, 1987). This method is well characterized, relatively noninvasive, produces highly reproducible infarcts, and allows for the selective placement of infarcts in well-defined cortical areas.
What Does It Model? As in the MCA/CCAO model, photothrombosis is intended to model poststroke neocortical epilepsy characterized by focal seizures with elementary clonic or inhibitory motor signs with or without secondary generalization (Engel, 2001).
Methods of Generation Animal Issues In developing the photothrombosis model for the study of poststroke epilepsy, we have used male Sprague-Dawley and F344 rats spanning ages from 2 to 30 months (Kelly et al., 2001a; Kharlamov et al, 2003). Our current studies use F344 animals at 4 and 20 months of age. Procedures Photothrombosis Photothrombosis is performed in our laboratory according to Watson et al. (1985) with modifications. Rats are anesthetized with ketamine and xylazine (9 : 1) and placed in a stereotaxic frame. A midline scalp incision is made, and the scalp is retracted laterally. Rose bengal (20 mg/kg; Sigma) is injected through a catheter into the left femoral vein over 2 minutes, as the brain is stimulated through intact skull for 10 minutes by an argon laser-activated light beam (Lexel model 75, class IV, 514.5 nm, 15 amp power supply, 150 mW output). The incident beam is ~5 mm wide and focused 1.8 mm posterior to the bregma and 2.8 mm lateral to the midline corresponding to the area of the left sensorimotor cortex. The laser stimulation protocol generates cortical infarcts of approximately 25 mm3 (Figure 8) in both 4- and 20-month-old animals. The skull is cooled by continuous airflow from a fan to prevent heat-mediated tissue injury. Body temperature is monitored and maintained at 37° C using a thermoregulated pad. After stimulation, the catheter is removed, and the wound is sutured. Animals receive a subcutaneous injection (5 ml) of lactated Ringer’s solution immediately after surgery and each day postoperatively until adequate hydration and nutrition are ensured. Electrodes In our initial study (Kelly et al., 2001a), subdural wire electrodes (1 mm end exposure) were placed ipsilaterally
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Monitoring Ease of Development Current monitoring procedures are the same as those described for the MCA/CCAO and are relatively easy to establish. Comprehensive review of video-EEG records, however, is time-consuming and labor intensive. Reliability 1 mm
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FIGURE 8 Photothrombosis. (A) Placement of an argon laser beam on the exposed skull of a rat overlying the left sensorimotor cortex. (B) 2,3,5-triphenyltetrazolium chloride (TTC)-stained coronal slice of brain performed 24 hours after lesioning of a 20-month-old animal. (See color insert.)
anterior and posterior (or medial and lateral) to the lesion using stereotaxic coordinates. A single subdural wire electrode was used as an indifferent electrode on the contralateral cortex positioned near the frontal pole. These electrodes were linked to a bipolar twist electrode placed in the posterior ventral hippocampus ipsilateral to the lesion (Figure 9). All electrodes were attached to Amphenol connectors and secured to the skull with jewelers’ screws and dental acrylic. Animals were allowed to recover for 1 week before monitoring. In our subsequent study (Kharlamov et al., 2003), only skull screw electrodes were placed, as described for the MCA/CCAO studies (Figure 1).
Monitoring protocols are standardized as described in the MCA/CCAO model. Standardized schedules of intermittent chronic video-EEG recordings minimize sampling errors and allow for a reasonably representative capture of ictal events and estimates of their frequency of occurrence. Occurrence of Spontaneous Seizures Seizures occur spontaneously in this model, including focal clonic contractions; motor arrest; and grade 4 convulsions (to be discussed). Remission In our study that monitored young Sprague-Dawley animals for 6 months following lesioning (Kharlamov et al., 2003), no evidence of remission was seen in epileptic animals.
Characteristics and Defining Features Behavioral Features Similar to the MCA/CCAO studies, most animals (shamoperated and lesioned) variably demonstrated 7- to 9-Hz SWD, which will not be described further.
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Seizure Severity Our initial study (Kelly et al., 2001a) was designed to explore whether the photothrombosis technique could be used to model cortical infarction and poststroke epileptogenesis. Several variables were evaluated in this study, including lesion size, lesion location, and animal age. Subdural and depth electrodes were used in these studies as described (Figure 9). The primary finding of the study was that epileptic seizures were recorded in postmature rats 2 months after lesioning the frontoparietal cortex with large photothrombotic infarcts that extended to the cortical–subcortical interface. These seizures were characterized behaviorally by motor arrest and electrically by ictal discharges that had distinct morphology, frequency, duration, and laterality, and appeared to originate in the peri-infarct area. Small cortical lesions were ineffective in producing seizures, except for one animal that demonstrated recurrent prolonged focal discharges unaccompanied by behavioral change. Grade 3 seizures were observed in a small number of mid-aged and aged animals lesioned with large infarcts in anterior frontal and frontoparietal areas. Examples of representative ictal discharges are described below. Focal Rhythmic 1-Hz Sharp- and Slow-Wave Discharges An animal lesioned with a small cortical infarct of the left midfrontal area at 6 months of age and recorded 4 months later demonstrated multiple ictal discharges from the subdural wire electrode positioned adjacent to the area of infarction. One such discharge lasted ~90 seconds and consisted of rhythmic 1-Hz sharp- and slow-wave activity that evolved with changing amplitude in a crescendo–decrescendo pattern before reverting to baseline activity (Figure 10). The animal was quiescent and no behavioral change was observed before, during, or after the ictal discharge. Focal Polyspike and Spike-wave Discharges Associated with Behavioral Arrest Animals lesioned with large cortical infarcts, which extended to the cortical–subcortical interface, could demonstrate frequent electrical seizures associated with motor arrest. Figure 11A shows EEG traces from an animal lesioned at 2 months and recorded 2 months later that demonstrated frequent ictal discharges associated with clear motor arrest. Recordings from the peri-infarct area demonstrate a burst of 6- to 10-Hz polyspike and spike-wave discharges of variable morphology and amplitude lasting ~13 seconds. This discharge is apparent in the contralateral cortex with reduced amplitude; the hippocampal electrode gives little evidence of the discharge. After several of these discharges, the animal displayed brief body jerks before resuming normal behavior. Figure 11B shows EEG traces
FIGURE 10 Photothrombosis. Subdural wire and depth electrode electroencephalogram (EEG) recordings of a 10-month-old animal lesioned with a small infarct in the midfrontal cortex at 6 months of age. The EEG demonstrated multiple ictal discharges from the subdural wire electrodes positioned adjacent to the area of infarction. (A) Rhythmic 1-Hz sharp and slow wave activity. (B) Compressed EEG activity seen in (A) that evolved with changing amplitude in a crescendo–decrescendo pattern before reverting to baseline activity after ~90 seconds.
from an animal lesioned at 6 months and recorded 2 months later. Recordings from the peri-infarct area demonstrate an ictal discharge lasting ~20 seconds, similar to that shown in Figure 11A. During this discharge, variable, slightly increased amplitude activity was seen in the hippocampal electrode. The recordings in Figure 11 demonstrate the general finding that the peri-infarct ictal discharge was always much more pronounced compared with the activities recorded in the contralateral cortex and ipsilateral hippocampus. During ictal discharges, an individual animal showed significant variability in the amount of changed electrical activity in contralateral cortex and hippocampus, ranging from little to none when compared with that of the peri-infarct area. Based on the results of this study, we developed the photothrombosis model in a subsequent study (Kharlamov et al., 2003) with the goal of characterizing the EEG and behavioral properties of young adult rats during extended videoEEG monitoring following lesioning. Two-month-old male Sprague-Dawley rats underwent photothrombosis of the left sensorimotor cortex (n = 10) or were used as controls (n = 9). Qualitative and quantitative EEG analysis was performed on digital video-EEG records obtained during 6 months of recording. The main finding of this study was that 5 of 10 (50%) lesioned animals developed focal epileptic seizures ipsilateral to the cortical infarct characterized by rhythmic spike-wave discharges with or without behavioral change. Importantly, electrical and behavioral characteristics common to both lesioned and control animals included generalized tonic–clonic seizures (one naïve control; one lesioned animal). Examples of representative ictal discharges are described below.
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FIGURE 11 Photothrombosis. (A) Subdural wire and depth electrode electroencephalogram (EEG) recordings of a 4-month-old animal lesioned with a typical infarct in the frontoparietal cortex at 2 months of age. The EEG demonstrated frequent ictal discharges associated with clear behavioral motor arrest. Recordings from the peri-infarct area (middle trace) demonstrate a mix of frequencies that change to a higher amplitude ictal discharge with variable waveforms lasting ~13 seconds. This discharge is apparent in the contralateral cortex with reduced amplitude; the hippocampal electrode gives little evidence of the discharge. (B) Similar recordings of an animal at 8 months of age lesioned with a 6-mm light beam in the frontoparietal cortex at 6 months of age. Recordings from the peri-infarct area demonstrate an ictal discharge lasting ~20 seconds associated with motor arrest similar to that shown in (A).
Focal Spike-wave Discharges Associated with Behavioral Arrest Focal rhythmic 5- to 9-Hz spike-wave discharges of the lesioned hemisphere lasted 2 to 3 seconds with maximal emphasis in either frontal or parietal areas. These events could occur without or with behavioral change (Figure 12). In general, a 5-Hz spike-wave discharge pattern was most frequent, occurred during wakefulness, and was typically associated with motor arrest of the animal, which could be accompanied by eye blinking and followed by brief facial clonus or a body jerk. During sleep, these spike-wave discharges were not accompanied by any observable change in behavior. These discharges could appear in repeated trains associated with motor arrest of the animal punctuated by brief movements during spike-wave–free intervals (Figure 13). A related ictal pattern was a focal 9-Hz spike-wave discharge with superimposed high beta-range activity that demonstrated a crescendo increase in amplitude (Figure 14); similar to 5-Hz spike-wave discharges, this discharge was followed immediately by animal arousal. Additionally, epileptic animals demonstrated increased delta, theta, and low beta range power ipsilateral to the infarct that reliably distinguished them from lesioned nonepileptic and control animals (Figure 15). Interestingly, the electrographic and behavioral aspects of these seizures share some similarity to those recorded in studies of traumatic brain injury (D’Ambrosio et al., 2004, 2005).
Generalized Tonic–Clonic Seizures Evolving generalized polyspike and spike-wave activity associated with tonic–clonic behavior was observed in one naïve control and one lesioned animal (Figure 16). These animals demonstrated repeated tonic–clonic seizures typically lasting 1 to 2 minutes. No apparent cause was determined for these seizures in the control animal. The lesioned animal began to demonstrate recurrent tonic–clonic seizures ~2.5 months following photothrombosis. Routine handling of this animal appeared to precipitate most, if not all, of the seizure activity. Although focal spike-wave seizures with onset ipsilateral to the lesion were first recorded in this animal 26 days after lesioning, no tonic–clonic seizure was noted to have focality at onset. Forebrain vs. Hindbrain Seizures Although the functional anatomy of recorded seizures has yet to be elaborated in this model, we believe that seizures may originate in forebrain neocortex. Electrographic and EEG Features Duration of Latency Period In the study by Kharlamov et al. (2003), the five lesioned animals demonstrating focal spike-wave discharges had latencies to the first recorded spike-wave discharge of 26,
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FIGURE 12 Photothrombosis. Focal 5-Hz spike-wave discharge lasting 3 seconds over left centroparietal cortex adjacent to the area of infarct. Vertical cursor indicates time of mid eyeblink during the brief seizure associated with motor arrest. Record obtained 2 months after lesioning of a 2-month-old animal. F3-C3 C3-P3 F3-P3 F4-C4 C4-P4 F4-P4 F3-F4 C3-C4 P3-P4 100 µV 1 sec
FIGURE 13 Photothrombosis. Repetitive focal 5-Hz spike-wave discharges, each lasting 2 to 3 seconds, occurring over left centroparietal cortex adjacent to the area of infarct. First vertical cursor indicates apparent motor arrest during a spike-wave discharge as shown in the picture at left. Second vertical cursor indicates brief behavioral arousal between spike-wave discharges as shown in the picture at right. Record obtained 2 months after lesioning of a 2-month-old animal.
47, 105, 178, and 181 days (mean 107.4 + 32.2 days). Because of significant practical constraints in animal monitoring in this study, recorded latencies may represent overestimates of actual latency durations.
approximately 2 and 20 seconds. Generalized tonic–clonic seizures in both control and lesioned animals lasted variably between 1 and 2 minutes.
Seizure Duration
Frequency
In our two studies (Kelly et al., 2001a; Kharlamov et al., 2003), durations of focal ictal discharges lasted between
In the study by Kharlamov et al. (2003), a total of 48 spike-wave discharges were recorded from the five animals
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FIGURE 14 Photothrombosis. A 9-Hz spike-wave discharge with high beta range component superimposed. Amplitude of the discharge increases from beginning to end. Cursor indicates the point of behavioral arousal. Record obtained 6 months after lesioning of a 2-month-old animal.
demonstrating spike-wave discharges. Following the first recognized spike-wave discharge of these animals, a total of 223 hours of recording time was obtained, yielding an average occurrence of focal spike-wave discharges of 0.215/hour (i.e., one seizure every 4.6 hours).
Neuropathology Cell Loss Infarct core. Infarct generation is highly reproducible in the photothrombosis model and cell loss within the infarct core is uniformly complete. Available evidence, however, indicates that infarct volumes can vary based on the age of the animal. A recent study using photothrombosis (rose bengal, 80 mg/kg) and F344 rats showed that infarct volumes in aged animals (20 and 27 months) were significantly larger than those in young adult animals (4 months) produced with identical stimulation parameters (Kharlamov et al., 2000). Our current photostimulation technique (rose bengal, 20 mg/kg; argon laser) results in infarcts of equal volume in 4- and 20-month-old F344 animals (unpublished data).
Ischemic penumbra. In general, photothrombosis is associated with a limited ischemic penumbra (~500 mm) surrounding the infarct core (Witte et al., 2000); beyond this area, the cortex appears intact and devoid of neuronal loss as assessed by hematoxylin-eosin, cresyl violet, and Nissl staining (Schroeter et al., 1999). Our own testing in young adult animals with Fluoro Jade-B (FJB) performed 2 days after laser-induced photothrombosis showed no positive staining outside of the cortical area destined to become the infarct core (unpublished data). Reactive Gliosis No significant gliotic process is seen following photothrombosis other than that which takes place in the limited ischemic penumbra of the infarct core. In the study by Kharlamov et al. (2000), aged animals demonstrated a decreased glial response in the infarct’s gliotic rim associated with greater lesion-induced neuronal loss. Plasticity To date, no evidence exists of recognizable neuronal plasticity following photothrombosis, including neurogenesis or sprouting within the hippocampus or dentate gyrus.
Photothrombosis
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Imaging and Metabolic Changes Several studies using photothrombosis in rats have detailed anatomic and physiologic changes in ipsilateral cortex that could conceivably predispose the animals to epilepsy. For example, paired-pulse inhibition in cortical slices was significantly reduced or absent within an area extending up to 5 mm from the center of a 2-mm cortical lesion. These changes were present on the first day after lesioning and persisted for at least 60 days (Domann et al., 1993). Intracellular recordings from cortical layers II/III were obtained 1.5 to 2.5 mm lateral to the lesion and demonstrated decreased resting potential, decreased conductances of early and late IPSP, and “epileptiform” postsynaptic potentials blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist AP-5. These results suggested changes in intrinsic membrane properties and decreased g-aminobutyric acid (GABA)-mediated synaptic inhibition in the surround of the lesion (Neumann-Haefelin et al., 1995). Neuronal hyperexcitability in the same area was demon-
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strated by in vivo multiunit recordings obtained 2 to 3 mm lateral to the lesion up to 4 months after lesioning (Schiene et al., 1996). Additionally, extracellular recordings of the peri-infarct area demonstrated that induction of long-term potentiation was facilitated consistent with an amplification of neuronal network plasticity in the area (Hagemann et al., 1998). In other studies of the peri-infarct area done 1 week after lesioning, a reverse transcription-polymerase chain reaction assay demonstrated an upregulation of GABAA receptor a1 and a2 subunit mRNA (Neumann-Haefelin et al., 1999); however, immunohistochemical studies of the same area demonstrated a downregulation of a1 and no change in a2 GABAA receptor subunits (Neumann-Haefelin et al., 1998), suggesting a translational block or posttranslational modification of GABAA receptor subunit expression. An immunohistochemical study (Redecker et al., 2002) reported a differential decrease of a1, a2, a5, and g2 subunits bilaterally up to 30 days after lesioning and a significant upregulation of the a3 subunit homotopic to the infarct. Intriguingly, all of these changes were blocked by the administration of MK-801 (2 mg/kg) 30 minutes before lesioning. All of the studies described used male Wistar rats of unspecified age (250 to 340 g), a lesion size of 2 to 3 mm in diameter, and a lesion site at or near the primary somatosensory (parietal) area. Although several of the findings described strongly suggest potential epileptogenicity of the peri-infarct area, no overt behavioral seizure activity was reported in any of the reported studies. Possible explanations include that the parietal location of the infarct was not effective in initiating seizures, or that nonmotoric seizures occurred but went undetected. Although it is attractive to speculate that seizures and epilepsy could develop from physiologic and anatomic changes in cortex ipsilateral and proximal to the lesion, photothrombosis can cause changes in areas remote from the lesion (diaschisis), including the contralateral hemisphere. For example, magnetic resonance imaging of male SpragueDawley rats demonstrated increased T2-weighted signal intensities consistent with vasogenic edema in the cortical lesion, corpus callosum, and bilateral external capsules 15 hours after photothrombosis; diffusion-weighted imaging, which reflects cellular damage and death, demonstrated increased signal intensities in and around the core of the lesion but not in white matter (Pierpaoli et al., 1993). In studies using male Wistar rats, photothrombosis-induced cortical-spreading depression that appeared to be triggered within or near the infarcted zone and spread to more distal sites ipsilaterally (Dietrich et al., 1994). Electrophysiologic recordings from neocortical slices obtained 6 to 7 days following photothrombosis demonstrated decreased paired– pulse inhibition in bilateral cortices, suggesting maintained hyperexcitability in widespread areas (BuchkremerRatzmann et al., 1996). Quantitative autoradiography per-
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FIGURE 16 Photothrombosis. Generalized high amplitude polyspike and spike-wave discharges during the beginning of a tonic–clonic seizure. Time of video insert indicated by cursor at the end of record. Record obtained 6 months after lesioning of a 2-month-old animal.
formed 1 week after lesioning demonstrated reduced [3H] muscimol binding to GABAA receptors (Qu et al., 1998) and increased [3H] baclofen binding to GABAB receptors (Que et al., 1999) in bilateral cerebral cortices. Importantly, a recent in situ hybridization study demonstrated no loss of GABAergic interneurons in bilateral cortices and hippocampal subsections up to 30 days after lesioning (Frahm et al., 2004). Given that most of the aforementioned studies were performed within hours to days after lesioning, it remains to be demonstrated whether any of the anatomic or functional alterations might have persisted, reverted to normal, or have an important role in establishing epileptogenesis.
systems following photothrombotic infarction appears to be a complex phenomenon that prominently involves tissue ipsilateral to the lesion. Although several studies have demonstrated widespread anatomic and physiologic changes following photothrombosis (Witte et al, 2000) suggest likely changes in the functioning of GABAA receptors and provide a theoretic basis of decreased GABAergic inhibition during epileptogenesis, it is not known whether changed GABAA receptor expression and function are influential or determinative in establishing poststroke epilepsy.
Genetic and Molecular Changes
Antiepileptic drug testing has not been performed in the photothrombosis model to determine whether these drugs can prevent epileptogenesis or limit seizure activity after it has been established.
Because the photothrombosis model has had limited use in the study of poststroke epilepsy, relatively little information is available on potential genetic and molecular changes associated with epileptogenesis in this model. A study from our laboratory (Liu et al., 2002), however, indicated that large photothrombotic infarcts of the neocortex could result in a long-lasting differential expression of GABAA receptor subunit mRNA in ipsilateral cortex variably associated with the epileptic state. Alteration in cortical GABAergic
Response to Antiepileptic Drugs and Usefulness in Screening Drugs
Limitations Ease of Development The photothrombosis method is an easy, straightforward, minimally invasive procedure that can produce cortical
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brain infarction in any desired location of the cerebral convexities. The photothrombosis model offers distinct practical advantages to the more technically demanding vascular occlusion models; however, significant limitations are seen of the photothrombosis model when considering the pathophysiologic mechanisms of stroke in humans. In contrast to human stroke, photothrombosis has the following characteristics: (1) it preferentially occludes small-diameter (<50 mm) pial cortical vessels, rather than mid-to-large size extracranial or intracranial arteries; (2) it is end-arterial in nature; (3) the thrombus formed as a result of endothelial damage by singlet oxygen contains virtually no fibrin; and (4) early blood–brain barrier opening and severe vasogenic edema consume the tissue bordering the core of infarction and thereby markedly limit the extent of the ischemic penumbra (Watson et al., 1985). Compared with the MCA/CCAO model, photothrombosis results in much smaller infarct volumes (~25 mm3 vs. ~100 mm3); it is not presently known whether this degree of difference in lesion size has an appreciable effect on the establishment of epileptogenesis. Mortality In general, mortality in our initial study (Kelly et al., 2001) varied with experimental conditions and animal age. Overall mortality during variable periods of evaluation was 34/110 animals (31%), including 8/34 control animals (24%) and 26/76 experimental animals (34%), which was not significantly different (Fisher’s exact test, two-sided P = .2725). Mortality in young and mid-aged animals was 16/80 (20%), and in aged animals (≥24 month) was 18/30 (60%), which was significantly different (P = .0001). Most deaths (19/34; 56%) occurred within 1 week of treatment, largely because of the prolonged effects of the anesthetic Equithesin (a mixture of water, chloral hydrate, magnesium sulfate, propylene glycol, ethanol, and sodium pentobarbital), acute renal toxicity from the relatively high dose of rose bengal (80 mg/kg), and our initial inexperience with caring for animals immediately following photothrombosis. In our subsequent study (Kharlamov et al., 2003), virtually no premature mortality was seen in young adult animals. This markedly improved result was likely owing to a combination of factors, including (1) substitution of ketamine and xylazine for Equithesin proved to be far superior for anesthetic control; (2) decreased rose bengal dosing from 80 mg/kg to 30 mg/kg; and (3) institution of a regimen of postoperative animal support that accelerated recovery from anesthesia, allowing for a quicker return of the animal to normal activities. In our current use of the photothrombosis model using 20 mg/kg of rose bengal in 4- and 20-monthold F344 animals, we continue to have negligible perioperative mortality and modest mortality during a 6-month period of monitoring following lesioning.
Reproducibility Lesions created by photothrombosis are highly reproducible, a hallmark of the technique. The precision of lesion localization and the limitation of infarction to cortical tissue allow focused study of restricted injury to selected areas of cortex. The severity of brain injury can be manipulated by changing the intensity or diameter of the activating light beam, the duration of irradiation, or the dose of rose bengal. High reproducibility of lesion formation is explained by the consistency of the tissue density of microvessels (diameter <50 mm), which are the main site of photochemically induced thrombosis (Watson, 1985). Age-relatedness Our studies have yet to demonstrate a clear dependence on age for the development of epileptic seizures. Our initial study (Kelly et al., 2001a) found that focal epileptic seizures could be generated in rats ≥2 months of age. Our subsequent study (Kharlamov et al., 2003) confirmed this finding in 2month-old animals. In our current studies, which use 4- and 20-month-old animals, preliminary results indicate that lesioned 20-month-old animals can generate recurrent grade 4 seizures similar to those shown in Figures 5, 6, and 16. Future Development The photothrombosis model has had limited development for the study of epileptogenesis and the epileptic state. Broad areas for future study include determining the principal elements of lesion-induced network changes, how excitatory and inhibitory receptors and their subunit compositions are regulated before and after the onset of epileptogenesis, the functional anatomy of seizure initiation and propagation, and the electrobehavioral properties of a potentially dynamically evolving epileptic state.
Insights Into Human Disorders Underlying Mechanisms As mentioned in the section on vascular occlusion models, the mechanisms that generate epileptogenesis following stroke are not known. Despite the clear differences in the mechanism of lesion formation in the photothrombosis model compared with human stroke, it is not known whether the mechanisms that establish epileptogenesis in this model are different than those operative in vascular occlusion models or in human stroke itself. Usefulness for Treatment Assessment, Development, and Screening As described above, antiepileptic drug testing has not been performed in the photothrombosis model to determine
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whether epileptogenesis can be prevented or whether established seizures can be limited. At its current level of development, the photothrombosis model requires substantial study to validate its basic findings before it can reasonably be applied to treatment assessments or screening of antiepileptogenic or antiseizure compounds.
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References dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats. Neuroscience 87: 871–879. Neumann-Haefelin, T., Bosse, F., Redecker, C., Mulller, H.W., and Witte, O.W. 1999. Upregulation of GABAA-receptor a1-and a2-subunit mRNAs following ischemic cortical lesions in rats. Brain Res 816: 234–237. Pierpaoli, C., Righini, A., Linfante, I., Tao-Cheng, J.H., Alger, J.R., and Di Chiro, G. 1993. Histopathologic correlates of abnormal water diffusion in cerebral ischemia: diffusion-weighted MR imaging and light and electron microscopic study. Radiology 189: 439–448. Qu, M., Buchkremer-Ratzmann, I., Schiene, K., Schroeter, M., Witte, O.W., and Zilles, K. 1998. Bihemispheric reduction of GABAA receptor binding following focal cortical photothrombotic lesions in the rat brain. Brain Res 813: 374–380. Que, M., Witte, O.W., Neumann-Haefelin, T., Schiene, K., Schroeter, M., and Zilles, K. 1999. Changes in GABA(A) and GABA(B) receptor binding following cortical photothrombosis: a quantitative receptor autoradiographic study. Neuroscience 93: 1233–1240. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32: 281–294. Redecker, C., Wang, W., Fritschy, J.M., and Witte, O.W. 2002. Widespread and long-lasting alterations in GABAA-receptor subunits after focal cortical infarcts in rats: mediation by NMDA-dependent processes. J Cereb Blood Flow Metab 22: 1463–1475. Schiene, K., Bruehl, C., Zilles, K., Qu, M., Hagemann, G., Kraemer, M., and Witte, O.W. 1996. Neuronal hyperexcitability and reduction of
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GABAA-receptor expression in the surround of cerebral photothrombosis. J Cereb Blood Flow Metab 14: 20–28. Schroeter, M., Jander, S., Witte, O.W., and Stoll, G. 1999. Heterogeneity of the microglial response in photochemically induced focal ischemia of the rat cerebral cortex. Neuroscience 89: 1367–1377. Sutherland, G.R., Dix, G.A., and Auer, R.N. 1996. Effect of age in rodent models of focal and forebrain ischemia. Stroke 27: 1663–1668. Wang, R.Y., Wang, P.S.G., and Yang, Y.R. 2003. Effect of age in rats following middle cerebral artery occlusion. Gerontology 49: 27–32. Watson, B.D., Dietrich, W.D., Busto, R., Wachte, M.S., and Ginsburg, M.D., 1985. Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol 17: 497–504. Williams, A.J., Lu, X.M, Slusher, B., and Tortella, F.C. 2001. Electroencephalogram analysis and neuroprotective profile of the N-acetylatedalpha-linked acidic dipeptidase inhibitor, GPI5232, in normal and brain-injured rats. J Pharmacol Exp Ther 299: 48–57. Williams, A.J., Tortella, F.C., Lu, X.C.M., Moreton, J.E., and Hartings, J.A. 2004. Anti-epileptic drug treatment of non-convulsive seizures induced by experimental brain ischemia. J Pharmacol Exp Ther 104.069146 (online). Witte, O.W., Bidmon, H.J., Schiene, K., Redecker, C., and Hagemann, G., 2000. Functional differentiation of multiple perilesional zones after focal cerebral ischemia. J Cereb Blood Flow Metab 20: 1149–1165. Yang, G.Y., and Betz, A.L. 1994. Reperfusion-induced injury to the blood brain barrier after middle cerebral artery occlusion in rats. Stroke 25: 1658–1665.
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FIGURE 3 9 - 2 Representative Timm silver sulfide staining demonstrating the presence of zinc-containing mossy fibers in the dentate gyms (A, square area, and CA3 circle). Rats were killed 15 days after a single injection into the left amygdalar region with 1.5 btl of 100mM ferric chloride. Animals show Racine (Racine, 1972) stage 4 behaviors. Note the mossy fiber sprouting (B) both in the suprapyramidal (closed arrows) and infrapyramidal blades (open arrows). (C) Arrowheads mark sprouting of mossy fibers in the CA3.
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41 Models Available for Infection-Induced Seizures JANET L. STRINGER
Equine encephalitis, St. Louis encephalitis, cytomegalovirus, and rabies have also been reported to cause seizures. Seizures occur in ~85% of children infected with Japanese encephalitis and up to 10% of adults with West Nile virus (Solomon, 2004). It has been estimated that 2% to 5% of HIV-infected patients have seizures caused by the primary infection of the brain by the virus (Labar and Harden, 1998). In general, viral infections of the brain are more likely to lead to seizures than infections with bacteria. Generalized convulsions, however, can occur with bacterial meningitis, along with the classic symptoms of fever, headache and stiff neck. Possible pathogens causing meningitis can be predicted by the age of the patient. Infections with Haemophilus influenzae (Labar and Harden, 1998) are more commonly associated with seizures, compared with other bacterial infections. Sometime the seizures are caused by the inflammatory response to the bacterial infection. Later in the course of bacterial meningitis, seizures and focal neurologic deficits are produced by thrombosis of meningeal veins. Convulsions occur in ~35% of patients with brain abscesses and in >50% of patients with subdural empyema (Labar and Harden, 1998). Other infections that involve the central nervous system have been reported to cause seizures. In developing countries, up to 50% of adult-onset epilepsy is caused by infection of the CNS with the cyst form of tapeworms (DeGiorgio et al., 2004). Approximately one third of cases of malaria have cerebral involvement and, in a minority of cases, seizures may be the presenting symptom (Labar and Harden, 1998). The incidence of acute seizures in neurosyphilis is 14% to 60% (Hooshmand, 1976). Infectious diseases in which seizures occur, but are not typically the presenting
INTRODUCTION Infections of the central nervous system (CNS) are associated with a significantly increased risk of seizures (Labar and Harden, 1998; Eisenschenk and Gilmore, 2001). Seizures may be the presenting symptom or only one manifestation of the infection. Common infections that can present with seizures include herpes simplex, cytomegalovirus, arbovirus, human immunodeficiency virus (HIV), neurocysticercosis, malaria, toxoplasmosis, bacterial meningitis, and brain abscess. During an infection, the seizures can be caused by (1) direct invasion of brain tissue by the infecting organism, (2) production of toxins by the organism or (3) production of inflammatory mediators by the brain. Systemic infections that result in hypoxia or other severe metabolic changes can also give rise to seizures. About 5% of patients with an infection of the CNS will experience a seizure (Annegers et al., 1988). Additionally, infections of the central nervous system account for 15% of all acute symptomatic seizures. Treatment for these seizures usually involves treatment of the underlying infection. Determining the mechanisms behind seizure initiation after infection of the CNS may provide insights into both normal brain function and how the brain responds to abnormal conditions. Viral encephalitis is a viral infection of brain parenchyma that is characterized by neuronal and glial degeneration, inflammatory infiltrate, edema, and tissue necrosis (Labar and Harden, 1998). Both focal and generalized seizures are seen in patients with viral encephalitis. Encephalitis caused by herpes simplex virus is the most common encephalitis associated with seizures (Eisenschenk and Gilmore, 2001). The seizures are both partial complex and generalized.
Models of Seizures and Epilepsy
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feature, include rubeola, schistosomiasis, trichinosis, paragonimiasis, echinococcosis, trypanosomiasis, typhus, and amebiasis. Animal studies have been done for two of the more common infectious causes of seizures—neurocysticercosis and herpes simplex encephalitis. Neurocysticercosis is the most com-mon parasitic infection of the brain and a leading cause of epilepsy in the developing world (DeGiorgio et al., 2004). Of the viral infections, herpes simplex is the most common cause of encephalitis that is associated with seizures (Eisenschenk and Gilmore, 2001).
SEIZURES IN A MODEL OF NEUROCYSTICERCOSIS General Description of Model Neurocysticercosis is a parasitic infection of the human central nervous system caused by the helminth Taenia solium. The most common clinical manifestation of neurocysticercosis is seizures (Carpio et al., 1998; Garcia and Del Brutto, 2000; White, 2000). Pigs serve as the intermediate hosts by allowing the parasite to reside within its tissues, primarily muscle. Humans become infected with the tapeworm form (taeniasis) by eating undercooked pork that is infected (White, 2000). After ingestion of eggs, the larvae hatch in the intestines and penetrate the intestinal mucosa, enter the bloodstream, and migrate to tissues where they mature into cysticerci. These viable parasites modulate and inhibit the host immune and inflammatory response (White et al., 1997). Although the cysticerci reach their mature size within a few weeks, there is typically a period of 4 to 5 years between infection and onset of symptoms. Thus, infection of the CNS alone does not explain symptoms. Individuals who have died of other causes, have been found at autopsy to have viable cysticerci in their CNS. Patients with seizures invariably have a prominent inflammatory infiltrate around the cysts, including the presence of proinflammatory cytokines and an altered blood–brain barrier (Alverez et al., 2002; RidauraSanz, CR, 1987; White, 2000). Thus, it has been hypothesized that seizures result not from parasitic infection per se, but from the host response initiated by dying cysts (White, 2000; Escobar, 1983; Gutierrez, 1990). The epileptogenic substance is not known, nor is it known whether the mediator of the seizures is a substance released by the dying parasite or one produced by the surrounding inflammatory cells. No animal model for neurocysticercosis exists, but a model has been developed to examine the seizures produced by the dying parasites (Stringer et al., 2003).
Method of Generation Taenia crassiceps cysticercosis in mice has been used as an experimental model for the human infection by Taenia
solium cysticercosis (Villa and Kuhn, 1996; Kunz et al., 1989; Sciutto et al., 1995). Intraperitoneal inoculation of Taenia crassiceps leads to infestation of the peritoneal cavity of the mouse (female BALB/c mice have been used) with a large number of cysts, but the tapeworm does not infect the CNS in the mouse. As in the human infection, live parasites are associated with little or no inflammation, whereas dying parasites initiate a chronic granulomatous reaction. Granulomas associated with parasites can be identified visually and removed from the peritoneal cavity 3 months after inoculation. The granulomas that surround the parasites have been shown to go through four stages of maturation (Robinson et al., 1997). Stage 1 granulomas have areas of histologically intact tegument of the parasite surrounded by host inflammatory cells. Stage 2 granulomas display no areas of normal tegument of the parasite, have infiltration with lymphocytes, but some intact parasite morphology remains, including a cyst cavity. Stage 3 granulomas have complete infiltration with host mononuclear cells, no remaining evidence of a cyst cavity within the parasite, but have remnants of degenerating parasite. Stage 4 granulomas reveal only host cells and debris without clearly identifiable parasite elements. Only stage 1 and 2 granulomas produce an extract that will cause seizures (Stringer et al., 2003). Because it is not possible to visually identify the stage of the granuloma when removing it from the mouse, a portion of each granuloma must be removed for staging (Robinson et al., 1997). To prepare the extract, each granuloma is homogenized in phosphate-buffered saline. The extract prepared from the granulomas is then injected into the brain to mimic neurocysticercosis. Testing of the extract in adult rats has been published (Stringer et al., 2003). Adult Sprague-Dawley rats (125 to 175 g) are anesthetized with a combination of ketamine, acepromazine, and xylazine (25 mg/kg, 0.8 mg/kg, 5 mg/kg, respectively) intraperitoneally and then placed in a stereotaxic frame. The extract can be injected into whatever brain region is targeted (hippocampus and amygdala injections will result in focal seizures; unpublished observations). For injection, a 6- to 8inch length of tubing is attached to the end of a 1-inch stainless steel tube (0.12 OD, 0.006 ID, Small Parts, Inc.) and then to a 10 ml syringe. Recording, if desired, can be carried out simply by using a 1-inch length of stainless steel, which is taped beside the injection tube, attached to an amplifier, and to a chart recorder. The assembly is placed in the brain region of interest using stereotaxic coordinates and then is fixed into place with dental cement. After time to allow the cement to harden, a baseline recording is obtained for 5 to 10 minutes. A single injection of 10 ml extract (containing 25 mg total protein), or control substance, is administered and the animal monitored continuously for at least 1 hour. Animals remained lightly anesthetized throughout the recording with repeated injections of small amounts of the anesthetic. Immediately after conclusion of the recording,
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Seizures in a Model of Neurocysticercosis
the animals should be perfused through the heart with 4% paraformaldehyde for histologic confirmation of the injection and recording site.
Characteristics and Defining Features Initial experiments determined that injection of 10 ml of phosphate-buffered saline had no effect on the recorded electrical activity (Stringer et al., 2003). From experiments with known convulsants, it was determined that the recorded electrical activity had to reach at least twice the amplitude of the baseline activity to be defined as epileptiform and the onset of the epileptiform activity should occur within 3 minutes to be considered a direct effect of the injected material. Each extract was tested in at least two animals (Figure 1). Extracts from stage 1 and 2 granulomas consistently produced an epileptiform discharge in the hippocampus. The onset of the activity varied somewhat, but usually started within 1 minute of injection. On average, the epileptiform activity lasted 44 seconds with a range of 25 to 76 seconds. In no animal did the epileptiform activity return after the initial activity had terminated, nor was any bursting activity seen beyond what is shown in Figure 1. Injections of extracts from nonstimulated host cells prepared from spleen of uninfected mice and homogenized viable parasites did not produce epileptiform activity. No behavioral seizures were observed after injection of extract, nor was any evidence seen of seizure spread out of the hippocampus. The seizure activity with the injections is so short that electrical recording is required for its accurate detection. It
is not known whether behavioral seizures would be observed in unanesthetized animals. No spontaneous seizures were ever recorded with this model. Examination of the Nisslstained sections after the injection and seizure activity showed no evidence of neuronal or glial cell loss. The seizures are relatively short, so no sprouting, reactive gliosis, or cell loss is expected. This model could be used to test the usefulness of antiepileptic drugs, or anti-inflammatory drugs, for use in neurocysticercosis.
Limitations Development of this model was very straightforward, assuming some familiarity with stereotaxic localization. The injections and subsequent seizure activity had no effect on survival of the animals. The injections were reliable with accurate staging of the granulomas. Age-dependent effects have not been tested.
Insights into Human Disorders Seizures in patients with neurocysticercosis are thought to be provoked seizures, occurring with active inflammation (Carpio et al., 1998). The seizures often resolve as the inflammation subsides, suggesting that one of the mediators of the inflammation is the cause of the seizures. The study by Stringer et al. (2003) provides direct evidence that a substance produced in early stage granulomas can initiate seizure activity. A number of bioactive substances have been shown to be present in the inflammatory granulomas. In humans, the inflammatory response is associated with
FIGURE 1 Epileptiform activity induced by local injections of granuloma extracts. Each part presents the chart recording from a single animal. The arrow on each tracing indicates the time of extract administration (volume of 10 ml). In D, an extract from normal mouse spleen was injected at the arrow. Reprinted from Stringer et al 2003 with permission from Elsevier.
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production of both Th1 and Th2 cytokines, including ginterferon, interleukin-18, interleukin-4, interleukin-13, and interleukin-10 (Restrepo et al., 1998, 2001). In the mouse model, it has been previously shown that there is sequential expression of Th1 cytokines in the early-stage granulomas, followed by Th2 cytokines in the later stages (Robinson et al., 1997). Evidence exists for an epileptogenic role of some cytokines, particularly IL-1b (Vessani et al., 2000). More recently, it has been hypothesized that neuropeptides may regulate the cytokine expression in the granulomas of cysticercosis (Robinson et al., 2002). This model should be useful for determining which inflammatory mediator(s) are epileptogenic in neurocysticercosis.
SEIZURES INDUCED BY INFECTION WITH HERPES VIRUS General Description of Model A patient with viral encephalitis has an increased risk for the development of unprovoked seizures (Hauser and Hesdorffer, 1990; Rocca et al., 1987; Annegers et al., 1988). Among viruses that can cause encephalitis, herpes simplex virus, type-1 (HSV-1) is the one most commonly linked to seizures. HSV-1, which is a neurotropic virus that can travel through the CNS by retrograde axonal transport, has been shown to have an affinity for the temporal region (Esiri, 1982). In patients with encephalitis caused by HSV-1, 38% will have seizures (Schmutzhard, 2001). The symptoms associated with HSV-1 infection are most likely influenced by both viral and host factors. HSV-1 infections are also characterized by the ability of the virus to become latent (Baringer and Pisani, 1994). Once the virus has become latent, a variety of external or systemic stimuli can reactivate HSV-1. Attempts have been made to produce an animal model of HSV-1 infection with adequate reflection of both the acute and chronic characteristics of the infection. Here are reviewed the models of HSV-1 infection that will result in seizures. The earliest attempts to infect animals with HSV-1 were done in rabbits (Stroop and Schaefer, 1986, 1989; Schlitt et al., 1986; Schlitt et al., 1988). Adult New Zealand white rabbits were anesthetized and the dura mater covering the left olfactory bulb was removed. An aliquot of viral suspension was injected into the bulb. Controls consisted of injection of viral culture medium. Mortality was 70% to 80% without evidence of a dose-response relationship. The most frequently observed seizures were tonic posturing for <30 seconds, followed by clonic movements of the mouth, hindlimbs, or both for ~30 seconds. Animals also had partial seizures consisting of isolated bilateral facial or unilateral forepaw clonic movements and circling behavior (Schlitt et al., 1988). Recordings from the animals that survived
showed evidence of electroencephalographic abnormalities in the posterior lateral cerebral hemispheres and corresponding inflammation. Some evidence showed that the infection could be reactivated (Stroop and Schaefer, 1986).
Methods of Generation Mice (Wu et al., 2003), rats (Beers et al., 1993), and rabbits (Schlitt et al., 1986; Schlitt et al., 1988) have been used in generating models of HSV-1 infection. The virus has to be grown in African green monkey kidney (VERO) or baby hamster kidney-21 (BHK) cells and then titrated by a plaque assay (Coan et al., 1985) or calculation of tissue culture infectious doses (Beers et al., 1993). Male BALB/c mice, 5- to 6-weeks old, have been infected by corneal scarification (2 ¥ 105 to 106 plaqueforming units; Chen et al., 1997) after being anesthetized with pentobarbital. Female Lewis rats have been inoculated intranasally with HSV-1 after being anesthetized with pentobarbital (Beers et al., 1993). The mortality in Lewis rats, with a dose of 1.4 ¥ 106 50% tissue culture infectious dose per ml, was 11%. Viral shedding was measured in ocular and nasal secretions from the infected rats. Nose and eye secretions were positive for HSV-1 in 72% and 28% of animals, respectively, indicating significant shedding of the infectious agent. No study reports a systematic study of gender, age, or stain differences in the infection or seizure rate. The presence of seizures has been determined by observation in rats (Beers et al., 1993) and by electrical recording in the hippocampus of mice (Wu et al., 2003). For monitoring of electrical activity in the BALB/c mice (Wu et al. 2003), silver chloride electrodes (A-M Systems, Inc. Carlsborg, WA, USA) were stereotactically implanted bilaterally into the hippocampus. A reference electrode was placed in the frontal sinus and bipolar electrical wires were passed into the subcutaneous tissue on the neck for electromyography recording. The electrodes were plugged into a connector that was then fixed to the skull with dental cement. Recordings were then obtained from freely moving mice.
Characteristics and Defining Features The BALB/c mice went through a characteristic progression of symptoms after inoculation with HSV-1 (Wu et al., 2003). In the first 4 days after infection, all animals appeared healthy. At 4 days postinfection, most animals had ruffled coats, labored breathing, hunched posture, and loss of appetite. The severity of the symptoms in infected mice was related to HSV-1 dosage and symptoms were graded as mild, moderate, or severe. In the severely affected group, all of the animals had interictal spikes, bursting and behavioral seizures and all of the animals died. In the animals with moderate disease, one third had interictal activity and 20% had bursting and behavioral seizures. Behavioral seizure
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References
activity included repetitive wet dog shakes, forelimb clonus with or without rearing, and generalized extension. The seizure duration, determined from the electrical recording, was from a few seconds to tens of seconds. In general, both interictal activity and bursts occurred predominantly in one hippocampus and more frequently in the mice with more severe symptoms. Status epilepticus was not reported in any animal. In mice that survived more than a month after inoculation, no spontaneous seizures were observed. Intracellular recordings from CA3 pyramidal cells in slices obtained from infected mice, however, displayed abnormal excitability, in that they had a more depolarized resting membrane potential, a lower threshold for generating bursts, and a decrease in amplitude of the afterhyperpolarization compared with controls. More severe symptoms in the mice (Wu et al., 2003) correlated with higher viral titers in frontal and limbic tissue and with focal hemorrhagic necrosis in the piriform cortex, hippocampus, amygdala, entorhinal cortex, and frontal cortex. The presence of reactive astrocytes or sprouting was not tested, but given the extent of neuronal damage after administration of HSV-1, one would predict both reactive astrocytes and sprouting. Of the Lewis rats inoculated with HSV-1 (Beers et al., 1993), 39% developed partial complex seizures that eventually generalized. Each seizure lasted approximately 30 to 60 seconds and they recurred every 30 minutes. Status epilepticus was not observed in any animal. In animals with seizure activity, inflammatory infiltrates and hemorrhagic lesions were detected in the trigeminal ganglia, olfactory bulb, amygdala, hippocampus, piriform cortex, entorhinal cortex, and the spinal trigeminal ganglia. Astrocytic hypertrophy was demonstrated by an increase in immunoreactivity for glial fibrillary acidic protein. The presence of sprouting was not measured, but would be predicted. In survivors of the acute infection, the histologic changes appear to resolve in 14 to 30 days after inoculation. This absence of residual pathology is distinct from the human infection. Chen et al. (2004) have developed a model of HSV-1 infection using hippocampal slice cultures. Cultures were prepared from 10- to 12-day-old Sprague-Dawley rats. HSV1 (105 plaque-forming units) was added to the culture medium at 14 days in vitro. The cultures were then washed and maintained in culture for an additional 1 to 14 days. HSV-1 infection induced epileptiform activity, neuron loss, and sprouting of mossy fibers as indicated by Timm’s stain. Addition of acyclovir 1 or 24 hours after infection with HSV-1 significantly reduced the neuronal damage. The neuronal damage in these cultures is more severe than that produced when the virus is given in vivo, suggesting that this is not a model of HSV-1 infection in vivo, but could be used to examine the mechanisms by which the virus causes cell loss and hyperexcitability. The effectiveness of antiepileptic drugs in these models has not been tested.
Limitations One of the biggest drawbacks to these models is the need to grow, propagate, and infect animals with a human pathogen, which is shed by the infected animals. This severely limits the ease with which the experiments can be done. Also, a high mortality rate occurs with the infection in rodents. In in vivo testing (Wu et al., 2003; Beers et al., 1993), the higher the titer of HSV-1 in the inoculation, the higher the incidence of seizure activity and the higher the morality rate. Thus, to develop a highly reliable model of seizures induced by viral infection, a way to improve mortality needs to be developed. In addition, the seizure activity that is produced is somewhat variable, most likely because of the different brain regions that are infected with the virus.
Insights into Human Disorders Rodents have been used successfully to model human infection with herpes simplex virus (Stroop and Schaefer, 1986). The models have evidence of inflammation and neuronal death, hyperexcitability, and behavioral seizures. Although they are reasonable models of the human disease, as experimental models the in vivo models are complicated by the high mortality rate and variability of the seizure activity. The in vivo models may be useful for testing drugs for the treatment of viral encephalitis, but are not well suited for testing antiepileptic drugs. The experiments done with acute infection in brain slices (Chen et al. 2004) have the potential to determine the mechanisms by which the virus directly increases excitability and causes neuronal death.
References Alverez, J.I., Colegial, C.H., Castano, C.A., Trujillo, J., Teale, J.M., and Restrepo, B.I. 2002. The human nervous tissue in proximity to granulomatous lesions induced by Taenia solium metacestodes displays an active response. J Neuroimmunol 127: 139–144. Annegers, J.F., Hauser, W.A., Beghi, E., Nicolosi, A., and Kurland, L.T. 1988. The risk of unprovoked seizures after encephalitis and meningitis. Neurol 38: 1407–1410. Baringer, J.R., and Pisani, P. 1994. Herpes simplex virus genomes in human nervous system tissue analyzed by polymerase chain reaction. Ann. Neurol 36: 823–829. Beers, D.R., Henkel, J.S., Schaefer, D.C., Rose, J.W., and Stroop, W.G. 1993. Neuropathology of herpes simplex virus encephalitis in a rat seizure model. J Neuropathol Exp Neurol 52: 241–252. Carpio, A., Escobar, A., and Hauser, W.A. 1998. Cysticercosis and epilepsy: a critical review. Epilepsia 39: 1025–1040. Chen, S-F., Huang, C-C., Wu, H-M., Chen, S-H., Liang, Y-C., and Hsu, K.S. 2004. Seizure, neuron loss, and mossy fiber sprouting in herpes simplex virus type 1-infected organotypic hippocampal cultures. Epilepsia 45: 322–332. Chen, S-H., Kramer, M.F., Schaffer, P.A., and Coen, D.M. 1997. A viral function represses accumulation of transcripts from productive-cycle genes in mouse ganglia latently infected with herpes simplex virus. J Virol 71: 5878–5884.
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Coan, D.M., Fleming, H.E. Jr, Leslie, L.K., and Retondo, M.J. 1985. Sensitivity of arabinosyladenine-resistant mutants of herpes simplex virus to other antiviral drugs and mapping of drug hypersensitivity mutations to the DNA polymerase locus. J Virol 53: 477–488. DeGiorgio, C.M., Medina, M.T., Duron, R., Zee, C., and Pietsch Escueta, S. 2004. Neurocysticercosis. Epilepsy Currents 4: 107–111. Eisenschenk, S., and Gilmore, R.L. 2001. Seizures associated with nonneurologic medical conditions. In The Treatment of Epilepsy: Principles and Practice. Ed. E. Wyllie 3rd. pp. 657–669. New York: Lippincott, Williams & Wilkins. Escobar, A. 1983. The pathology of neurocysticercosis. In Cysticercosis of the Central Nervous System. Ed. E. Palacios, J. Rodriguez-Carbajal, J. Taveras. Springfield: Charles C. Thomas. Esiri, M.M. 1982. Herpes simplex encephalitis immunohistological study of the distribution of viral antigen within the brain. J Neurol Sci 54: 209–226. Garcia, H.H., and Del Brutto, O.H. 2000. Taenia solium cysticercosis. Infect Dis Clin North Am 14: 97–119. Gutierrez, Y. 1990. Diagnostic Pathology of Parasitic Infection with Clinical Correlation. pp. 432–459. Philadelphia: Lea & Febiger. Hauser, W.A., and Hesdorffer, D.C. 1990. Epilepsy: Frequency, Causes and Consequences. New York: Demos Publications. Hooshmand, H. 1976. Seizure disorders associated with neurosyphilis. Diseases of the Nervous System 37: 133–136. Kunz, J., Kalinna, B., Watschke, V., and Geyer, E. 1989. Taenia crassiceps metacestode vesicular fluid antigens shared with the Taenia solium larval stage and reactive with serum antibodies from patients with neurocysticercosis. Zentralbl Bakteriol 27: 510–520. Labar, D.R., and Harden, C. 1998. Infection and inflammatory diseases. In Epilepsy: A Comprehensive Textbook. Ed. J. Engel, Jr. and T. A. Pedley. pp. 2587–2596. New York: Lippincott, Williams & Wilkins. Restrepo, B.I., Alvarez, J.I., Castano, J.A., Arias, L.F., Restrepo, M., Trujillo, J., Colegial, C.H., et al., 2001. Brain granulomas in neurocysticercosis patients are associated with a Th1 and Th2 profile. Infect Immuno 69: 4554–4560. Restrepo, B.I., Llaguno, P., Sandoval, M.A., Enciso, J.A., and Teale, J.M. 1998. Analysis of immune lesions in neurocysticercosis patients: central nervous system response to helminth appears Th1-like instead of Th2. J Neuroimmunol 14: 64–72. Ridaura-Sanz, C.R. 1987. Host response in childhood neurocysticercosis: some pathological aspects. Childs Nerv Syst 3: 206–207. Robinson, P., Atmar, R.L., Lewis, D.E., and White, A.C. 1997. Granuloma cytokines in murine cysticercosis. Infect Immun 65: 2925–2931. Robinson, P., White, A.C., Lewis, D.E., Thornby, J., David, E., and Weinstock, J. 2002. Sequential expression of the neuropeptides substance P
and somatostatin in granulomas associated with murine cysticercosis. Infect Immun 70: 4534–4538. Rocca, W.A., Sharbrough, F.W., Hauser, W.A., Annegers, J.F., and Schoenberg, B.S. 1987. Risk factors for complex partial seizures: a populationbased case-control study. Ann Neurol 21: 22–31. Schlitt, M., Bucher, A.P., Stroop, W.G., Pindak, F., Bastian, F.O., Jennings, R.A., Lakeman, A.D., et al. 1988. Neurovirulence in an experimental focal herpes encephalitis: relationship to observed seizures. Brain Res 440: 293–298. Schlitt, M., Lakeman, A.D., Wilson, E.R., To, A., Acoff, R.W., Harsh, G.R. III, and Whitley, R.J. 1986. A rabbit model of focal herpes simplex encephalitis, J Infect Dis 153: 732–735. Schmutzhard, E. 2001. Viral infections of the CNS with special emphasis on herpes simplex infections. J Neurol 248: 469–477. Sciutto, E., Fragoso, G., Baca, M., De la Cruz, V., Lemus, L., and Lamoyi, E. 1995. Depressed T-cell proliferation associated with susceptibility to experimental Taenia crassiceps infection. Infect Immun 63: 2277– 2281. Solomon, T. 2004. Flavivirus encephalitis. N Engl J Med 351: 370–378. Stringer, J.L., Marks, L.M., White, A.C. Jr., and Robinson, P. 2003. Epileptogenic activity of granulomas associated with murine cysticercosis. Exp Neurol 183: 532–536. Stroop, W.G., and Schaefer, D.C. 1986. Production of encephalitis restricted to the temporal lobes by experimental reactivation of herpes simplex virus. J Infect Dis 153: 721–731. Stroop, W.G., and Schaefer, D.C. 1989. Neurovirulence of two clonally related herpes simplex virus type 1 strains in a rabbit seizure model. J Neuropathol Exp Neurol 48: 171–183. Vessani, A., Moneta, D., Conti, M., et al. 2000. Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice. Proc Natl Acad Sci U S A 97: 11534–11539. Villa, O.F., and Kuhn, R.E. 1996. Mice infected with the larvae of Taenia crassiceps exhibit a Th2-like immune response with concomitant anergy and downregulation of Th1-associated phenomena. Parasitology 112: 561–570. White, A.C., Jr. 2000. Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis, and management. Ann Rev Med 51: 187–206. White, A.C. Jr., Robinson, P., and Kuhn, R. 1997. Taenia solium cysticercosis: host-parasite interactions and the immune response. Chem Immunol 66: 209–230. Wu, H.M., Huang, C.C., Chen, S.H., Liang, Y.C., Tsai, J.J., Hsieh, C.L., and Hsu, K.S. 2003. Herpes simplex virus type 1 inoculation enhances hippocampal excitability and seizure susceptibility in mice. Eur J Neurosci 18: 3294–304.
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42 Brain Tumor and Epilepsy: A New Neurophysiologic and Neuropathologic Ex Vivo In Vitro Model ALI GORJI AND ERWIN-JOSEF SPECKMANN
An estimated 127,000 new cases of cancer of the brain and the central nervous system (CNS) are diagnosed worldwide each year (Parkin et al., 1999). The most common medical problems in patients with brain tumor involve the management of seizures, peritumoral edema, medication side effects, and venous thromboembolism. Among these, epileptic seizures are common in patients with primary brain tumors or brain metastases. Seizures are often the first symptom of intracranial tumors. They frequently can predate other symptoms or diagnosis by many years and can occur when computed tomography (CT) scan is normal and a magnetic resonance image (MRI) may show only subtle changes (Whittle and Beaumont, 1995; Whittle et al., 1992). Seizures can occur with any primary or secondary intracerebral tumor and also are not uncommon in extracerebral intracranial tumors. Primary brain tumors cause seizures in 20% to 45% of patients and are often associated with lesion reduction or progression (Liigant et al., 2001). In general, the incidence of seizures is higher in conjunction with primary brain tumors than with metastases. Seizures are observed in about 90% of oligodendroglioma, in 60% of astrocytoma, up to 30% of high grade glioma, 30% to 40% of meningioma, approximately 10% of primary CNS lymphoma, 20% to 40% of brain metastases, and up to 15% of leptomeningeal or dural metastases (Table 1). Gliomas are the most common among epilepsy-related tumors, accounting for 72% to 88% of the tumors (Spencer et al., 1984), whereas meningiomas and metastatic tumors are the leading causes of seizures in late-onset epilepsy (Perez-Lopez et al., 1995). Furthermore, CNS infections, metabolic disorders, and drug toxic effects can increase the severity and the frequency of preexisting
Models of Seizures and Epilepsy
epileptic seizures in these patients (Beaumont and Whittle, 2000; Hildebrand, 2004). Tumor-associated epileptic seizures are essentially focal, although secondary generalization is common and it can occur so quickly that, in certain patients, the focal phase passes unnoticed. The main factor predicting tumor-related epilepsy is cortical location. Often, a good relation is seen between the tumor location and the type of seizure experienced. Complex partial seizure with different types of aura was reported as the most common seizure type in temporal lobe tumoral epilepsy (Zaatreh et al., 2003). Partial seizures are frequent with neoplasm in either the supplementary motor cortical area or the second somatic sensory region. Generalized tonic–clonic type seizures can occur either as secondary generalization after a partial seizure or as a primary seizure type (Ramamurthi et al., 1980; Cascino, 1990). Seizure frequency and incidence also depend on neoplasm location and its pathologic characteristics. The occipital lobe tumor has the highest threshold for producing seizures, whereas the frontal parasagittal region of the supplementary motor area and the opercula and insula regions of the secondary somatic sensory area have a much lower threshold (Mahaley and Dudka, 1981). The higher incidence observed in low- versus high-grade glioma could be related to the much longer survival of patients with grade 2 glioma. Several histologic changes have been observed in peritumoral cortex, but it remains unclear how these factors relate to epileptogenesis. Study of hippocampal neuronal density in temporal lobe epilepsy, with and without gliomas, revealed significant differences. Epileptic patients without tumor generally had the features of mesial temporal
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TABLE 1 Brain Tumors and Seizure Attacks. The Incidence of Seizures for Differing Tumor Types Tumor type Oligodendroglioma
Seizure incidence 90%
Astrocytoma
60%
High grade glioma
30%
Meningioma Primary CNS lymphoma Brain metastases
30–40% 10% 20–40%
sclerosis, with between 43% and 68% less neurons in the CA1 and CA4 areas, when compared with the hippocampi of epileptics with tumor. The numbers of neurons in the latter group were 35% to 50% less than normal humans. This study indicates that, although both groups demonstrate similar clinical manifestation, they rely on different histochemical or morphologic patterns and pathophysiologic mechanisms. (Kim et al., 1990). Somatostatin- and gaminobutyric acid (GABA)-immunoreactive neurons were identified and counted in epileptic cortex associated with low-grade gliomas. Although significant difference was seen in the overall cell count, this study revealed a decrease of GABAergic and somatostatin immunoreactive neurons and suggested a loss of inhibitory interneurons (Haglund et al., 1992). The role of ion level disturbances in tumoral and peritumoral regions in the pathophysiology of tumor-associated epileptic seizures is not entirely understood. Some preliminary studies, however, used microdialysis and ion exchanger methods in human brain tissues and showed an increase of extracellular potassium concentrations ([K+]o) accompanied by reduction of extracellular chloride levels ([Cl-]o) in peritumoral regions of glioblastoma (authors non published data). The relationship of potassium concentration and potassium clearance mechanisms to epileptogenicity of neuronal tissue has been well studied in different animal models (Pedley et al., 1976; D’Ambrosio et al., 1999). Alteration of [K+]o, either elevation or reduction, increases the susceptibility of neuronal tissue to generate epileptiform burst discharges. Changes of [K+]o lead to excitation of neuronal networks because of the reduction or elevation of the electromotive driving force, the impairment of GABA receptormediated activity, and the perturbation of the extracellular space (McBain et al., 1993; Moddel et al., 2003). Hyperpolarizing inhibitory postsynaptic potentials in cortical and hippocampal neurons are generated by the influx of chloride ions (Misgeld et al., 1986; Thompson et al., 1988; Thompson and Gühwiler, 1989). Because GABA receptor-mediated inhibitory current is determined by the difference between the equilibrium potential for chloride and the neuronal mem-
brane potential (Thompson, 1994), reduction of [Cl-]o would be expected to cause a depolarizing shift in the GABA reversal potential, leading to hyperexcitability and interictal-like spiking (Schwartzkroin and Prince, 1980). Microdialysis findings during electrocorticography (ECoG) from tumor-associated epileptic seizures and other epileptic patients have been compared with normal brain, revealing significant differences in levels of glutamine, alanine, isoleucine, valine, and phosphoethanolamine (Hamberger et al., 1991). Analysis of human glioma biopsy specimens for the amino acid neurotransmitters and glutamine has shown that gliomas associated with epilepsy have a higher concentration of glutamine (Bateman et al., 1988). Autoradiography of peripheral benzodiazepine receptors in human U251 glioblastoma cultures, which have GABAergic properties, demonstrated an increased number of binding sites, as compared with the normal brain (Olson et al., 1992). Whereas it is plausible that amino acids as well as ion imbalances contribute to epileptogenesis, it may, however, be difficult to distinguish whether such changes represent cause or consequence. Certain derangements in enzyme pathways can be observed both in epileptic and neoplastic tissues. These include increases in glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, choline acetyltransferase, acetylcholinesterase, lactate dehydrogenase (LDH), camp phosphodiesterase, and hexokinase. These elevations may represent primary causative lesions, or more likely upregulation responses to increased amino acid turn over (McCormick et al., 1990; Nishioka et al., 1992). When brain tumors occur with seizures as the first clinical sign of the disease, anticonvulsant therapy is widely recommended. Double, or even triple, agent therapy may be required. The response to drug therapy is unpredictable, and some epileptics attain little or no relief. Because antiepileptic drugs (AED) seem to have negative effects on mechanisms of tumor control, intrinsic antineoplastic effects of some AED (e.g., valproic acid [VPA]) would be beneficial. An antineoplastic effect in various cancer cell lines has been reported for VPA. Mechanisms involved include up-regulation of gene expression in the proapoptotic ERK-AP-1 pathway, inhibition of glycogen synthase kinase-3-, downregulation of protein kinase C, activation of peroxisome proliferators-activated receptors and blocking of histone deacetylase (Blaheta et al., 2002). Based on the report of a subcommittee of the American Academy of Neurology, however, prophylactic administration of AED is not recommended in patients with primary or metastatic brain tumors who have never presented with epileptic seizures (Glantz et al., 2000). AED use in patients with neoplastic lesions is complicated by several problems, including CNS toxicity, drug interferences, blood toxicity, and possible enhancement of hypersensitivity to AED by irradiation (Hildebrand 2004). Long-term follow-up of patients with intractable tumoral
Experimental Investigations of Tumor-Associated Epilepsy
epilepsy suggests good response of seizures to surgery (Zaatreh et al., 2003). In some patients, however, particularly those with poorly controlled multiple complex partial seizures, even extensive resections guided by ECoG can produce disappointing postoperative results (Goldring 1986). Two main problems are recurrence or persistence of seizures after surgery, whereas some patients do not experience seizures until after resective surgery.
EXPERIMENTAL INVESTIGATIONS OF TUMOR-ASSOCIATED EPILEPSY Knowledge of the causative abnormalities of tumorassociated epilepsy is prerequisite for precise and effective treatment of this seizure disorder. The largest obstacle to progress is the difficulty in distinguishing whether observed changes, either in vivo or in vitro, are direct cause of epileptiform activity or a consequence of such disturbances. Progress also awaits the development of an appropriate and reproducible animal model of tumoral epileptic seizures. Although tumoral epileptic seizures are not confined to humans (Palmer, 1972), no cases have been reported of seizures in animal with experimentally induced tumors. An ideal model for the analysis of tumor-associated epileptogenesis would require (1) the histologic appearance closely resembles the pattern of tumor, including interaction of tumor cells with non-neoplastic neurons and glia; (2) origin and local spread of ictal form activity can be electrophysiologically mapped with respect to the tumor and its invasion area; and (3) the tissue studied by electrophysiology can be subsequently analyzed by histopathologic techniques to strictly correlate electrophysiologic and morphologic and molecular abnormalities. To develop a model useful for elucidating the electrophysiologic, molecular, and structural basis of gliomaassociated epileptogenesis, rat C6 glioma cells stably transfected with a green fluorescence protein (GFP) gene were transplanted into rat hindlimb or parietal neocortex, giving rise to diffusely invading gliomas histologically resembling human glioblastomas. Then, 2 ¥ 106 C6 cells in a total volume of 10 ml, after trypsinized from a confluent culture flask, were injected into the neocortex in hindlimb or parietal regions of the neocortex in depth of 2 to 3 mm. Using this method, the implantation occurred approximately at the granular layer of the neocortex. GFP promotes identification of tumor cells during electrophysiologic investigation as well as in neuropathologic analysis. The animals were then reared under normal conditions for 2 weeks. After this period, the tumors were macroscopically evident on inspection of the brain surface. The tumors had a volume of ~8 mm3, being roughly spherical or conical, with diameters of 1 to 3 mm and intracortical penetration depths of ~2 mm. In some cases, tumors expanded into the subarachnoid space, occasionally
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leading to large extracerebral masses and brain deformation. After preparing of cerebral slices and staining with the voltage-sensitive dye, optical fluorescence imaging techniques were used to detect the origin and propagation of spontaneous bioelectrical activity elicited by omission of Mg2+ from superfusate. Bioelectrical field potentials were also recorded from layers II/III in close proximity to the tumor using conventional glass microelectrodes. Intracerebral C6-GFP transplants showed a diffuse growth pattern, extensive perivascular invasion, and large areas of necrosis. Tumors were established at the site of implantation of glioma cells into the neocortex. The tumors were macroscopically evident on inspection of the brain surface. The tumors had a volume of ~8 mm3, being roughly spherical or conical, with diameters of 1 to 3 mm and intracortical penetration depths of ~2 mm. In some cases, tumors expanded into the subarachnoid space, occasionally leading to large extracerebral masses and brain deformations (Figure 1). On washout of Mg2+ from the perfusate of the slices, spontaneous epileptiform activity arose within 20 to 40 minutes from layers II/III. These were usually ictalform, with durations >1 to 2 seconds, and intercalated shorter, interictal-type discharges. Using optical imaging method, the initiation site was pointed to a location just adjacent to the tumor, in the supragranular layers of the lateral neocortex. Spread of activity could be deduced from temporal latencies between onset of field potential changes and, more precisely, also from fluorescence changes. When activity initiated close to the tumor (~200 to 800 mm from the tumor border), these sites being located both medially and laterally to the tumor (Figure 2), no activity was revealed within the tumor. These findings were in line with multichannel magnetoencephalographic (MEG) and electroencephalographic (EEG) studies of glioma patients, which showed that epileptiform activity was usually localized at border of the tumor (Patt et al., 2000). Histologically, tumor extension could be observed using the same slices that were used before for activity measurements. In these slices, the tumor covered an area of ~2.5 mm2. The main invasion zone into normal brain amounted to 200 mm, with single tumor cells being found even at a distance of 2.7 mm as a result of migration along blood vessels in gray and white matter. Immunohistochemical NeuN staining of the region of activity initiation revealed cortical neurons surrounded by single invading glioma cells. The fluorescence marker GFP stably expressed by C6 glioma cells served two purposes. First, it enabled exact tumor localization within the slice to record activity from intratumoral, peritumoral, and remote areas. Second, GFP was used in frozen and paraffin section to identify diffusely invading tumor cells on histologic examination. Origin and spread of epileptiform activity was determined with optimal imaging of slice preparations. Finally, subsequent histologic
FIGURE 1 C6-SP-green fluorescence protein (GFP) cells in vitro and after transplantation in rat cortex. A: Identification of GFP using conventional fluorescence microscopy. Cytoplasms are stained using antivimentin antibody and Cy3-conjugated secondary antibody, whereas nuclei are stained with Hoechst 33258. B: Intracerebral transplants exhibiting GFP fluorescence in native cortical slices of 500-mm thickness 2 weeks after transplantation of 2 ¥ 106 cells; asterisks indicate cerebral ventricle (compare with Figure 2, A2). C: Histology of C6-SP-GFP transplants showing areas of necrosis with pseudopalisading; hematoxylin and eosin (HE) staining, 500-mm slice fixed in Zamboni solution, embedded in paraffin, and sectioned at 2 mm. D: Section parallel to C in fluorescence imaging. Note that areas of necrosis are negative for GFP fluorescence. Diffuse invasion is highlighted by arrows (2-mm paraffin section). E: Zones of single (diffuse) tumor cell infiltration can be observed in greater detail using confocal laser scanning microscopy (50-mm cryo section). F: Double fluorescence showing invading tumor cells interacting with MAP2-stained axons in the invasion zone (2-mm paraffin section, axons stained with anti-MAP2 antibody, and Cy3-conjugated secondary antibody). G: Histology of a cortical slice after electrophysiologic analysis. Tumor localization can be correlated with the focus of epileptiform activity (compare with Figure 2 B1). Remote tumor cell nests are seen (arrows), resulting from migration along blood vessels (50-mm cryo section prepared from the same 500-mm slice used for bioelectrical measurement shown in Figure 2, stained with HE). H: Distribution of neurons in the invasion rim of tumor. Neurons are labeled both within the tumor (arrows) as well as outside of the compact tumor mass encircled from perivascularly arranged invading tumor cells (arrowhead) (50-mm cryo section adjacent to the section shown in G processed for immunohistochemical staining of neurons using anti-NeuN antibody, corresponding to the rectangle in G. I: Region generating the start of epileptiform signals is shown, corresponding to the dot in G and Figure 2 B1. Only single perivascular tumor cells encircling NeuN-labeled neurons are seen (arrows). Bar 1 mm in B–D, G; 100 mm in F, I; 50 mm in A, E, H (From Senner, V. et al., with permission). (See color insert.)
Experimental Investigations of Tumor-Associated Epilepsy
531
FIGURE 2 Imaging of a glioma growing in a neocortical slice preparation after in vivo implantation of 2 ¥ 106 cells in a total volume of 10 ml into the neocortex of rats 14 days before slice preparation (A) and bioelectric activity in glioma-invaded slice preparation (B). A: Digital photograph of a tumor-invaded slice preparation in the recording chamber under normal (1) and GFP fluorescence illumination (2, excitation: 475 nm band pass; emission: 500 nm long pass). B1: Recording of spontaneous bioelectric activity after withdrawal of Mg2+ from the perfusate. Hexagons represent two positions of the photodiode array for optical imaging superimposed on the slice, corresponding to the positions shown in B2. Position 2 was recorded ~5 minutes after position 1. Positions of the field potential (FP) electrodes, location of tumor mass (dashed lines), focus of activity initiation (large dot), and corresponding directions of activity spread (arrows) are indicated. B2: Spontaneous ictaform activity in field potential discharges (FP) at two recording positions (see B1) and corresponding fluorescence changes of neocortical tumor-invaded slice preparation are shown (B3). Optical signals correspond to fluorescence changes picked up by selected diodes represented by small dots in B1 (spatial orientation in B1 and B2 is identical). Note the time lag between field potential recordings (FP2 close to tumor before FP1 away from tumor). This latency (~85 ms) is reflected in the time-lag between optical signals (see also dotted lines), indicating that activity spreads smoothly from the initiation site (lower recordings) to more medial parts of the slice. Indeed, the slope of the dotted lines indicates a time lag of 62 and 75 msec between bottom and top diodes (positions 1 and 2, respectively), corresponding to a linear propagation speed of about 0.05 m/s. This matches with the propagation speed between the field potential recordings in position 1 amounting to 0.051 m/s. (See color insert).
analysis of the slices enabled correlations of electric activity and histopathology. Based on the finding that glioma cells can spike, it has been hypothesized that epileptic activity is generated by tumor cells themselves (Labrakakis et al., 1997). The data here as well as some from other investigations, however, showed the absence of activity within the tumor mass. The finding that epileptic activity is generated in the invasion
area of gliomas is of interest for both pathogenesis and therapy, because the treatment strategy of tumor-related epilepsy is controversial, with some authors advocating lesionectomy alone, and others recommending additional extirpation of the surrounding cortex (Duffau et al., 2002). The above-mentioned model of glioma provides the possibility to study both electrophysiologic and morphologic parameters using the same tissue with high resolution. This
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Chapter 42/Models Available for the Infection-Induced Seizures
FIGURE 2 (Continued) B3: Optical recording of bioelectric activity shown as succession of pseudocolor images. Activity is color coded from blue (normoand hyperpolarization) to red (depolarization). Scaling in position 1 and 2 is identical. Each image represents one set of data recorded by the photodiode array (hexagons here and in B1) at one point in time. Start of the images is ~40 to 50 msec before signal in FP2 in B2; intervals between frames are 25 msec (position 1) and 35 msec (position 2) (different intervals to cover entire signal in both cases). Note that full spread of signal (and at least of initial signal in position 2) is reached within approximately three frames (or 75 to 105 msec), which again matches with the values derived from B1. Asterisk in B2, position 2, indicates that, although activity apparently spreads into the field from lateral parts, the strongest signal is seen distal to this position in more medial parts of the slice (third photodiode from top, and row 4 in B3). The spread of this type of signal is apparently less linear, as it appears only later in the field potential signal (large negativity in FP2) recorded outside of the diode field (From Senner, V. et al., with permission). (See color insert).
should be particularly useful for analyzing the molecular features underlying glioma-associated epileptogenesis.
References Bateman, D.E., Hardy, J.A., McDermott, J.R., Parker, D.S., and Edwardson, J.A. 1988. Amino acid neurotransmitter levels in gliomas and their relationship to the incidence of epilepsy. Neurol Res 10: 112–114. Beaumont, A., and Whittle, I.R. 2000. The pathogenesis of tumour associated epilepsy. Acta Neurochir (Wien) 142: 1–15. Blaheta, R.A., and Cinatl, J. Jr. 2002. Anti-tumor mechanisms of valproate: a novel role for an old drug. Med Res Rev 22: 492–511. Cascino, G.D. 1990. Epilepsy and brain tumors: implications for treatment. Epilepsia. 31 (Suppl 3): S37–S44. D’Ambrosio, R., Maris, D.O., Grady, M.S., Winn, H.R., and Janigro, D. 1999. Impaired K(+) homeostasis and altered electrophysiological properties of post-traumatic hippocampal glia. J Neurosci. 19: 8152– 8162. Duffau, H., Capelle, L., Lopes, M., Bitar, A., Sichez, J.P., and Effenterre, R. 2002. Medically intractable epilepsy from insular low-grade gliomas: improvement after an extended lesionectomy. Acta Neurochir 144: 563–573. Glantz, M.J., Cole, B.F., Forsyth, P.A., et al. 2002. Practice parameter: Anticonvulsant prophylaxis in Patients with newly diagnosed brain tumors. Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 54: 1886–1893. Goldring, S., Rich, K.M., and Picker, S. 1986. Experience with gliomas in patients presenting with a chronic seizure disorder. Clin Neurosurg 33: 15–42.
Haglund, M.M., Berger, M.S., Kunkel, D.D., Franck, J.E., Ghatan, S., and Ojemann, G.A. 1992. Changes in gamma-aminobutyric acid and somatostatin in epileptic cortex associated with low-grade gliomas. J Neurosurg 77: 209–216. Hamberger, A., Nystrom, B., Larsson, S., Silfvenius, H., and Nordborg, C. 1991. Amino acids in the euronal microenvironment of focal human epileptic lesions Epilepsy Res 9: 32–43. Hildebrand, J. 2004. Management of epileptic seizures. Curr Opin Oncol 16: 314–317. Kim, J.H., Guimaraes, P.O., Shen, M.Y., Masukawa, L.M., and Spencer, D.D. 1990. Hippocampal neuronal density in temporal lobe epilepsy with and without gliomas. Acta Neuropathol (Berl) 80: 41– 45. Labrakakis, C., Patt, S., Weydt, P., Cervos-Navarro, J., Meyer, R., and Kettenmann, H. 1997. Action potential-generating cells in human glioblastomas. J Neuropathol Exp Neurol 56: 243–254. Liigant, A., Haldre, S., Oun, A. et al. 2001. Seizure disorders in patients with brain tumors. Eur Neuro 45: 46–51. Mahaley, M.S. Jr., and Dudka, L. 1981. The role of anticonvulsant medications in the management of patients with anaplastic gliomas. Surg Neurol 16: 399–401. McBain, C.J., Traynelis, S.F., Dingledine, R., and Schwartzkroin, P.A. 1993. Epilepsy: Models, Mechanisms, and Concepts. pp. 437–461. Cambridge: Cambridge University Press. McCormick, D., McQuaid, S., McCusker, C., and Allen, I.V. 1990. A study of glutamine synthetase in normal human brain and intracranial tumours. Neuropathol Appl Neurobiol 16: 205–211. Misgeld, U., Deisz, R.A., Dodt, H.U., and Lux, H.D. 1986. The role of chloride transport in postsynaptic inhibition of hippocampal neurons. Science 232: 1413–1415.
References Moddel, G., Gorji, A., and Speckmann, EJ. 2003. Background potassium concentrations and epileptiform discharges. I. Electrophysiological characteristics of neuronal activity. Brain Res 959: 135–148. Nishioka, T., Oda, Y., Seino, Y. et al. 1992. Distribution of the glucose transporters in human brain tumors. Cancer Res 52: 3972–3979. Olson, J.M., McNeel, W., Young, A.B., et al. 1992. Localization of the peripheral type benzodiazepine binding site to mitochondria of human glioma cells. J Neurol Oncol 13: 35–42. Palmer, J.J. 1972. Haemangioblastomas. A review of 81 cases. Acta Neurochir (Wien) 27(3):125–148. Parkin, D.M., Pisani, P., and Ferlay, J. 1999. Global cancer statistics. CA Cancer J Clin 49: 33–64. Patt, S., Steenbeck, J., Hochstetter, A. et al. 2000. Source localization and possible causes of interictal epileptic activity in tumor-associated epilepsy. Neurobiol Dis 7: 260–269. Pedley, T.A., Fisher, R.S., Futamachi, K.J., and Prince, D.A. 1976. Regulation of extracellular potassium concentration in epileptogenesis. Fed Proc. 35: 1254–1259. Perez Lopez, J.L., Longo, J., Quintana, F., Diez, C., and Berciano, J. 1985. Late onset epileptic seizures. A retrospective study of 250 patients. Acta Neurol Scand 72: 380–384. Ramamurthi, B., Ravi, B., and Ramachandran, V. 1980. Convulsions with meningiomas: incidence and significance. Surg Neurol 14: 415– 416. Schwartzkroin, P.A., and Prince, D.A. 1980. Changes in excitatory and inhibitory synaptic potentials leading to epileptogenic activity. Brain Res 183: 61–76.
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Senner, V., Kohling, R., Puttmann-Cyrus, S., Straub, H., Paulus, W., and Speckmann, E.-J. 2004. A new neurophysiological/neuropathological ex vivo model localizes the origin of glioma-associated epileptogenesis in the invasion area. Acta Neuropathol (Berl) 107: 1–7. Spencer, D.D., Spencer, S.S., Mattson, R.H., and Williamson, P.D. 1984. Intracerebral masses in patients with intractable partial epilepsy. Neurology 34: 432–436. Thompson, S.M., Deisz, R.A., and Prince, D.A. 1988. Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons. J Neurophysiol 60: 105– 124. Thompson, S.M., and Gahwiler, B.H. 1989. Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl- in hippocampal CA3 neurons. J Neurophysiol 61: 512–523. Thompson, S.M. 1994. Modulation of inhibitory synaptic transmission in the hippocampus. Prog Neurobiol 42: 575–609. Whittle, I.R., and Beaumont, A. 1995. Seizures in patients with supratentorial oligodendroglial tumours. Clinicopathological features and management considerations. Acta Neurochir (Wien) 135: 19–24. Whittle, I.R., Sellar, R., and Ironside, J.W. 1992. Epileptogenic anaplastic astrocytoma imaged only by T2-weighted magnetic resonance studies: clinical and surgical implications. Br J Neurosurg 6: 537–542. Zaatreh, M.M., Firlik, K.S., Spencer, D.D., and Spencer, S.S. 2003. Temporal lobe tumoral epilepsy: characteristics and predictors of surgical outcome. Neurology 61: 636–641.
FIGURE 4 2 - 1 C6-SP-green fluorescence protein (GFP) cells in vitro and after transplantation in rat cortex. A: Identification of GFP using conventional fluorescence microscopy. Cytoplasms are stained using antivimentin antibody and Cy3-conjugated secondary antibody (red), whereas nuclei are stained with Hoechst 33258 (blue). B: Intracerebral transplants exhibiting GFP fluorescence in native cortical slices of 500-gm thickness 2 weeks after transplantation of 2 × 106 cells; asterisks" indicate cerebral ventricle (compare with Figure 2, A2). C: Histology of C6-SP-GFP transplants showing areas of necrosis with pseudopalisading; hematoxylin and eosin (HE) staining, 500-gm slice fixed in Zamboni solution, embedded in paraffin, and sectioned at 2 Jam. D: Section parallel to C in fluorescence imaging. Note that areas of necrosis are negative for GFP fluorescence. Diffuse invasion is highlighted by arrows (2-Jam paraffin section). E: Zones of single (diffuse) tumor cell infiltration can be observed in greater detail using confocal laser scanning microscopy (50Jam cryo section). F: Double fluorescence showing invading tumor cells (green) interacting with MAP2-stained axons (red) in the invasion zone (2-Jam paraffin section, axons stained with anti-MAP2 antibody, and Cy3-conjugated secondary antibody). G: Histology of a cortical slice after electrophysiologic analysis. Tumor localization can be correlated with the focus of epileptiform activity (compare with Figure 2 B1). Remote tumor cell nests are seen (arrows), resulting from migration along blood vessels (50-Jam cryo section prepared from the same 500-Jam slice used for bioelectrical measurement shown in Figure 2, stained with HE). It: Distribution of neurons in the invasion rim of tumor. Neurons are labeled both within the tumor (arrows) as well as outside of the compact tumor mass encircled from perivascularly arranged invading tumor cells (arrowhead) (50-Jam cryo section adjacent to the section shown in G processed for immunohistochemical staining of neurons using anti-NeuN antibody, corresponding to the rectangle in G. I: Region generating the start of epileptiform signals is shown, corresponding to the red dot in G and Figure 2 B1. Only single perivascular tumor cells encircling NeuN-labeled neurons are seen (arrows). Bar 1 mm in B-D, G; 100Jam in F, I; 50 Jam in A, E, H (From Senner, V. et al., with permission).
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FIGURE 4 2 - 2 Imaging of a glioma growing in a neocortical slice preparation after in vivo implantation of 2 x 106 cells in a total volume of 10~tl into the neocortex of rats 14 days before slice preparation (A) and bioelectric activity in glioma-invaded slice preparation (B). A: Digital photograph of a tumor-invaded slice preparation in the recording chamber under normal (1) and GFP fluorescence illumination (2, excitation: 475 nm band pass; emission: 500 nm long pass). BI: Recording of spontaneous bioelectric activity after withdrawal of Mg 2+ from the perfusate. Hexagons represent two positions of the photodiode array for optical imaging superimposed on the slice, corresponding to the positions shown in B2. Position 2 was recorded ~5 minutes after position 1. Positions of the field potential (FP) electrodes, location of tumor mass (dashed lines), focus of activity initiation (red dot), and corresponding directions of activity spread (arrows) are indicated. B2: Spontaneous ictaform activity in field potential discharges (FP) at two recording positions (see B1) and corresponding fluorescence changes of neocortical tumor-invaded slice preparation are shown (B3). Optical signals correspond to fluorescence changes picked up by selected diodes represented by blue dots" in B1 (spatial orientation in BI and B2 is identical). Note the time lag between field potential recordings (FP2 close to tumor before FP1 away from tumor). This latency (-85 ms) is reflected in the time-lag between optical signals (see also dotted lines), indicating that activity spreads smoothly from the initiation site (lower recordings) to more medial parts of the slice. Indeed, the slope of the dotted lines indicates a time lag of 62 and 75 msec between bottom and top diodes (positions 1 and 2, respectively), corresponding to a linear propagation speed of about 0.05 m/s. This matches with the propagation speed between the field potential recordings in position 1 amounting to 0.051 m/s. B3: Optical recording of bioelectric activity shown as succession of pseudocolor images. Activity is color coded from blue (normo- and hyperpolarization) to red (depolarization). Scaling in position 1 and 2 is identical. Each image represents one set of data recorded by the photodiode array (hexagons here and in B1) at one point in time. Start of the images is ~40 to 50 msec before signal in FP2 in B2; intervals between frames are 25 msec (position 1) and 35 msec (position 2) (different intervals to cover entire signal in both cases). Note that full spread of signal (and at least of initial signal in position 2) is reached within approximately three frames (or 75 to 105msec), which again matches with the values derived from B1. Asterisk in B2, position 2, indicates that, although activity apparently spreads into the field from lateral parts, the strongest signal is seen distal to this position in more medial parts of the slice (third photodiode from top, and row 4 in B3). The spread of this type of signal is apparently less linear, as it appears only later in the field potential signal (large negativity in FP2) recorded outside of the diode field (From Senner, V. et al., with permission).
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43 An Animal Model of Rasmussen’s Encephalitis1 JAMES O. MCNAMARA
histopathology of the cortex of the ill rabbits and humans with RE (Rogers et al., 1994; He et al., 1998). Here, we consider the features of this animal model.
INTRODUCTION Rasmussen’s encephalitis (RE) is a medically refractory epilepsy that usually begins in the first decade of life and is characterized by progressive degeneration of a single cerebral hemisphere. The progressive atrophy is associated with progressively worsening unilateral seizures and unihemispheric neurologic problems (e.g., language deficits, hemiparesis, and hemisensory loss). Histologic features of resected brain tissue include multifocal inflammatory changes localized primarily to the cortex of the affected hemisphere, including microglial nodules, perivascular lymphocytic cuffing, meningeal infiltrates, neuronal loss, and gliosis (Rasmussen et al., 1958). Because the seizures are unresponsive to standard antiseizure pharmacotherapy, hemispherectomy is the standard treatment, typically leaving the child free of seizures but with a severe neurologic deficit (Rasmussen et al., 1958; Oguni et al., 1991; Robitaille, 1991; Hart, 2004). Although the etiology and pathogenesis of RE and the mechanism limiting the process to one hemisphere are incompletely understood, a series of recent studies demonstrated that a humoral autoimmune mechanism contributes to the pathogenesis of this disease. The key finding that ultimately led to this demonstration was that immunization of rabbits with the glutamate receptor subunit GluR3 unexpectedly resulted in epileptic seizures and neurologic deficits, demonstrating that an autoimmune mechanism could produce some features of the syndrome. The link to RE, in particular, emerged from the similarity of the
METHODS OF GENERATION Construction of Plasmid and Purification of Fusion Protein GluR3 protein was expressed as a fusion protein linked to glutathione S-transferase (GST) using the GST bacterial expression system (Pharmacia). A Bam H1-Sma1 restriction fragment encoding a portion of the N-terminal extracellular domain (residues 246 to 455) of rat GluR3 was subcloned into pGEX-KT vector and used to transform Escherichia coli. As a control, the GST protein alone was obtained by transforming bacteria with the pGEX-KT vector itself. The GST-GluR3 fusion protein was purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and visualized by soaking the gel in 0.3 M CuCl2 for 10 minutes. GST protein was purified either as described for GST-GluR3 or using glutathione-sepharose 4B according to the manufacturer’s protocol; equivalent results were obtained with animals immunized with GST prepared by either method. Purified proteins were aliquoted and frozen at -80° C until use.
Immunization of Rabbits White New Zealand male rabbits weighing 2.5 kg were injected subcutaneously with 100 mg GSTGluR3 or GST in complete Freund’s adjuvant, with subsequent injections in incomplete Freund’s adjuvant. The first boost was given 2
1 This work was supported by a grant (NS 36808) from the National Institute of Neurological Disease and Stroke.
Models of Seizures and Epilepsy
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Copyright © 2006, Elsevier Inc. All rights of reproduction in any form reserved.
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Chapter 43/An Animal Model of Rasmussen’s Encephalitis
weeks after the initial immunization and the remaining boosts given at 4-week intervals. Plasma was obtained by collecting blood in sodium heparin (1000 units/ml).
CHARACTERISTICS AND DEFINING FEATURES Repeated immunization with GSTGluR3 was associated with a distinctive neurologic disorder in two of five rabbits (He et al., 1998). Each of the two rabbits was alert and responsive to crude sensory stimuli, such as air puff to the face or gentle pinching of extremities. Interestingly, each of the two rabbits exhibited motor incoordination while ambulating, either in an open space or in its home cage; this incoordination occurred in the absence of overt weakness of hindlimbs or forelimbs. In addition to the incoordination, each rabbit exhibited occasional epileptic seizures manifested as repetitive tonic or clonic movements of all four extremities lasting ~20 to 40 seconds. In one of the animals, this tonic–clonic seizure was immediately preceded by head nodding associated with unresponsiveness, behaviors typical of class 2 seizures in rats (Racine, 1972). These seizures were followed by obtundation for several minutes before resumption of normal activities. The illness began 2 weeks after the second immunization in one animal and 3 days after the third immunization in the other. Each animal exhibited reduced food and water intake and lost ~25% of its body weight. Persistent obtundation, together with weight loss, eventually necessitated killing both of these rabbits. No illness was evident in three other rabbits despite five to seven immunizations with GSTGluR3 over several months. Likewise, no illness was found in seven rabbits receiving multiple immunizations with GST nor in two rabbits receiving no immunizations. These findings (He et al., 1998) confirmed and extended findings of our initial report (Rogers et al., 1994) in which the same neurologic disorder occurred after the fourth immunization with a TrpEGluR3 fusion protein (including amino acids 245 to 458 of rat GluR3) in two of three rabbits. Immunization of more than 50 rabbits with other fusion proteins containing GluR1, 2, 5, 6, or nAChR subunits did not induce this disorder. Inspection of hematoxylin- and eosin-stained sections revealed mononuclear cell infiltrates in circumscribed regions of cerebral cortex and surrounding blood vessels in the cortex of both cerebral hemispheres of the two sick rabbits, but in none of the other twelve rabbits (data not shown), confirming our previous observations (Rogers et al., 1994). Electroencephalographic (EEG) recording electrodes were not implanted in any of these rabbits. Examination of the brains of the ill, GluR3-immunized animals revealed no gross abnormalities. Microscopic examination of hematoxylin-eosin stained sections revealed mul-
tifocal inflammatory abnormalities consisting of microglial nodules and perivascular lymphocytic cuffing, principally in cerebral cortex, and also lymphocytic infiltration in the meninges of the two ill, GluR3-immunized rabbits but in none of the healthy GluR3 or the GST immunized rabbits (He et al., 1998), confirming our earlier study (Rogers et al., 1994). In contrast to the human disorder, inflammatory infiltrates of cerebral cortex occur bilaterally in the animal model. Might the immunization with GluR3 induce a humoral response that contributed to the neurologic disorder? IgG that selectively bound GluR3, but not GluR1 or GluR6, was detected in the plasma of all five GluR3-immunized rabbits as detected by Western blotting (He et al., 1998). Furthermore, application of anti-GluR3 IgG isolated from the two ill rabbits to cultured cortical cells maintained in primary culture destroyed these cells by a complementmediated mechanism. By contrast, anti-GST IgG isolated from the control-immunized rabbits was not cytotoxic. If the anti-GluR3 antibody contributed to the illness, why was the illness evident in only two of the four rabbits with the “cytotoxic” antibodies (cytotoxicity mediated by complement)? Interestingly, immunohistochemical and Western blotting experiments revealed IgG, including anti-GluR3 IgG, staining cortical neurons of the two ill but not in healthy GluR3-immunized rabbits, suggesting that the anti-GluR3 antibodies gained access to the GluR3 in vivo in the ill, but not in the healthy, GluR3-immunized rabbits.
LIMITATIONS Although the information forthcoming from this animal model has provided valuable guidance of direct studies of the human disorder, this model has multiple limitations. Use of rabbit as the species for study is a serious limitation. In comparison with rats or mice, rabbits are much more costly for purchase and maintaining. Countless reagents available for study of rats or mice (e.g., antibodies, cDNA) are not available for rabbits. The greater size of the rabbits renders them more difficult to handle than a small rodent and this contributed to the lack of EEG data. Rabbits have not been subjected to genetic manipulation as have mice. Together, this led to our efforts to establish this model in mice. We immunized more than 10 distinct lines of mice with the GST-GluR3 fusion protein; despite successfully triggering a humoral immune response in most of these strains as detected by Western blotting, none exhibited weight loss or the neurologic disorder evident in rabbits. The inability to induce an animal model in a mouse is not surprising, considering that robust models of other autoimmune diseases (e.g., myasthenia gravis) have been difficult to establish in mice.
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References
INSIGHTS INTO HUMAN DISEASE As noted in the Introduction, the etiology and pathogenesis of RE are not fully understood. That said, the unexpected discovery that immunization of rabbits with GluR3 resulted in epileptic seizures and neurologic deficits and a histopathology similar to RE led to studies aimed at determining whether an autoimmune mechanism contributed to the pathogenesis of RE. A subset of patients with RE were found to have circulating anti-GluR3 IgG (Rogers et al., 1994). Importantly, seizure severity and frequency are reduced in some patients with RE treated with plasmapheresis or IgG-selective immunoadsorption, in parallel with reduction of GluR3 antibody titers (Rogers et al., 1994; Andrews et al., 1996; Antozzi et al., 1998). Although not all patients with RE have anti-GluR3 antibodies at all times of their illness, these findings—triggered by the animal model—provide compelling evidence for a humoral autoimmune mechanism in some patients with RE. Demonstration of a humoral autoimmune mechanism, in turn, provided the rationale for investigation of potential cellular autoimmune mechanisms in RE and led to the discovery that, as with multiple sclerosis, cellular autoimmune mechanisms are likely operative in the pathogenesis of RE in some cases (Bien et al., 2002). In addition to implicating a humoral autoimmune mechanism in RE, the studies of the animal model led to recognition of potential mechanisms by which the humoral autoimmunity is effected. Some antibodies collected from immunized rabbits or humans with RE elicit excitatory currents in cultured neurons (Twyman et al., 1995), leading to the idea that enhanced activation of glutamate receptors by antibodies may trigger seizures and also destroy neurons by an excitotoxic mechanism. As noted, analyses of sera isolated from the animal model revealed that circulating antiGluR3 can destroy primary mixed neuronal or glial cultures by a complement-dependent mechanism (He et al., 1998). Additional support for IgG and complement in the pathogenesis of RE emerged from immunohistochemical studies revealing IgG and complement deposition on neurons in resected tissue from a subset of RE, but not complex, partial epilepsy patients (He et al., 1998; Whitney et al., 1999). These findings led to subsequent discoveries elucidating the cellular targets of the IgG and complement attack (Whitney
and McNamara, 2000) and interactions between excitotoxic and complement-mediated attacks of cortical neurons. In summary, recognition of similarities of the unexpected GluR3-immunization–induced neurologic disorder and RE ultimately established evidence for an autoimmune mechanism in the pathogenesis of RE. Studies of the animal model have also shed light on mechanisms by which the humoral autoimmune attack may be effected.
References Andrews, P.I., Dichter, M.A., Berkovic, S.F., Newton, M.R., and McNamara, J.O. 1996. Plasmapheresis in Rasmussen’s encephalitis. Neurology 46: 242–246. Antozzi, C., Granata, T., Aurisano, N. et al. 1998. Long-term selective IgG immunoadsorption improves Rasmussen’s encephalitis. Neurology 51: 302–305. Bien, C.G., Bauer, J., Deckwerth, T.L. et al. 2002. Destruction of neurons by cytotoxic T cells: a new pathogenic mechanism in Rasmussen’s encephalitis. Ann Neurol 51: 311–318. Hart, Y. 2004. Rasmussen’s encephalitis. Epileptic Disord 6: 133–144. He, X.P., Patel, M., Whitney, K.D., Janumpalli, S., Tenner, A., and McNamara, J.O. 1998. Glutamate receptor GluR3 antibodies and death of cortical cells. Neuron 20: 153–163. Oguni, H., Andermann, F., and Rasmussen, T.B. 1991. The natural history of the syndrome of chronic encephalitis and epilepsy: a study of the MNI series of 48 cases. In Chronic Encephalitis and Epilepsy Ed. F. Andermann. pp 7–35. Boston: Butterworth-Heinemann. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalography & Clinical Neurophysiology 32: 281–294. Rasmussen, T., Olszweski, J., and Lloyd-Smith, D. 1958. Focal seizures due to chronic localized encephalitis. Neurology 8: 435–455. Robitaille, Y. 1991. Neuropathologic aspects of chronic encephalitis. In Chronic Encephalitis and Epilepsy Ed. F. Andermann. pp 79–110. Boston: Butterworth-Heinemann. Rogers, S.W., Andrews, P.I., Gahring, L.C. et al. 1994. Autoantibodies to glutamate receptor GluR3 in Rasmussen’s encephalitis. Science 265: 648–651. Twyman, R.E., Gahring, L.C., Spiess, J., and Rogers, S.W. 1995. Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site. Neuron 14: 755–762. Whitney. K.D., Andrews, P.I., and McNamara, J.O. 1999a. Immunoglobulin G and complement immunoreactivity in the cerebral cortex of patients with Rasmussen’s encephalitis. Neurology 53: 699–708. Whitney, K.D., and McNamara, J.O. 1999b. Humoral autoimmunity and modulation of synaptic transmission. Annu Rev Neurosci 22: 175–195. Whitney, K.D., and McNamara, J.O. 2000. GluR3 autoantibodies destroy neural cells in a complement-dependent manner modulated by complement regulatory proteins. J Neurosci 20: 7307–7316.
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44 Therapeutic Assays for the Identification and Characterization of Antiepileptic and Antiepileptogenic Drugs H. STEVE WHITE, MISTY SMITH-YOCKMAN, AJAY SRIVASTAVA, AND KAREN S. WILCOX
nine clinically effective drugs, including felbamate (1993), gabapentin (1993), lamotrigine (1994), fosphenytoin (1996), topiramate (1996), tiagabine (1997), levetiracetam (1999), zonisamide (2000), and oxcarbazepine (2000). Despite this apparent success, a significant need remains for more efficacious and less toxic AEDs. This is particularly true for that population of patients whose seizure disorder falls in that category often referred to as “therapy resistant” (Loscher, 2002b). Clearly, the more predictive the animal model for any given seizure type or syndrome, the greater the likelihood that an investigational AED will demonstrate efficacy in human clinical trials. The models employed in the early phase of AED discovery are highly predictive of subsequent efficacy in easy-to-manage generalized and partial epilepsy. Given that ~25% to 40% of patients with partial epilepsy fail to achieve satisfactory seizure control, one could easily argue that these early discovery models lack sufficient predictability for refractory seizures. Furthermore, a paucity of model systems is predictive of the hard-to-treat catastrophic epilepsies and epileptic syndromes of childhood. This is not to say that the new drugs are without value. In fact, most of these second generation AEDs have provided significant benefit to patients with partial epilepsy in the form of greater efficacy, better tolerability, more favorable pharmacokinetics, and greater long-term safety. Two of these second generation AEDs, however, have subsequently been found to possess potential long-term safety concerns (e.g., felbamate and vigabatrin) that were not predicted by initial preclinical studies. For patients who continue to experience uncontrolled seizures at the expense of intolerable adverse drug reactions, a clear and immediate need remains for more efficacious and better tolerated AEDs. It is within
INTRODUCTION Epilepsy affects more than 50 million persons worldwide and consists of more than 40 clinical syndromes (Jacobs et al., 2001). Currently, treatment strategies are symptomatic in nature and aimed at the suppression of clinical seizures with one or more of the available AEDs. In the 1990s, nine new AEDs were approved for the add-on treatment of partial seizures. Of these, two have received approval for monotherapy of newly diagnosed epilepsy (i.e., oxcarbazepine and topiramate) or for conversion to monotherapy (i.e., lamotrigine). Clearly, this was an exciting era for the physician treating patients suffering from intractable seizure disorders. Likewise, for the patient with epilepsy these new drugs provided renewed hope for complete seizure control and lessening of their AED-associated side-effect profile. Never before had so many new and novel AEDs been available for the management of epilepsy. A significant need remains, however, for more effective therapies for patients with refractory partial epilepsy. Ultimately, all patients with epilepsy will benefit from the development of safer, bettertolerated therapeutic options. The discovery and development of a new AED relies heavily on the preclinical employment of animal models to establish efficacy and safety prior to their introduction in human volunteers. The current era of AED discovery was ushered in by Merritt and Putnam when they demonstrated the feasibility of using the maximal electroshock seizure (MES) model to identify the anticonvulsant potential of phenytoin (Putnam and Merritt, 1937). A number of animal models have since been employed in the search for more efficacious and more tolerable AEDs. Since 1993, this approach has contributed to the successful development of
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this realm that the AEDs discovery process confronts its greatest challenges and ignites significant debate. This chapter reviews the current approach to AED discovery employed by the Anticonvulsant Drug Development Program at the University of Utah and other laboratories and discusses some of the inherent advantages and limitations of the primary animal models employed in this process. Table 1 summarizes the various models to be discussed in this review. This table is not meant to be an exhaustive list of all of the models that could be employed in the search for new therapies. Indeed, if one were to be all-inclusive, the table would include all of the models that are discussed in this volume. What might be suggested from a review of Table 1 is that an appropriate evaluation of an investigational therapy should include a battery of studies. For example, to fully assess the potential of a given therapy in today’s environment, it would be appropriate to include an assessment of efficacy against pediatric and adult models of partial and generalized seizures. Moreover, it would also be appropriate to include an assessment against spontaneous seizures in one of the postinsult or genetic models. Lastly, future evaluation might include assessing whether a given therapy might possess disease-modifying properties in one or more models. Subsequent discussion will focus on some of the emerging models that may be more likely to identify the truly novel AED for the treatment of pharmacoresistent partial epilepsy and the prevention of epilepsy. The basic characteristics of each model system will not be reviewed because they have been discussed in detail by others in this edition.
CHARACTERISTICS OF THE IDEAL MODEL SYSTEM As summarized in the 2002 National Institutes of HealthNational Institute of Neurological Disorders and StrokeTABLE 1 List of Animal Seizure and Epilepsy Models Discussed in this Chapter Localization-related (partial, focal) epilepsies — Kindling — 6 Hz psychomotor seizures Generalized seizures — Maximal electroshock — Pentylenetetrazol — Genetic absence epilepsy rat from Strasbourg (GAERS) — lh/lh mouse — Gamma hydroxybutyrate Neonatal and pediatric seizure models — Hypoxia — Hyperthermia — Flurothyl — Kindling
American Epilepsy Society (NIH/NINDS/AES) Models II Workshop, the “ideal” epilepsy model should reflect similar pathophysiology and phenomenology to human epilepsy. In addition, seizures should evolve spontaneously following a postinsult latent period or in a developmental time frame consistent with the human condition. Furthermore, because new drugs are needed to treat the therapy-resistant population, the ideal model should display a pharmacologic profile that is resistant to at least two of the existing AEDs (Stables et al., 2003). In addition, it would be preferable if a given model were also amenable to high-throughput screening. Inasmuch as human epilepsy is a heterogeneous neurologic disorder encompassing many seizure phenotypes and syndromes, it is highly unlikely that any one animal model will ever predict the full therapeutic potential of an investigational AED. This necessitates the current evaluation of an investigational AED in several syndrome-specific model systems. If, in the future, specific syndrome- or seizurephenotype models are developed (and validated clinically), they could be utilized to identify more effective antiseizure and potentially antiepileptic therapies capable of preventing, delaying, or modifying the disease. It is important to note that clinical validation requires the successful development of a new drug and is considered the “Holy Grail” of any animal model.
THE CURRENT ERA OF ANTIEPILEPTIC DRUG DISCOVERY Since 1974, the National Institutes of Neurological Disorders and Stroke has played a pivotal role in stimulating the discovery and development of new chemical entities for the symptomatic treatment of human epilepsy. The efforts of the NINDS have largely been heralded by the Anticonvulsant Screening Project, which, since its inception, has accessioned ~25,000 investigational AEDs from academic and pharmaceutical chemists worldwide. Through a contract with the University of Utah Anticonvulsant Drug Development Program, the initial characterization of their anticonvulsant and behavioral toxicity profile has been established using a battery of well-defined animal models (White et al., 1995; White et al., 1998; Kupferberg, 2001; White et al., 2002). The screening protocol of the Anticonvulsant Drug Development Program is constantly evolving to include well-characterized models that might provide more clinically relevant information.
Early Identification of Anticonvulsant Activity As shown in Figure 1, the University of Utah Anticonvulsant Drug Development Program employs three primary screens in their initial identification studies. They include the maximal electroshock (MES), subcutaneous
The Current Era of Antiepileptic Drug Discovery
MES & scPTZ tests inactive
6 Hz test
ACTIVE ACTIVE
Quantification
inactive
-TPE -ED50 and TD50
Differentiation -scBic -scPic -Frings AGS -Kindled rat
Stop testing
FIGURE 1 Schematic diagram depicting the initial screen of the University of Utah Anticonvulsant Drug Development Program. An investigational antiepileptic (AED) is initially screened for efficacy in both the maximal electroshoick (MES) and subcutaneous pentylenetetrazol (scPTZ) tests. The activity of those compounds with demonstrated efficacy and minimal behavioral toxicity is subsequently quantitated (ED50 and TD50) at the time to peak anticonvulsant effect. Compounds found inactive in the MES and scPTZ tests are then evaluated in the levetiracetam-sensitive 6Hz seizure test. For those compounds that are found to be active in the 6Hz test, their activity is quantitated at their respective time to peak effect. All compounds found to be active in one or more of these three identification screens are then differentiated on the basis of their activity in the subcutaneous bicuculline (scBic) test, the subcutaneous picrotoxin (scPic) test, the Frings audiogenic seizure-susceptible (AGS) mouse, and the hippocampal kindled rat model of partial epilepsy. (From White, H.S. 2004. Antiepileptic Durg Discovery. In The Treatment of Epilepsy Ed. S.D. Shorvon, D.R. Fish, E. Perucca, and W.E. Dodson. pp. 89–95. Massachusettes: Blackwell Science, Inc., with permission.)
pentylenetetrazol (scPTZ), and 6-Hz psychomotor seizure tests. Each of these evoked seizure models provides valuable information regarding the potential anticonvulsant spectrum of an investigational AED. Maximal Electroshock and Subcutaneous Pentylenetetrazol Tests The MES and scPTZ seizure models continue to represent the two most widely used animal seizure models employed in the search for new AEDs (White et al., 2002). Merritt and Putnam (1937) successfully employed the MES test in a systematic screening program to identify phenytoin. The subsequent success of phenytoin in the clinical management of generalized tonic–clonic seizures and partial epilepsy provided the validation necessary to consider the MES test as a reasonable model of human generalized tonic clonic seizures.
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Everett and Richards (1944) demonstrated that seizures induced by the g-aminobutyric acid-A (GABAA)-receptor antagonist PTZ could be blocked by trimethadione and phenobarbital, but not by phenytoin. A year later, Lennox demonstrated that trimethadione was effective in decreasing or preventing petit mal (i.e., absence epilepsy) attacks in 50 patients but was ineffective or worsened grand mal (i.e., generalized tonic clonic seizures) attacks in 10 patients (Lennox, 1945). Trimethadione’s success in the clinic and its ability to block threshold seizures induced by PTZ provided the necessary correlation to establish the PTZ test as a model of generalized absence seizures. With these observations, the current era of AED screening using the MES and scPTZ tests was launched. The pharmacologic profile of the MES and scPTZ tests does provide some insight into the potential clinical utility of drugs that are found to be active in one or both of these tests. For example, the pharmacologic profile of the MES test clearly supports its utility as a predictive model for human generalized tonic–clonic seizures. To date, all of the drugs that have demonstrated efficacy in the MES test and subsequently evaluated in the clinic have been found to possess activity against generalized tonic–clonic seizures. In contrast, the lack of any demonstrable efficacy by tiagabine, vigabatrin, and levetiracetam in the MES test argues against the utility of this test as a predictive model of partial seizures. Consistent with this conclusion is the observation that N-methyl-d-aspartate (NMDA) antagonists are effective against tonic extension seizures induced by MES; however, they were found to be without benefit in patients with partial seizures (Loscher and Honack, 1991). The positive results obtained in the scPTZ seizure test were historically considered suggestive of potential clinical utility against generalized absence epilepsy. This interpretation was based largely on the finding that drugs active in the clinic against spike-wave seizures (e.g., ethosuximide, trimethadione, valproic acid, the benzodiazepines) could block clonic seizures induced by scPTZ, whereas drugs such as phenytoin and carbamazepine, which were ineffective against spike-wave seizures, were also inactive in the scPTZ test. Based on this argument, phenobarbital, gabapentin, and tiagabine (TGB) should all be effective against spike-wave seizures and lamotrigine should be inactive against spikewave seizures. Clinical experience, however, has demonstrated that this is an invalid prediction. For example, the barbiturates, gabapentin, and tiagabine all aggravate spikewave seizure discharge, whereas lamotrigine has been found to be effective against absence epilepsy. As such, the overall utility of the scPTZ test in predicting activity against human spike-wave seizures is limited. Thus, from a drug-development perspective, positive results in the scPTZ test should be corroborated by positive findings in other models of absence such as the g-butyrolactone (Snead, 1992) seizure
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test, the genetic absence epileptic rat of Strasbourg (GAERS) (Marescaux and Vergnes, 1995), and the lethargic (lh/lh) mouse (Hosford et al., 1992; Hosford and Wang, 1997) before any conclusion concerning potential clinical utility against spike-wave seizures is made. As summarized in Table 2, the pharmacologic profile of these three additional models more reasonably predicts efficacy against spike-wave seizures than the scPTZ test. Another important advantage of all three of these models is that they accurately predict the potentiation of spike-wave seizures by drugs that elevated GABA concentrations (e.g., vigabatrin and TGB), drugs that directly activate the GABAB receptor, and the barbiturates. What, if any, advantages do the MES and scPTZ tests provide in the current AED discovery process? First, both tests provide some insight into the ability of a given drug to penetrate the blood–brain barrier and exert a central nervous system (CNS) effect. Furthermore, both models are nonselective with respect to mechanism of action. As such, they are well suited for the early evaluation of anticonvulsant activity because neither model assumes that a particular drug’s pharmacodynamic activity is dependent on its molecular mechanism of action. Lastly, both model systems display clear and definable seizure endpoints and require minimal technical expertise. This, coupled with lack of
dependence on molecular mechanism, makes them ideally suited to screen large numbers of chemically diverse entities. Beyond being amenable to high-volume screening, however, the MES and scPTZ tests fail to meet any of the remaining criteria described for the ideal model system. In addition, there are now several examples wherein the pharmacology of AEDs can be affected by the disease state and, because the MES and scPTZ tests are conducted in pathologically normal rodents, there is no guarantee that identified AEDs will be equally effective in pathologically abnormal rodents. For example, the MES and scPTZ tests failed to identify levetiracetam’s anticonvulsant activity. Subsequent investigations demonstrated that levetiracetam was active in pathologically abnormal models of partial and primary generalized seizures (Gower et al., 1992; Loscher and Honack, 1993; Gower et al., 1995; Klitgaard et al., 1998; Loscher et al., 1998). In this regard, levetiracetam appears to represent the first truly novel AED identified in recent years. The identification and subsequent development and launch of levetiracetam as an efficacious AED for the treatment of partial seizures demonstrates the need both for flexibility when screening for efficacy and to incorporate levetiracetam-sensitive models into the early evaluation process.
TABLE 2 Correlation between Anticonvulsant Efficacy and Clinical Utility of the Established and 2nd-Generation AEDs in Experimental Animal Modelsa Clinical Seizure Type
Experimental Model MES (Tonic Extension)b sc PTZ (Clonic Seizures)b Spike-Wave Dischargesd
Tonic and/or clonic generalized seizures
Myoclonic/generalized absence seizures
Generalized absence seizures
Partial seizures
CBZ, PHT, VPA, PB [FBM, GBP, LTG, TPM, ZNS] ESM, VPA, PBc, BZD [FBM, GBP, TGBc, VGBc] ESM, VPA, BZD [LTG, TPM, LVT]
Electrical Kindling (Focal Seizures)
CBZ, PHT, VPA, PB, BZD [FBM, GBP, LTG, TPM, TGB, ZNS, LVT, VGB]
Phenytoin-Resistant Kindled Rate
[LVT, GBP, TPM, FBM, LTG]
6 Hz (44 mA)f
VPA [LVT]
a BZD, benzodiazepines; CBZ, carbamazepine; ESM, ethosuzimide; FBM, felbamate; GBP, gabapentin; LTG, lamotrigine; LVT, levetiracetam; PB, phenobarbital; PHT, phenytoin; TGB, tiagabine; TPM, topiramate; VPA, valproic acid; ZNS, zonisamide; VGB, vigabatrin. b Data summarized from White et al., 2002. c PB, TGB, and VGB block clonic seizures induced by sc PTZ but are inactive against generalize absence seizures and may exacerbate spike wave seizures. d Data summarized from Snead, 1992; Marescaux and Vergnes, 1995; Hosford et al.; and Hosford and Wang, 1997. e Data summarized from Losher, 2002a. f Data summarized from Barton et al., 2001. [] 2nd-generation AEDs
The Current Era of Antiepileptic Drug Discovery
Low-Frequency (6 Hz) SeizureTest Levetiracetam clearly exemplifies why a need continues to identify and characterize new screening models to minimize the risk of missing other potentially novel AEDs. To this end, the Anticonvulsant Drug Development Program at the University of Utah is currently utilizing the 6-Hz psychomotor seizure model in its early identification studies (Figure 1; Barton et al., 2001; Barton et al., 2003). When the low-frequency (6 Hz), long-duration (3 seconds) corneal stimulation model was developed, the authors were attempting to validate the 6-Hz model as a screening test for partial seizures; however, the pharmacologic profile was not consistent with clinical practice (Brown et al., 1953). For example, phenytoin was found to be inactive in the 6-Hz seizure test. Given the observation that the 6-Hz model was no more predictive of clinical utility than the other models available at the time (i.e., the MES and scPTZ tests), it was virtually abandoned. Subsequent investigations in our laboratory confirmed the relative insensitivity of the 6-Hz test to phenytoin and extended the observation to include carbamazepine, lamotrigine, and topiramate (Barton et al., 2001). The relative resistance of some patients to phenytoin and other AEDs in today’s clinical setting and the lack of sensitivity of the MES and scPTZ to levetiracetam, prompted additional studies to reevaluate the 6-Hz seizure test as a potential screen for therapy-resistant epilepsy (Barton et al., 2001). Results from these studies suggest that the 6-Hz seizure test may offer some advantage over the MES and scPTZ tests. In particular, the pharmacologic profile of the 6-Hz seizure test has been demonstrated to differentiate itself from other acute seizure models (Barton et al., 2001). For example, as the stimulus intensity is increased from the CC97 (convulsive current required to evoke a seizure in 97% of the mice tested) to twice the CC97, the pharmacologic profile shifted from being relatively nondiscriminating to being highly discriminating (Table 3). For example, at the CC97 (22 mA), all TABLE 3 Effect of Stimulus Intensity on the Anticonvulsant Efficacy of Phenytoin, Lamotrigine, Ethosuximide, Levetiracetam, and Valproic Acid in the 6 Hz Seizure Texta ED50 (mg/kg, i.p.) and 95% C.I.b Antiepileptic drug
22mA
32mA
44mA
Phenytoin
9.4 (4.7–14.9)
>60
>60
Lamotrigine
4.4 (2.2–6.6)
>60
>60
167 (114–223)
>600
Ethosuximide
86.9 (37.8–156)
Levetiracetam
4.6 (1.1–8.7)
19.4 (9.9–36.0)
1089 (787–2650)
Valproic acid
41.5 (16.1–68.8)
126 (94.5–152)
310 (258–335)
a b
From Barton et al., 2001 with permission Confidence interval shown in ( )
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of the AEDs tested (phenytoin, lamotrigine, ethosuximide, levetiracetam, and valproic acid) were active at doses devoid of behavioral toxicity. At a current intensity 1.5 times the CC97 (32 mA), the 6-Hz seizure was resistant to phenytoin and lamotrigine, but sensitive to ethosuximide, levetiracetam, and valproic acid. As the stimulus current is further increased to 44 mA, the 6-Hz seizure becomes insensitive to ethosuximide and less responsive to levetiracetam and valproic acid. As such, the 6-Hz test may represent a potential therapy-resistant model wherein seizures can be acutely evoked in normal mice. Such a model would provide an inexpensive alternative to the extremely labor-intensive and expensive chronic models such as kindling.
Differentiation of Anticonvulsant Activity Once the efficacy of an investigational AED is established using either the MES, scPTZ, or 6-Hz seizure test, the NINDS-sponsored Anticonvulsant Drug Development Program at the University of Utah utilizes a battery of tests to characterize further the anticonvulsant potential of an active investigational AED. These include assessing the ability of the investigational AEDs to block audiogenic seizures in the Frings audiogenic seizure-susceptible mouse, limbic seizures in the hippocampal kindled rat, and acute clonic seizures induced by the GABAA receptor antagonist bicuculline and the Cl--channel blocker picrotoxin (White et al., 1995; White et al., 1998; White et al., 2002). This approach serves to define whether an active compound possesses a narrow or broad spectrum of activity and provides the sponsor with a sense of how its compound compares with prototype marketed compounds. Of the multitude of tests that might be conducted, the kindled rat is the only chronic model currently employed by most AED discovery programs. As summarized in Table 2, the kindled rat model offers perhaps the best predictive value of all of the tests described thus far. For example, it is the only model that adequately predicted the clinical utility of the first and second generation AEDs including tiagabine and vigabatrin. Furthermore, the kindled rat is the only model that accurately predicted the lack of clinical efficacy of NMDA antagonists (Loscher and Honack, 1993). Given its predictive nature, why is the kindled rat not utilized as a primary screen rather than a secondary screen for the early identification and evaluation of novel AEDs? The answer is primarily one of experimental logistics. Any chronic model such as kindling is extremely labor intensive and requires adequate facilities and resources to surgically implant the stimulating–recording electrode, to kindle, and to house sufficient rats over a chronic period of time. Furthermore, unlike the acute seizure models, the time required to conduct a drug study with a chronic model far exceeds the time required to conduct a similar study with any of the acute seizure models (e.g., MES, scPTZ, or 6-Hz seizure
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tests), thereby severely limiting the number of AEDs that can be screened in a timely manner. It is important to keep in mind that seizures are the ultimate clinical expression of any given patient’s epileptic disorder and, thus, prevention of seizures as an endpoint for any initial AED discovery program is not inappropriate. As such, the utilization of the MES, scPTZ, or any other acute seizure model to screen a structural series of candidate AEDs to identify and optimize a lead compound should be considered acceptable. To address whether the lead compound differentiates itself in terms of better tolerability, safety, and efficacy against therapy-resistant seizures requires additional testing in more appropriate model systems that would necessarily include kindling and refractory seizure models (see Chapter 45).
tional MES and subcutaneous Metrazol tests, yet it demonstrates excellent efficacy in the GAERS model of primary generalized seizures and in the kindled rat model (Klitgaard et al., 1998). Likewise, the efficacy of tiagabine and vigabatrin against human partial seizures was not predicted by the MES test, but by the kindled rat model (Rogawski and Porter, 1990; Suzdak and Jansen, 1995). Furthermore, as mentioned, exacerbation of spike-wave seizures would not have been predicted by the scPTZ test but by the other models (i.e., GHB, GAERS, and the lh/lh mouse) wherein both drugs have been shown to increase spike-wave discharges (Hosford and Wang, 1997). These examples serve to illustrate the limitations of some of the animal models while emphasizing their overall utility in predicting both clinical efficacy and potential seizure exacerbation. What is clear is the need to evaluate each investigational AEDs in a variety of seizure and epilepsy models. In addition, consider whether a given therapeutic displays age-dependent efficacy by utilizing an appropriate pediatric model or is effective in one or more of the emerging genetic epilepsy models. Only then will it be possible to gain a full appreciation of the overall spectrum of activity for a given investigational drug.
Moving Beyond the Current Approach Activity of a test substance in one or more of the electrical and chemical tests described provides some insight into the overall anticonvulsant potential of the compound. A concern voiced in recent years, however, is that the continued use of the MES and scPTZ tests in the early evaluation of an investigational AED are likely to identify “me too” drugs and are unlikely to discover those drugs with different mechanisms of action. A review of the data summarized in Table 2 clearly demonstrates the importance of employing multiple models in any screening protocol when attempting to identify and characterize the overall potential of a candidate AED substance. For example, levetiracetam is inactive in the tradi-
FUTURE OF AED DISCOVERY AND DEVELOPMENT Spontaneous Seizure Models As summarized in Table 4, a number of animal epilepsy models display spontaneous seizures several days to weeks
TABLE 4 Animal Models of Epileptogenesis and Secondary Hyperexcitability Neuronal degeneration
Mossy fiber sprouting
Chronic hyper-excitability
Latent period
Kindlinga
present
present
yes
nob
no
Status Epilepticusc (i.e., kainic acid, pilocarpine, Li+-pilocarpine, PPSd, SASe)
marked
marked
yes
days-weeks
yes
Traumatic brain Injury Cortical undercutf FeCl2g Fluid percussionh
yes yes yes
? ? yes
yes yes yes
yes yes yes
yes yes yes
Neonatal Hypoxiai
no
minimal
yes
yes
yes
Neonatal
no
no
yes
yes
yes
Model
Spontaneous seizures
Hyperthermiaj a
Data summarized from Goddard, 1967; Loscher, 1997; Dalby and Mody, 2001; McNamara, 1995 Spontaneous seizures do develop with prolonged kindling stimulation c Data summarized from Ben-Ari et al., 1979; Sloviter, 1991; Hellier and Dudek, 1999; Lado et al., 2000; Sankar et al., 2000; Dalby and Mody, 2001; Andre et al., 2001 and 2002; Pitkanen and Sutula, 2002 d Perforant path stimulation e Sustained amygdala stimulation f Data summarized from Sharpless and Halpern, 1962; Echling and Battista, 1963; Prince and Jacobs, 1998; Bush et al., 1999 i Data summarized from Jensen et al., 1992; Lauer and McIntosh, 1999; Santhakumar et al., 2001; Golari et al., 2001; Kharatishvili et al., 2004 j Data summarized from Baram et al., 1997; Toth et al., 1998; Dube et al., 2000; Lado et al., 2000; Baram et al., 2004 b
Future of AED Discovery and Development
postinsult. In this regard, these models fulfill an important characteristic of the ideal model system (i.e., spontaneous recurrent seizures [SRS] following a species-appropriate latent period). As discussed, all of the AEDs developed to date were initially identified using one or more of the established evoked seizure models. In some cases, the active drug may have been evaluated in the kindled rat model of partial seizures. None of the AEDs available to date, however, were tested against SRS in animal models of partial epilepsy until well after their subsequent development (Leite and Cavalheiro, 1995; Glien et al., 2002; Brandt et al., 2004; Grabenstatter and Dudek, 2004). The first study to evaluate the pharmacology of SRS was that of Leite and Cavalheiro (1995). These investigators demonstrated that high doses of phenobarbital (40 mg/kg), carbamazepine (120 mg/kg), phenytoin (100 mg/kg), and valproic acid (600 mg/kg), but not ethosuximide (400 mg/kg), blocked spontaneous recurrent seizures in the pilocarpine poststatus model when administered over a 14-day period. Albeit an interesting and heroic study given the time commitment required, their results are not consistent with clinical experience wherein 25% to 40% of patients are refractory to these and other AEDs. Whether the efficacy that was observed was the result of the high doses employed is difficult to state; nonetheless, the central goal of therapy is to obtain complete seizure control at nontoxic doses. Two subsequent studies in two different temporal lobe seizure models (i.e., pilocarpine and sustained amygdala stimulation) showed that individual animal responses to two different drugs, levetiracetam and phenobarbital, differed markedly. For example, when plasma drug levels were maintained within the same range in all rats, they observed complete control of SRS in some rats, whereas no effect was seen in others (Glien et al., 2002; Brandt et al., 2004). Results from yet another study (published in abstract form) using a repeated-measures, cross-over treatment protocol, supports the idea that the evaluation of an AED against SRS may be not only feasible, but yield valuable information to support the clinical development of an investigational AED (Grabenstatter and Dudek, 2004). In this particular study, the investigational AED RWJ-333369 was found to dose-dependently reduce the relative seizure frequency of SRS in the postkainate status model of temporal lobe epilepsy at nontoxic doses. Furthermore, an excellent correlation was noted between degree of seizure protection and measured plasma concentration. Perhaps of even more interest was the finding that RWJ-333369 was more efficacious than the marketed AED topiramate when tested using the same model and methods (Grabenstatter et al., 2005) (i.e., RWJ-333369 produced a greater reduction in seizure frequency than topiramate). A dose of 30 mg/kg topiramate reduced relative seizure frequency by approximately 50%, whereas a dose of 30 mg/kg RWJ-333369 suppressed 75% of the convulsive seizures. Moreover, a greater percentage
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of seizure freedom was also observed in the RWJ-333369 versus the topiramate group. These data support the hypothesis that a repeated-measures, cross-over protocol is an effective method for testing AEDs in animal models with spontaneous seizures and may be useful in identifying new AEDs for pharmacoresistant epilepsy and in developing adjunct therapies (Grabenstatter and Dudek, 2004). Collectively, these examples clearly suggest that it is possible to establish a model wherein the pharmacology of the preclinical model mimics the human situation (e.g., the existence of drug responders and nonresponders). They further suggest that such models may be used to evaluate newer AEDs for efficacy against difficult-to-treat seizures. Although costly and labor intensive, this approach should be considered when attempting to differentiate a new drug from those that are currently on the market.
Genetic Epilepsy Models In addition to the two genetic spike-wave seizure models already discussed (i.e., GAERS and lh/lh mouse), a number of other potentially useful genetic models could be used in the search for novel therapies. These are discussed in detail in chapters 16, 17, 18, 19, 20, and 21. In addition to those models that evolved spontaneously, a number of genetic mutations have been identified to date which result in an epileptic phenotype or an altered seizure threshold. Most of these mutations lie in genes that encode receptor-gated and or voltage-sensitive ion channels. The ability to use genetic tools to create transgenic mice singlegene models of epilepsy present excellent opportunities to test molecular mechanisms thought to underlie the development of epilepsy and to test specific therapies against seizures induced by a known molecular defect. With the exception of perhaps the GAERS rat, none of the genetic models available today are routinely utilized in the systematic search for new therapies. Until they do become more widely utilized for the drug-development process, it is difficult to say which is most predictive of human epilepsy. It is highly likely that each will provide unique information; however, the big question is whether any one model will lead to the development of a truly novel antiepileptic drug.
Pediatric Animal Models The mechanisms underlying seizures in the developing brain can be different than in the adult. In addition, therapies that may be effective in treating seizures in the adult brain may not be appropriate in the developing brain. Furthermore, many types of catastrophic epilepsies of children are resistant to currently available anticonvulsants. Therefore, as was stressed in a recent NIH sponsored workshop, “Workshop on Pediatric Epilepsy Models,” held May 13 to
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14, 2004, an important, unmet need remains to utilize, as well as to continue to develop, pediatric models of epilepsy while investigating novel approaches to the treatment of epilepsy. It is anticipated that progress in identifying genes that underlie a variety of pediatric epilepsies (e.g., tuberosclerosis, Benign Familial Neonatal Convulsions) will eventually lead to a better understanding of the etiology of these seizure disorders as mouse models of the disease are developed using such techniques as conditional knockouts, knockins, and transgenic animals. In addition to the emerging genetic mouse models, however, currently a few rodent models of pediatric seizures might prove useful in identifying novel therapeutic approaches to the symptomatic treatment of epilepsy. One such animal model of pediatric seizure disorders is the flurothyl inhalation model (Velisek et al., 1995b). Following inhalation, generalized clonic seizures can be induced in rats as early as postnatal day 9. Therefore, this model represents a simple, yet useful screening model for therapies effective in juvenile forms of generalized epilepsy. Although the mechanism through which flurothyl induces seizures is still unknown, it is a validated screening model in that anticonvulsants that are effective against flurothyl seizures are also effective in human epilepsy. Interestingly, the pharmacology of the flurothyl model demonstrates several examples in which the efficacy of certain anticonvulsants (e.g., phenytoin and ganaxolone) is altered throughout the course of development (Velisek et al., 1995b; Liptakova et al., 2000). These findings clearly indicate the need to investigate the efficacy of anticonvulsants through a range of ages. The kindling model has also been utilized in juvenile rats and reliable kindling can occur after about postnatal day 7 (Velisek et al., 1995a). Kindling under these circumstances must be rapidly induced via multiple stimulations in a single day because the young rat grows so quickly that the relative placement of the stimulating–recording electrodes are not conserved through development. Using this approach, Lado and colleagues (2001) demonstrated that gabapentin exerts an anticonvulsant effect in the immature rat against the fully expressed kindled seizure at doses that do not affect motor function. This study demonstrates the potential utility of the rapid amygdala kindling of juvenile rats for the early screening of investigational AEDs. In addition, two age-specific models of hyperexcitability are worthy of mention (i.e., neonatal hyperthermia- and neonatal hypoxia-induced seizures) (Jensen et al., 1995; Toth et al., 1998; Dube et al., 2000; Lado et al., 2000). These particular models have provided valuable insight into the functional and pathologic consequences associated with an age-specific insult. Little is currently known about their pharmacologic profile and potential utility for therapy discovery.
Development of Pharmacoresistant Epilepsy Models A need remains to identify therapies that will be more effective for therapy-resistant seizures. As such, a need also continues to identify and incorporate more appropriate models of refractory epilepsy into the AED screening process. Currently, several model systems could be suggested, including the phenytoin-resistant kindled rat (Loscher et al., 2000), the carbamazepine-resistant kindled rat (Nissinen et al., 2000), the 6-Hz psychomotor seizure model (Brown et al., 1953; Barton et al., 2001), poststatus epilepticus-induced spontaneous seizures (Glien et al., 2002; Brandt et al., 2004), and the in vitro low magnesium hippocampal slice preparation (Armand et al., 2000). It will take the successful clinical development of a drug with demonstrated clinical efficacy in the management of refractory epilepsy before any one of these (or other) model systems will be clinically validated. Nonetheless, this should not prevent the community at large from continuing the search for a more effective therapy using the models that are available. In fact, until a validated model exists, it becomes even more important to characterize and incorporate several of the available models into the drug discovery process while continuing to identify new models of refractory epilepsy. (See chapter 45 for a more detailed discussion of the utility of existing models of pharmacoresistance for therapy discovery.)
Disease Modification Currently, no known therapies can modify the course of acquired epilepsy. Attempts to prevent the development of human epilepsy following febrile seizures, traumatic brain injury, and craniotomy with the older established drugs have been disappointing (Temkin, 2001). A greater understanding of the pathophysiology of acquired epilepsy at the molecular and genetic level, however, may lead to the development of new therapeutic approaches that reach beyond the symptomatic treatment of epilepsy to modify the progression, or possibly even prevent, the development of epilepsy in the susceptible patient. The realization of such possibilities will necessitate a change in our current AED discovery approach. As discussed, the existing approach is effective for identifying drugs that are effective for treating seizures, but is not likely to be effective in identifying the disease-modifying therapeutic. This, in part, is because the community at large is just beginning to utilize existing models of acquired epilepsy to address whether a particular drug might show diseasemodifying properties (e.g., slow or prevent the development of epilepsy following a given insult or prevent the emer-
547
References
gence of therapy resistance or cognitive decline associated with epilepsy) (Loscher, 2002b, 2002a; Walker et al., 2002; Pitkanen, 2004). Several animal models of epileptogenesis share many similarities with human epilepsy (Table 4). In addition to their value for understanding the molecular biology of epilepsy, these animal models are also extremely valuable for assessing the potential disease-modifying potential of existing and future drugs. Currently, several of the chronic animal models display spontaneous seizures secondary to a particular (Loscher, 2002b, a; White et al., 2002). Although we know a lot about the models themselves, little is known about their overall relevance to the human condition. According to the recommendations set forth from the 2002 NIH/NINDS/AES Models II Workshop, the existing and future models should be utilized to (1) verify the lack of efficacy reported in human trials; (2) determine intervention points in the models that are clinically relevant and realistic (e.g., after an injury that has a high risk of resulting in epilepsy and after the first seizure, when the chances for development of chronic epilepsy are the highest); (3) utilize the models to define outcome measures (e.g., prevention of epilepsy, or improved behavioral or cognitive outcomes, and the prevention of pharmacoresistance or neuronal loss); and (4) develop treatment protocols that will provide a consistent approach by all investigators (Stables et al., 2003). If we are to be successful in identifying a novel, disease-modifying therapy in the near future, we must become intentional in our efforts to characterize and incorporate such models of epileptogenesis into our current screening protocols. As summarized in Table 4, specific models of epileptogenesis include kindling (McNamara, 1995; Loscher, 1997; Dalby and Mody, 2001), status epilepticus (Sloviter, 1991; Andre et al., 2000; Lado et al., 2000; Sankar et al., 2000; Andre et al., 2001; Dalby and Mody, 2001; Pitkanen and Sutula, 2002), and traumatic brain injury (Sharpless and Halpern, 1962; Echlin and Battista, 1963; Willmore et al., 1978; Lowenstein et al., 1992; Laurer and McIntosh, 1999; Engstrom et al., 2001; Golarai et al., 2001; Santhakumar et al., 2001; Kharatishvili et al., 2004). All three of these models display varying degrees of cell loss, synaptic reorganization, network hyperexcitability, and a latent period that is followed by the expression of spontaneous seizures, thereby each fulfill some important characteristics of a model of acquired partial epilepsy. Clearly, comparative data are urgently needed from these and other models of acquired epilepsy before any conclusions regarding their overall utility can be assessed. In this regard, it is important to note that each model system will bring certain strengths to epilepsy research and therapy development for both the symptomatic treatment of seizures and epilepsy disease modification.
SUMMARY This chapter has focused on the current process used by the University of Utah Anticonvulsant Drug Development Program to evaluate the anticonvulsant efficacy of an investigational AED submitted to the NINDS Anticonvulsant Screening Project for the symptomatic treatment of epilepsy. The brief overview of the model systems utilized by this program are by no means meant to suggest that this is the only approach that can be used when screening for anticonvulsant efficacy. In this review, an attempt has been made to identify and discuss the advantages and limitations of this approach and the various animal model systems that are currently employed. An effort has also been made to offer a look into the future at how the incorporation of additional models might lead to the identification of a truly novel AED. In this regard, an ever-pressing need exists to continue the search for drugs that will be more effective for the patient with therapy-resistant epilepsy. This latter approach requires the identification and characterization of numerous model systems and the subsequent development of a highly efficacious therapy in the pharmacoresistant patient population before any recommendations regarding appropriate models of pharmacoresistance can be offered. Lastly, therapeutics designed to modify the course of epilepsy or prevent the development of epilepsy in the susceptible patient requires testing in appropriate animal models. Numerous models of acute status epilepticus display many similarities with the human condition, including pathology, a species-appropriate latent period, and the development of spontaneous seizures following the latent period. Among the various syndrome-specific models, several subtle and gross differences necessitate their continued evaluation to access their validity to the human condition and their appropriateness for therapy discovery. True validation of any given model of epileptogenesis, however, necessitates the development of an effective therapy that prevents or delays the development of epilepsy or secondary hyperexcitability in the human condition and whose activity was successfully predicted by preclinical testing.
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45 Animal Models of Drug-Refractory Epilepsy WOLFGANG LÖSCHER
In at least 30% of patients with epilepsy, the seizures persist despite the choice of an adequate antiepileptic drug (AED) and carefully monitored treatment. In most of these patients, neither monotherapy with other AED nor combination therapy with two or more AED leads to seizure control, so that these patients are considered resistant to AED therapy. The mechanisms underlying such pharmacoresistence are only poorly understood. Animal models of epilepsy allowing selection of pharmacoresistant and pharmacosensitive subgroups of animals would be a valuable tool to study mechanisms of intractability and to develop more efficacious treatment strategies. Currently, only one model with these characteristics has been extensively explored (i.e., amygdala-kindled Wistar rats) from which phenytoin-resistant and phenytoin-sensitive rats can be selected by repeated testing with phenytoin. Phenytoin-nonresponders are also resistant to various other AED, thus paralleling the clinical situation of multidrug-resistant temporal lobe epilepsy. One major drawback of this model, however, is that kindled rats do not exhibit spontaneous recurrent seizures, so that elicited seizures have to be used for drug studies. This is also true for the 6-Hz “psychomotor” seizure model in mice, which has been proposed to provide a useful model of therapy-resistant limbic seizures. Some recent studies have indicated that rats with spontaneous recurrent seizures developing after a status epilepticus differ in their individual response to AED, thus allowing selection of drug-refractory and drug-responsive subgroups. This group of models has the greatest parallels with a common form of human drug-resistant epilepsy, the mesial temporal lobe or limbic epilepsy syndrome. Thus, a number of potentially interesting models of drug-refractory epilepsy are available, but these models require further characterization and validation.
Models of Seizures and Epilepsy
INTRODUCTION More than 30% of patients with epilepsy have inadequate control of seizures despite the choice of an adequate antiepileptic drug (AED) and carefully monitored treatment (Regesta and Tanganelli, 1999; Kwan and Brodie, 2000; Stables et al., 2003). Although the terms—drug-refractory, pharmacoresistant, or medically intractable—lack a precise definition, most clinicians would consider as pharmacoresistant an epilepsy that had not been completely controlled by any of two to three first-line AED usually prescribed for a given epilepsy syndrome. Although partial suppression of seizures can be considered a statistical success, it often does not result in significant benefit to the patient’s overall condition or quality of life (Stables et al., 2003). The probability of intractability largely depends on the type of seizures and epilepsy, with complex partial seizures such as occurring in temporal lobe epilepsy (TLE) having the poorest prognosis of all seizure types in adults (Regesta and Tanganelli, 1999). Drug-intractable epilepsy is a major health problem, associated with increased morbidity and mortality, and accounting for much of the economic burden of epilepsy (Regesta and Tanganelli, 1999). The problem of intractable or difficult-to-treat seizures has not been changed to any significant extent by the recent introduction of various new AED, although drug treatment has become better tolerable for a number of patients (Regesta and Tanganelli, 1999; Löscher, 2002a). A major obstacle in developing new strategies for treatment of drug-refractory epilepsy is that mechanisms of refractoriness are only poorly understood. Some clinical features are associated with pharmacoresistance, including early onset of seizures (before 1 year of age), high
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Chapter 45/Animal Models of Drug-Refractory Epilepsy
seizure frequency before onset of treatment, a history of febrile seizures, the type of seizures (~60% of patients with intractable epilepsy suffer from partial seizures) or of epilepsy, structural brain lesions, and malformations of cortical development (Regesta and Tanganelli, 1999). Relatively little research, however, has been undertaken into the basis of these associations. Many possible causes of refractory epilepsy exist, so that it is likely to be a multifactorial process (Regesta and Tanganelli, 1999; Sisodiya, 2003). Genetic factors (e.g., gene polymorphisms) may be important and explain why two patients with the same type of epilepsy or seizures can differ in their response to AED. Disease-related factors are certainly important, including the etiology of the seizures, progression of epilepsy under treatment with AED, alterations in drug targets, or alterations in drug uptake into the brain. Furthermore, drug-related factors are most likely involved in insufficient seizure control, including loss of anticonvulsant efficacy during treatment (i.e., development of tolerance) or ineffective mechanisms of action of currently available AED in patients with medically intractable epilepsy. An important characteristic of pharmacoresistant epilepsy is that most patients with refractory epilepsy are resistant to most, and often all, AED (Regesta and Tanganelli, 1999; Kwan and Brodie, 2000). As a consequence, patients whose epilepsy is not controlled on monotherapy with the first AED have a chance of only ~10% to 15% to have it controlled by other AED, even when using AED that act by diverse mechanisms. This argues against epilepsy-induced alterations in specific drug targets as a major cause of pharmacoresistent epilepsy, but rather points to nonspecific and possibly adaptive mechanisms, such as decreased drug uptake into the brain by seizure-induced overexpression of multidrug transporters in the blood–brain barrier (Löscher and Potschka, 2002; Sisodiya, 2003). Despite the problem of intractable epilepsy, only few animal models are specifically dedicated to identifying effective therapeutic agents for resistant epilepsy or to studying mechanisms of drug resistance (Löscher, 1997; Löscher, 2002b; White, 2003). An animal model of epilepsy allowing selection of subgroups of animals with drug-refractory and drug-responsive seizures could be a valuable tool to study why and how seizures become intractable and to develop more effective treatment strategies. We became interested in developing such an animal model almost 20 years ago (cf., Löscher, 1986), leading to the discovery of phenytoin-resistant subgroups of amygdala-kindled Wistar rats (Löscher and Rundfeldt, 1991). Other potentially promising, but as yet not extensively characterized, models of drug-refractory seizures include the 6-Hz corneal stimulation seizure model, originally described as a model of “psychomotor seizures” by Toman (1951) and recently proposed as a model of drug-resistant partial epilepsy (Barton et al.,
2001), and post-status epilepticus (SE) models of mesial TLE, in which part of the animals develop spontaneous recurrent seizures not responding to AED (Glien et al., 2002; Brandt et al., 2004). This chapter primarily deals with these three types of models of drug-refractory partial epilepsy, but a number of additional models will also be described. With respect to the models described and discussed, it is important to consider that the term “pharmacoresistant” applied in the context of animal models, based on experience in patients with epilepsy, can be minimally defined as persistent seizure activity not responding or with very poor response to monotherapy with at least two current AED at maximal tolerated doses (Stables et al., 2003). A goal should be to develop animal models of drug-refractory epilepsy that reflect the most common types of intractable or difficult-totreat epilepsy in humans. Almost all of the currently available models are models of TLE.
ANTIEPILEPTIC DRUG-RESISTANT KINDLED RATS Kindling refers to the phenomenon that animals chronically implanted with a stimulation electrode in one structure of the limbic system or other brain areas (the amygdala being among the most responsive structures) develop focal and secondarily generalized seizures of increasing severity and duration on periodic electrical stimulation with an initially subconvulsive current. Since its introduction in 1969 by Goddard et al., kindling has become one of the most widely used animal model of epilepsy, particularly because the mechanisms involved in kindling are thought to be relevant for development of TLE, the most common and difficult-to-treat type of epilepsy in adults. We first proposed amygdala-kindling as a model to investigate intractable epilepsy in 1986 (Löscher, 1986; Löscher et al., 1986). By directly comparing standard AED in the amygdala-kindling model and the standard maximal electroshock seizure (MES) test in age-matched female Wistar rats, we found that kindled seizures were less sensitive to anticonvulsant treatment than primarily generalized seizures as produced in the MES test. Furthermore, in the kindling model, focal seizure stages were found to be much less responsive to AED than secondarily generalized seizures, which is consistent with clinical experience. We proposed that search for novel compounds with high potency in the amygdala-kindling model may be a promising strategy in the development of new AED for patients with intractable epilepsy (Löscher, 1986; Löscher et al., 1986; Löscher and Schmidt, 1988). In follow-up studies in the amygdala-kindling model, using female rats of the Wistar outbred strain, we found that the individual response of fully kindled rats to phenytoin differs, that is that kindled seizures in some animals consis-
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Antiepileptic Drug-Resistant Kindled Rats
tently respond and others never respond to phenytoin (Rundfeldt et al., 1990). This finding was systematically explored by determining the effect of phenytoin on the threshold for induction of afterdischarges (ADT) (i.e., the threshold for induction of focal seizure activity in kindled rats). Phenytoin was repeatedly tested in large groups of fully kindled rats to prove the reproducibility of its effect on ADT in individual animals. In a first study with 52 female kindled rats (Löscher and Rundfeldt, 1991), phenytoin reproducibly increased the ADT in 21% (phenytoin responders), induced variable effects (i.e., an increase in one trial but no increase in another trial, or vice versa) in 58% (variable responders), and never increased the ADT in another 21% of kindled rats (phenytoin nonresponders). The difference in response to phenytoin between responders and nonresponders was dramatic in that average ADT increases induced by phenytoin in responders at plasma concentrations of 25 to 30 m/ml were between 400% and >1000% above individual predrug control values, whereas no increase or even slight decreases in ADT were determined in nonresponders at the same phenytoin plasma concentration range. Interestingly, phenytoin responders and nonresponders did not differ in kindling acquisition or the severity and duration of their fully kindled seizures. No prognostic measure was found by which it could be predicted if a given kindled rat would respond or not respond to treatment with phenytoin. In a subsequent prospective study, it was investigated if the original finding of phenytoin-resistant and nonresistant kindled animals was reproducible among another group of 68 fully amygdala-kindled Wistar rats (Löscher et al., 1993). Phenytoin was tested four times in each fully kindled animal at intervals of at least 5 days. Of the rats, 12% were nonresponders, another 12% were responders, and the remaining animals were variable responders. Thus, we could reproduce the original finding, although the frequency of responders
and nonresponders differed between the two studies. Because all rats had had the same number of kindled seizures before phenytoin application, the difference in sensitivity to phenytoin between responders and nonresponders was certainly not caused by the period of time over which the animals had experienced repetitive seizures without treatment. We concluded that kindled rats with phenytoin resistance are a unique resource for the investigation of mechanisms for drug resistance in epilepsy, particularly because pathophysiologic processes in phenytoin-resistant rats can be directly compared with those of kindled rats that reproducibly respond to this drug (Löscher and Rundfeldt, 1991; Löscher et al., 1993). In recent years, we have repeated the selection of phenytoin responders and nonresponders in kindled female Wistar rats several times, using either phenytoin or its prodrug fosphenytoin for selection. Average data from more than 200 rats show a consistent anticonvulsant response to phenytoin in only 16% of the animals, no anticonvulsant response in 23%, and a variable response in the remaining 61% (Löscher, 1997; Löscher, 2002b). This subgroup selection does not depend on gender, but phenytoin responders and nonresponders could also be selected from male Wistar rats. A schematic illustration of the experimental protocol used for selection of responders and nonresponders is shown in Figure 1. An example for ADT data and plasma drug levels from such subgroups is shown in Figure 2. Based on our data, we suggest that the three subgroups of amygdala-kindled rats model three different clinical scenarios. The nonresponder subgroup models drug-refractory patients with TLE in whom AED treatment does not significantly reduce seizure frequency. The variable responder group models patients in whom AED treatment reduces seizure frequency but does not achieve complete control of seizures. The responder subgroup models patients who achieve complete control of seizures during AED treatment.
Drug trial in fully kindled Post-surgical
rats
recovery (~2 weeks)
~2 weeks
~3 weeks
Implantation of kindling electrodes
Daily kindling stimulations
Fully kindled state
~ 4-6 weeks
Repeated
Repeated
determination
testing of phenytoin
of control ADT
on ADT (once/week) Blood samples for drug analysis in plasma
Phenytoinresponders and nonresponders
Use of drug-refractory and drug-responsive subgroups for additional experiments
FIGURE 1 Schematic illustration of selection of drug-refractory (nonresponders) and drug-responsive (responders) rats from the kindling model by repeated testing with phenytoin.
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Chapter 45/Animal Models of Drug-Refractory Epilepsy
200 150
*
*
*
100 50 0
C Focal seizure threshold (µA)
Saline Phenytoin
1. Trial
2. Trial
150 100 50
1. Trial
2. Trial
200
3. Trial
*
*
*
150 100 50
D
1. Trial
2. Trial
3. Trial
Plasma levels of phenytoin Responders Nonresponders
50
Saline Phenytoin
200
Responders (n = 16)
250
0
3. Trial
Nonresponders (n = 10)
250
0
Focal seizure threshold (µA)
250
B
All rats (n = 52)
Drug plasma levels (µg/ml)
Focal seizure threshold (µA)
A
40 30 20 10 0
1. Trial
2. Trial
3. Trial
FIGURE 2 Example of a selection of drug-refractory (nonresponders) and drug-responsive (responders) rats from the kindling model by repeated testing with the phenytoin prodrug fosphenytoin. A total of 52 fully kindled, female Wistar rats were used in this experiment. The anticonvulsant effect of a maximal tolerated intraperitoneal (i.p.) dose of fosphenytoin (83.5 mg/kg) was tested by single-dose administration once per week by determining the focal seizure threshold (ADT) in each rat 1 hour after drug injection. Control thresholds were determined in each rat before each drug trial, using i.p. injection of saline. All data are shown as means ± SEM. Significant differences to control threshold are indicated by asterisk (P < .001). After each drug injection, blood was sampled for drug analysis in plasma. Only drug trials in which phenytoin plasma levels were within or near to the therapeutic range in patients with epilepsy (~25 to 30 mg/ml) were used for final evaluation of data. Of the 52 rats shown in A, 16 rats always responded with a significant ADT increase (B; responders), whereas 10 rats never showed such an anticonvulsant effect (C; nonresponders). The remaining 26 rats exhibited variable effects to phenytoin (not illustrated). As shown in D, plasma levels of phenytoin were not different between responders or nonresponders. ADT, afterdischarge threshold. Data from Löscher, W., et al, 2002b.
For all subsequent studies to be described, we only used the responder and nonresponder subgroups of kindled Wistar rats.
Pharmacoresistent Kindled Rats As a Tool To Evaluate Antiepileptic Drugs Following the identification of phenytoin-resistant kindled Wistar rats, most clinically available AED were tested in such animals. Results are shown in Table 1. Except the novel drug, levetiracetam, all AED were significantly less efficacious or not efficacious at all in phenytoin nonresponders compared with phenytoin responders, demonstrating that the phenytoin resistance of a subgroup of kindled Wistar rats extends to various other old and new AED. This reflects
the clinical situation in patients with TLE, because most patients who are refractory to one AED are also resistant to other AED, including newly developed drugs (Regesta and Tanganelli, 1999; Kwan and Brodie, 2000). Whether levetiracetam has advantages in this respect remains to be determined, but a recent clinical trial in 120 patients with drug-resistant epilepsy, who had tried at least three to four other AED before levetiracetam was instituted, demonstrated an impressive and sustainable seizure freedom rate (32%) under treatment with levetiracetam (Betts et al., 2003). The high efficacy of levetiracetam in phenytoinresistant kindled rats suggests that the mechanisms underlying the AED refractoriness in this model do not affect the antiepileptic activity of levetiracetam, which will be discussed in more detail.
Antiepileptic Drug-Resistant Kindled Rats
TABLE 1 Anticonvulsant efficacy of old and new antiepileptic drugs in kindled Wistar rats selected by their response to phenytoin into nonresponders and responders After kindled rats had been selected into responders and nonresponders by repeated testing with phenytoin as shown in Fig. 1, each of the other drugs shown in the table was tested in at least two different doses in phenytoin-responders and -nonresponders. The anticonvulsant effect was determined by increase in focal seizure threshold (ADT) compared to control threshold in the same rats. Groups of 8–10 rats were used for each drug trial. All drugs shown in the table significantly increased ADT in phenytoin-responders. Loss of efficacy in nonresponders is indicated by comparing the drug-induced ADT increase in nonresponders with that obtained in responders. Except levetiracetam, all drugs were less efficacious (by at least 50%) in phenytoin-nonresponders compared to responders.
Drug Phenytoin
Loss of anticonvulsant efficacy against focal seizures in nonresponders (in % compared to responders)
Reference
100%
Löscher and Rundfeldt, 1991
Carbamazepine
51%
Löscher and Rundfeldt, 1991
Phenobarbital
85%
Löscher et al., 1993
Valproate
79%
Löscher et al., 1993
Vigabatrin
46%
Löscher et al., 2000
Lamotrigine
60%
Ebert et al., 2000
Felbamate
75%
Ebert et al., 2000
Topiramate
61%
Reismüller et al., 2000
52%
Löscher et al., 2000a
No loss of efficacy
Löscher et al., 2000a
Gabapentin Levetiracetam
Effects of Chronic Drug Administration in the Kindling Model All drug studies in amygdala-kindled rats described above were performed with administration of single doses of AED, and ADT was determined at one time point after acute drug administration. In patients with epilepsy, drugrefractoriness of seizures is usually seen during chronic administration of AED. This prompted us to study the effects of chronic treatment with phenytoin in kindled rats with different responses to acute administration of phenytoin (Rundfeldt and Löscher, 1993). To allow maintenance of therapeutic plasma levels during chronic treatment in rats, the striking differences in pharmacokinetics of AED between humans and rodents have to be considered (Löscher and Schmidt, 1988). Phenytoin exhibits dose-dependent elimination in different species, including rats, so that a true half-life cannot be calculated. For instance, elimination halflife of phenytoin is ~1.5 hours after intraperitoneal (i.p.)
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administration of 50 mg/kg in female Wistar rats, but ~4 hours after i.p. administration of 75 mg/kg (Rundfeldt and Löscher, 1993). Because of long-lasting saturation of phenytoin-metabolizing enzymes, elimination rate is significantly decreased during repeated drug administration (Rundfeldt and Löscher, 1993). Based on these observations, we developed a dosing regimen that allows maintenance of effective phenytoin concentrations in female Wistar rats (Rundfeldt and Löscher, 1993). Treatment was started with administration of 75 mg/kg on day 1, followed by 50 mg/kg once daily on all subsequent days of the treatment period. During a treatment period of 2 weeks, some but not all rats that responded to phenytoin at onset of drug administration developed tolerance to phenytoin’s anticonvulsant effect during chronic treatment (Rundfeldt and Löscher, 1993). Individual differences were seen in the chronic efficacy of phenytoin that were not secondary owing to pharmacokinetics. Nevertheless, most phenytoin responders remained protected throughout the chronic treatment period (Rundfeldt and Löscher, 1993), indicating that the initial response to phenytoin was a predictor of chronic anticonvulsant efficacy.
Mechanisms of Drug Resistance in Kindled Rats Various factors that could be important for our finding of pharmacoresistent subgroups of amygdala-kindled Wistar rats were studied since the first description of this model (Löscher, 1997; Ebert et al., 1999; Löscher, 2002b). These studies showed that pharmacoresistence is not caused by differences in the location of the kindling electrode in the amygdala, drug pharmacokinetics, seasonal variations in drug response, or the sex of the animals (i.e., phenytoin nonresponders could also be selected from male Wistar rats). To examine the possible involvement of genetics in the differential effects of phenytoin found in kindled rats, we undertook breeding studies with phenytoin responders and nonresponders, using male and female Wistar rats (Ebert and Löscher, 1999). Altogether, four generations of kindled Wistar rats were studied. The data suggest that the ability to respond or not to respond to phenytoin is genetically determined, although it does not follow a simple scheme of inheritance (Ebert and Löscher, 1999). The involvement of genetics in the different drug sensitivity in responders and nonresponders selected from kindled Wistar rats was substantiated by a study in which we tested phenytoin in six other outbred and inbred rat strains (i.e., Sprague-Dawley, Wistar-Kyoto, Lewis, Fischer 344, ACI, and Brown Norway) (Cramer et al., 1998; Löscher et al., 1998). The only strain in which nonresponders could be selected was Brown Norway. In contrast to Wistar outbred rats, however, no responders could be selected in the Brown Norway strain, so that Wistar rats were the only
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strain allowing selection of nonresponders and responders. All subsequent experiments, thus, were done in the Wistar strain. Besides genetics, another possible explanation for the development of different pharmacosensitivity in kindled rats would be the kindling process itself. Epileptic patients often initially respond to an AED, but this effect may be lost with increasing duration of the disease (i.e., when epilepsy becomes chronic). To address the influence of kindling on the anticonvulsant response to phenytoin, we tested phenytoin’s anticonvulsant effect on ADT before and after kindling in the same rats. Following kindling, rats were repeatedly tested with phenytoin to allow subgroup selection. Unexpectedly, in rats that were nonresponders after kindling, phenytoin exerted a significant anticonvulsant effect before kindling (Löscher et al., 2000b). This study indicates that kindled phenytoin nonresponders become nonresponders, at least in part, through the kindling process (i.e., kindling-induced brain alterations). In view of the results of our breeding studies in Wistar rats (Ebert and Löscher, 1999), the genetic background of an individual rat seems to determine whether it becomes a responder or nonresponder by kindling-induced limbic epileptogenesis. Which kindling-induced brain alterations could determine whether a rat becomes a responder or nonresponder to an AED such as phenytoin? We currently concentrate on the three following possibilities. Alterations in Brain Targets of Antiepileptic Drugs It has been proposed that the mechanisms underlying pharmacoresistence in TLE most likely involve the functional and morphologic changes developing in regions such as the hippocampus in the course of the disease (Heinemann et al., 1994). Drugs of primary choice for treatment of TLE (e.g., phenytoin or carbamazepine) are thought to act via modulation of voltage-activated sodium and calcium channels (Rogawski and Löscher, 2004). It was previously shown that the properties of these channels change in the hippocampus of patients with therapy-refractory TLE (Beck et al., 1997; Beck et al., 1998; Reckziegel et al., 1998), which could explain the loss of therapeutic efficacy of major AED. Indeed, Wadman et al. found an impaired modulation of sodium current inactivation by carbamazepine in hippocampal CA1 neurons from patients with pharmacoresistant TLE that was associated with hippocampal sclerosis (Vreugdenhil et al., 1998). More recently, Remy et al. (2003a) reported that the mechanism of action of carbamazepine, use-dependent block of voltage-dependent sodium channels, is completely lost in dentate gyrus granule cells from carbamazepine-resistant patients with epilepsy. A similar loss of carbamazepine’s effect was also found in the pilocarpine model in rats (Remy et al., 2003b). Similarly, a significant reduction in carbamazepine’s effect on sodium
channels was determined in hippocampal CA1 neurons of kindled rats (Vreugdenhil and Wadman, 1999). The reduction in carbamazepine’s effect was only transient in kindled rats (Vreugdenhil and Wadman, 1999). Furthermore, carbamazepine is a very potent and efficacious anticonvulsant when systemically administered in kindled rats (Löscher et al., 1986; Hönack and Löscher, 1989) so that the transiently reduced carbamazepine response of sodium channels described in the hippocampus of kindled rats by Vreugdenhil and Wadman (1999) is not associated with resistance to the drug’s anticonvulsant effect in vivo. Similarly, at high doses, carbamazepine blocks spontaneous recurrent seizures in the pilocarpine model in rats (Leite and Cavalheiro, 1995), so that the loss of carbamazepine’s effect on sodium channels of hippocampal cells in this model reported by Remy et al. (2003b) is not associated with resistance to the drug’s anticonvulsant effect in vivo. As shown by our experiments in amygdala-kindled Wistar rats, however, a small subgroup of kindled rats is resistant to AED, which could be caused by alterations in the sensitivity of cellular targets of such drugs. To directly address the possibility that neuronal sodium currents in the hippocampus play a crucial role in the pharmacoresistence of TLE, we selected amygdala kindled rats with respect to their in vivo anticonvulsant response to phenytoin into responders and nonresponders and then compared phenytoin’s effect on voltage-activated sodium currents in CA1 neurons (Jeub et al., 2002). Furthermore, in view of the potential role of calcium current modulation in the anticonvulsant action of phenytoin, the effect of phenytoin on highvoltage activated calcium currents was studied in CA1 neurons. Electrode-implanted, but not kindled, rats were used as sham controls for comparison with the kindled rats. In all experiments, the interval between last kindled seizure and ion channel measurements was at least 5 weeks. In kindled rats with in vivo resistance to the anticonvulsant effect of phenytoin (phenytoin nonresponders), in vitro modulation of sodium and calcium currents by phenytoin in hippocampal CA1 neurons did not differ from respective data obtained in phenytoin responders (i.e., phenytoin resistance was not associated with a changed modulation of the sodium or calcium currents by this drug). Compared with sham controls, phenytoin’s inhibitory effect on sodium currents was significantly reduced by kindling without difference between the responder and nonresponder subgroups. These findings cast doubt on the hypothesis that pharmacoresistance is solely related to a reduced pharmacologic sensitivity of sodium or calcium currents. Alterations in Brain Uptake of Antiepileptic Drugs Because a patient being resistant to one AED is often resistant to other AED, involving drugs with different mechanisms, it seems unlikely that functional changes at target
The 6-Hz Psychomotor Seizure Model of Partial Epilepsy
sites for AED, (e.g. voltage-dependent sodium channels) can fully explain the pharmacoresistence in such a patient. A more likely explanation to account for medical intractability is that AED do not reach sufficiently high brain levels, despite adequate plasma levels within the therapeutic range. Tishler et al. (1995) were the first to report that brain expression of the multiple drug resistance gene (MDR1), which encodes the multidrug efflux transporter P-glycoprotein (Pgp), is markedly increased in most patients with medically intractable epilepsy. In line with enhanced MDR1 expression, immunohistochemistry for P-gp showed increased staining in capillary endothelium and astrocytes (Tishler et al., 1995). Tishler et al. (1995) proposed that P-gp may play a clinically significant role by limiting access of AED to the brain parenchyma, so that increased MDR1 expression may contribute to the refractoriness of seizures in patients with pharmacoresistent epilepsy. Until recently, however, it was not known whether AED are transported to any significant extent by P-gp or other multidrug transporters in the brain. P-gp is thought to constitute a defense mechanism limiting brain accumulation of naturally occurring toxins and xenobiotics (Begley, 2004). In the brain, P-gp is thought to be primarily located in the luminal cell membrane of endothelial cells of the blood–brain barrier (BBB) and is involved in transferring certain drugs back into blood after they have entered endothelial cells from blood, thus limiting penetration of such drugs into brain parenchyma (Begley, 2004). In a large series of in vivo experiments in rats, we recently demonstrated that extracellular brain concentrations of various AED, including phenytoin, can be markedly enhanced by Pgp inhibitors, suggesting that these AED are substrates for P-gp in the BBB (cf., Löscher and Potschka, 2002). Based on these data, we studied whether the expression of P-gp is enhanced by kindling and whether differences in P-gp expression exist between phenytoin responders and nonresponders in kindled Wistar rat populations. We found that kindled rats have lower extracellular brain levels of phenytoin than age-matched controls (Potschka and Löscher, 2002), and that phenytoin-nonresponders differ from responders in a marked overexpression of P-gp in the kindled amygdala (Potschka et al., 2004b). Thus, overexpression of P-gp is a likely explanation for the multidrug resistance in phenytoin-refractory kindled Wistar rats. The recent finding that levetiracetam seems not to be a substrate of P-gp (Potschka et al., 2004a) would explain that levetiracetam, in contrast to all other AED examined in this model, exerts the same antiepileptic efficacy in AED-refractory and AED-responsive kindled rats (Löscher, 2002b). Genetic Polymorphisms The breeding studies and experiments in various rat strains described above strongly indicated that genetic
557
factors are involved in the individual differences in the efficacy of AED in the amygdala-kindling model. Such genetic factors could also explain the increased expression of P-gp in the kindled amygdala of phenytoin-nonresponders (Potschka et al., 2004b) (e.g., by assuming a polymorphism affecting the promotor region of the P-gp gene). Hitherto, more than 50 single nucleotide polymorphisms (SNP) and insertion or deletion polymorphisms in the human MDR1 gene have been reported, and mutations at positions 2677 and 3435 were associated with alteration of P-gp expression or function (Brinkmann and Eichelbaum, 2001; Eichelbaum et al., 2004). In a recent study in 315 patients with epilepsy, classified as drug-resistant in 200 and drug-responsive in 115, patients with drug-resistant epilepsy were more likely to have the CC genotype at the MDR1 C3435T polymorphism, which is associated with increased expression of Pgp (Siddiqui et al., 2003). To our knowledge, it is not known whether such functionally relevant genetic polymorphisms also occur in the two genes (mdr1a: mdr1b:) that encode for Pgp in the brain of rodents (Demeule et al., 2002). We have therefore started to search for polymorphisms in the gene (mdr1a:) that encodes for Pgp in brain capillary endothelial cells in rats of phenytoin-refractory and phenytoin-responsive subgroups of kindled rats.
Advantages and Limitations of the Model A major advantage of the AED-resistant kindled rat model is obviously that it allows direct comparison of AEDrefractory and AED-responsive rats from the same strain, which is an important benefit for studies on mechanisms of pharmacoresistence. Because rats have to be implanted with electrodes, followed by subsequent kindling, and then several weeks of repeated testing of phenytoin’s effects on ADT, before AED-refractory and AED-responsive rats are obtained for further studies (Figure 1), however, the model is very time and labor intensive. On the other hand, because seizures are induced at will, the model does not require any continuous EEG or video monitoring of seizures. A real step forward would be to find rat inbred strains which either are nonresponders or responders to AED, thus avoiding the laborious selection and low yield of these subgroups within one outbred rat strain, such as Wistar. We still search such inbred strains, although our initial experiments in this respect failed to obtain a strain consisting of either 100% responders or nonresponders (see above).
THE 6-Hz PSYCHOMOTOR SEIZURE MODEL OF PARTIAL EPILEPSY Another interesting model of drug-refractory epilepsy is the 6-Hz psychomotor seizure model in mice (Barton et al., 2001). The latter model, also termed “minimal electroshock
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seizure threshold (6-Hz EST) model” (Swinyard, 1972), was developed more than 50 years ago by Toman (1951a,b) and designated as the “psychomotor” seizure test because the seizures evoked by low frequency stimulation were thought to resemble those seen clinically in psychomotor (partial or limbic) epilepsy, both in their overt manifestations and in their electroencephalographic (EEG) pattern. Toman et al. suggested that the 6-Hz model may differ from other assays, such as the MES or pentylenetetrazol (PTZ) models of generalized seizures, in being potentially useful in screening candidate drugs for clinical usefulness in human complexpartial seizures (Everett and Toman, 1951; Toman, 1951a,b; Toman et al., 1951, 1952; Goodsell et al., 1952). This suggestion was largely based on the fact that phenacemide (phenylacetylurea; Phenurone), the most effective agent available at this time for this type of seizures, was the only compound found by them to block the 6-Hz seizures in mice at nondepressant dose levels. Subsequently, Swinyard et al. (Brown et al., 1953) repeated and extended the pharmacologic experiments of Toman et al. in the 6-Hz model.
Evaluation of Antiepileptic Drugs in the 6-Hz Model in Mice The 6-Hz model is based on the fact that electrical stimulation by low-frequency (6-Hz) rectangular pulses of 0.2 msec duration delivered through corneal electrodes for a relatively long duration (3 seconds) induces less seizure spread than the widely used high-frequency (50- or 60-Hz) MES model. 6-Hz seizures are characterized by immobility (stun reaction), forelimb clonus, and automatic, stereotyped behaviors reminiscent of human limbic epilepsy (Swinyard, 1972). The median convulsive current required to produce such psychomotor seizures in 50% (CC50) of the mice tested was 8 mA (Brown et al., 1953). Pharmacologic testing was conducted by Brown et al. (1953) at a current intensity of 32 mA ( i.e., four times CC50). At this current, several AED available at the time (phenobarbital, paramethadione, mephenytoin, mephobarbital, phenacemide, trimethadione) were effective to block the seizures, whereas phenytoin and phethenylate (Thiantoin) were ineffective (Brown et al., 1953). This pharmacologic profile led Brown et al. (1953) to conclude that the 6-Hz model does not have more predictive value in the laboratory screening and assay of potentially useful antipsychomotor drugs than has any other testing procedure employed at this time. The model, therefore, was abandoned shortly after its description. Given the resistance of many patients with complexpartial seizures to phenytoin and the initial observation by Swinyard and colleagues that phenytoin is ineffective in the 6-Hz psychomotor seizure model, Steve White and colleagues at the University of Utah became interested in the
6-Hz model as a potential screen for therapy-resistant epilepsy (Barton et al., 2001). They studied its pharmacologic profile in male CF1 mice at varying current intensities, demonstrating that the level of protection afforded by a particular AED in this model is dependent on the stimulus intensity (Barton et al., 2001). At 22 mA, the convulsant current in 97% of mice (CC97), all AED tested (phenytoin, lamotrigine, ethosuximide, levetiracetam, valproate) blocked the seizures, demonstrating that the model did not discriminate between clinical classes of AED at this current. When the current was increased to 32 mA (50% above CC97), several AED (phenytoin, lamotrigine, topiramate) lost their anticonvulsant efficacy, but various other AED, including carbamazepine, phenobarbital, felbamate, tiagabine, ethosuximide, and levetiracetam, blocked the seizures. At 44 mA (twice the CC97), however, most AED lost their efficacy, and only levetiracetam and valproate allowed complete protection against the 6-Hz seizures (Barton et al., 2001). The effective doses in 50% of mice (ED50) needed to block 6Hz seizures by valproate (310 mg/kg) or levetiracetam (1089 mg/kg) were much higher at 44 mA compared with the lower stimulation intensities. Based on these observations, Barton et al. (2001) suggested that the 6-Hz stimulation may provide a useful model of therapy-resistant limbic seizures.
Seizure Spread in the 6-Hz Model To study the spread of seizure activity after 6-Hz stimulation, Barton et al. (2001) used c-Fos immunohistochemistry, showing that the 6-Hz seizure model displays a different pattern of seizure-induced neuronal activation than that observed in the MES and PTZ models of generalized seizures. In contrast to the widespread c-Fos staining observed with the two latter models, intense c-Fos staining from 6-Hz stimulation remained localized to the amygdala and piriform cortex and, at 44 mA, the dentate gyrus of the hippocampal formation (Barton et al., 2001). Barton et al. (2001) suggested that the recruitment of the dentate gyrus at 44 mA may account for the decrease in potency of levetiracetam and valproate at this stimulation intensity.
Advantages and Limitations of the Model When used with a high stimulus intensity of 44 mA, the 6-Hz model is resistant to most AED. Interestingly, as for the AED-resistant kindled rat model, levetiracetam differs from most other AED examined in the 6-Hz model in that it is effective to protect against the otherwise refractory seizures. A clear difference to phenytoin-resistant kindled rats is valproate, which is efficacious in the 6-Hz model but not in phenytoin-refractory rats. A real advantage of the 6Hz model compared with the kindling model is its simplicity, allowing screening of several compounds over a
Post-Status Epileptic Models of Temporal Lobe Epilepsy
relatively short time. In contrast to the kindling model of TLE, in which chronic brain alterations develop on kindling acquisition thereby altering the pharmacologic responsiveness of the animals (Löscher and Schmidt, 1988), normal mice are used for the 6-Hz model. Thus, the 6-Hz model may be an interesting approach for AED testing, but is obviously not suited to study the mechanisms leading to chronic epilepsy. Based on the extensive experiments of Barton et al. (2001), the 6-Hz model was introduced as a secondary screen to the screening approach currently employed by the Anticonvulsant Screening Project at the University of Utah, to examine those compounds that are found inactive in the MES and PTZ tests (Barton et al., 2001). Based on this renewed interest in the 6-Hz model, several new, promising compounds have been tested recently in this model (e.g., Isoherranen et al., 2001; Isoherranen et al., 2002; Isoherranen et al., 2003; Kaminski et al., 2004). It remains to be determined whether this approach will lead to the identification of additional novel AED subsequently found to possess clinical activity in patients with therapy-resistant epilepsy (Barton et al., 2001).
POST-STATUS EPILEPTIC MODELS OF TEMPORAL LOBE EPILEPSY In both the kindling model in rats and the 6-Hz model in mice, seizures are elicited by electrical stimulation for drug testing (i.e., the animals do not exhibit chronic epilepsy with spontaneous recurrent seizures). Although it is not known whether this is a disadvantage for evaluating the therapeutic potential of novel compounds for the treatment of epilepsy or for studying mechanisms of drug resistance, a model of pharmacoresistent epilepsy mimicking the human condition (i.e., inadequate control of spontaneous recurrent seizures) would certainly be an important development. To our knowledge, such model has not been characterized or validated as yet. Because of the similarity expected in eliciting an epilepsylike state with spontaneous recurrent seizures, either chemically or electrically induced models of SE in rats are valuable candidates in the search for models of pharmacoresistent epilepsy (Stables et al., 2003). The pharmacology of spontaneous recurrent seizures in such models, however, is largely unknown. Drug trials in rats with spontaneous recurrent seizures are laborious and timeconsuming, which is the most likely explanation that only few studies on AED testing in such models are available. Leite and Cavalheiro (1995) studied the anticonvulsant effect of conventional AED (i.e., phenobarbital, phenytoin, carbamazepine, valproate, and ethosuximide) against spontaneous recurrent seizures in the pilocarpine model in rats and found that phenobarbital (40 mg/kg/d), carbamazepine
559
(120 mg/kg/d), phenytoin (100 mg/kg/d), and valproate (600 mg/kg/d) effectively blocked the seizures when administered over a period of 2 weeks. They concluded that spontaneous recurrent seizures developing after pilocarpine-induced SE may be a useful model for finding new AED with better efficacy against complex partial seizures (i.e., the most difficult-to-treat seizures in adult patients with epilepsy). Possibly because of the high doses of AED administered, however, almost all rats were protected from spontaneous seizures by phenobarbital, phenytoin, carbamazepine, and valproate in the experiments of Leite and Cavalheiro (1995). This is not consistent with the clinical situation of TLE, where >50% of patients do not have their epilepsy adequately controlled by drug treatment (Leppik, 1992). Because plasma drug levels were not determined in the study of Leite and Cavalheiro (1995), it is not possible to compare directly the rat data with human data, because pharmacokinetics of AED markedly differ between rats and humans (Löscher and Schmidt, 1988). Most AED are much more rapidly eliminated in rats than in humans (Löscher and Schmidt, 1988), which makes maintenance of active drug levels by conventional routes of application (e.g., oral or i.p.) almost impossible. In their study in the pilocarpine model in rats, Leite and Cavalheiro (1995) administered all AED (except phenobarbital) two to three times a day i.p. at high doses to counteract the problem of rapid elimination. This, however, leads to marked fluctuation in plasma concentrations between drug administrations and to peak drug plasma levels far exceeding the therapeutic plasma concentration range in humans. Furthermore, the high doses of AED administered in the study of Leite and Cavalheiro (1995) induced marked adverse effects, such as sedation and muscle relaxation. In this regard, it is important to consider that pharmacoresistence in patients with epilepsy is usually defined as no clinically relevant improvement of seizures at maximal tolerated dose of standard AED (Regesta and Tanganelli, 1999). This definition of drug resistance should also be used in animal models, because an anticonvulsant effect of a drug at a toxic dose is without relevance in terms of prediction of clinical efficacy. In the study of Leite and Cavalheiro (1995) in the pilocarpine model, loss of anticonvulsant efficacy (i.e., tolerance) was observed for phenobarbital, carbamazepine, and valproate in that spontaneous seizures were more effectively blocked in the first week of treatment compared with the second week. Thus, such development of tolerance has to be considered when planning and interpreting studies in animal models of epilepsy. In our recent study, described below, with levetiracetam in the pilocarpine model (Glien et al., 2002), we tried to resolve some of the problems of chronic drug testing in rats with spontaneous recurrent seizures.
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Selection of Responders and Nonresponder from the Pilocarpine Model of Temporal Lobe Epilepsy Stimulated by the findings in the kindling model, we began about 5 years ago to study whether pharmacoresistent rats can also be selected from TLE models with spontaneous recurrent seizures. In contrast to most of our experiments in kindled rats, which were performed with acute (single-dose administration) of AED and seizure induction at a defined time (e.g., 30 minutes) after drug administration, drug efficacy testing in rats with spontaneous recurrent seizures is much more laborious and time-consuming, because AED have to be administered over a prolonged period and rats have to be recorded continuously during this period for the occurrence of seizures. A major problem of such chronic drug trials in rats is the difference in pharmacokinetics between rats and humans. As pointed out, most AED are much more rapidly eliminated in rats (and mice) than in humans (Löscher and Schmidt, 1988), which makes maintenance of effective drug levels by conventional routes of application (e.g., oral or i.p.) almost impossible. Continuous drug administration via the drinking water or food is not an alternative, because rodents drink and eat mostly during the night, so that drug levels rapidly decrease during the day (Löscher and Schmidt, 1988). Thus, if plasma drug levels are not controlled during chronic drug trials in rats, a rat (or rat model) may be considered pharmacoresistent just because seizures occur in the absence of effective drug concentrations in it. As a consequence, pharmacokinetics of AED in rats have to be used as a basis for drug administration. Only few AED (e.g., phenobarbital) have half-lives long enough to maintain effective drug levels by conventional routes of application (e.g., oral or i.p.) in rats. Phenytoin has a short, dose-dependent half-life in rats, but the half-life increases during chronic drug administration because of metabolic enzyme saturation (Rundfeldt and Löscher, 1993). For drugs with a half-life of <3 to 4 hours in rats, which is the case for most other AED, continuous drug administration via subcutaneously implanted osmotic minipumps is the only way to maintain anticonvulsant drug levels throughout the period of the drug trial (Löscher and Schmidt, 1988). In addition to pharmacokinetic problems which have to be dealt with when planning and interpreting drug studies in rats with spontaneous recurrent seizures, development of metabolic or functional tolerance resulting in loss of efficacy during prolonged drug treatment may form a significant bias for chronic drug studies. For instance, as described above, Leite and Cavalheiro (1995), using the pilocarpine model of TLE, observed tolerance to the anticonvulsant effect of phenobarbital, carbamazepine, and valproate in that spontaneous seizures were more effectively blocked in the first week of treatment compared with the second week. For most
AED, tolerance can be overcome by increasing the drug dosage, so that tolerance should not be misinterpreted as pharmacoresistance (Frey et al., 1986). Based on these considerations, we chose the pilocarpine model of TLE in Wistar rats to study the anticonvulsant efficacy of levetiracetam on spontaneous recurrent seizures (Glien et al., 2002). The drug was chronically administered via subcutaneously (s.c.) implanted minipumps at an infusion rate providing plasma drug levels within the maximal plasma concentration range known from clinical trials with levetiracetam. Rather than calculating all drug effects as means per group of treated rats (as for instance done in the study of Leite and Cavalheiro [1995] in the pilocarpine model), we closely examined the individual data, because, based on our previous kindling studies, our hypothesis was that large interindividual differences in anticonvulsant efficacy of AED exist when an outbred rat strain such as Wistar rats is used. The Wistar rats were subjected to pilocarpine-induced SE and recorded for spontaneous recurrent seizures in the months following pilocarpine treatment. A group of eight rats with frequent spontaneous seizures was used for the drug trial with levetiracetam. The experimental protocol for drug testing in these rats was as follows (Figure 3). For 2 weeks, rats received s.c. implantation of osmotic minipumps filled with saline (predrug control period), followed by a 2-week period with implantation of levetiracetam-filled minipumps (drug period), after which pumps were replaced by drug-free pumps for another 2 weeks (postdrug control period). The levetiracetam concentration in the pumps during the drug period was adjusted to give daily doses resulting in the maximal plasma concentration range determined previously in patients with TLE during chronic treatment with levetiracetam. During the 6 weeks of the experiment in epileptic rats, seizures were recorded by video monitoring. Average seizure frequency during the pre- and postdrug control period in the eight epileptic rats was 21 and 25 seizures. This was reduced to an average seizure frequency of eight seizures during the 2 weeks of treatment with levetiracetam. The individual response of rats to levetiracetam, however, varied markedly from complete seizure control to no effect at all, although plasma drug levels were within the same range in all rats. When seizure frequency was separately calculated for the first and second week of treatment, the significant anticonvulsant effect determined in the first week was partially diminished in the second week, suggesting that tolerance may have developed in some of the rats (Glien et al., 2002). Our study demonstrated that rats with spontaneous recurrent seizures exhibit a marked interindividual variability toward anticonvulsant drug effects, which is similar to the clinical situation in patients with TLE. Thus, although the average seizure frequency was reduced by treatment with levetiracetam, the individual drug response ranged from
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Drug trial in rats with spontaneous seizures Latency period (~4 weeks)
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seizu res DRUG
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FIGURE 3 Schematic illustration of selection of drug-refractory and drug-responsive rats from poststatus epilepticus models of temporal lobe epilepsy by prolonged administration of antiepileptic drugs.
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FIGURE 4 Effect of levetiracetam (LEV) on spontaneous recurrent seizures (SRS) in rats. SRS developed after a status epilepticus that had been induced by intraperitoneal (i.p.) administration of pilocarpine. Once frequent SRS had been determined over several weeks in these rats, they were used for drug testing as follows. SRS were recorded over a period of 2 weeks before onset of LEV treatment (predrug control), followed by drug treatment via osmotic minipumps for 2 weeks, and then a 2-week postdrug control period as shown in Figure 3. All data are shown as means ± SEM. A, B, and C show the average number of seizures recorded over the three 2-week period; D illustrates the average plasma concentration of LEV during the treatment period. In A, average seizure data from the eight rats of this experiment are given; B shows respective data from the three responders and C data from the three nonresponders of this group (see text for definitions). Data are from Glien, M., et al, 2002.
complete seizure control to no drug response. Because plasma levels were within the same range in all rats, the interindividual variation in drug response cannot be explained by differences in pharmacokinetics. As shown in Figure 4, three of eight rats (38%) were responders with complete or almost complete seizure control, three of eight
(38%) were nonresponders, and two of eight (25%) could not clearly be included in either group because of variation between pre- and postdrug control seizure frequency. Interestingly, the percentages of responders and nonresponders are in a similar range as those previously reported by us in the kindling model in Wistar rats (see above).
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Our study with levetiracetam shows that interesting results can be obtained by drug testing in epileptic rats, which are much closer to data from clinical evaluation of this drug in patients with epilepsy than data from acute drug testing in traditional seizure models, in which only elicited seizures are examined. Problems of the pilocarpine model of TLE for chronic drug studies, however, include the high mortality associated with a pilocarpine-induced SE and the behavioral alterations in rats surviving the SE (Goodman, 1998). Although we could reduce mortality by repeated low-dose treatment of rats with pilocarpine for SE induction (Glien et al., 2001), most of the epileptic rats arising from this model are difficult to handle because of hyperexcitability and aggression, so that repeated drug administration in conscious rats is almost impossible. This prompted us to compare rats from the pilocarpine model with epileptic rats from other models with chemical or electrical SE induction. Based on this comparison, we recently developed a new model in which spontaneous recurrent seizures develop after different types of SE induced by sustained electrical stimulation of the basolateral amygdala (BLA) in Wistar or Sprague-Dawley rats (Brandt et al., 2003). In contrast to rats from chemical SE models, rats from the BLA model do not exhibit any obvious aggressive behavior or extreme hypersensitivity to handling (Brandt et al., 2003), so that these rats are better suited for chronic drug administration. A first study with chronic administration of phenobarbital in epileptic SpragueDawley rats from the BLA model indicates that, similar to the pilocarpine model, responders and nonresponders can be selected from this model (Brandt et al., 2004), which will be described.
Selection of Responders and Nonresponders from a Post-status Epilepticus Model of Temporal Lobe Epilepsy Resulting from Prolonged Electrical Stimulation of the Basolateral Amygdala Based on our previous experience in different rat strains and genders, female outbred rats of the Sprague-Dawley strain are used for drug studies in this model because prolonged stimulation of the BLA nucleus results in most of these rats in a generalized convulsive SE, which induces development of spontaneous recurrent seizures in >90% of the animals (Brandt et al., 2003). The rats are stereotactically implanted with a bipolar electrode into the right BLA in the same way as for amygdala-kindling (Brandt et al., 2003). About 2 weeks after electrode implantation, the rats are electrically stimulated via the BLA electrode for induction of a self-sustained SE. The following stimulus parameters are chosen: stimulus duration 25 minutes; stimulus consisting of 100 msec trains of 1 msec alternating positive
and negative square wave pulses. The trains are given at a frequency of two per second and the intratrain pulse frequency is 50 per second. Peak pulse intensity is 700 mA. In this way, about 90% of rats develop a self-sustained SE with generalized convulsive seizures. After 4 hours, SE is interrupted by diazepam (10 mg/kg i.p.) in all rats. If necessary, the application of this dose of diazepam is repeated, but in most rats seizure activity is terminated after the first diazepam injection. By using continuous video- and EEGrecording for up to 24 hours after injection of diazepam, we recently demonstrated that diazepam completely terminated all behavioral seizure activity and the EEG alterations associated with this behavior and also blocked any reappearance of seizures over 24 hours after injection (Brandt et al., 2003). Starting about 4 weeks later, the rats are monitored by EEG-video recordings for up to 2 months until the first spontaneous seizures are detected as described recently (Brandt et al., 2003; 2004). Rats with frequent spontaneous recurrent seizures are used for drug studies as shown in Figure 3. To evaluate whether epileptic rats from this model can be selected into drug-refractory and drug-responsive animals by AED testing, a first study was performed with phenobarbital in a group of 11 rats with spontaneous recurrent seizures (Brandt et al., 2004). Phenobarbital was chosen because it is an efficacious AED in rat models of TLE with a sufficiently long half-life to allow maintenance of therapeutic drug levels during prolonged treatment (Löscher and Hönack, 1989; Leite and Cavalheiro, 1995; Brandt et al., 2004). As described in detail recently (Brandt et al., 2004), several preliminary experiments were performed to develop a dosing protocol allowing maintenance of plasma drug concentrations within or above the therapeutic range (10 to 40 mg/ml; Baulac, 2002) over 24 hours per day, 7 days per week. Furthermore, we were interested in administering phenobarbital at maximal tolerated doses, so that rats were closely observed for adverse effects. Based on these preliminary experiments, a dosing protocol with an i.p. bolus dose of 25 mg/kg in the morning of the first treatment day, followed 10 hours later by an administration of 15 mg/kg, and then twice daily 15 mg/kg for the 13 subsequent days, was used in rats with spontaneous recurrent seizures. Before onset of drug treatment, baseline seizure frequency was determined over 2 weeks (predrug control period), then phenobarbital was administered over 2 weeks, followed by a postdrug control period of 2 weeks. Blood was sampled 10 hours after the first drug injection and 12 hours after the last drug injection for drug analysis in plasma. In each of the 6 weeks of the experiment, seizures were continuously (24 hours per day, 7 days per week) monitored by video or EEG recording as described in detail recently (Brandt et al., 2004). A schematic illustration of the drug trial is shown in Figure 3. Detailed results of selection with phenobarbital have been described recently (Brandt et al., 2004), so that respond-
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ers and nonresponders will be only briefly characterized here. All rats received phenobarbital at maximal tolerated doses as indicated by the marked sedation, which was observed in all rats during treatment. Analysis of plasma drug concentrations showed that drug concentrations within the therapeutic range (10 to 40 m/ml) were maintained in all rats throughout the period of treatment (Figure 5). In 6 of 11 rats with spontaneous recurrent seizures, complete control of seizures was achieved and another rat exhibited a >90% reduction of seizure frequency. These seven rats were considered responders (Figure 5). Three animals of the remaining four rats showed no anticonvulsant response but
A
Seizure frequency All rats (n = 11)
an increase of seizure frequency during drug treatment. The fourth rat showed only moderate (<50%) reduction in seizure frequency. These four rats, therefore, were considered nonresponders (Figure 5). Plasma drug concentrations did not differ significantly between responders and nonresponders (Figure 5). Two of the nonresponders exhibited an extremely high seizure frequency during the control and treatment periods of the experiment, whereas the other two nonresponders did not differ from responders in seizure frequency during the control periods. The type of spontaneous recurrent seizures was the same in all responders and nonresponders (i.e.,
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FIGURE 5 Effect of phenobarbital (PB) on spontaneous recurrent seizures (SRS) in rats. SRS developed after a status epilepticus that had been induced by prolonged electrical stimulation of the basolateral amygdala. About 5 months after the status epilepticus, SRS were recorded over a period of 2 weeks before onset of PB treatment (predrug control), followed by drug treatment for 2 weeks, and then a 2-week postdrug control period as shown in Figure 3. All data are shown as means ± SEM. A, B, and C show the average number of seizures recorded over the three 2-week period; D illustrates the average plasma concentration of PB from the blood samples taken at the end of the treatment period. In A, average seizure data from the 11 rats of this experiment are given; B shows respective data from the 7 responders and C data from the 4 nonresponders of this group (see text for definitions). In the responder group, PB significantly suppressed SRS compared with the pre- and postdrug periods (P < .005; indicated by asterisk). Data from Brandt, C., et al, 2004.
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Chapter 45/Animal Models of Drug-Refractory Epilepsy
generalized convulsive seizures, resembling stage 4 or 5 seizures of the Racine scale) (Racine, 1972). Furthermore, the severity of the initial, electrically induced SE was not different between responders and nonresponders, indicating that the same severity and duration of SE produces two subgroups of epileptic rats, AED responders and nonresponders. We currently study the mechanisms that may be involved in this finding.
monitoring of spontaneous recurrent seizures. Such drugrefractory rats seem to be ideal models for pharmacoresistant epilepsy and could be used for evaluating the therapeutic potential of novel compounds for the treatment of epilepsy. Furthermore, such rats would be useful to study the mechanisms underlying pharmacoresistence.
OTHER POTENTIALLY PROMISING MODELS Advantages and Limitations of Post-status Epilepticus Models of Temporal Lobe Epilepsy Because of the similarity expected in eliciting an epilepsylike state (spontaneous recurrent seizures), chemically or electrically induced models of SE are considered relevant models of the human condition (Stables et al., 2003). As shown by our recent drug studies in chemical and electrical post-SE models of TLE, it is possible to select rats with AED-refractory or AED-responsive spontaneous seizures from such models, although more studies with additional AED are needed to evaluate whether these rats are really drug-refractory. To avoid that rats are falsely considered drug-refractory because of pharmacokinetic factors (e.g., too rapid drug elimination), drug administration protocols have to be based on AED pharmacokinetics in rats, so that anticonvulsant drug levels are maintained in plasma throughout the treatment period. Female rats are known to eliminate several AED more slowly than males, which is an advantage for chronic drug studies (Löscher and Schmidt, 1988). Interpretation of results from drug testing in rats with spontaneous recurrent seizures can be performed in a similar way as done in patients with epilepsy with endpoints such as seizure frequency and seizure severity. Because of the need to continuously monitor spontaneous recurrent seizures over a period of at least 6 weeks (Figure 3), however, drug testing in post-SE models of TLE is technically difficult, timeconsuming, and expensive. Automated seizure recording systems, including EEG monitoring via miniaturized telemetric devices, and new methods of prolonged drug delivery, it is hoped will soon make chronic studies in such models less labor-dependent and increase the use of epileptic animals for evaluation of drug effects (Stables et al., 2002; 2003). In conclusion, although chemically or electrically induced models of SE are widely used to study mechanisms of epileptogenesis and to identify targets for antiepileptogenic therapies, these models have not yet been characterized to any extent as models of pharmacoresistent epilepsy. As suggested by our recent studies in post-SE models of TLE in Wistar and Sprague-Dawley rats, it is possible to identify rats with drug-refractory spontaneous seizures by prolonged AED administration and continuous EEG/video
More recently, several additional models of drugrefractory seizures have been proposed (cf., White, 2003), but these models are less well pharmacologically characterized than the models described above. Using the amygdalakindling model in male Sprague-Dawley rats, Robert Post’s group reported that exposure to lamotrigine during kindling development leads to a reduced subsequent response to the drug in fully kindled animals (Postma et al., 2000). Weiss and Post (1991) previously reported the same phenomenon for carbamazepine in that rats that had been treated with carbamazepine during kindling were subsequently unresponsive to carbamazepine, whereas fully kindled seizures are normally very responsive to this AED. This prompted Steve White’s group to assess whether this phenomenon generalizes to PTZ kindling in male Sprague-Dawley rats (Srivastava et al., 2003). Similar to amygdala kindling in the study of Postma et al. (2000), lamotrigine (5 mg/kg before every PTZ challenge) did not affect kindling development (Srivastava et al., 2003). In vehicle-treated, PTZ-kindled rats, lamotrigine (15 mg/kg) blocked the fully kindled seizures, whereas this effect was lost in rats that had been treated with lamotrigine during kindling development. Lamotrigine-refractory PTZ-kindled rats were also resistant to carbamazepine, but not to valproate (Srivastava et al., 2003). The authors suggested that lamotrigine-resistant kindled rats may serve as a model of drug-refractory epilepsy (Srivastava et al., 2003). Seizure activity associated with cortical dysplasia (CD) is often resistant to commonly used AED (Zupanc, 1996; Crino et al., 2002). Although a number of animal models for CD exist, the response of these animals to common AED has not been systematically investigated. This prompted Smyth et al. (2002) to investigate the effects of AED in SpragueDawley rats exposed to methylazoxymethanol acetate (MAM) in utero, an animal model featuring nodular heterotopia with hyperexcitability within dysplastic brain regions. In MAM-exposed rats, valproate failed to increase the latency to kainate-induced seizures, whereas it significantly increased seizure latency in age-matched controls (Smyth et al., 2002). In acute hippocampal slices from MAM-exposed rats, epileptiform bursting induced by 4-aminopyridine was resistant to treatment with phenobarbital, carbamazepine, valproate, ethosuximide, and lamotrigine, whereas these
References
AED significantly suppressed bursts in control slices (Smyth et al., 2002). The authors concluded that MAM-exposed rats exhibit a dramatically reduced sensitivity to commonly prescribed AED, mimicking the clinical situation reported in dysplasia-associated epilepsy (Smyth et al., 2002). Apart from animal models, a number of in vitro models of drug-refractory seizures (e.g., the low magnesium entorhinal-hippocampal slice preparation) have been developed and used for pharmacologic testing of novel AED (Heinemann et al., 1994; Pfeiffer et al., 1996; Dreier et al., 1998; Armand et al., 2000). As with all other models described in this chapter, a major problem of validating these in vitro models is the lack of a positive control (i.e., a novel AED with clinical efficacy in the management of refractory epilepsy). As a consequence, whether novel compounds that are found to be active in models of drugrefractory seizures subsequently will be found to be effective for the management of clinically refractory epilepsy is not presently known (White, 2003).
CONCLUSIONS Drug-resistant epilepsy represents a challenge for both experimental and clinical research to gain new insights and perspectives of its mechanisms to improve future rational management approaches. As shown in this chapter, a number of potentially useful animal models of drug-refractory epilepsy exist, which can be used to study mechanisms of drug resistance. Such models should be added to the late preclinical phases of AED development to prove at a relatively early stage if a new drug exhibits advantages towards standard AED in drug resistant seizure types. A continuing effort should be made to validate existing models and identify new models of different types of refractory epilepsy. It is hoped that further investigation and exploitation of such models will contribute to our understanding of the processes through which epilepsy becomes intractable. Investigation of the basis of drug resistance in epilepsy should offer new rational approaches to treatment, for instance by design of novel AED that are not targets for brain-expressed resistance mechanisms.
References Armand, V., Rundfeldt, C., and Heinemann, U. 2000. Effects of retigabine (D-23129) on different patterns of epileptiform activity induced by low magnesium in rat entorhinal cortex hippocampal slices. Epilepsia 41: 28–33. Barton, M.E., Klein, B.D., Wolf, H.H., and White, H.S. 2001. Pharmacological characterization of the 6 Hz psychomotor seizure model of partial epilepsy. Epilepsy Res 47: 217–228. Baulac, M. 2002. Phenobarbital and other barbiturates: clinical efficacy and use in epilepsy. In Antiepileptic Drugs. 5th Ed. Eds. R.H. Levy, R.H., Mattson, B.S. Meldrum, and E. Perucca. pp. 514–521. Philadelphia: Lippincott Williams & Wilkins.
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Beck, H., Steffens, R., Heinemann, U., and Elger, C.E. 1997. Properties of voltage-activated Ca2+ currents in acutely isolated human hippocampal granule cells. J Neurophysiol 77: 1526–1537. Beck, H., Steffens, R., Elger, C.E., and Heinemann, U. 1998. Voltagedependent Ca2+ currents in epilepsy. Epilepsy Res 32: 321–332. Begley, D.J. 2004. ABC transporters and the blood-brain barrier. Curr Pharm Des 10: 1295–1312. Betts, T., Yarrow, H., Greenhill, L., and Barrett, M. 2003. Clinical experience of marketed Levetiracetam in an epilepsy clinic—a one year follow up study. Seizure 12: 136–140. Brandt, C., Glien, M., Potschka, H., Volk, H., and Löscher, W. 2003. Epileptogenesis and neuropathology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats. Epilepsy Res 55: 83–103. Brandt, C., Volk, H.A., and Löscher, W. 2004. Striking differences in individual anticonvulsant response to phenobarbital in rats with spontaneous seizures after status epilepticus. Epilepsia 45: 1488–1497. Brinkmann, U., and Eichelbaum, M. 2001. Polymorphisms in the ABC drug transporter gene MDR1. Pharmacogenomics J 1: 59–64. Brown, W.C., Schiffman, D.O., Swinyard, E.A., and Goodman, L.S. 1953. Comparative assay of antiepileptic drugs by “pychomotor” seizure test and minimal electroshock threshold test. J Pharmacol Exp Ther 107: 273–283. Cramer, S., Ebert, U., and Löscher, W. 1998. Characterization of phenytoin-resistant kindled rats, a new model of drug-resistant partial epilepsy: Comparison of inbred strains. Epilepsia 39: 1046–1053. Crino, P.B., Miyata, H., and Vinters, H.V. 2002. Neurodevelopmental disorders as a cause of seizures: neuropathologic, genetic, and mechanistic considerations. Brain Pathol 12: 212–233. Demeule, M., Regina, A., Jodoin, J., Laplante, A., Dagenais, C., Berthelet, F., Moghrabi, A. et al. 2002. Drug transport to the brain: key roles for the efflux pump P-glycoprotein in the blood-brain barrier. Vascul Pharmacol 38: 339–348. Dreier, J.P., Zhang, C.L., and Heinemann, U. 1998. Phenytoin, phenobarbital, and midazolam fail to stop status epilepticus-like activity induced by low magnesium in rat entorhinal slices, but can prevent its development. Acta Neurol Scand 98: 154–160. Ebert, U., Rundfeldt, C., Lehmann, H., and Löscher, W. 1999. Characterization of phenytoin-resistant rats, a new model of drug-resistant partial epilepsy: influence of experimental and environmental factors. Epilepsy Res 33: 199–215. Ebert, U., and Löscher, W. 1999. Characterization of phenytoin-resistant kindled rats, a new model of drug-resistant partial epilepsy: influence of genetic factors. Epilepsy Res 33: 217–226. Ebert, U., Reissmüller, E., and Löscher, W. 2000. The new antiepileptic drugs lamotrigine and felbamate are effective in phenytoin-resistant kindled rats. Neuropharmacology 39: 1893–1903. Eichelbaum, M., Fromm, M.F., and Schwab, M. 2004. Clinical aspects of the MDR1 (ABCB1) gene polymorphism. Ther Drug Monit 26: 180–185. Everett, G.M., and Toman, J.E.P. 1951. Experimental Psychomotor Seizures and Antiepileptic Drugs. Federation Proceedings 10: 293. Glien, M., Brandt, C., Potschka, H., Voigt, H., Ebert, U., and Löscher, W. 2001. Repeated low-dose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy. Epilepsy Res 46: 111–119. Glien, M., Brandt, C., Potschka, H., and Löscher, W. 2002. Effects of the novel antiepileptic drug levetiracetam on spontaneous recurrent seizures in the rat pilocarpine model of temporal lobe epilepsy. Epilepsia 43: 350–357. Goddard, G.V., McIntyre, D.C., and Leech, C.K. 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 25: 295–330.
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Goodman, J.H. 1998. Experimental models of status epilepticus. In Neuropharmacology Methods in Epilepsy Research. Ed. S.L. Peterson, and T.E. Albertson. pp. 95–125. Boca Raton: CRC Press. Goodsell, J.S., Everett, G.M., and Toman, J.E.P. 1952. Evaluation of the psychomotor seizure as an anticonvulsant screening method. J Pharmacol Exp Ther 106: 390. Heinemann, U., Draguhn, A., Ficker, E., Stabel, J., and Zhang, C.L. 1994. Strategies for the development of drugs for pharmacoresistent epilepsies. Epilepsia 35 (Suppl. 5): S10–S21. Hönack, D., and Löscher, W. 1989. Amygdala-kindling as a model for chronic efficacy studies on antiepileptic drugs: experiments with carbamazepine. Neuropharmacology 28: 599–610. Isoherranen, N., Woodhead, J.H., White, H.S., and Bialer, M. 2001. Anticonvulsant profile of valrocemide (TV1901): a new antiepileptic drug. Epilepsia 42: 831–836. Isoherranen, N., White, H.S., Finnell, R.H., Yagen, B., Woodhead, J.H., Bennett, G.D., Wilcox, K.S. et al. 2002. Anticonvulsant profile and teratogenicity of N-methyl-tetramethylcyclopropyl carboxamide: a new antiepileptic drug. Epilepsia 43: 115–126. Isoherranen, N., Yagen, B., Woodhead, J.H., Spiegelstein, O., Blotnik, S., Wilcox, K.S., Finnell, R.H. et al. 2003. Characterization of the anticonvulsant profile and enantioselective pharmacokinetics of the chiral valproylamide propylisopropyl acetamide in rodents. Br J Pharmacol 138: 602–613. Jeub, M., Beck, H., Siep, E., Ruschenschmidt, C., Speckmann, E.J., Ebert, U., Potschka, H. et al. 2002. Effect of phenytoin on sodium and calcium currents in hippocampal CA1 neurons of phenytoin-resistant kindled rats. Neuropharmacology 42(1): 107–116. Kaminski, R.M., Livingood, M.R., and Rogawski, M.A. 2004. Allopregnanolone analogs that positively modulate GABA receptors protect against partial seizures induced by 6-Hz electrical stimulation in mice. Epilepsia 45: 864–867. Kwan, P., and Brodie, M.J. 2000. Early identification of refractory epilepsy. N Engl J Med 342: 314–319. Leite, J.P., and Cavalheiro, E.A. 1995. Effects of conventional antiepileptic drugs in a model of spontaneous recurrent seizures in rats. Epilepsy Res 20: 93–104. Löscher, W. 2002a. Current status and future directions in the pharmacotherapy of epilepsy. Trends Pharmacol Sci 23: 113–118. Löscher, W. 2002b. Animal models of drug-resistant epilepsy. Novartis Found Symp 243: 149–160. Löscher, W. 1997. Animal models of intractable epilepsy. Prog Neurobiol 53: 239–258. Löscher, W. 1986. Experimental models for intractable epilepsy in nonprimate animal species. In Intractable Epilepsy: Experimental and Clinical Aspects. Ed. D. Schmidt, and P.L. Morselli. pp. 25–37. New York: Raven Press. Löscher, W., and Hönack, D. 1989. Comparison of the anticonvulsant efficacy of primidone and phenobarbital during chronic treatment of amygdala-kindled rats. Eur J Pharmacol 162: 309–322. Löscher, W., and Potschka, H. 2002. Role of multidrug transporters in pharmacoresistence to antiepileptic drugs. J Pharmacol Exp Ther 301: 7–14. Löscher, W., and Rundfeldt, C. 1991. Kindling as a model of drug-resistant partial epilepsy: selection of phenytoin-resistant and nonresistant rats. J Pharmacol Exp Ther 258: 483–489. Löscher, W., and Schmidt, D. 1988. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical considerations. Epilepsy Res 2: 145–181. Löscher, W., Cramer, S., and Ebert, U. 1998. Selection of phenytoin responders and nonresponders in male and female amygdala-kindled SpragueDawley rats. Epilepsia 39: 1138–1147. Löscher, W., Jäckel, R., and Czuczwar, S.J. 1986. Is amygdala kindling in rats a model for drug-resistant partialepilepsy? Exp Neurol 93: 211–226.
Löscher, W., Reissmüller, E., and Ebert, U. 2000a. Anticonvulsant efficacy of gabapentin and levetiracetam in phenytoin-resistant kindled rats. Epilepsy Res 40: 63–77. Löscher, W., Reissmüller, E., and Ebert, U. 2000b. Kindling alters the anticonvulsant efficacy of phenytoin in Wistar rats. Epilepsy Res 39: 211–220. Löscher, W., Rundfeldt, C., and Hönack, D. 1993. Pharmacological characterization of phenytoin-resistant amygdala-kindled rats, a new model of drug-resistant partial epilepsy. Epilepsy Res 15: 207–219. Pfeiffer, M., Draguhn, A., Meierkord, H., and Heinemann, U. 1996. Effects of gamma-aminobutyric acid (GABA) agonists and GABA uptake inhibitors on pharmacosensitive and pharmacoresistent epileptiform activity in vitro. Br J Pharmacol 119: 569–577. Postma, T., Krupp, E., Li, X.L., Post, R.M., and Weiss, S.R. 2000. Lamotrigine treatment during amygdala-kindled seizure development fails to inhibit seizures and diminishes subsequent anticonvulsant efficacy. Epilepsia 41: 1514–1521. Potschka, H., and Löscher, W. 2002. A comparison of extracellular levels of phenytoin in amygdala and hippocampus of kindled and non-kindled rats. Neuroreport 13: 167–171. Potschka, H., Baltes, S., and Löscher, W. 2004a. Inhibition of multidrug transporters by verapamil or probenecid does not alter blood-brain barrier penetration of levetiracetam in rats. Epilepsy Res 58: 85–91. Potschka, H., Volk, H.A., and Löscher, W. 2004b. Pharmacoresistence and expression of multidrug transporter P-glycoprotein in kindled rats. Neuroreport 19: 1657–1661. Racine, R.J. 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph Clin Neurophysiol 32: 281–294. Reckziegel, G., Beck, H., Schramm, J., Elger, C.E., and Urban, B.W. 1998. Electrophysiological characterization of Na+ currents in acutely isolated human hippocampal dentate granule cells. J Physiol (Lond) 509: 139–150. Regesta, G., and Tanganelli, P. 1999. Clinical aspects and biological bases of drug-resistant epilepsies. Epilepsy Res 34: 109–122. Reissmüller, E., Ebert, U., and Löscher, W. 2000. Anticonvulsant efficacy of topiramate in phenytoin-resistant kindled rats. Epilepsia 41: 372–379. Remy, S., Gabriel, S., Urban, B.W., Dietrich, D., Lehmann, T.N., Elger, C.E., Heinemann, U., et al. 2003a. A novel mechanism underlying drug resistance in chronic epilepsy. Ann Neurol 53: 469–449. Remy, S., Urban, B.W., Elger, C.E., and Beck, H. 2003b. Anticonvulsant pharmacology of voltage-gated Na+ channels in hippocampal neurons of control and chronically epileptic rats. Eur J Neurosci 17: 2648– 2658. Rogawski, M.A., and Löscher, W. 2004. The neurobiology of antiepileptic drugs. Nat Rev Neurosci 5: 553–564. Rundfeldt, C., Hönack, D., and Löscher, W. 1990. Phenytoin potently increases the threshold for focal seizures in amygdala-kindled rats. Neuropharmacology 29: 845–851. Rundfeldt, C., and Löscher, W. 1993. Anticonvulsant efficacy and adverse effects of phenytoin during chronic treatment in amygdala-kindled rats. J Pharmacol Exp Ther 266: 216–223. Siddiqui, A., Kerb, R., Weale, M.E., Brinkmann, U., Smith, A., Goldstein, D.B., Wood, N.W., et al. 2003. Association of multidrug resistance in epilepsy with a polymorphism in the drug-transporter gene ABCB1. N Engl J Med 348: 1442–1448. Sisodiya, S.M. 2003. Mechanisms of antiepileptic drug resistance. Curr Opin Neurol 16: 197–201. Smyth, M.D., Barbaro, N.M., and Baraban, S.C. 2002. Effects of antiepileptic drugs on induced epileptiform activity in a rat model of dysplasia. Epilepsy Res 50: 251–264. Srivastava, A.K., Woodhead, J.H., and White, S. 2003. Effect of lamotrigine, carbamazepine, and sodium valproate on lamotrigine-resistant kindled rats. Epilepsia 44 (Suppl. 9): 42.
References Stables, J.P., Bertram, E.H., White, H.S., Coulter, D.A., Dichter, M.A., Jacobs, M.P., Löscher, W. et al. 2002. Models for epilepsy and epileptogenesis: report from the NIH workshop, Bethesda, Maryland. Epilepsia 43: 1410–1420. Stables, J.P., Bertram, E., Dudek, F.E., Holmes, G., Mathern, G., Pitkänen, A., and White, H.S. 2003. Therapy discovery for pharmacoresistent epilepsy and for disease-modifying therapeutics: summary of the NIH/NINDS/AES models II workshop. Epilepsia 44: 1472– 1478. Swinyard, E.A. 1972. Electrically induced convulsions. In Experimental models of epilepsy: A Manual for the Laboratory Worker. Ed. D.P. Purpura, J.K. Penry, D. Tower, D.M. Woodbury, and R. Walter. pp. 433–458. New York: Raven Press. Tishler, D.M., Weinberg, K.T., Hinton, D.R., Barbaro, N., Annett, G.M., and Raffel, C. 1995. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 36: 1–6. Toman, J.E.P. 1951a. Neuropharmacologic considerations in psychic seizures. Neurology 1: 444–460. Toman, J.E.P. 1951b. Experimental Psychomotor Seizures. Electroencephalography and Clinical Neurophysiology 3: 253.
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Toman, J.E.P., Fine, E.A., Everett, G.M., and Henrie, L.M. 1951. Experimental psychomotor seizure in laboratory animals. Electroencephalography and Clinical Neurophysiology 3: 102. Toman, J.E.P., Everett, G.M., and Richards, R.K. 1952. The search for new drugs against epilepsy. Texas Reports on Biology and Medicine 10: 96–104. Vreugdenhil, M., Vanveelen, C.W.M., Vanrijen, P.C., Dasilva, F.H.L., and Wadman, W.J. 1998. Effect of valproic acid on sodium currents in cortical neurons from patients with pharmaco-resistant temporal lobe epilepsy. Epilepsy Res 32: 309–320. Vreugdenhil, M., and Wadman, W.J. 1999. Modulation of sodium currents in rat CA1 neurons by carbamazepine and valproate after kindling epileptogenesis. Epilepsia 40: 1512–1522. Weiss, S.R., and Post, R.M. 1991. Development and reversal of contingent inefficacy and tolerance to the anticonvulsant effects of carbamazepine. Epilepsia 32: 140–145. White, H.S. 2003. Preclinical development of antiepileptic drugs: past, present, and future directions. Epilepsia 44 (Suppl 7): 2–8. Zupanc, M.L. 1996. Update on epilepsy in pediatric patients. Mayo Clin Proc 71: 899–916.
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46 Monitoring for Seizures in Rodents EDWARD H. BERTRAM
are developing new approaches (or variations in old techniques) that are suited to their particular needs. Common themes and issues, however, exist to all of these variations. In this chapter are described some of these different approaches, primarily with a view toward the varying needs of the researchers. In addition, we will touch on the issues and goals that the investigator should consider before deciding on a particular approach to documenting seizures. The documentation of seizures, often difficult enough in cooperative people, is significantly more difficult in small animals that are not inclined to cooperate with investigators who are concerned about a problem that is of no significance to the animal itself. This chapter describes the questions that researchers ask, the technical problems that are encountered, and the different technical approaches that can be taken. Although we describe a number of technical possibilities, what we really hope to present is a set of principles, some examples of solutions based on common research questions, and a path to allow a researcher to choose the approach to seizure documentation that meets the needs of the particular issues faced in a research project. Over the last decade, it has become clear that multiple approaches are available based on the desired level of detail that the investigator wishes to have. At no time should the information be viewed as complete or absolute, but rather a presentation of potential solutions to common problems and of frequently encountered pitfalls.
This chapter focuses on the techniques to document spontaneous seizures in rodents using video or electroencephalography (EEG), or a combination of the two. Over the history of epilepsy-related research using animal models, a key component of the research has been the physiologic documentation of the seizures through EEG recordings. In the beginning, the models in use were induced seizures that occurred only when the appropriate seizure-inducing stimulus was applied. Recording EEG was a relatively easy task because the investigator knew, with relative certainty, when the seizure would occur so that it was only necessary to record around the time of the expected seizure. More recently epilepsy research has been strengthened by the development of a number of models with spontaneous seizures, a development that provides this field of research something more closely akin to human epilepsy. Although this development has been greatly welcomed by the research community, it has brought with it the increased problem of documenting seizures that occur in a less-predictable pattern, sometimes less than once a day. With the truly explosive growth in transgenic mouse models that have epilepsy, or potentially have epilepsy, the need to document an often very subtle epileptic phenotype, or sometimes an overt behavioral phenotype that may or may not be epilepsy, has also increased the need for seizure documentation. Our first descriptions of prolonged EEG monitoring in rats (Bertram and Lothman, 1991; Bertram et al., 1997) were easy to prepare, because little was available on this subject. Most rodent work was done with acute seizures, and the EEG requirements were generally limited to a few hours at a time. With the growing numbers of animal models with spontaneous seizures, the need for prolonged monitoring has also grown, and now a number of laboratories (and vendors)
Models of Seizures and Epilepsy
REASONS FOR MONITORING Many reasons are given to monitor animals for possible epilepsy, and some of the more common ones are outlined
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in Table 1. Although these reasons are obvious to anyone involved in epilepsy, they are important issues because knowing which of those to address will determine the approach to monitoring that will be taken in any particular experiment and help determine the overall effort on the part of laboratory personnel, a frequently underestimated and underappreciated aspect of monitoring. Reviewing data and maintaining the system can easily take a half day for every day of monitoring, especially for an inexperienced reviewer. The two methods most commonly used are video recording for behavioral seizures and EEG recording to determine the physiologic patterns. Depending on the relationship between the behavior and seizure, using only one of the techniques may be more than adequate for the purpose. The evaluation of possible epilepsy can take several approaches. If the seizures are behaviorally overt (e.g., prominent motor behaviors), and at some point these behaviors have been documented with EEG as true seizures, video recording for later visual review is often more than adequate. If the behavior is consistent from seizure to seizure and the behavior is obvious, video recording can also be used to determine approximate seizure frequency. On the other hand, if the seizures are behaviorally more subtle, or if it is unclear whether altered behaviors are seizures, EEG recordings will be needed. Similarly, if the questions focus on the physiology of the seizure or pattern of onset, or if the nature of the behavioral changes is less clear, recording EEG is necessary and video recordings become less important. The message, therefore, is, before embarking on seizure monitoring in rodents, determine precisely what information is needed and choose an approach that will answer the questions with sufficient certainty so that little doubt remains regarding the results or the conclusions. It cannot be overemphasized, however, that at some point it is necessary to have a correlation between behavior and physiology (EEG). Our experience suggests that some repetitive, stereo-
TABLE 1 Reasons for Seizure Monitoring in Rodents Documentation of epilepsy/seizures Determination of phenotype in transgenic animals Frequency of seizures Behavioral nature of seizures Differentiating seizure related from non seizure related behaviors Severity of seizures Duration of seizures Effect of therapy Electrophysiology of the seizure Functional anatomy of the seizure Natural history of a particular form of epilepsy Chronobiology of spontaneous seizures
typed, and, at times, bizarre behaviors have no EEG correlation that would indicate seizure, whereas sometimes when what appear to be clear-cut ictal discharges on EEG turns out to be artifact from the environment or from some normal activity (e.g., head scratching).
EQUIPMENT Four components comprise video EEG monitoring: the video recording system, the EEG recording system, the components that connect the animals to the EEG system, and cages in which the animals are housed while the EEG and video are being recorded. In the last decade, with the increased sophistication of electronics, the number of possible solutions has increased significantly. In this section, we present some of the possible approaches to seizure documentation in the context of experimental questions, including the strengths and weaknesses of each of the approaches.
Video Recording At the moment, there are two broad categories for video recording: analog and digital. By analog, we mean cameras connected to videotape records (e.g., VHS or super VHS) and, by digital, we mean the direct recording to a digital storage format usually on the computer hard disk. The division between analog and digital refers to the storage format of the video recordings. Cameras Cameras, whether analog or digital, come in a great variety of formats and resolutions, as well as levels of light sensitivity. Regardless of the nature of the actual recording process, the camera must have the appropriate output for the desired recording or storage format (e.g., either NTSC or PAL, if videotape recorders will be used). In general, it is advisable to use cameras of higher resolution, especially if multiple animals will be recorded simultaneously or if a need exists to see subtle behavioral changes. With regard to light sensitivity and color, the choice is driven by the expected needs and recording conditions. For most applications, black and white video is sufficient, although most cameras available now are color, and usually little advantage is seen in storage needs if recording the video digitally. Light sensitivity, on the other hand, is a very important issue, especially if 24-hour recordings under light and dark conditions are planned. If the recordings will be made under steady conditions of light, most standard video cameras are sufficiently sensitive, sometimes to relatively low levels of ambient light. On the other hand, if a more typical 12-hour light, 12-hour dark cycle is used, then it may be necessary to use a camera that has sensitivity in the infrared range and
Equipment
use a source of infrared illumination to obtain usable nighttime images. These cameras are often black and white, which should be no problem. Some cameras switch between color and black and white, depending on the ambient light conditions. In addition to the camera itself, a lens is necessary. Three things need to be considered in selecting the appropriate camera lens: the field of view, the variability in the illumination of the subject, and the constancy of the subject’s distance from the camera. The desired field of view (and to some extent the subject’s distance from the camera) will help determine the focal length of the lens (also known as telephoto, wide angle, and so forth). The constancy of illumination is extremely important in determining whether a manual or automatic iris is chosen. The iris controls the amount of light that gets to the sensor and, in general, it is easier to choose an automatic iris that adjusts to ambient light conditions. The constancy of the distance from camera to subject will help determine whether to get an autofocus or manual focus lens. In general, because the animals are confined to cages that have a depth (in relation to the camera) of less than 12 inches, a manual focus is sufficient and may, in fact, be preferable because the autofocus may focus on cage walls or other objects that result in the animals’ frequently being out of focus. Recorders The two broad categories of video recording devices are analog tapes and digital media, in this case computers. Each has its own advantages and disadvantages, so once again, before deciding for one format, the investigator should consider carefully how the video will be used. Videotape recorders (VHS, Super VHS) have been available for many years and are now relatively inexpensive. When used at the slowest recording setting of a tape (depending on the length of the tape), they can provide 6 to 8 hours of continuous recording. If 16 or 24 hours of recording is desired without the need for tape changes by the investigators, two or three recorders can be linked (video out of one to video in of the next). The internal clocks and timers can then be set so that the next tape starts just as the previous one is ending. Using such a setup, the investigators only need to change tapes once a day. To imprint a date and time on the tape, a video clock or titler can be used to embed these data on the videotape. The titler is placed in line between the camera and the VCR. Other simpler choices to document time include placing a clock somewhere in the field of view. The time and date are primarily useful when trying to determine whether events are occurring in a particular diurnal pattern or if one wishes to link the video recording to another recording that also has a clock (e.g., a digital EEG) to determine how the behavior and EEG correlate.
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The advantages of the video recorder are that it is a simple, independent recording system that is not tied to a particular EEG system, the technology is widely available, and it requires no special expertise. It also has the advantage in that data can be reviewed by fast-forwarding the tapes while still maintaining reasonable visual resolution. The primary disadvantages arise in linking the tapes to the EEG recordings and in moving to a particular time on tape that might match with an EEG event. In the latter situation, it is necessary to move the tape by forwarding to an approximate time and then moving more slowly to the desired time. The videotapes work well as stand-alone systems, in which only behavioral data are desired. With this approach, the video recordings are replayed in fast-forward mode and the reviewer documents overt seizures, such as limbic motor seizures (Racine class 4 or 5, see Chapters 28 and 48). On the other hand, less overt seizures are frequently missed by fast-forward review, which leads to an underestimate of seizures in some animals or a complete failure to recognize seizures in others. Digital video, whether recorded with a digital camera or with an analog camera and converted at the computer to a digital file, is becoming the dominant method of recording seizure behavior. It has some clear advantages over videotape recordings: the EEG and the digital video recording can be easily synchronized so that, unlike videotape recordings, it is possible to go essentially instantly to the point in the recording that one wishes to see, as opposed to having to fastforward to the desired spot on the tape. Digital recordings can easily have segments saved for digital presentations. As with tape recordings, digital video comes in a variety of resolutions, so that investigators need to know what resolution serves their interests best, as the higher quality digital recordings (the higher resolution recordings can consume one or more gigabytes of storage per hour of recording). If recording multiple animals and wanting to discern the more subtle (or sometimes less than subtle) behavioral changes, then the higher resolution formats are likely worth the increased storage needs. Seeing video segments of the desired laboratory setup can help the investigator determine which is the best choice. Although digital video is often the format of choice, specific situations arise when the tape recordings have a clear advantage. Perhaps the time when tape recordings are most useful is when the investigator is using video only to determine whether animals are having behavioral seizures and how often. The fast-forward mode of the videotape provides better visual resolution at a higher review speed than digital video. With digital, if the fast review speed exceeds about twice normal, a significant degradation of the image quality occurs, whereas image quality (for purposes of review) can be maintained on the tape playback at several times higher speeds. Once again, before deciding on what method to acquire, know what your requirements are and, if possible, try it out first.
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Electroencephalographic Recording Although the concept of connecting a single awake animal to an EEG machine appears straightforward and often is when the recording is for only a few hours, the technical issues of obtaining a quality signal from multiple animals for days or weeks present a number of problems that must be taken into account. Connecting multiple individuals to a single recording device may reduce the quality of the signal to the point that the data are unusable. Maintaining animals on a recording system for prolonged periods of time requires a system design that allows for relative normal activity levels while maintaining a good connection to the EEG. To resolve these issues, it is necessary to design modifications in recording setups, connections, and housing that will meet the goals with a minimum of interference with the animal’s activities. No one EEG system is best for the recording of multiple rodents at one time. Almost all have been developed initially for clinical use and then adapted for the laboratory. Some laboratories have obtained turn-key systems from a single vendor, others have put together systems using components from a variety of vendors. In the latter scenario, the choices were often driven by equipment that was available and familiar to the investigator and what could be added to the equipment that was on hand to make the systems work. Various laboratories have coupled amplifiers from Axon (Cybercamp, Molecular Devices Corporation, Union City, CA) (Gorter et al., 2001), WPI (World Precision Instruments, Sarasota, FL) (Nissinen et al., 2000), or Grass (Astro-Med, Inc., West Warwick, RI) (Bertram et al., 1997, Kharlamov et al., 2003) with software from Stellate Systems (Monitor or Harmonie). Others have installed complete digital systems from Stellate (Stellate Systems, Montreal, Quebec) (Zhang et al., 2004) or Biologic (Bio-logic Systems Corp., Mundelein, Illinois) (N. de Lannerolle, personal communication). Some of the technical aspects of using these systems are discussed later in the chapter.
Radiotelemetry Another approach to monitoring favored by some is radiotelemetry EEG (e.g., the system from Data Science International, Arden Hills, MN). In telemetry, EEG electrodes are implanted and connected to a combination amplifier and radio transmitter. The amplified signal is transmitted to a receiver that is placed at the bottom of, or just below, the cage, which in turn is connected to the computer. The advantage of such a system is clear: the animals (rats or mice) have complete freedom of movement because the device is completely implanted with no external tethers. With the progressive miniaturization of electronic equipment, these devices have been made small enough to implant in mice. Disadvantages, however, exist as well. Most impor-
tantly, the current systems have a very limited number of implantable electrodes for EEG (usually two), which only allows for a single channel of EEG. For many applications, this limitation is not a problem; if, for example, the need is only to document seizures. In some cases, the transmission can be interrupted if the animal moves too far from the receiver (as in standing on its hind legs). The systems also tend to have higher start-up costs for similar numbers of EEG channels. The value of such a system lies in its ability to record EEG continuously over several months with no need to deal with cables. If the need for EEG data is limited (as in several hours per day, over a few days, this approach may be unnecessary, but for prolonged recordings of a single channel, radiotelemetry may be the best choice.
Hardwired Encephalography We will now discuss systems that use a cable to connect the animal to the EEG machine, of which there are two broad categories: analog and digital. Both are more than adequate for recording quality EEG in this setting, but each has its own advantages and disadvantages. Originally, analog machines were designed to drive pen writers on a paper medium, and they can still be used this way, although it is now more common to port the amplified signal to a digital recording system. Digital machines digitize the signal early in the pathway, sometimes at the input box, and can either convert the signal to an analog format for a paper write-out or, more commonly, send it directly to a computer for storage and display on a monitor. Perhaps the biggest difference between the machines is how the signal is brought into the system. In analog machines, each electrode acts as a separate input and can be matched with any other electrode to create a single channel of EEG signal. The creation of these montages occurs at the level of the machine by combining the inputs of the desired electrodes. One issue that is often considered is whether a benefit exists in using a preamplifier on the animal’s headset or at the headset end of the cable. We do not use them; other laboratories do. In theory, they can help improve the signal to noise ratio, but only if the source of noise comes after the preamplifier. If, for example, the source of noise is at the pins on the headset and the preamplifier is on the cable, one will likely amplify the noise. On the other hand, noise that comes later in the system could be minimized by the use of a preamplifier. We have found that most of the noise comes around the headset–cable junction, so that most animals would require a preamplifier on the headset to improve EEG quality. Because preamplifiers would require two of a swivel’s electrical connections to supply the power and because we have believed (perhaps falsely) that the benefit would be minimal, we have chosen not to use them. Others find them useful.
Equipment
Using Analog Encephalographic Machines Analog EEG amplifiers have been in use for decades and reliably amplify EEG signal. Because most of rodent EEG will be either from the cortex directly or from intracerebral electrodes, the amplitudes of the original signal will usually be sufficiently large (0.1 to 4 mV, unless a technical problem with the electrodes occurs or the electrode is placed in white matter or a ventricle) so that great amounts of amplification are not necessary. Most machines create specific mechanical electrode pairings at the level of the machine by linking the desired electrodes through central switches. Although it is possible to record all electrodes against a common reference, unlike in digital machines, it is not required. Thus, a number of rats can be connected to the machine and each channel acts independently. Using 32-channel machines (Grass Model 12), we have simultaneously recorded up to 15 rats, each with two electrode pairs, without difficulty. Although rarely used today, EEG can be recorded on paper. The primary problem with this approach is that once the signal is written out, the time base, gain, or filters can no longer be changed to see the signal in greater or lesser detail. For the conversion of the analog signal to digital for storage and display on a computer, most EEG machines have an analog out port that will connect the machine to an analog to digital converter of a computer. The software can then record, display, amplify, and filter the signal for best visualization on screen and for printouts for publication. The pin order for the analog out and A-D converter are often machine and software specific, so that the assistance of the vendor in making sure that the connections are correct is valuable. The primary disadvantage of the analog EEG machines is that they rarely come together with recording software, so if the machines are to be used to create computer EEG files, it is necessary to obtain the software from another vendor and link the machine to the software, which requires either knowledge on the part of the investigator or a cooperative software vendor. On the other hand, not having everything come from a single vendor has the advantage of not being tied to one vendor for everything.
Digital Electroencephalographic Machines and Their Special Considerations Today, the overwhelming majority of new EEG machines sold are digital, and most digital EEG machines come as a complete package with amplifiers, A-D converters, digital filters, software, and often digital video as well. This bundling obviously makes the acquisition of the system much simpler. The advantage to the system is the great flexibility in the recording and display of the signal after acquisition. Previously, however, questions have arisen about the requirement to record the EEG from all electrodes (in the case of multiple animals, all animals) against a single
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common reference. This requirement led to concerns about the ability to record multiple independent animals against a single reference electrode and still maintain a reasonable recording quality. Initial personal communications from investigators attempting to record multiple animals from a digital system supported this concern. In the last few years, other investigators have acquired digital systems and report no such problems. These digital EEG machines either allowed for multiple independent references or all of the animals were linked to a common reference. Further details are provided below in the description of several systems in use today. Perhaps the best approach to use when considering the acquisition of a digital EEG system is to consider exactly how many animals will be recorded at one time, and ask the vendor to demonstrate that the machine will work satisfactorily in the desired configuration. For digital machines, there is a common reference electrode, against which all other electrodes recorded. The pairing of electrodes is a mathematical calculation that is performed by the machine’s computer. The digital approach has the advantage in that many possible electrode connections (montages) can be created from the same primary data. The disadvantage is the common reference electrode that is required for the recordings. Although this technique works well when only one animal is being recorded, the common reference electrode for many rats is a significant problem when trying to record and create montages from multiple animals at one time. The use of a common reference from a single animal when recording from many can introduce significant amounts of noise into the system, to the point that the data are not interpretable. What several investigators have tried (with reported success) is taking a reference electrode from each of the animals and linking them so that a common reference can be used. This tactic and the grounding of cages or cage bottoms (see below under “Cages” for more details) can allow good recordings from multiple animals on a digital machine. Digitization Frequency Another issue that must be considered in choosing a particular system (analog or digital) when storing the data digitally is the digitization frequency of the A-D converter and the frequency range the investigator wishes to study. Most systems come with a standard 200- to 400-Hz digitization frequency per channel, which is more than adequate for good visualization of basic seizure patterns and activity up to 100 to 150 Hz. This range, however, may not be adequate for investigators who wish to study the very high frequency oscillations in the high gamma range (200 Hz or higher). For these individuals, a digitization frequency of 1,000 Hz per channel may be necessary. The only real disadvantage to this higher sampling rate is the increased storage requirements for the collected data. Sampling at this frequency also can
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increase the intrusion of a variety of types of environmental noise, a problem that could increase with the addition of more animals to the system, so that recording a very limited number of animals at one time may become necessary if the goal is to record high-quality fast frequencies.
Connecting the Animal to the Machine For recordings of short duration (several hours), during which an investigator will intermittently check the animal, very little is really needed in way of special accommodations. A cable can go directly to the input of the EEG from the animal, although placing the cable so that it is directly overhead and centered over the cage allows the animal reasonable short-term motility. If recording from mice, the cable should be light and flexible enough to allow reasonable mobility without undo stress on the animal or the headset, which can detach quite easily from the thin skulls of mice. For longer recordings, anything beyond daytime working hours, a number of accommodations must be made. The following sections describe possible solutions to the many problems presented when trying to record from rodents over prolonged periods of time.
Swivels Generally, for longer recordings the use of electrical swivels greatly facilitates longer duration recordings. The swivels come in a variety of designs but, in general, consist of a center rotating spindle with conductor rings that are in contact with fixed conductor brushes. The primary points that distinguish the swivels of the different vendors (besides price) is the number of contacts available, the materials used, the ease of turning, the nature of the cabling connections, and whether the swivel is electrical only, electrical and fluid in one, or electrical but capable of mating with a separate fluid swivel. Our own experience suggests that most swivels are capable of providing an electrically quiet connection. An additional issue to consider is the ease of turning. In this regard, we have found considerable variation from vendor to vendor and, at times, from shipment to shipment from a single vendor. Ideally, it should take no to minimal effort to turn the swivel, because any greater turning resistance will place strain on the headset, which could lead to its premature loss. This issue is especially true in young rats and in mice of any age. Many manufacturers and vendors of swivels exist, including Stoelting (Wood Dale, IL), Plastics One (Roanoke, VA), Harvard Apparatus (Holliston, MA), Kent Scientific (Torrington, CT) (Rattus, et al.), and Dragonfly (Ridgeley, WV). Some of the swivels we use have been in service for more than 10 years, so that the initial investment of $500 or more (in some cases, over $1,000) has been rewarded with significant longevity. For recording mice, we have had the greatest experience with Dragonfly swivels, initially with mercury filled acrylic
swivels, and, more recently, metal brush swivels that use ball bearings to reduce torque. These products have an extremely low torque turning resistance, which places essentially no stress on the headsets. For adult rats, most of the products will likely be successful, but it is a good idea to check them physically before committing to one or the other, especially if a larger number is obtained. Placing a single swivel above one cage can be accomplished in a variety of ways: attached to the edge of the cage, held in place by a ring stand and clamp, or placed in a frame that is suspended above the cage. Building a system with multiple cages for chronic video EEG recording, on the other hand, presents a number of problems for connecting animals to the EEG, so that the placing and modifying of the swivels is a major component in the design of a successful system. Attaching the swivel in a chronic recording setup (and its associated cable connections) has a number of conditions that must be considered. All the connections to and from the swivel need to be accessible for easy maintenance. The end of the swivel that has the cable connection to the animals must be accessible enough to allow easy connections and disconnections of the animal while being far enough away from the rat or mouse so that it cannot destroy the connections. For rats, this usually means a connection or swivel that is at least 30 cm above the bottom of the cage, but usually a bit higher. The type of cage and how the swivel and its connections can be accessed partly determines the positioning. For a cage that can be opened in the front, the swivel could be placed even in the cage, whereas for solid cages (e.g., cylinders), the swivel should be placed 5 to 8 cm above the cage top to give the researcher sufficient working space. Several approaches have been taken over the years for attaching the swivel to the recording setup. A number of investigators have chosen to allow the swivel to move freely up and down and somewhat horizontally by attaching the swivel to either a counterbalanced arm or to a pulley system. The advantage here is clear: the cable is always under a slight tension so that it does not bend and, thus, is kept out of the animals’ reach. In addition, because the cable can move more freely with the animal, it is less likely to place stress on the headset or the animal itself. The primary disadvantage is that these setups are mechanically more complicated and are susceptible to their own failures. The other approach to swivel placement is to fix them to a position that is separate from the cage. We have built an acrylic tray that holds up five swivels. The tray is attached to the bottom of the overlying shelf (or otherwise suspended above the cages). The connections to the rats pass through the bottom of the tray, while the connections to the amplifiers are supported and protected by the tray (Figure 1). This approach is simple and essentially maintenance free, but does not allow for easy access to the swivel, and no overhead room is available to add a fluid swivel for microdialysis or infusions. To make the swivel more accessible, we
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FIGURE 1 Swivels in acrylic tray. These swivels from Stoelting are secured to an acrylic tray (built in the laboratory). Holes are drilled in the shelf of the tray, which is made rigid by the addition of the two side rails. Cables from the swivels are run along the top of the tray to the connector pin board, which can be used to connect to the EEG machine or to a stimulator. The ends of the swivels seen have Ampenol connectors for attaching the cable to the animals. (From Bertram et al., 1997, with permission.)
have started using the same metal rod and connector frame systems used in chemistry laboratories. A double rod primary support is placed across the back of the cage shelf (two rods are needed to give sufficient rigidity to the support). At fixed intervals, short rod pairs extend over the cages, where the swivels are attached by a small acrylic plate (Figures 2 and 3). If desired, a fluid swivel can be attached independently above the electrical swivel. This arrangement allows free access to essentially all parts of the swivel, providing easy maintenance and connections. Connecting the cables from the swivels to the EEG recorders is a fairly simple matter of plugging the cables into the EEG headbox or other connection to the amplifier. Only one major caveat: if the cables go directly from the swivel to the amplifier, the possibility always exists that the cables will be pulled loose from the swivel because of an unintended movement. To reduce this possibility, we have used a connector board that is hard-wired to the swivels in a predetermined pattern. On the other side of the board are sockets for connecting cables to the amplifiers. This approach also makes the system a bit more modular and easier to maintain. A photo of this intermediate board is in Figure 4.
FIGURE 2 Swivel setup. This detailed photo shows a Dragonfly swivel with the primary connections to the cable and back to the EEG machine. We have modified the swivel by placing an acrylic armature to the bottom aspect of the swivel that serves as a fixed support for connecting the cable from the swivel to the cable to the animal. In the background the support rods are seen where they enter the acrylic block to which the swivel is attached. This swivel can be linked to a fluid swivel, which is seen on top. We have also modified the device by placing small umbrellas beneath the fluid swivel to prevent damage in case of leaks.
Cables to the Animals The cables connecting the animal to the swivel are the most vulnerable and fragile part of the recording system. First, there is a continual wear and tear on the cable, from the constant, at times forceful, activity of the rodent. In addition, there is the natural curiosity of the animal combined with the tendency to chew on chewable objects that makes the connecting cables vulnerable to failure. What has been
FIGURE 3 Swivel setup. Two swivels side-by-side demonstrate more clearly the rod support system as well as the different cable and tubing connections.
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FIGURE 4 Connector board. Attached at the end of an acrylic tray, but can be set up independently. Wires from swivels (ribbon cables) are attached (usually soldered) to the backs of the connectors. Separate wires go from the connector board to the EEG machine, or from a stimulator to the connectors.
essential for these chronic recordings to succeed is that the cable between the swivel and the rat be readily reparable or replaceable. Most of the swivels come supplied with a fixed cable that is often cut to a custom length, with a standard connector. These will work well until the wear and tear of animal activity or accidental chewing make the cable unusable, at which point the swivel may have to return to the manufacturer for replacement, with the resultant loss of use of the swivel for a significant period of time. For this reason, a key component to the long-term success of the recording system is a replaceable cable. Start by modifying the fixed cable supplied with the swivel, keep it short, and build a custom replaceable segment to connect the animal to the system. This modification usually involves placing a connector plug or socket that will connect to the cable to the animal. The key aspect for the connecting cable is that it is flexible so as not to place undue strain on the animal’s headset. As noted in a previous publication (Bertram et al., 1997), a variety of ways are available to make the cable, including purchasing standard shielded cable with the desired number and diameter of individually insulated wires. These can then be cut to any length desired and the ends finished with the appropriate connectors. The primary problem with many of these cables is that they are rather stiff, so that they have to be long enough to provide sufficient flexibility, which for some cables can be 50 cm or
more. Cables of this length are not a problem as long as sufficient overhead space exists. As we previously described, we have had great success by using lighter weight wires (24 gauge or smaller) that we braid and place through the center of a door spring. In addition to using individual wires to construct the cables, we have also used flat four- or sixconductor telephone cable, ribbon cable, and shielded or unshielded multiconductor cables with up to ten individual wires. These latter cables are relatively stiff and, thus, require longer lengths for sufficient flexibility. The construction with the spring exterior is relatively fast to create, is flexible, even with lengths as short as 30 cm, and, because the spring is unpleasant for the rats to chew, is essentially chew proof. Cable length should be just enough to reach the edges of the cage so that the animal can lie down in any position but not so long that when the animal rises up, there is enough bend in the cable that it can get to the cable. When the swivels are attached to a weighted counterbalance arm that goes up and down, always applying a very slight upward pull on the cable, flexibility and an exact length are less of an issue.
Cages Housing of the animals is an extremely important aspect in video EEG monitoring, especially if the animal is going to be monitored for days or weeks. The basic principles are that the cage should allow unrestricted movement of the animal and the cable that connects it to the EEG machine. There should be no to minimal overhead height limitations so that the animal can have unrestricted ability to rear up and explore without hitting its head and, thus, electrode assembly on a cage top closure. If the video monitoring is to be performed from the side, then the side of the cage to the camera must be transparent. If video monitoring will be performed from above, the cage tops must either be clear or absent. Cage dimensions must meet (or preferably exceed) the requirements set forth by the regulatory authorities, with the primary goal of giving the animals sufficient room to move and turn freely while keeping the space small enough so that the rat never places a significant strain on the cable by having the option to move beyond the limits imposed by the length of the cable. Ideally, the cage would have a circular floor, and the original cages we designed and built were circular, but many of the available cages are not, so, unless the investigator wishes to build custom cages (recommended for some purposes), no perfect match will exist between the length of the cable and the dimensions of the cage. We have also modified our original cylindrical cages for rats to the dimensions needed for mice (Figures 5 and 6). Circular cages with the only opening at the top have the advantage of a relatively simple design and construction.
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FIGURE 7 Multiple rat monitoring cages. Row of four cages with overFIGURE 5 Circular cage for mouse monitoring. Cage made of 5-inch diameter acrylic tubing, 6 inches in height. A plastic funnel, with the spout removed, serves as a cage top. The bottom is made of 1/4-inch stainless steel mesh that is attached with a silicon adhesive. Mice can be recorded in such a chamber for at least several weeks without interruption.
head electrical and fluid swivels. Cages are built from sheet acrylic and have front opening doors, either attached with hinges or sliding up and down in a groove.
They also give the animal a fairly good range through which it can move. The primary disadvantage is that the access to the animal is only from above, which at times can be challenging, especially if the rat has fluid lines as well for infusions or dialysis. A number of laboratories have developed tall square cages with a front door that is either hinged or which slides up and down through slots (Figures 7 and 8). This design gives direct access to the animal and its lines at any point, which allows for easy corrections to problems, often with minimal disturbance to the animal.
FIGURE 6 Mouse with headset and support belt. Cable attached to mice is modified because mouse skulls will not support the stresses of direct attachment to cables over prolonged periods (days or weeks). The cable is attached to a support rod that is on the Velcro belt that goes around the animal’s thorax and chest. The pins from the mouse are connected to freefloating wires that go to the headset. Mice have been monitored continuously for up to several weeks with this or slightly modified cable connections.
FIGURE 8 Rat cage showing more detail with cable that just reaches floor of cage, together with fluid line in separate spring protector. Animals can be kept on chips with slide-out tray when not monitoring or on grounded steel mesh insert (not shown) when undergoing EEG monitoring.
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The electrical environment in and around the cage can have a significant influence on the recording quality, especially if multiple animals are recorded. As more fully explained in the following section on electrical artifact, the greatest challenge arises from trying to record multiple animals at one time. The amplifiers of most EEG machines are designed with a common mode rejection that removes electrical artifact that is common at all electrodes, a relatively easy task when limited to a single head, but potentially more difficult when recording from multiple animals of varying activity schedules, housed in separate environments within each cage. The proper cage design can reduce many of these problems. Our original cages had wire mesh bottoms that all rested on the same metal shelving. Thus, all cages were grounded to one another. Although noise problems from other sources remained a problem, electrical isolation of the animals from one another did not occur and was thus minimized with this cage design. Recently, we built cages with a new design that had an entry door on the front. The cages were built entirely of acrylic (including the bottom that had a slide-out tray for bedding) and, thus, they electrically isolated the animals from one another. As a consequence, we had a significant amount of noise. We added a wire mesh bottom just above the litter tray, used wires to connect these bottoms to one another and to the underlying metal shelves. The noise was significantly reduced. Other investigators who had developed an earlier variation of this design had built the side and back walls from sheet metal, which also served to provide a uniform electrical environment for the animals.
Electrical Artifact Perhaps the greatest problem in recording multiple animals (or even a single animal) is the constant struggle with electrical artifact, which, as in clinical EEG, can come from a variety of sources. Although artifact is usually quite obvious, completely hiding the underlying EEG signal, it can also mimic interictal or ictal activity to the point that it is impossible to tell real brain activity from signal that originates outside the skull. In this section, we outline the common and some of the less-common causes of artifact and how to diagnose and fix the problem. Perhaps the most common source of artifact, especially if it involves a single animal is a loose connection or broken wire. A key tool for locating the source of this type of artifact is an ohmmeter for measuring resistances at selected points in the lines that connect the animals to the machine. A good ohmmeter with a 9-V battery (e.g., a Simpson Model 260) is necessary when measuring intracerebral electrode resistances, which can range between 5,000 and 20,000 Ohms with normal stainless steel electrodes. Although concerns about using direct current in the brain can arise, the animal does not notice when the current is applied,
likely because it is quite small. The higher voltage of this ohmmeter is necessary to obtain reliable and consistent readings in a volume conductor (the brain). Measuring resistances in a logical sequence from the level of the electrode pins, the swivel channels and at the cables for the continuity of individual wires and potential crosstalk between wires should be a routine procedure whenever suspicious activity is seen in one or more channels. As mentioned in the section on cage design, if multiple animals housed in individual cages are recorded on a single system, a real potential for noise exists if the animals are electrically isolated from one another. To prevent this type of problem, we recommend using cages with wire mesh bottoms or sheet metal sides, followed by grounding the metal components of the cages together and then to the shelving. If metal housing connects to the electrical swivels, it may be helpful to link those casings to a common ground. Finally, link all implanted animal ground electrodes to a single common input in the EEG machine (frequently known as isoground). This approach may not completely eliminate noise, but usually it significantly reduces it. Incomplete grounding of cages and animals is often obvious because the problem is seen in all channels, but sometimes the source of the noise is not associated with the channel(s) that is causing it, because the noise may appear in all of the channels except the one(s) associated with the real cause. When confronted with noise (and just about anything, including activity resembling seizures can be noise), think first about the common sources, which include bad grounding, loose connections (between connectors, between pins, and wires anywhere from the intracerebral electrodes to the pins going to the EEG input), animal activity (e.g., head scratching or chewing), ungrounded cages, and unmatched impedances among the intracerebral electrodes. Some less commonly recognized sources of noise include open (and turned on) EEG channels with crosstalk to other channels, bad connectors in headbox, and crosstalk at the digitizer. Localizing the problem can take time, and requires a logical stepwise approach to rule in or out potential sources sequentially. In general, the first thing to consider when seeing intriguing and potentially exciting recordings is that one is seeing noise from biological or electronic sources. Only after excluding noise as a source can one feel a little excited about the results on the EEG.
SURGICAL AND IMPLANTATION ISSUES A variety of approaches can be used to implant and fix electrodes to the skull for chronic recordings, and they are largely determined by the electrodes chosen, but some basic issues are common across all types of electrodes. Four basic components are necessary: small stainless steel or plastic screws (0 gauge or smaller) for fixing the headset plastic to
Specific Issues for Age and Species
the skull, the plastic for covering the skull and fixing the electrodes in place (dental acrylic: methyl methacrylate, fast setting), the electrodes for implantation, and the connector between the electrode and the cable. Of the four components, the one that has the greatest variability in choice is the electrode. Many are fabricated in the laboratory from wire and connector pins, but others are purchased already built. Most of the locally built electrodes are single or twisted pair wires (stainless steel or, less commonly, platinum iridium) that are insulated with a thin coating of Teflon. Other materials (e.g., silver or copper) are not recommended because they cause toxic local reactions in the tissue. The single wires are usually placed in the cortex. Deeper placement of a single wire for monopolar recording is more difficult because the singles are less stiff, and accurate stereotactic placement is less certain. In placing the electrodes in the superficial cortex when electrical stimulation is considered, be careful in stripping the insulation so that the dura, which is quite pain sensitive, is not inadvertently stimulated. The twisted pair electrodes are used for bipolar recordings and stimulations in deeper structures. To prevent breaks in the insulation that could lead to inadvertently recording from or stimulating other structures, do not twist the wires too tightly. Gold pins or other connectors can be soldered before implantation to the extracranial ends of the wires. Concentric bipolar electrodes (which are more commonly purchased) are best for recording from welldefined and restricted regions. Similarly, these electrodes are useful if very precise and restricted focal electrical stimulation is the goal. For the induction of afterdischarges or status epilepticus, however, they are less useful in part because the area of stimulation is frequently too restricted. A variety of commercially available connectors are available that hold the electrode wires for connection to the cable, and the ones from Plastics One are used by a number of laboratories. They are complete systems and have a secure connection between the headset and the cable. We have used Amphenol plastic strip connectors over the years, in part because of long habit, and in part because we can customize the connectors in a variety of ways. The connectors are designed to hold the WPI220 series of pins and sockets. We cut the strips for a defined number of pins or sockets, and then add one or two additional holes on the strip to serve as ports for the screws that will hold the cable and headset together. We have found that hex head screws work best for this purpose because they can be screwed in more easily than flat-head (single-slot) screws. As a precautionary note, although it is easy to connect the headset and cable in normal animals, it is more difficult to do the same in the poststatus epilepticus animals, in part because they are less likely to cooperate. For these animals, we have found that it is less stressful on the animal, the investigator, and the headset to anesthetize the animals lightly with an inhalational anesthetic before attempting to attach the cable.
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Two issues are extremely important when performing the surgeries to implant the electrodes for long-term recordings: skull preparation and screw placement. The two aspects to skull surface preparation are removing all soft tissue and preventing infections in the space between the skull and headset. Remnants of soft tissue can begin to regrow and eventually lift up the headset or act as a conduit for infection. The best approach that we have found is using gauze moistened with an iodine antiseptic, followed by a similar vigorous scrub with a 2% hydrogen peroxide solution. This latter step is very effective at moving small remnants of soft tissue and at slightly roughening the skull surface, which provides for a better adhesion between the skull and the dental acrylic of the headset. The basic principle behind screw placement in the skull is to prevent rocking of the headset, either front to back or side to side, plus a few extra screws for good measure. Thus, several should be placed a few millimeters (or more) from the anterior and posterior borders as well as from the sides. For adult rats, we have found that placing screws on the lateral aspect of the skull helps keep the skull secure longer. To achieve this placement, the attachment of the masseter muscles to the lateral ridges of the skull must be lifted up (usually with a very gentle scraping with a fine rounded forceps’ tip, at which point a small pocket forms between the muscle and the skull. The screw can then be placed a millimeter or two below the lateral ridge, and the liquid plastic dripped into the space, covering the screw. This approach has significantly improved the longevity of the headsets.
SPECIFIC ISSUES FOR AGE AND SPECIES The basic issues for monitoring (EEG recordings, video monitoring) are the same regardless of the species or age of the animal. There are differences, however, in how one attaches a 25-g mouse to the system as compared with a 500-g rat. The rat is obviously large and strong enough and the skull thick enough to move around with a cable and swivel that offer some slight resistance or weight. For the mouse, on the other hand, there can really be no significant strain on the headset, otherwise it will quickly detach. In addition, the mouse has much less room on its skull for screws and electrodes. Thus, for the mouse, specific changes must be made in the standard rat approach to allow for successful long-term monitoring (for up to several weeks at least). Perhaps the single most important accommodation for mouse recording is in the selection of the swivel. Most of the commercially available electrical swivels have a greater or lesser resistance to turning and, in many cases, the effort required to turn the swivel is greater than the mouse can generate. In our own experience with swivels from a number of
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manufacturers (but certainly not all), we have found that the Dragonfly swivels, first the liquid mercury based and more recently the ball bearing containing, have essentially no turning resistance, making them ideal for mice. The other major aspect is the effect of the cable itself, which should place minimal to no weight on the animals. Making the cable slightly shorter, so that it remains suspended well above the mouse by a centimeter or two is the first step. In the early stages of developing the mouse recording system, we attached the cable end to a rigid wire that extended from the back of a thoracic harness (see Figure 6). The mouse had a traditional plastic headset that connected to the socket on the cable. This configuration worked well, except that it was difficult to build and the mice would occasionally get out of the belt. More recently, we have simplified the design greatly, so that the cable terminal hangs freely and no plastic strip headset holds the electrode pins. Instead, the electrode wires come out of the dental acrylic and stand freely above the animals. These pins are placed directly into the sockets on the cable (using forceps) and friction holds them in place. The flexibility of the loose electrode wires reduces strain on the headset but the wires are stiff enough to turn the swivel. The quality of the EEG has not been affected by this approach and we have been able to maintain these headsets for up to 4 weeks. An even bigger challenge is the recording of rat pups over a period of time. At this time, it can be simply said that the technology does not really exist. The pups are too small for an implantable device, and, because they cannot be away from the mothers for more than several hours until weaned, prolonged cable recordings are not an option because the mother will likely damage the recording cable. The problem is further complicated by the rapid growth in the animal’s head, so that where an electrode is implanted may not be where it is a few days later. For the moment, we will have to content ourselves to intermittent recordings from young animals for a few hours at a time until the technology develops either to maintain the pups away from the mother for prolonged periods or for truly miniaturized devices.
SUMMARY Video and EEG monitoring, especially over the long term can be useful in the examination of a number of questions, but it takes more effort than most people are aware or willing to expend. Many of the questions can be answered with relatively simple approaches, including video monitoring, but to determine if certain novel behaviors (or behaviors with several potential interpretations) are truly seizures, a combination of video and EEG is usually necessary. Reducing, ruling out, and correcting artifact is a continuous problem that cannot be underestimated or ignored.
SPECIFICS ABOUT SOME DIGITAL SYSTEMS Bio-Logic CEEGgraph The success of recording multiple animals from this system is based on the use of a separate reference electrode for each animal. The Bio-Logic CEEGraph intracranial monitoring equipment can record up to 128 channels. The 128 channels are divided into 8 sets of 16 channels at the headbox. At the headbox, a separate ground electrode and reference electrode can also be connected from each set of 16 EEG leads. The EEG signals for a given set of 16 channels are amplified with respect to the reference signal for that set of channels. Up to eight animals can thus be recorded on this equipment if the electrodes from the animals are connected to the equipment such that only one animal’s EEG leads are connected to a given set of 16 channels. Each set of 16 channels acts in essence like a separate EEG acquisition system, although with one difference. The difference is that although a separate ground connection is provided on the headbox for each set of 16 channels, all ground connections on the headbox are connected to a single ground point on the amplifiers. If multiple animals are connected to the equipment, the ground electrode on each animal, in essence, acts like a separate connection to ground from the equipment. This in itself does not negate the possibility of recording from multiple animals, but it is important that all the animals be in the same electrical environment. It is also important to avoid the possibility of ground loops by ensuring that there are not separate pathways from different animals to ground.
Stellate Systems The recording system (32 channels) uses a single common reference electrode for creating montages later. Other researchers report that the system can work well by linking all of the indifferent electrodes from all of the rats into a single electrode (a series of linked sockets with a single output connection) that will go to the reference input on the headbox. A similar linking of ground electrodes is also necessary, but that communal linking is true for most systems. Several investigators (personal communication) have reported success with four to six rats, but it is unclear whether additional animals will create noise problems that are difficult to overcome.
QUESTIONS TO ASK IN DECIDING WHICH METHODS TO USE TO DOCUMENT SEIZURES IN RODENTS This section is intended to serve as a way for an investigator who is considering using some form of video EEG monitoring to acquire a system that will fit the needs now
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and in the near future. No weighting or point system is available to help with the process because the decision is often based on a complex interplay of factors. If the final answer is that your needs are of a very high level, but your need for the technique is small and intermittent, you may also wish to consider the following question: Whom do I know who is already monitoring animals regularly so that I might borrow some monitoring time on his system? It could save you much effort and headache. The answers to these questions will help an investigator make the necessary choices for a video only approach, for one or two animals for a few hours at a time with basic video and EEG monitoring or for a complex system designed for monitoring multiple animals for days or weeks at a time. Before starting down this path, consider precisely what is needed to answer the scientific questions in a way that truly supports the conclusions that will be drawn. Are you monitoring to confirm that a suspected behavior is a seizure? In the absence of prior seizure documentation, you will need a combination of EEG and video obtained together. Some strange and stereotyped behaviors are often not seizures and some seizures are barely perceptible behaviorally. Are you trying to define an epileptic phenotype in a transgenic animal? The answer to the first question applies. If you have only one or two genetic lines you need checked, then it may be worth collaborating with someone who has monitoring experience. If the creation of new lines is an ongoing activity, then having your own system may be worthwhile. How much effort can you/will you commit to this activity? If monitoring for seizures will be an active and continued part of your research, then the investment in equipment and manpower is likely worthwhile. What is your budget? A new video EEG recording system can cost $30,000 or more, with additional costs for swivels, cages, and a review station. A further cost is of technician time to review the data and maintain the system. What kind of compatibility do you need? Are you going to share files with other programs or investigators now or in the future? If building around available equipment, what do you need to add? The most expensive piece is usually the amplifier, so if you have an old EEG machine, a vendor may be able to provide the software and appropriate connections. What kind of animals will you monitor? Size and age will determine what kind of housing, cabling, and swivels you may need. How many animals will you be monitoring? At one time? Overall?
The answer will determine how many channels of EEG you will need, as well as the number of swivels and cages. If the total needs are small, you may consider outsourcing the work. Are you just trying to confirm whether the animal that is taken from a population that is susceptible to develop epilepsy (and epilepsy is confirmed from previous video EEG monitoring) is epileptic? Are the seizures overt to the casual observer? In some well-established epilepsy models (as in the poststatus epilepticus models), the behavioral seizures are stereotyped, usually obvious, and generally have a good correlation with the EEG. In such cases, if the documentation that the animal is having seizures is all that is needed, video monitoring alone may be sufficient. If seizures are less apparent or undocumented, EEG will likely be necessary as well. What kind of facilities do you have for this activity? A corner in a laboratory? A dedicated vivarium room? Current regulations will likely require you to perform chronic (as in more than 12 hours) recordings in a vivarium. You should keep this need in mind when planning for monitoring. How many hours per days or days per week will you monitor the animals? Will you need swivels for longer recordings? How much effort will laboratory staff have to expend with monitoring? Laboratory or vivarium? How many research staff can you dedicate to this operation? What is their level of skill? Chronic monitoring has significant upkeep requirements as well as the need for regular data review and reporting. Gaining the skills in EEG recording, surgery, and troubleshooting can take a number of months before one is proficient. Can you interrupt the recordings to review the data or do you need to transfer the data to a central reading station to minimize interruptions in recordings? A separate reading station, away from the vivarium is very efficient, but adds significantly to the cost. What are the light conditions in the recording area, and how will that affect the video recordings? Do you need to consider infrared low light recordings? How will you synchronize the video and EEG recordings? With a clock titler or a clock in the picture on videotape or with synchronized digital video and EEG recordings. Do you need special housing for the animals? Special caging can add to the time and cost in setting up a system, but is generally worth the effort.
Acknowledgments The author would like to thank Dr. Nihal de Lanerolle for his description on the use of the Biologic CEEGraph digital EEG system as well as John Williamson for his many invaluable insights and technical creativity
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during the evolution of our monitoring systems. This work was supported in part by NINDS grants NS25605 and NS49617.
References Bertram, E., and Lotham, E. 1991. Ambulatory EEG cassette recorders for prolonged electroencephalographic monitoring in animals. EEG Clin Neurophysiol 79: 510–512. Bertram, E.H., Williamson, J.M., Cornett, J.C., Spradlin, S., and Chen, Z.F. 1997. Design and Construction of a Long-term Continuous Video-EEG Monitoring Unit for Simultaneous Recording of Multiple Small Animals. Brain Research Protocols 2: 85–97.
Gorter, J.A., van Vliet, E.A., Aronica, E., and Lopes da Silva, F.H. 2001. Progression of spontaneous seizures after status epilepticus is associated with mossy fibre sprouting and extensive bilateral loss of hilar parvalbumin and somatostatin-immunoreactive neurons. Eur J Neurosci 13: 657–669. Kharlamov, E.A., Jukkola, P.I., Schmitt, K.L., and Kelly, K.M. 2003. Electrobehavioral characteristics of epileptic rats following photothrombotic brain infarction. Epilepsy Res 56: 185–203. Nissinen, J., Halonen, T., Koivisto, E., and Pitkanen, A. 2000. A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Res 38: 177–205. Zhang, G., Raol, Y.H., Hsu, F.C., Coulter, D.A., and Brooks-Kayal, A.R. 2004. Effects of status epilepticus on hippocampal GABAA receptors are age-dependent. Neuroscience 125: 299–303.
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47 Imaging Approaches in Small Animal Models ASTRID NEHLIG AND ANDRE OBENAUS
published in the late 1970s, for the measurement of local cerebral metabolic rates for glucose (LCMRglc) by the [14C]2-deoxyglucose (2DG) technique (Sokoloff et al., 1977) and local cerebral blood flow (LCBF) rates using the [14C]iodoantipyrine (IAP) technique (Sakurada et al., 1978). All theoretic and practical issues concerning these two techniques can be found in the articles cited above. Both techniques were adapted to the adult mouse (Bouilleret et al., 2000a; Jay et al., 1985, 1988) and to the immature rat (Nehlig et al., 1988, 1989). These techniques allow mapping of cerebral glucose utilization or blood flow in as many regions of interest as needed in conscious, freely moving animals in physiologic, pharmacologic, behavioral, or pathologic situations. The spatial resolution of these techniques, which is excellent, is based on the revelation of the silver grains on autoradiographic films, which are excited by the electrons emitted from the 14C-labeled molecule that has accumulated in all brain regions. The optical density of the regions of interest can be measured (e.g., on serial coronal brain sections) and compared with that of calibrated [14C]methylmethacrylate plastic standards. The final step is the calculation (by means of specific operational equations) of the relation of 14C concentration to rates of cerebral glucose utilization or blood flow in each specific brain region of interest using an image processing system. Many software systems are now available for these procedures, including free software offered on a National Institutes of Health (NIH) website. The main technical difficulty in both techniques relates to the fact that the insertion of polyethylene catheters, usually into the femoral artery and vein, is needed for quantification. The venous catheter allows for the intravenous (i.v.) injection (2DG) or infusion (IAP) of the tracer, whereas the arterial catheter is needed to withdraw timed blood samples for the quantification of the temporal evolution of
INTRODUCTION The use of neuroimaging for assessment and treatment of neurologic disease in animal models is rapidly increasing. Advances in imaging are driven primarily by our improved understanding of the physiological bases of these techniques and by access to more sophisticated imaging modalities. The main limitations of these approaches relate to the fact that most animal models of epilepsy have been developed in rodents and, therefore, require the miniaturization of the techniques, which, for the most part, have been developed to monitor human brain structure and function. This limitation is particularly acute for studies in immature rats and mature or immature mice. The first part of this chapter is devoted to autoradiographic techniques that have been used to study animal models for about three decades and have provided a better understanding of the pathophysiology of several epileptic syndromes (Dubé et al., 2000b, 2001b; Ingvar, 1986; Ingvar and Siesjö, 1990; Meldrum, 1983). The second part is devoted to magnetic resonance imaging (MRI) that has recently become the imaging technique of choice in clinical studies. The superb anatomic results obtainable with MRI, combined with its ability to observe, serially, biophysical tissue changes in vivo, have already provided evidence of alterations within the small animal brain following seizures and epilepsy (Jackson, 1994; Sitoh and Tien, 1998; Stears and Spitz, 1996).
AUTORADIOGRAPHY OF CEREBRAL METABOLISM AND BLOOD FLOW Technical Considerations and Limitations The autoradiographic techniques allowing for the measurement of cerebral functional activity in the rat were first
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the specific activity of the tracer in blood. In the adult rat, the insertion of catheters does not present any technical difficulty and the volume of blood is sufficient to allow for repetitive sampling with no significant consequences on the physiologic status of the animals. However, in mice or immature rats, the catheters are difficult to insert; this difficulty is compounded in immature animals where the small size of the vessels is combined with the immaturity of the vessel (especially the vein) wall. Moreover, because of the small body blood volume, it is necessary to reduce the volume and number of the samples. For these reasons, it is clear that quantification is virtually impossible to achieve in immature mice. Another difficulty revolves around the issue of temporal resolution. While LCBF can be measured with IAP over very short durations, ranging between 20 and 60 seconds, the quantification of LCMRglc requires a 45-minute period of stable glucose metabolism, which is impossible to achieve with events such as short seizures. This limitation has led many authors to use the 2DG technique to map brain metabolism during seizures in a nonquantitative manner (Albala et al., 1984; Ben Ari et al., 1981; Handforth and Ackerman, 1988; Handforth and Treiman, 1995a,b; McIntyre et al., 1991; Tremblay et al., 1984). In this type of approach, the optical density of a structure is compared with standards and converted to a ratio of the optical density of the structure of interest over that of a structure that is supposed to remain unchanged, most often the corpus callosum or the cerebellum (Collins et al., 1983; McIntyre et al., 1991). However, this type of qualitative approach may lead to large errors and needs to be applied with great care. In the original [14C]2DG method, as described by Sokoloff et al. (1977), the tracer is injected as a bolus at the onset of the experiment. The 45 min experimental time period allows for the clearance of most of the unmetabolized 2DG from the circulating blood. This procedure minimizes potential errors due to a large proportion of free 2DG present in blood and brain tissue at the time of sacrifice—which is the case in short duration experiments. Arterial blood sampling, at predetermined times over the 45 min experiment, allows the investigator to extrapolate, from the temporal evolution of the plasma concentration of glucose and [14C]2DG, the integrated specific activity of the radioactive tracer in the brain. Plasma concentration of [14C]2DG itself depends on various parameters, mainly body blood flow rates and peripheral metabolic activity, which greatly influence the final value of the brain integrated specific activity of the tracer. Indeed, in a given paradigm, the final value of integrated specific activity of [14C]2DG in brain may vary by 3-fold. These factors are not controlled in qualitative experiments, and may obviously lead to large errors. If it is desirable to map short events, it is preferable to choose alternate methods like the autoradiographic measurement of LCBF which can be quantified over very short durations (Sakurada et al., 1978).
However, if the [14C]2DG method is applied, one should not work over too short durations. The first 20 min of the [14C]2DG experiment are the most critical since they represent the largest part of the integral of the specific activity of the tracer in plasma. Thus, in some cases, it is better to prolong the experimental time, up to 30-35 min, which is possible if the event of interest leads to large increases in brain activity. Using this compromise, one can still carry out quantification with relatively small risks of errors in calculation of LCMRglc. Finally, if none of these procedures is compatible with the seizure event under study, then the experimenter should map brain activity, within a single animal at a time, using for comparison the activity of that brain structure that is not expected to change. In the following part, we will summarize how the use of autoradiographic imaging techniques has helped to outline seizure circuits, to characterize age-related responses to seizures, and to understand the relationship between metabolism, circulatory changes and brain damage.
Autoradiographic Measurement of Cerebral Glucose Use During Seizures Metabolic Mapping in Adult Animals with Seizures: Correlation with Brain Damage The 2DG autoradiographic technique has been extensively used to map metabolic changes induced by various types of seizures. This technique has been helpful in identifying the circuits involved in seizures induced by bicuculline, flurothyl, and kainate, and has contributed to our understanding of the role of metabolic factors in the genesis of brain damage. These studies showed that strong, rapidlyoccurring regional hyper-metabolism, followed by a marked metabolic depression correlates with the development of neuronal damage induced by sustained seizures in adult rodents and primates (Folbergrova et al., 1981; Ingvar, 1986; Ingvar et al., 1987; Meldrum, 1983; Siesjö et al., 1983). The late phase of metabolic depression coincides with the onset of neuronal damage (Ingvar and Siesjö, 1990), which appears to be independent from the deterioration of systemic factors like blood gases and acidosis. Indeed, damage occurs even in anesthetized, artificially ventilated rats in which systemic factors are maintained in the normal range (Meldrum, 1983; Nevander et al., 1983). Hypermetabolic regions that do not undergo the secondary depression phase—for example, the hypothalamic nuclei, caudate nucleus and globus pallidus in the lithium-pilocarpine model of SE—do not undergo marked damage (Clifford et al., 1987; Handforth and Treiman, 1995b). The quantitative 2DG technique was also applied to the measurement of LCMRglc in genetic models of epilepsy like the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). When measured in the basal situation (hence,
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representing a mixture of ictal and interictal events), LCMRglc shows widespread increases (relative to nonepileptic animals) in all brain regions, although the seizure circuit is limited to the thalamo-cortical loop (Nehlig et al., 1991). This finding is consistent with the results from human studies reporting generalized increases in brain metabolism of patients with childhood absence epilepsy (Engel et al., 1985; Theodore et al., 1985). The use of drugs aggravating or preventing the occurrence of spike-and-wave discharges in GAERS demonstrated that the metabolic increase occurs during the interictal phases rather than during seizures (Nehlig et al., 1993), as further confirmed by continuous recording of CBF by means of Laserflow Doppler (Nehlig et al., 1996). In fact, during the spike-and-wave discharges, neuronal activity does not exceed the normal range of neuronal excitatory or inhibitory states—a finding quite different from that seen during paroxysmal depolarizations that occur during convulsive seizures (Gloor, 1979). The reason why brain metabolism is increased over normal levels during interictal states remains unclear. Age-Related Changes in Cerebral Metabolism During Seizure Activity Studies performed on two models of status epilepticus (SE) identified the age-related maturation of the structures involved in the circuits underlying seizures. In animals less than three weeks old and undergoing kainate-induced seizures, the pattern of brain activation is limited to the hippocampus and septum; this pattern reflects the distribution of paroxysmal discharges recorded in the hippocampus at that age. Starting at the end of the third week, when the toxin leads to the occurrence of limbic motor seizures, other regions involved in the seizure’s circuit are also activated, namely the amygdaloid complex, thalamus, piriform, and entorhinal cortex. At that time in maturation, metabolic maps become similar to adult maps; the pattern of metabolic activation reflects mainly the maturation of the limbic circuitry, which allows the propagation of the seizures to extrahippocampal and subcortical sites (Albala et al., 1984; Tremblay et al., 1984). In the lithium-pilocarpine model of SE, LCMRglc also varies with postnatal age. In PN10 rats, pilocarpine-induced SE involves more regions than kainateinduced SE, and increases in LCMRglc occur in piriform cortex, lateral septum, amygdala, hippocampal CA1 area, and hilus, but not in hypothalamic and thalamic areas. In PN21 and adult rats, increases in LCMRglc are generalized and quite dramatic in the cerebral cortex, mainly entorhinal and piriform, the hippocampus, amygdala, septum, and thalamus (Figure 1 and Fernandes et al., 1999). The activation of a limited number of brain areas in animals younger than 3 weeks is associated with no or limited brain damage (Dubé et al., 2001; Fernandes et al., 1999; Priel et al., 1996; Sankar et al., 1998; Tremblay et al.,
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1984). In rats older than 3 weeks, kainate, pilocarpine, and lithium-pilocarpine seizures lead to widespread brain damage mainly in hippocampus, amygdala, septum, entorhinal, and piriform cortex where largest metabolic increases are recorded (Dubé et al., 2001; Fernandes et al., 1999; Nicketa et al., 1984; Sperk, 1994; Turski et al., 1989). The relation between large regional increase of LCMRglc associated with seizures and development of neuronal damage has also been reported in other models of seizures in adult rodents and primates (Meldrum, 1983; Ingvar, 1986; Nevander et al., 1985). In the pentylenetetrazol (PTZ) model of SE induced by repetitive low doses of the convulsant (El Hamdi et al., 1992), the pattern of metabolic activation varies also with age. The PN10 rat shows widespread, general activation of all brain structures. The largest metabolic increases occur in brainstem regions involved not only in seizure circuits but also in the control of autonomic functions essential for survival. In the PN21 rat, LCMRglc also showed the greatest increase in most brainstem regions but were either similar to or lower than control values in most anterior brain regions. In this model, long-term decreases in LCMRglc are recorded (Hussenet et al., 1995), but no brain damage is seen (Pereira de Vasconcelos et al., 1992; Pineau et al., 1996). This result is surprising, given the relationship between metabolic changes during prolonged seizures and brain damage detailed above; that is, the regions that are already metabolically depressed during the prolonged PTZ seizures in PN21 rats should undergo brain damage. Thus, it appears that the PN21 brain is able to withstand prolonged seizures (induced by the GABAA antagonist) that lead to metabolic decreases, without undergoing neuronal damage—which is opposite to what occurs in the adult brain. Circuits Involved in Acute Limbic Seizures The 2DG technique has been used to map the progression of the circuit involved in seizure activity, for example during the development of lithium-pilocarpine SE (as defined by EEG changes). During initial entry into SE, cycles of variable convulsive severity are accompanied by an alternation of metabolic increases between small and large brain structural domains. In the later stages of SE entry, these cycling mechanisms become ineffective, leading to steady-state maximal forebrain recruitment. During the later stages of SE, the transition from fast spiking to periodic epileptic discharges proceeds through successive stages characterized first by a reduction of cerebral glucose utilization within the forebrain and then by a reduction of the anatomic substrate of the seizures (Handforth and Treiman, 1995a,b). Likewise, during the development of kainateinduced SE, an initial increase in 2DG uptake is observed in hippocampal CA3 region, subiculum and lateral septum. When convulsions become severe, the entire limbic system
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FIGURE 1 [14C]2-deoxyglucose autoradiographs of rat brain sections taken at the level of the hippocampus, thalamus, and substantia nigra in P10, P21, and adult control and lithium-pilocaprine-treated rats. Measurements of LCMRglc were performed during the second hour of SE (see Fernandes et al., 1999). Note the low and homogeneous LCMRglc in P10 control rats. In P21 control rats, rates of glucose utilization are increased in all brain areas compared to P10 rats, and they become more differentiated especially in the cortex, hippocampus and medial geniculate body of adult control animals. As can be seen on the color scale indicating values of glucose utilization, during lithiumpilocarpine-induced SE, LCMRglc is largely increased over control levels in all cortical regions and hippocampus of P21 and adult animals, while metabolic increase is moderate in most subcortical regions. In P10 rats, there is a moderated increase in LCMRglc in the hippocampus and a marked increase only in the substantia nigra. (See color insert.)
and its subcortical projections are metabolically active while sensory pathways and neocortex are depressed (Collins et al., 1980). Correlation between the clinical and EEG pattern of SE with the extension of the seizure circuit was also mapped by the 2DG technique during progressive states of limbic SE induced by amygdala or entorhinal cortex stimulation. In the amygadala stimulation model, mapping of metabolic activity showed that there is a systematic progression of seizure activity. It originates in the amygdala, then spreads to some direct amygdala projection areas; from there, it spreads to a restricted network of interconnected ipsilateral limbic nuclei which then recruits most of the remaining limbic structures, first ipsilaterally and then contralaterally (Handforth and Ackerman, 1988). The 2DG technique has also helped to define the brain areas involved in different behavioral manifestations (i.e., ambulation, mastication, immobility or generalized activity) of SE induced by amygdala stimulation. In the ambulatory phase, brain activation is mostly unilateral and limited to amygdala and its projections. In the masticatory phase, the recruitment of brain regions is similar to the
previous phase but bilateral and includes the hippocampus. The immobile phase is characterized by weak and restricted 2DG activity. The phase of generalized activity involves recruitment of all brain areas, with strong bilateral activity in striatum, neocortex and thalamus (McIntyre et al., 1991; White and Price, 1993a,b). During entorhinal cortex stimulation, the dentate gyrus initially restricts the entry of seizures from entorhinal cortex to the rest of hippocampus. When this blockade breaks down, there is rapid bilateral spread through the hippocampal formation, with passive interruption of normal behavior. With prolonged seizure discharges, amygdala is involved together with subcortical extrapyramidal and thalamic nuclei associated with behavioral convulsions (Collins et al., 1983).
Long-term Metabolic Changes Induced by Acute Sustained Seizures Long-term metabolic changes have been studied in models of temporal lobe epilepsy, mainly the pilocarpine
Autoradiography of Cerebral Metabolism and Blood Flow
and lithium-pilocarpine models, both during the latent (Dubé et al., 2000a,b, 2001b) and the chronic phases (Dubé et al., 2000b, 2001a,b; Scorza et al., 1998). Age-related differences in the long-term consequences of SE can be distinguished. In PN10 rats in which no overt damage develops. LCMRglc is similar to control rats at 14 days and 2 to 6 months after SE. In PN21 rats, LCMRglc is decreased during the latent phase (i.e., at 14 days after SE) in the hippocampus, amygdala, thalamus, and entorhinal cortex, even though the SE-related damage is limited to the lateral thalamus and hilus at that age. In adult rats, the pattern of metabolic decreases during the latent period is similar to that seen in PN21 rats, but there is a good correlation between the distribution of metabolic decreases and neuronal damage. In rats that underwent SE at PN21 or as adults, brain metabolism is decreased during the interictal phases of the chronic period (i.e., at 3-6 months after lithiumpilocarpine-induced SE) in the same regions as during the latent period. The most striking feature, however, is the mismatch between the number of neurons surviving in the hilus, which is very low at both ages, and the level of metabolic activity, which is close to normal at both ages; a similar mismatch can be found in the piriform cortex of rats subjected to SE as adults (Dubé et al., 2001a,b). This mismatch suggests a major role of the hilus (and the piriform cortex in adult rats) in the initiation and maintenance of epilepsy. A similar lack of concordance between the extent of hypometabolism and neuronal loss has also been reported in human studies (Chassoux et al., 2004; Lamusuo et al., 2001). Progressive changes in the seizure circuit were also studied by means of 2DG autoradiography in a model of temporal lobe epilepsy induced by unilateral intrahippocampal injection of kainate in mice. In that model, epileptogenesis was shown to involve bilateral cortical reactivity and the participation of limbic (hippocampus, amygdala, and thalamus) and motor structures. When hippocampal sclerosis is fully developed, hypometabolism is limited to the structures directly connected to the damaged hippocampus and most likely involved in the circuit of spontaneous seizures (Bouilleret et al., 2000a).
Autoradiographic Measurement of Local Cerebral Blood Flow Measurement of Cerebral Blood Flow To Map Brain Functional Activity In human epilepsy, single photon emission computed tomography (SPECT) of LCBF measurement is usually used to define the circuit of the seizures during presurgical examination. In animal studies, the autoradiographic measurement of CBF with [14C]IAP is used as an alternative to the LCMRglc measurement when the events studied occur over
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periods too short to allow the use of the 2DG technique (e.g., short seizures). For example, this technique allows for the mapping of LCBF changes elicited by audiogenic seizures in Wistar AS rats (Audiogenic Sensitive, Marescaux et al., 1987). During these seizures, LCBF rates increase in all brain areas, but rats are mainly in brainstem regions known to be involved in the expression of audiogenic seizures. When Wistar AS is kindled (20 to 40 sound stimulations), the generalized LCBF increase is of lower amplitude in the brainstem regions and similar to that of naïve rats in the forebrain. This redistribution of LCBF rates may reflect the evolution of the initial tonic seizure located in the brainstem into tonic-clonic seizures also involving the forebrain (Nehlig et al., 1995). Likewise, we mapped LCBF changes at different times during and after a seizure induced by maximal electroshock. In this paradigm, LCBF rates sequentially increase in brain regions, first in brainstem reflecting the major tonic component of the seizure and then peaking in forebrain regions during the later clonic component of the seizure. Thus, the LCBF response correlates with the clinical manifestations, clonic versus tonic, of seizure activity, suggesting that the measurement of LCBF is a sensitive method for detecting ictal hyperperfusion (André et al., 2002). Coupling of Cerebral Blood Flow and Metabolism: Relation With Brain Damage In adult and immature animals and humans, LCMRglc and LCBF are tightly coupled in most situations (Baron et al., 1984; Chiron et al., 1992; Chugani et al., 1986, 1987; Kuschinsky, 1996; Nehlig et al., 1988, 1989). In adult animals, LCBF rates and LCMRglc usually increase to a similar degree during the early phase of seizures. In vulnerable structures (e.g., the hippocampus, cerebral cortex, and thalamus), this early increase is followed by a pronounced decrease in LCBF accompanied by elevated LCMRglc by about 2 h of SE (Ingvar and Siesjö, 1983, 1990; Siesjö et al., 1983; Ingvar, 1986). In immature animals, the relationship between circulatory and metabolic changes during SE and subsequent cerebral damage is less clear. In PN21 and adult rats subjected to lithium-pilocarpine SE, a marked mismatch is noted between LCBF and LCMRglc restricted to the forebrain structures (Figure 2 and Pereira de Vasconcelos et al., 2002). These regions are known to exhibit neuronal damage in adult rats, but only limited damage in PN21 rats (Cavalheiro et al., 1987; Priel et al., 1996; Sankar et al., 1998). In these structures (i.e., entorhinal and piriform cortices, hippocampus, amygdala, septum, and thalamus), metabolic increases are very large, whereas LCBF rates increase much less, sometimes not at all. Interestingly, all these regions undergo neuronal damage in adult rats, whereas damage is limited to the hilus, medial amygdala, layer II of entorhinal cortex, and
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lateral thalamus in PN21 rats (Dubé et al., 2001a, b). On the contrary, no mismatch between LCBF and LCMRglc is seen in structures where no damage occurs after SE (i.e., mainly posterior, midbrain, and cerebellar regions) (Pereira de Vasconcelos et al., 2002). Thus, in the lithium-pilocarpine model, marked hypoperfusion during SE correlates well with neuronal damage in adult rats but correlates only partially in PN21 rats. Conversely, during PTZ-induced seizures that do not lead to neuronal damage in PN21 rats, only a moderate mismatch is noted between LCBF and LCMRglc in a limited number of structures (Pereira de Vasconcelos et al., 1995). In PN10 rats, the large increases in LCMRglc are not matched by concurrent increases in LCBF in both the PTZ and lithium-pilocarpine models (Figure 2 and Fernandes et al., 1999; Pereira de Vasconcelos et al., 1992, 1995, 2002). At this age, the mismatch between blood flow and metabolism does not, however, lead to any neuronal damage (Dubé et al., 2001; Fernandes et al., 1999; Pineau et al., 1999). Likewise, newborn marmoset monkeys subjected to bicuculline seizures show a clear mismatch in the cerebral cortex and hippocampus, with relative LCMRglc increases higher than those of LCBF (Fujikawa et al., 1986, 1989; Wasterlain et al., 1984); here too, this flow–metabolism mismatch does not lead to neuronal damage, even after more than 4 hours of SE.
Thus, the immature brain is able to undergo pronounced mismatches between blood flow and metabolism without any subsequent damage. This result indicates that the resistance of the immature brain to the metabolic and circulatory consequences of seizures is by far greater than that of the mature brain. The mechanisms involved are unknown.
Autoradiographic Measurement of Blood–Brain Barrier Permeability The vasogenic origin of brain damage caused by seizures can be explored by autoradiographic techniques that use [14C]a-aminoisobutyric acid, an amino acid that crosses an intact blood–brain barrier (BBB) very slowly. The regional permeability of the BBB has been measured in the PTZ (Padou et al., 1995), lithium-pilocarpine (Leroy et al., 2003) and kainate (Saija et al., 1992) models of seizures. In the PTZ model, there is a generalized increase in BBB permeability during seizures in both PN10 and PN21 rats (Padou et al., 1995), but no brain damage (Pineau et al., 1999). This increase most likely reflects the rapid increase in systemic blood pressure induced by PTZ tonic-clonic seizures (Padou et al., 1995). In the lithium-pilocarpine model, increases in BBB permeability occur both in structures that will undergo damage (thalamus, septum, amygdala) and structures that
FIGURE 2 [14C]2-deoxyglucose and [14C]iodoantipyrine autoradiographs of rat brain sections taken at the level of the dorsal hippocampus and median thalamus in P10, P21 and adult control rats and rats subjected to during lithiumpilocarpine SE. Radioactive tracers were injected 50 and 70 minutes after the beginning of SE for metabolic and blood flow studies, respectively (see Fernandes et al., 1999 and Pereira de Vasconcelos et al., 2002). Note the relatively lower and quite homogeneous tracer distribution in blood flow images compared to higher rates and heterogeneous distribution of the radioactive tracer in metabolic images in the three ages groups, indicating the mismatch between rates of LCBF and LCMRglc at all three ages.
Autoradiography of Cerebral Metabolism and Blood Flow
will not be injured (globus pallidus, hypothalamus) (Leroy et al., 2003). In the kainate model, generalized increases in BBB permeability are seen with a low dose of convulsant which does not lead to increases in metabolic activity, but not with a high dose of kainate which leads to brain damage (Saija et al., 1992). Thus, opening of the BBB is not reliably associated with neuronal injury in any model. At least in the kainate and lithium-pilocarpine models, brain damage depends on excitotoxic mechanisms caused by major neuronal hyperexcitability rather than on vasogenic alterations.
Further Technical Developments Three-Dimensional Autoradiography of Brain Metabolism We recently developed a robust, fully automated algorithm for the registration of serially acquired, coronal autoradiographic sections. This procedure results in a threedimensional (3D) reconstruction of the brain metabolic levels, and provides improved insight into the structures and circuits involved in seizures (Nikou et al., 2003). Moreover, we have superimposed this metabolic reconstruction onto the MRI of the animal (generated before the 2DG experiment) and aligned the autoradiographic and MRI images. This approach has now been applied to the measurement of the changes in brain metabolism during the various phases of the epilepsy induced by lithium-pilocarpine, and has already allowed us to visualize the circuit of the seizures at different times after the onset of SE (Figure 3; Roch, Namer
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and Nehlig, unpublished data). This type of approach will be useful to gain a better insight into the nature of the structures involved in the epileptogenic process and the development of the seizure circuit. Use of MicroSPECT and MicroPET Positron emission tomography (PET) is an imaging technique that detects positron emissions from selected radionucleotides. The miniaturization of clinical PET devices has led to the development of a commercially available micro-PET (Jacobs and Cherry, 2001). This imaging device generally requires a cyclotron capable of delivering the required radionucleotides. In recent years, the clinical utility of PET has spawned the commercial availability of longer half-life radionucleotides such as [18F] fluorodeoxyglucose (FDG). The question under study often dictates the choice of radionucleotide. Because of the relatively long half-life of 18-FDG (110 min) and its ability to acutely reflect metabolic changes, it has been the most commonly used agent for PET imaging. However, because of the high instrumentation costs associated with this technique (PET imager and cyclotron), many metabolic studies in animal models use the more traditional method of autoradiography, as described above. Although micro-PET imaging has been used in a variety of animal models, there are no studies dedicated to animal models of epilepsy. A recent report described the application of micro-PET to a variety of models of neuronal repair and plasticity and showed a dramatically elevated level of
FIGURE 3 Tridimensional reconstruction of [14C]2-deoxyglucose autoradiographs of rat brain taken during the second and the seventh hour of lithium-pilocarpine SE. The 3D images were generated by scanning the totality of the sections obtained from one animal, reconstructed using the algorithm developed in our laboratory (Nikou et al., 2002). In the left part of the figure, the images obtained in the three planes show the generalized increases in LCMRglc during the second hour of SE. In the right part of the figure, the images obtained by subtraction between glucose consumption in the control brain and in the brain studied between 6 and 7 hours after SE show the circuit that remains activated at that time, mainly involving the hippocampus and parahippocampal cortices. This part of the figure shows also an example of the superimposition of the MRI image and 2DG autoradiography of the rat. (See color insert.)
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metabolic activity within the hippocampus after kainateinduced seizures. Discrete events such as vibrissae stimulation can also be detected and result in modest increases in FDG uptake in the contralateral neocortex (Kornblum et al., 2000; Kornblum and Cherry, 2001). Although the spatial resolution of micro-PET images is currently less than desirable (>2 mm), the concurrent acquisition of MR data sets can provide useful anatomic resolution of the structures of interest (Figure 4). MicroSPECT has already been used in preclinical studies (Chapman et al., 2001) but to the best of our knowledge, not yet for following blood flow changes in epileptic animals. The major limitation in the use of microSPECT to measure CBF in rats is its poor definition (slightly inferior to microPET and much poorer than autoradiography). However, the resolution of both microSPECT and microPET technologies will undoubtedly improve, and should provide much-needed methods for following (over time) the evolution of seizure circuits in the same animal.
MAGNETIC RESONANCE IMAGING Methodological Considerations Magnetic resonance imaging is based on the visualization of molecules, usually protons, within a homogenous strong magnetic field. More accurately, the nuclear spins or the resonance phenomena of atomic nuclei are detected to create structural images. Whereas various nuclei (e.g., phosphorus) can be used, most MRI studies observe protons from water molecules. After placement of the subject within a magnet, a radiofrequency (RF) pulse is delivered to synchronize the nuclear spins of the protons within the area of interest. This synchronization of spins can be used to select the brain region(s) or slice(s) that are desired. As the protons relax after excitation, alternate types of image contrast can be obtained (see below). In planning animal imaging experiments, it is important to note the limitations of physical and instrumentation parameters.
FIGURE 4 These images were obtained from a single rat that has undergone pilocarpine-induced status epilepticus and exhibited moderate seizures. This plate illustrates the ability to use multiple imaging modalities in understanding the physiological alterations after seizures. The same animal was imaged serially for 7 days using first MR imaging and then PET. The T2 images do not reveal any altered signal within the brain until 4 days (arrow) after the status epilepticus event. In contrast the DWI series can clearly delineate the ongoing neuronal cell death within the piriform cortex at 24 hours after SE. Also note the increased signal in the thalamus at this time point. Interestingly, it is only at 4 days that the hippocampus begins to have an altered signal. At 7 days the DWI images do not show overt alterations. The same animal from the MR images underwent repeated PET imaging using 18FDG to evaluate metabolic activity. All images here were scaled for similar dose of the radiotracer. At 24 hours it is clear that the brain has become hypo-metabolic, and that there is then an increase in hippocampal and cortical signal. By 7 days the whole brain now has become hypermetabolic, suggesting that there is tissue remodeling and repair.
Magnetic Resonance Imaging
Instrumentation The primary components of an MRI system for image acquisition are composed of the following: (1) a magnet for generation of the static field, (2) a gradient system for position encoding within the magnet, (3) a RF coil for transmission of RF pulses, (4) a RF coil for receiving the MR signal, and (5) software for image reconstruction from the acquired data. Identification of the static magnet is often the first decision point and, in most instances, can dictate the resolution of the acquired data set. Current imaging magnets, ranging from clinical 1.5 Tesla instruments to research machines up to and exceeding 11.7 Tesla, can be purchased from exist-
FIGURE 5 A sample of the small animal imaging devices located within the Non-Invasive Imaging Laboratory at Loma Linda University that can be used for studying epilepsy. A) This Bruker MRI is a horizontal 4.7T with a 30 cm bore. The larger gradient set (arrow) supports a smaller micro-gradient set (a) for high resolution imaging. B) This high field Bruker 11.7T vertical bore imager has a bore size of 89mm that provides high resolution images of mouse and pediatric rodents. The arrow indicates a probe with coil inserted into the magnet. C) This Imtek micro-CT is fully enclosed and shields the operator from potentially harmful X-rays. A motorized animal bed system (arrow) moves the animal into the CT scanner where the X-ray source and detector rotate around the animal. D) A Concorde Microsystems PET imager (R4 model). The animal is positioned on a motorized bed system (arrow) that centers the animal within the detectors (a).
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ing MR vendors (e.g., Figure 5). Since higher field strength generally translates into greater resolution, it is usually desirable to purchase a high field strength magnet, despite higher instrument costs. A gradient system provides the means to select the slice of interest within the brain. Better performance characteristics of the gradient set can provide faster data collection and better image resolution. The software for reconstruction of the final images typically is proprietary software from the instrument manufacturer; the user interface can range from simple to sophisticated packages. Finally, RF coils for transmitting and receiving MR signals are commercially available but can also be customized by the user. Customization may be particularly
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important in imaging small animals. RF coils can range from very simple loops (Helmholtz style) to quadrature coils (Figure 6). Selection of the appropriate coil for data collection is often dependent on the research question. Although an exhaustive review of these components is beyond the scope of this chapter, additional information can
be found in several excellent reviews, (Klaunberg and Lizak, 2004; Paulus et al., 2001; Scarabino et al., 2003). In small animal imaging, the correct choice of animal model is important so that it is amenable to the imaging system at hand. Some issues for consideration are: bore size and orientation (i.e., horizontal or vertical); whether and how to incorporate temperature control and EEG monitoring; and selection of anesthetics (some of which may have profound effects for functional imaging). In general, it is preferential to use as high a field strength as possible, as small a bore as will accommodate the animal (and monitoring equipment), and an RF coil that is as small and as close to the skull/brain as possible. Software Most manufacturers provide the basic software for reconstruction of data sets into visible images. Additional features (e.g., region of interest analysis, quantitative map generation, and volumetric reconstruction) are now becoming available from most vendors. Most research laboratories, however, continue to develop and use their own in-house or customized data analysis software. Programming environments (e.g., Matlab and Interactive Data Language [IDL]) provide a conduit for rapid and simplified programming for specific research needs. Third-party software vendors have made great strides providing simplified and easy-to-use software for complex analyses; however, the costs for additional software choices can be significant. Animal Handling
FIGURE 6 Sample animal handling devices and coils used for small animal imaging. A) Probe head and coil assembly for use with a high field MR imager. Note the relatively small working space for these animals. aanimal cradle to support body, b- small stereotactic device with a small nose cone for delivery of volatile anesthetics, c- custom designed and built water heater for animal temperature maintenance, d- small RF coil where the animal head is placed in the center. B) Similar cradle device in use with a 4.7T MR imager. a- RF coil (note considerably larger bore size), b-small balloon that is placed over the nose for delivery of volatile anesthetics, canimal cradle to support body, d- sled system for rapid and accurate insertion into the bore of the magnet which allows for high throughput imaging. C) Similar design of a rat holder that can be used in the MRI, CT, and PET instruments without removing the animal. a- base plate for use with CT and PET, b- body cradle that will work with the coil and sled design in B, cear bars for accurate animal positioning, d- small balloon for delivery of volatile anesthetics, e- anesthesia delivery and evacuation lines.
Animal handling for MR research involves careful design of anesthesia delivery, animal restraint, and ability to deliver drugs or take biological samples all within a magnetic environment (Figure 6). Purchase of MR-compatible anesthesia systems can alleviate many issues; however, these systems often are expensive. In addition, the emergence and development of MR-compatible equipment for electroencephalography (EEG) now makes it possible to record electrical activity simultaneously or interleaved during the MR data acquisition. One area of customization is the use of animal restraint systems to minimize motion of the research subject. Most MR manufacturers now have options that allow the user to purchase rodent restraint systems, but these are also often very expensive. Research groups usually develop their own animal restraint systems that are customized to the specific research needs. Our own laboratory has a range of animal restraint systems (Figure 6) that are used with specific custom-designed RF coils and for specific research questions. These are usually made of plastic materials that are easy to manufacture, nonferrous (i.e., non-magnetic), and easy to clean. The primary purpose of immobilization is to
Magnetic Resonance Imaging
prevent movement artifacts during the MR acquisition. Immobilization can normally be achieved with the use of stereotaxic restraint systems combined with the correct choice of anesthesia. When simple restraint of the rodent head is insufficient to prevent movement, most MR systems allow gated data acquisition. This process involves the use of a physiological monitoring system (i.e. respiration) that then triggers data acquisition during specific epochs in the respiratory cycle. More involved systems allow for monitoring of heart rate, respiration, and other physiologic parameters during data acquisition and can be purchased from third-party vendors.
Epilepsy and Image Contrast One advantage of MR versus other imaging modalities is the ability to extract different levels of contrast (tissue information) with good anatomical and physiological resolution. While it is beyond the scope of this chapter to provide the physical basis for these contrast levels, we will review relevant concepts concerning animal studies of epilepsy. In particular we will focus on the three most commonly used contrast levels: T1-, T2- and diffusion-weighted imaging (DWI) T1-Weighted Imaging T1-weighted imaging (T1WI; T1 relaxation, spin-lattice relaxation) is defined as when the proton has “relaxed” to 63% of its original magnetization. Efficiency of energy transport between proton spins and lattice, in this case brain tissue, determines the T1 relaxation time. In addition, factors that can influence T1 are molecular motion (temperature) and the structure and physical state of the tissue (T1 is higher in solids and pure liquids). In addition, as MR imagers move to higher and higher field strength, T1 tissue values become longer. Very few studies have investigated the T1-related changes after seizures or epilepsy in animal models. Brain volumetric studies of kainic acid (KA)-induced epileptiform activity utilized T1- and T2WI to obtain high-resolution volumetric MRI at 10 days after stage four seizures (Wolf et al., 2002b) and demonstrated a reduction in hippocampal volumes with a concomitant increases in ventricular volumes. Another common for T1WI is with use T1 shortening contrast agents, such as gadolinium (Gd). This and other agents are often used to assess BBB breakdown after an insult. Roch and colleagues showed that at 2 hours after lithium-pilocarpine induced SE there was contrast enhancement within the thalamus, which resolved by 6 hours (Roch et al., 2002). Thus, for a short time period some leakage of the BBB occurred following SE.
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Currently, no reports have been published on quantitative T1 values in the brain of rodent models of epilepsy after seizures. T2-Weighted Imaging T2-weighted imaging (T2WI; T2 relaxation, spin-spin relaxation) occurs when 37% of the proton signal has fully relaxed. The interaction of proton spins determines the T2 relaxation rate. Solids and large molecules have short T2 relaxation times; conversely, small molecules (e.g., water) have long T2 relaxation times. Finally, the presence of macromolecules in solution can also shorten T2, but T2 relaxation does not depend on magnetic field strength. T2WI has been used extensively in the study of epilepsy in animal models, often in conjunction with other imaging modalities (e.g., DWI). Many of the studies have used T2WI for ventricular (Wolf et al., 2002a) and lesion volumes (Roch et al., 2002), edema formation (Bouilleret et al., 2000; Wall et al., 2000b), altered T2 signal (Roch et al., 2002, Bouilleret et al., 2000; Pirttila et al., 2001) (Figure 4) and quantitative T2 maps (Fabene et al., 2003; Pirttila et al., 2001). Altered T2 signal intensities, specifically the regional occurrence and duration of the signal intensity, are greatly dependent on model of seizure induction, severity of seizures, region investigated, and time course of MR investigation. Taking these variables into account, T2WI can provide many insights into the underlying physiology and neuropathology. Fabene and co-investigators examined the T2 changes 16 hours after pilocarpine-induced seizures and reported increased hyperintensities within most of the cortical regions and hippocampus (Fabene et al., 2003). Quantitative maps further showed that these signal increases were highest within the hippocampus (201%), whereas cortical regions had 42% to 125% increases in T2 relaxation, which were thought to correlate with edema formation. Wall and colleagues described the temporal evolution of T2 relaxation after pilocarpine-induced seizures. Increased T2 values were observed in the piriform cortex, amygdala, and thalamus as early as 3 hours after seizure induction and, in many cases, increased T2 relaxation persisted up to 7 days, the final imaging time point used in this study (Wall et al., 2000). An interesting observation was the transient T2 alteration within the retrosplenial cortex. Histologic evaluation confirmed that the T2 value elevation at 12 hours was caused by edema formation. Other studies have used T2 (visual and quantitative measures) to discriminate SE severity in the chronic phases of epilepsy (Roch et al., 2002). The chronic phase appears related to tissue sclerosis and shrinkage, as evidenced by increased ventricular signals. Other studies have used the T2 signal as the basis for postprocessing of data sets to detect nonvisible T2 alterations (Yu et al., 2002). Such analysis might be useful in the long-term study of rodents that develop chronic epilepsy.
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Diffusion-Weighted Imaging Diffusion-weighted imaging has rapidly evolved as the noninvasive method of choice for detecting acute lesions caused by epilepsy and SE (Wall et al., 2000). In DWI, contrast is modulated by molecular water diffusion. Factors influencing random water movement include the viscosity of the matrix, the presence of reflective boundaries, and local temperature. Cellular membranes and other normal tissue barriers restrict the free movement of water. Also important is the viscosity differential that exists between the intra- and extracellular compartments. Together, these factors produce an environment where diffusion is variably restricted and where the measurement technique must be crafted to distinguish apparent from real changes (Thomas et al., 2000). In addition, the distance that a water molecule diffuses in one direction will be different from its diffusion in another direction. Thus, there is directionality of diffusion within the brain, which is termed “anisotropic” diffusion. Ordered white matter tracts and fiber bundles will influence diffusion preferentially in the direction of the tract and restrict diffusion in the perpendicular direction. This phenomenon has been exploited with great success in diffusion tensor imaging (DTI) (Beaulieu, 2002; Horsfield and Jones, 2002; Xue et al., 1999). This method has been used for study of dysmyelination syndromes (Song et al., 2002) and in an epilepsy model (Li et al., 2003). When water molecules reflect from a surface, the mean diffusion path is effectively reduced, resulting in an apparent decrease in diffusion. Such a change might occur if pathologic processes (e.g., excitotoxic injury) produced neuronal swelling, which decreases the extracellular space. This effect can be seen after acute cerebrovascular infarction. Alternately, the injury cascade might include cell shrinkage and lysis, increasing the extracellular space, producing an increase in apparent diffusion caused by decreased tortuosity, such as that seen after infarction (van der Toorn et al., 1996). Increases and/or decreases in diffusion can often be seen within the same tissues, and is dependent upon the time frame in which the measurement is made. This time-dependence is true in ischemia, where early (<12 hours) after infarct induction there is a decrease in the apparent diffusion coefficient (ADC) but later (>24 hours) is associated with a long lasting increase in ADC. Real changes in diffusion will follow reallocation of water from a highly diffusing extracellular space to a more restrictive, diffusing intracellular environment (van der Toorn et al., 1996). The window for observing changes is important. Downstream from a neurotoxic event such as epilepsy, proliferating glial populations (Roper et al., 1992; Obenaus et al., 1993) may also contribute to an apparent diffusion coefficient decrease (see below) by increasing intracellular tortuosity causing restricted water movement. The quantitative measure of DWI is the computed ADC value. A change in ADC implies that the water diffusion is
altered as a result of biophysical changes; these changes report maximal sensitivity to injury rather than isolated individual contributions (Szafer et al., 1995). Thus, the sensitivity of DWI to early ongoing pathology makes it the imaging modality of choice for studying evolving injury. However, for established chronic pathology T2WI is often preferred. During evolving injury there is altered water mobility as cells die and glial cells try to mitigate the injury. After the injury has been established and tissue characteristics have stabilized, it is often more difficult to visualize altered water movement. T2WI can in these situations assist in delineation of the region of injury. One of the first published reports of DWI in an animal model of epilepsy was that by Zhong and colleagues (Zhong et al., 1993). After bicuculline injection they demonstrated a moderate decrease in ADC (14%) with no reported alterations in T2- or T1WI. These alterations were similar to those reported after ischemia and stroke. A series of reports using DWI in various animal models of epilepsy followed (Nakasu et al., 1995; Righini et al., 1994; Wang et al., 1996; Zhong et al., 1995). These reports demonstrated the sensitivity of DWI for visualization and quantification of ADC values after seizures. Given the variability between seizure models, it was somewhat surprising to find that ADC values decreased early after epileptiform activity in almost all cases. Furthermore, since many studies were limited to 1 to 7 days after seizure activity when most acute DWI changes resolve to control levels (Nakasu et al., 1995; Wall et al., 2000; Wang et al., 1996) the changes are likely to have been even larger at earlier post-seizure time points. In many of these studies, correlative histology revealed altered cellular composition after seizures. Our own laboratory has used DWI and ADC values to study the temporal effects of epilepsy in a number of model systems (Figure 4) (Bhagat et al., 2000; Eidt et al., 2004; Kendall et al., 2002; Wall et al., 2000). In the pilocarpine model of epilepsy, we have demonstrated within the piriform cortex a rapid decrease in ADC values within 3 hours after seizure onset that then slowly resolved to control values by 7 days (Wall et al., 2000). The hippocampus, a limbic structure that is not as acutely affected, showed a slower increase in ADC values over the 7-day time course. The high anatomic resolution of MRI offers the means for obtaining accurate region of interest analysis. Thus, cellular changes after epilepsy can be monitored in a region-by-region fashion that provides a more comprehensive view of ongoing alterations. In summary, the overall finding in most DWI studies of epilepsy has been a reduction in ADC (or increased signal on diffusion-weighted images) early after seizure induction, often with a return to control values (Eidt et al., 2004; Liu et al., 2000; Zhong et al., 1993). The time course of these changes varies with the model studied and may show many similarities with the time course of infarction. The question of what such a decrease in ADC means, pathologically, is
Magnetic Resonance Imaging
still unresolved. There is some consensus that, at least in the case of seizures, an early decrease in ADC reflects cell death and tissue swelling (thus restricting diffusion). Subsequent return to ADC control values appears to reflect glial migration into the injured tissue. However, there are emerging data (Obenaus, unpublished) that suggest that slowly evolving neuronal injury may be associated with an increased ADC. Thus, the cellular bases for increased or decreased ADC may depend on the type, location, speed, and severity of the tissue change. As with all MRI techniques, new ADC methods are rapidly emerging for quantifying DWI. Callaghan described the concept of Q-space imaging, whereby extremely high gradients (b-values) were applied to dissect out tissue constructs and describe the movement of water within a tissue as precisely as possible (Callaghan, 1991). These biophysical constraints, however, cannot be met with current animal imaging systems, even at very high magnetic field strengths. These concepts have been recently extended in studies by Assaf and Cohen in which high-resolution data sets were acquired and the data extrapolated to very high b-values (Assaf et al., 2000, 2002; Assaf and Cohen, 2000; Cohen and Assaf, 2002). Additional physiologic and diagnostic information, particularly regarding water movement in various disorders such as spinal cord injury, has been reported (Assaf et al., 2000). Our own laboratory has recently published a similar study using Q-space to define altered cellular populations within the piriform cortex after pilocarpine-induced seizures (Eidt et al., 2004). Such studies, when combined with histologic analysis, can provide diagnostic information about different altered cellular (neuronal versus glial) populations after epilepsy. For example, this technique may be useful in evaluation of pharmacological compounds that are being tested for amelioration of seizure-related neuropathology or for prevention of onset of seizures. Although DWI is certainly a powerful technique for the study of epilepsy and seizure events, it is important to note a very important limitation. Currently, there is considerable debate about the source of these ADC and DWI signals. Studies in other model systems have attempted to delineate whether signals arise from intra- or extracellular environments (Duong et al., 1999). These discussions are beyond the scope of this chapter; however, it is important to note that it is difficult to ascertain the source of the altered DWI and ADC signals. Future work addressing this issue will be important; correlative histology, in many instances, can assist in making this determination.
Other Imaging Methods Magnetic Resonance Spectroscopy Another powerful MR technique is MR spectroscopy (MRS). MRS uses frequency shifts of various metabolites
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within the brain or brain regions to determine regional concentrations. Depending on the field strengths (high-field strengths enable better resolution of metabolites), one can detect common metabolites such as N-acetyl-aspartate (NAA), an amino acid present in neurons; choline, a lipid membrane component; creatine, a measure of energy metabolism; a Glx peak that at higher field strengths can be resolved into glutamate, glutamine, and gamma-aminobutyric acid (GABA); and lactate, reflecting anaerobic glycolysis. Although MRS has been used clinically to evaluate many diseases, few have used it to study epilepsy in small animals (Connelly et al., 1994). In one study of kainate-induced epilepsy, decreased NAA and creatine levels were found by 14 days after seizures (Tokumitsu et al., 1997). These altered metabolite levels coincided with increased ADC and suggested neuronal loss that was confirmed with histology. Another study that examined the hippocampus after ictal, postictal, and interictal phases, reported that NAA increased during the ictal period but declined during the postictal period (Najm et al., 1997). The ultimate decline in NAA levels is thought to reflect neuronal loss. Lactate levels increased during the ictal and postictal periods and returned to control levels by 7 days. No changes were reported in choline or creatine levels. In summary, MRS can provide additional quantitative data on the cellular function after seizures (Novotny, 1995). Although MRS is difficult to perform (magnet shimming and magnetic field homogeneity issues), it can provide direct indices of cellular metabolic alterations within select brain regions. For example, recent human MRS studies of patients on the ketogenic diet have demonstrated the technique’s capability for monitoring— non-invasively—GABA and other metabolite levels after therapy (Wang et al., 2003)
Functional Magnetic Resonance The recent introduction of a new technique, fMRI, that reflects altered metabolic neuronal activity has been extended to the study of epilepsy in animal models. fMRI utilizes increased blood flow as a reporter of increased neuronal activity. Blood-oxygen-level-dependent (BOLD) fMRI uses the oxygenation status of hemoglobin as the “contrast’’ reporter (Ogawa et al., 1990). This methodology has been used widely to study normal and abnormal human brain function (Kim and Ogawa, 2002; Menon, 2001; Kannurpatti et al., 2003). Increased BOLD signal changes after administration of gamma-hydroxybutyric acid (GHB) has been reported in the thalamus with mixed positive and negative changes observed in the cortices (Tenney et al., 2003). The positive BOLD changes within the thalamus likely reflect increased neuronal activity, thought to be associated with seizure activity. Regional cortical changes are thought to reflect increased
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(positive) and decreased (negative) neuronal activation. These results were described in restrained awake animals. A more recent study using a genetic rat model of absence seizures has reported similar results (Tenney et al., 2004). Whereas this method of neuroimaging holds great promise, few animal studies have used this technique to study epilepsy. Special consideration must be given to animal handling techniques, analysis methodologies that are not yet routine, and interpretation of the results. fMRI may provide additional information on how pharmacotherapy alters neuronal activity the addition of a T1- or T2WI MRI sequence can provide anatomical localization of functional changes.
Computed tomography has been the mainstay of clinical imaging for a variety of acute diseases, most notably trauma and ischemia. Neither clinical nor small animal studies use CT as the primary reporter for epilepsy-induced alterations. The superior contrast resolution of MRI has precluded the use of CT for the experimental study of epilepsy. CT, however, can provide better spatial resolution than MRI. A further limitation is that micro-CT scans can often deliver a significant radiation dose that can alter the outcome of physiologic events under study. This is particularly true if repeated imaging is desired (Obenaus and Smith, 2004). The development of new CT imaging contrast agents may assist in future studies of epilepsy in small animal models.
Relevance to Neuropathology Computed Tomography X-ray computed tomography (CT) is a procedure in which an x-ray source and digital detector system rotates around the subject of interest. Recent advances in miniaturization of these devices have resulted in animal scale CT devices (Paulus et al., 2001). These instruments report primarily tissue density levels within the region studied. Brain tissue, which is reasonably homogenous in density, is often difficult to study using this imaging modality.
Do these imaging modalities reflect the physiologic and cellular alterations that have been described in the literature? Most of the current studies cited have used correlative histology to provide a link between the imaging findings and those seen on histochemistry. Ex vivo imaging can also provide exquisite anatomic detail and allow for the generation of three-dimensional neuropathology maps (Figure 7). As more and more neuroimaging modalities are used to study epilepsy, the need for correlative histology will
FIGURE 7 This figure illustrates the utility and quantitative data that can be extracted from ex vivo imaging. These images are from the same animal that contributed in vivo MR and PET scans in figure 3. The T2 and DWI images demonstrate a marked loss of signal within the piriform cortex region (arrows). In the DWI, the hippocampus also shows altered signal suggestive of altered tissue characteristics. The proton density (PD) image also shows some altered tissue density but this change is clearly less conspicuous than the changes seen in the T2 and DWI images. Finally using a 3D data set, we were able to reconstruct in 3D the volume of altered tissue changes seen with the MR.
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References
decrease to supplement our understanding of the cellular correlates of the imaging changes. In summary, just as clinical imaging of epilepsy can provide referential information about altered cellular composition, imaging of small animal models of epilepsy can also provide the necessary histologic correlation. This capability is particularly important as we begin to use repeated longitudinal observations to evaluate potential pharmacologic strategies.
Advantages and Limitations Small animal imaging has many significant advantages: (1) longitudinal studies in the same animal, (2) ability to noninvasively visualize anatomic and physiologic alterations, (3) multiple imaging contrast levels, (4) ability to collect a full three-dimensional data set, and (5) potential for fusing images from multiple imaging modalities. Limitations exist, however, that may preclude these imaging methodologies from gaining wider use. These include, but are not limited to (1) cost of the imaging equipment (ranging from $500,000 to several million dollars), (2) technical challenges of equipment operations (i.e., MRI), (3) need for highly trained technical support staff, (4) costly operating expenses (i.e., cryogens, RF coils), (5) need for dedicated support equipment (particularly true for PET imaging, where use of short-lived isotopes requires an onsite cyclotron), and (6) need for dedicated software for postprocessing and extraction of quantitative information. In addition, there are some limitations on the temporal and spatial resolution that one can achieve with in vivo imaging methods. The spatial limits are often those imposed by the imaging device (i.e., magnetic field strength, coil configuration, etc.). In the case of MRI, spatial improvements may be achieved at the expense of signal-to-noise ratio (SNR); e.g., spatial resolution increases but the SNR decreases when increasing the matrix size. This decreased SNR can be overcome by increasing imaging time; however, practical research constraints often minimize this option. Even given these limitations, it is still eminently desirable to perform imaging research in epilepsy if the scientific question(s) can be meaningfully addressed with these imaging modalities. Among the research questions that may benefit from imaging techniques are: a) What are the interrelationships between metabolic and anatomical changes during seizure induction? b) Can sprouting be detected on an MR signal, and can one follow this plasticity after the onset of spontaneous seizures? c) Does pharmacological intervention prevent MR-observable changes in tissue characteristics? and d) Do clinically-subthreshold seizures alter the underlying physiological and anatomical characteristics seen with imaging modalities? The imaging field for small animals is rapidly expanding. New devices and technical
advances give the investigator powerful new tools with which to investigate the mechanisms and treatment of epilepsy.
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Turski, L., Ikonomidou, C., Turski, W.A., Bortolotto, Z.A. and Cavalheiro, E.A. 1989. Review: Cholinergic mechanisms and epileptogenesis. The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy. Synapse 3: 154–171. van der Toorn, A., Sykova, E., Dijkhuizen, R.M., Vorisek, I., Vargova, L., Skobisova, E., van Lookeren Campagne, M. et al. 1996. Dynamic changes in water ADC, energy metabolism, extracellular space volume, and tortuosity in neonatal rat brain during global ischemia. Magn Reson Med 36: 52–60. Wall, C.J., Kendall, E., and Obenaus, A. 2000. Rapid alterations in diffusion-weighted images with anatomical correlates in a rodent model of status epilepticus. Am J Neuroradiol 21: 1841–1852. Wang, Y., Majors, A., Najm, I., Xue, M., Comair, Y., Modic, M., and Ng, T.C. 1996. Postictal alteration of sodium content and apparent diffusion coefficient in epileptic rat brain induced by kainic acid. Epilepsia 37: 1000–1006. Wang, Z., Bergqvist, C., Hunter, J., Dongzhu, J., Wang, D.-J., Wehrli, S., and Zimmerman, A. 2003. In vivo measurement of brain metabolites using two-dimensional double-quantum MR spectroscopy–exploration of GABA levels in ketogenic diet. Mag Resonance Imaging 49: 615–619. Wasterlain, C.G., Dwyer, B.E., and Fujikawa, D. 1984. Metabolic studies of neonatal seizures in newborn marmoset monkeys: a possible role in the pathogenesis of brain damage for mismatch between flow and metabolism. In Cerebral Blood Flow, Metabolism and Epilepsy Ed. M. Baldy-Moulinier, D.H. Ingvar, and B.S. Meldrum, B.S. pp. 121–129. London: John Libbey. White, LE., and Price, JL. 1993a. The functional anatomy of limbic status epilepticus in the rat. I. Patterns of 14C-2-deoxyglucose uptake and Fos immunocytochemistry. J Neurosci 13: 4787–4809. White, L.E., and Price, J.L. 1993b. The functional anatomy of limbic status epilepticus in the rat. II. The effects of focal deactivation. J Neurosci 13: 4810–4830. Wolf, O. T., Dyakin, V., Patel, A., Vadasz, C., de Leon, M.J., McEwen, B.S., and Bulloch, K. 2002a. Volumetric structural magnetic resonance imaging (MRI) of the rat hippocampus following kainic acid (KA) treatment. Brain Res 934: 87–96. Wolf, O.T., Dyakin, V., Vadasz, C., de Leon, M.J., McEwen, B.S., and Bulloch, K. 2002b. Volumetric measurement of the hippocampus, the anterior cingulate cortex, and the retrosplenial granular cortex of the rat using structural MRI. Brain Res Brain Res Protoc 10: 41–46. Xue, R., van Zijl, P. C. M., Crain, B. J., Solaiyappan, M., and Mori, S. 1999. In vivo three-dimensional reconstruction of rat brain axonal projections by diffusion tensor imaging. Magn Reson Med 42: 1123–1127. Yu, O., Roch, C., Namer, I.J., Chambron, J., and Mauss, Y. 2002. Detection of late epilepsy by the texture analysis of MR brain images in the lithium-pilocarpine rat model. Magn Reson Imaging 20: 771–775. Zhong, J., Petroff, O.A.C., Prichard, J.W., and Gore, J.C. 1993. Changes in water diffusion and relaxation properties of rat cerebrum during status epilepticus. Magn Reson Med 30: 241–246. Zhong, J., Petroff, O.A.C., Prichard, J.W., and Gore, J.C. 1995. Barbituatereversible reduction of water diffusion coefficient in flurothyl-induced status epilepticus in rats. Magn Reson Med 33: 253–256.
Li-Pilo SE: substantia nigra
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FIGURE 4 7 - 1 [~4C]2-deoxyglucose autoradiographs of rat brain sections taken at the level of the hippocampus, thalamus, and substantia nigra in P10, P21, and adult control and lithium-pilocaprine-treated rats. Measurements of LCMRglc were performed during the second hour of SE (see Fernandes et al., 1999). Note the low and homogeneous LCMRglc in P10 control rats. In P21 control rats, rates of glucose utilization are increased in all brain areas compared to P10 rats and they become more differentiated especially in the cortex, hippocampus and medial geniculate body of adult control anials. As can be seen on the color scale indicating values of glucose utilization, during lithium-pilocarpineinduce SE, LCMRglc is largely increased over control levels in all cortical regions and hippocampus of P21 and adult animals, while metabolic increase is moderate in most subcortical regions. In P10 rats, there is a moderated increase in LCMRglc in the hippocampus and a marked increase only in the substantia nigra.
FIGURE 4 7 - 3 Tridimensional reconstruction of [HC]2-deoxyglucose autoradiographs of rat brain taken during the second and the seventh hour of lithium-pilocarpine SE. The 3D images were generated by scanning the totality of the sections obtained/Yore one animal, reconstructed using the algorithm developed in our laboratory (Nikou et al., 2002). In the left part of the figure, the images obtained in the three planes show the generalized increases in LCMRglc during the second hour of SE. The absolute rates of cerebral glucose utilization are given on the color scale. In the right part of the figure, the images obtained by subtraction between glucose consumption in the control brain and in the brain studied between 6 and 7 hours after SE show the circuit that remains activated at that time, mainly involving the hippocampus and parahippocampal cortices. This part of the figure shows also an example of the superimposition of the MRI image and 2DG autoradiography of the rat.
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48 Behavioral Characterization of Seizures in Rats JANA VELÍSˇKOVÁ
mental conditions and seizure expression using various models are described in other chapters in this book.
INTRODUCTION In humans, clinical manifestations of an epileptic seizure involve a variety of phenomena, depending on seizure origin (focal vs. generalized), brain structure involved, and age. Seizures can present as short loss of awareness (staring and disruption of ongoing normal activity), automatisms (undressing, chewing, repeated movement, and so on), sensations (visual, sensory, auditory, olfactory, or gustatory), or motor phenomena. The motor events can be either uncontrolled rhythmic movements in face or limbs (myoclonic seizures), stiffening (tonic seizures), or sudden loss of muscle tone (atonic seizures). In rats or mice (the most frequently used experimental animals in seizure testing), behavioral manifestations of seizures have many characteristics of clinical seizure expression in humans. Depending on seizure model, changes in behavior include episodes of staring, head bobbing, automatisms (e.g., wet dog shakes, sniffing, repeated washing, scratching), or excessive orientation behavior with repeated rearing. Motor seizures involve myoclonus of facial and forelimb muscles, violent running and bouncing, body stretching or curling (tonus), and myoclonus of all four limbs with loss of the righting reflex. Seizure expression in developing animals is influenced by maturity of networks responsible for individual seizure types. Thus, certain seizure manifestations occur as a function of age, either appear and disappear during development or occur later in life, depending on maturity of the brain. This chapter focuses on behavioral manifestations of seizures, with special emphasis on developmental differences, whereas details about seizure induction in experi-
Models of Seizures and Epilepsy
NORMAL BEHAVIOR AND FACTORS INFLUENCING THE BEHAVIOR Normal behavior in adult rats involves initial orientation lasting 2 to 3 minutes when the rat is placed in a new environment. This includes exploring the cage, sniffing, occasional rearing, or washing. Following this period, the rat finds a darker corner (if available) to rest. In developing rats, normal behavior is changing as a function of age. Up to postnatal day (PN) 12, pups have occasional jerking and short-lasting head and body tremor as a part of their normal behavior. Locomotor hypoactivity predominates. From the end of the second postnatal week, the behavior is not too different from adult rats. For any behavioral testing, including seizure behavior, it is important to describe all conditions to which the animal has been exposed before testing. 1. Light–dark cycle: Rats are nocturnal animals, thus it is natural for them to sleep or rest during the day and be active during night. Factors such as receptor binding depend on the circadian rhythm and may influence the seizure outcome. Thus, whether the light is on during the day and off during the night or vice versa is an important factor, which must be taken into consideration in the experimental design and discussed as the part of data presentation. 2. Number of rats within individual cages: Rats are naturally living in groups. Studies testing the influence
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Chapter 48/Behavioral Characterization of Seizures in Rats
of environmental enrichment show that the behavior depends on the contact and interactions with other animals and leads to changes in the brain (e.g., increased neoneurogenesis) and affects seizure threshold (Young et al., 1999). Therefore, seizure behavior may be changed in rats housed isolated individually in small cages. Stress: Rats should not be tested on the day of delivery from the vendor, rather housed for some period of time (2 to 3 days) in the institutional facility before the testing. Seizure threshold can change during different seasons or extreme weather conditions, although the animals are housed under strictly controlled conditions. In developing rats, an important factor influencing the behavior is the separation from the dam. As little as 1 hour separation repeated for 3 consecutive days, causes long-lasting behavioral and other physiologic impairments (Kehoe and Bronzino, 1999). Temperature: Seizure threshold or other outcomes to be tested (i.e., seizure-induced damage) can be influenced by the body temperature. Developing animals cannot control their body temperature up to the third postnatal week. Thus, a heating pad with controlled temperature set at 34° C, natural temperature of the nest (Blumberg et al., 1992), should be used during seizure testing. Sex and the hormonal changes: Sex differences exist in many brain regions and can influence seizure susceptibility (Thomas, 1990; Kawata, 1995; Pericic and Bujas, 1997; Velísˇková and Moshé, 2001). Besides the sexual dimorphism of the brain, sex hormones can affect the seizure threshold (Holmes and Weber, 1984; Schwartz-Giblin et al., 1989; Edwards et al., 1999; Velísˇková et al., 2000). Stages of the female rat 4-day estrous cycle should be monitored before behavioral testing and correlated with the data obtained. Strain differences: Strain differences in seizure susceptibility have been reported (Loscher et al., 1998; Brandt et al., 2003). One of the reasons may be maturity of different systems depending on the length of gestation, which is variable among different strains (i.e., the length of gestation of Wistar rats is 21 days, whereas for Sprague-Dawley rats it is 23 days [Kábová et al., 2000]).
HOW TO RECOGNIZE A BEHAVIORAL SEIZURE Motor Seizure Clonic Seizures (Synonyms: Myoclonic, Minimal, Forebrain Seizures) The origin of clonic seizures is in the forebrain (Browning and Nelson, 1986). During this type of seizure,
the righting reflex is preserved. Some authors also refer to these seizures as “limbic seizures” because clonic seizures occur with higher doses of kainic acid, a model with seizure origin in limbic structures (Ben-Ari et al., 1981; Nadler, 1981). This terminology may be misleading, however, because the occurrence of clonic seizures rather represents secondary generalization than the typical limbic seizures. This is confirmed by electrical stimulations of different limbic structures during kindling development and 2deoxyglucose studies. In the models of limbic seizures, paroxysmal activity spreads from the original structure to other limbic regions corresponding to behavioral arrest, head nodding, and automatisms in the kainic acid– pilocarpine models or stages 1 and 2 of kindling (Lothman and Collins, 1981; Handforth and Ackermann, 1988; Browning, 1994). The occurrence of forelimb clonus is a sign of activation of structures beyond the limbic system, namely thalamus, neocortex, and basal ganglia (Engel et al., 1978; Ackermann et al., 1986; Handforth and Ackermann, 1988; Browning, 1994; Velísˇková et al., 2005). In adult rats, clonic seizures involve rhythmic movements of forelimbs often accompanied by facial clonus and lasting seconds to tens of seconds. The forelimb clonus can be either unilateral or coordinated bilateral clonus with or without rearing or tail erection (Straub tail). The hind limbs are widely abducted and the position of an animal appears as a kangaroo position (Ono et al., 1990; Velísˇek et al., 1992). The movements are well synchronized. In some seizure models (e.g., flurothyl), a tonic component can be observed, consisting of tonic twist of the trunk and forelimbs. The hind limbs sometimes can also be involved and the rat may lose balance for a short period (Velísˇková, 1999). There is almost immediate active effort of the rat to get back to the upright position. In contrast to tonic–clonic seizures following this type of tonus, forelimb clonus with preserved righting reflex follows. Clonic seizures can be elicited in all developmental stages. During the first 2 postnatal weeks, however, clonic seizures in most models are rapidly progressing into tonic–clonic seizures (Figure 1). Thus, in early developmental stages, clonic seizures may be masked by tonic–clonic seizures (Velísˇková et al., 1990). This “masking” effect may be observed also in adult rats, when a high dose of the convulsive agent is used. The forelimb clonus can be either unilateral or bilateral with or without rearing. Rearing, however, is less common in rats younger than PN15. Synchronization of clonic movements greatly depends on developmental stage. Developing rats synchronize poorly. Thus, during the first 2 postnatal weeks, clonic seizures look like unilateral tapping of a forelimb or uncoordinated bilateral swimming movements with occasional hind limb clonus. The righting reflex is always preserved.
How to Recognize a Behavioral Seizure
FIGURE 1 Changes in the interval duration between the clonic and tonic–clonic flurothyl-induced seizures as a function of age. During the first two postnatal weeks (PN9–PN15), a clonic seizure progresses almost immediately into the tonic–clonic seizures without a clear separation between the two types of seizures. During the third and fourth postnatal week, animals experience a single clonic seizure clearly separated from the tonic–clonic seizure. From the end of the fourth postnatal week, animals usually experience two or more clonic seizures before the tonic–clonic seizure ensues (Velísˇková, 1999).
Tonic–Clonic Seizures (Synonyms: Brainstem, Major Seizures) The anatomic substrates responsible for tonic–clonic seizures involve the brainstem structures (Browning, 1985; Browning and Nelson, 1986). It should be noted here that few models exist of seizures with primary origin in the brainstem structures, such as maximal electroshock, genetically prone animals (GEPR-3 and GEPR-9), N-methyl-daspartate (NMDA), or strychnine-induced seizures. In these models, forelimb clonus with rearing represents spreading of seizure activity from brainstem to forebrain structures (Jobe et al., 1991). On the other hand, in most models (especially involving the g-aminobutyric acid [GABA] system or limbic seizure models), tonic–clonic seizures represent a spread of paroxysmal activity from the forebrain to the brainstem (Browning and Nelson, 1986). During this type of seizures, the righting reflex is lost. The rat falls down on the cage floor and makes no active effort to regain balance. Tonic–clonic seizures are generalized seizures consisting of three phases: (1) Initial wild run or a jump followed by fall accompanied by loss of the righting reflex. (2) Next, tonic phase follows involving tonic flexion and then extension of forelimb, hind limb, or both with variable duration. The tonic extension of hind limbs represents the most severe seizure phase (Swinyard, 1973). (3) After the tonic phase, long-lasting clonus of all limbs develops. These three phases can be observed regularly in adult animals in different models of generalized seizures.
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During development, tonic–clonic seizures are elicited regularly by most convulsants. The occurrence of individual phases, however, is variable depending on the seizure model. During the first 2 postnatal weeks in most models, the initial phase of wild running is short and consists often of poorly coordinated slow swimminglike movements, which increase in frequency with age. The tonic phase has variable duration and includes tonic flexion and extension of forelimbs and flexion of hind limbs. Interestingly, in some models, the tonic extension of hind limbs does not regularly occur before the second postnatal week (Schickerová et al., 1984). This phenomenon, however, may rather depend on the seizure model than on the lack of ability to develop tonic extension of hind limb at the early development. The longlasting, slow uncoordinated swimminglike clonic phase follows with interruption by periods of gasping and opisthotonus (de Feo et al., 1985; Velísˇek et al., 1992; Velísˇková and Velísˇek, 1992).
Atypical Behavior and Automatisms (Stereotypies) Atypical behavior is a type of behavior that normally does not belong to normal behavioral repertoire. Automatisms, on the other hand, are repetitive movements that originally may serve a purpose; however, as a response to epileptogenic stimulus they are useless. To consider the atypical behavior or the automatism as a seizure, it should be correlated and confirmed by an electrographic recording. An inexperienced observer may miss this type of behavior in experimental animals as he might in humans. Different seizure models present with distinct features of atypical behavior or automatisms and also this changes as a function of age. Atypical behavior is characterized in adult rats by behavioral arrest and episodes of staring and is usually observed in models of absence seizures (electroencephalography [EEG] clearly displays spindles of spike and wave activity during the staring periods [Marescaux et al., 1984; Maresˇ et al., 1997a]). In the developing rats, atypical behavior consists of tremor with tail stiffening or erection and can be demonstrated in models of limbic seizures. Behavioral arrest and staring, as well as the correlation with spike and wave EEG discharges typical for absence seizures, can be reliably demonstrated from the third postnatal week (Schickerová et al., 1984). Automatisms (stereotypies) are characteristic for limbic seizures. Depending on the drug, automatisms can be presented as excessive washing, water licking, sniffing, orientation, rearing, scratching, circling, or wet dog shakes (WDS). The WDS however, may not always represent a seizure. In the kainic acid model, WDS are often one of the first signs of seizures and occur when EEG discharges are present in the entorhinal cortex (Ben-Ari et al., 1981). On
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the other hand, in the kindling model, WDS are often observed at the end of a kindling seizure at the moment of an afterdischarge cessation and, thus, in this case may not represent a seizure behavior. In any case, the WDS are a sign of hippocampal involvement because destruction of granule cells of the dentate gyrus prevents the occurrence of WDS (Frush and McNamara, 1986; Grimes et al., 1990). In immature rats, scratching is a dominant automatism in the kainic acid model. The WDS are not present during the first 2 postnatal weeks, but increase to occur with age (Velísˇková et al., 1988). In other models (intrahippocampal tetanus toxin injections), however, WDS can also develop in very young rats (PN10) (Anderson et al., 1997), suggesting that the age is not a limiting factor.
WHAT SEIZURE PARAMETERS TO TEST Several parameters can be used to characterize a seizure model. These parameters are used to compare seizure susceptibility. 1. Seizure threshold can be measured by the time necessary for a seizure to occur, as the amount of convulsant drug necessary to induce a seizure, or as the number of stimulations required to induce seizure. Lowering the seizure threshold corresponds to a proconvulsant effect, whereas increasing the seizure threshold means an anticonvulsant effect. 2. Seizure severity is determined by a scoring system specifically developed for a particular type of seizures. The scoring system design is based on specific characteristics of a model, how the seizures progress owing to gradual involvement of distinct networks, and based on age-specific expression of seizures. A different scoring system has to be used for focal seizures (e.g., kindling, Tables 1 and 2; limbic seizures, Table 3), generalized seizures originating in the forebrain (minimal electroshock or convulsants affecting GABA neurotransmission (i.e., pentylenetetrazol [PTZ], bicuculline, picrotoxin, and so forth, Table 4), or generalized seizures with brainstem origin (e.g., maximal electroshock or audiogenic seizures, Table 5). Also, a distinct scoring system can be used for an acute and a chronic convulsant drug administration (e.g., PTZ kindling) (Ono et al., 1990). Some models may have similar characteristics and the same scoring system can be used; other models or developing animals may require some modifications from the suggested original scoring system (Tables 1–5). 3. Incidence of seizures can be a parameter of drugs efficacy against distinct types of seizures (clonic vs. tonic–clonic) and the ability of a drug to block the progression of a seizure either from the forebrain to the brainstem or vice versa, depending on the seizure origin.
TABLE 1 Scoring system for focal seizures with secondary generalization I: Kindling; adult rats (Racine et al., 1973; Pinel and Rovner, 1978) Seizure stage
Behavioral expression
Righting reflex
Structure involved
1
Mouth and facial movement
Preserved
Forebrain
2
Head nodding
3
Contralateral forelimb clonus
4
Symmetrical forelimb clonus with rearing Lost
Brainstem
5
Rearing and falling
6*
Wild running, jumping, rolling and vocalization
7*
Tonic posturing
8*
Spontaneous seizures
*Denotes stages described by Pinel and Rover beyond regular kindling stages, which occur after delivering over 200 stimulations.
TABLE 2 Scoring system for focal seizures with secondary generalization II: Kindling; developing rats (Haas et al., 1990) Seizure stage
Behavioral expression
0
Behavioral arrest
1
Mouth clonus
2
Head bobbing
3
Unilateral forelimb clonus
3.5
Alternating forelimb clonus
4
Bilateral forelimb clonus
5
Bilateral forelimb clonus with rearing and falling
6
Wild running and jumping with vocalization
7
Tonus
Righting reflex
Structures involved
Preserved
Forebrain
Lost
Brainstem
TABLE 3 Scoring system for focal seizures with secondary generalization III: Kainic acid-/pilocarpine-induced seizures Seizure stage
Behavioral expression
Righting reflex
Structures involved
1
Staring with mouth clonus
Preserved
Limbic Structures
2
Automatisms (WDS, scratching*)
3
Unilateral forelimb clonus
4
Bilateral forelimb clonus
5
Bilateral forelimb clonus with rearing and falling
6
Tonic-clonic seizure
Other forebrain regions (neocortex, thalamus, etc.)
Lost
Brainstem
*Stages occurring more often in developing animals than adults.
Distinct Behavioral Seizure Expression Based on the Neurotransmitter System
TABLE 4 Scoring system for primary generalized seizures with forebrain origin (Pohl and Maresˇ, 1987; Velísˇková et al., 1990) Seizure stage
Behavioral expression
0
No changes in behavior
0.5
Abnormal behavior (sniffing, extensive washing, orientation)
1
Isolated myoclonic jerks
2
Atypical (unilateral or incomplete) clonic seizure
3
Fully developed bilateral forelimb clonus
3.5
Forelimb clonus with a tonic component and twist of body
4
Tonic-clonic seizure with suppressed tonic phase; only clonus of all limbs
5
Fully developed tonic-clonic seizure
Righting reflex
Structures involved
Preserved
Forebrain
Lost
Brainstem
TABLE 5 Scoring system for generalized tonic-clonic seizures originating in the brainstem (Jobe et al., 1973) Seizure stage
Behavioral expression
0
No response
1
Running only; no seizure
2
Two running phases separated by a refractory period; generalized clonus involving forelimbs, hindlimbs, pinnae and/or vibrissae Same as 2, but only one running phase and no refractory period
3 4
Brainstem
two running phases separated by a refractory period; tonic flexion of neck, trunk, and forelimbs with clonus of hindlimbs
5
Same as 4, but only one running phase and no refractory period
6
Two running phases separated by a refractory period; convulsive endpoint similar to 4, except that hindlimbs are in partial tonic extension (i.e., tonic extension of thighs and legs with clonus of feet)
7
Same as 6, but only one running phase and no refractory period
8
Two running phases separated by a refractory period; convulsive endpoint similar to 4, except that hindlimbs are in complete tonic extension Same as 8, but only one running phase and no refractory period
9
Structure involved
The clonic seizure involving forelimbs may occur as an additional stage due to the seizure progression into the forebrain.
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DISTINCT BEHAVIORAL SEIZURE EXPRESSION BASED ON THE NEUROTRANSMITTER SYSTEM g-Aminobutyric Acid Antagonists and adverse agonists to GABAA receptors or blockade of glutamate acid decarboxylase (GAD) induce both nonconvulsive and motor seizures: 1. Behavioral arrest occurs in adult animal, with EEG displaying spindles of spike and wave activity, and can be observed following the administration of lower doses of convulsant drugs affecting GABA. As mentioned, this phenomenon is age-dependent. In developing rats, restlessness and locomotor hyperactivity occur as the first signs of seizures. 2. Myoclonic twitches are usually whole-body twitches. Sometimes they look like the animal has been suddenly stopped or pushed back from the forward motion or quietness. Accumulation of myoclonic twitches frequently leads to occurrence of clonic seizures. 3. Clonic seizures (forebrain seizures) can be unilateral or bilateral with preserved righting reflex. The transition from clonic to tonic–clonic seizures can be regularly demonstrated in immature rats up to the end of third postnatal week, but does not occur so often (Figure 1) in older rats. 4. Tonic–clonic seizures (hindbrain seizures) start with wild running followed by a loss of righting reflex. After the fall, tonic flexion and extension usually occurs on forelimbs and hind limbs. After the tonic phase, which may or may not occur, clonus of all four limbs regularly develops and lasts for minutes to tens of minutes. This seizure is often lethal.
Excitatory Amino Acids Excitatory amino acids (EAA) hardly cross the blood–brain barrier (BBB) (Johnston, 1973) and, thus, the most sensitive are developmental animals with immature BBB (Joo, 1987). Glutamate produces forebrain seizures with progression into tonic–clonic seizures and seizures can be reliably induced up to PN18 (Maresˇ et al., 2000). On the other hand, seizures induced by individual EAA receptor agonists seem to have a focal origin rather than a primary generalized (Velísˇek et al., 1995) and have special features characteristic for individual agonist (Maresˇ et al., 2004). Often, high doses of EAA agonists need to be used in older rats (from third postnatal week) (Schoepp et al., 1990). The origin of seizures induced by EAA depends on the type of EAA receptor involved. The non-NMDA receptor agonists (kainate or AMPA) produce seizures originating in limbic structures (Nadler, 1981; Thurber et al., 1994) with automatisms progressing into myoclonic seizures, whereas NMDA
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seizures originate in the brainstem structures and automatisms and myoclonic seizures cannot be demonstrated (Maresˇ and Velísˇek, 1992). This is related to the distribution of individual EAA receptor types within the central nervous system (CNS). Highest densities of kainate and AMPA receptors are in the forebrain structures (Insel et al., 1990; Miller et al., 1990), whereas NMDA receptors have highest density in the hindbrain and cerebellum (Palmada and Centelles, 1998). Automatism(s) or immobility followed by hyperactivity is usually the first sign of seizures induced by EAA and is EAA receptor type specific (Maresˇ and Velísˇek, 1992). Quisqualic acid, as an AMPA receptor agonist, induces circling behavior and immobility in the initial phases following the injection (Turski et al., 1990; Thurber et al., 1992; Thurber et al., 1994). Following kainic acid administration, rats become hypoactive with prominent age-specific expression of automatisms, but ataxia does not occur (Albala et al., 1984; Velísˇková et al., 1988; Stafstrom et al., 1992). In adult rats, WDS are a hallmark automatism, whereas in immature rats WDS are replaced by vigorous scratching, especially during the first 2 postnatal weeks (Velísˇková et al., 1988) (Figure 2). On the other hand, immobility and ataxia followed by extreme locomotor hyperactivity occur following NMDA, homocysteic acid, homocysteine, and glutamate (Maresˇ and Velísˇek, 1992; Kubová et al., 1995; Maresˇ et al., 1997b; Folbergrová et al., 2000; Maresˇ et al., 2000). Additionally, one of the first signs of seizures following NMDA administration involves special tail movements. The tail is formed into an “S” shape and the movements are whiplike. Twitches or tremor (usually in immature rats) often precede the motor seizures. Clonic and tonic–clonic seizures: Higher doses of these drugs evoke motor seizures representing the generalization. The transition from clonic to tonic–clonic seizures
FIGURE 2 Ontogenetic development of behavioral seizure phenomena in rat: N-methyl-D-aspartate (NMDA) and kainic acid (KA) seizures.
can be regularly demonstrated in immature rats up to the end of the third postnatal week, but does not occur so often in older rats (Velísˇková et al., 1988). It should be emphasized that in this category, clonic and tonic–clonic seizures occur with behavioral features as described above; however, the occurrence of both types is drug- and age-specific (Albala et al., 1984; Velísˇková et al., 1988; Maresˇ et al., 2004). Clonic seizures are the hallmark of secondary generalized seizures following kainic acid or quisqualic acid, whereas tonic–clonic seizures are characteristic for the NMDA model, although following higher doses of kainic acid, tonic–clonic seizures occur, especially in developing animals (Velísˇková et al., 1988; Maresˇ and Velísˇek, 1992; Thurber et al., 1994). Special types of seizure behavior: Additionally, special seizure patterns can occur, particularly during development. One of these patterns involves flexion seizures (emprosthotonus; Figure 3). These seizures are highly age-specific and can be observed up to the third postnatal week (Maresˇ and Velísˇek, 1992; Kábová et al., 1999). These flexion seizures are typical for drugs acting as agonists at NMDA receptors such as NMDA or homocysteic acid (Maresˇ and Velísˇek, 1992; Maresˇ et al., 1997b). Interestingly, homocysteine induces flexion seizures only in rats up to P12 (Kubová et al., 1995). Flexion seizures occasionally can also be observed following kainic acid administration until P12 (Velísˇková, unpublished observation).
Acetylcholine System Seizures induced by activation of acetylcholine system (pilocarpine) originate in the limbic structures similarly to kainic acid-induced seizures. Although seizures induced by kainic acid seem to start in the entorhinal cortex and propagate via perforant path into the hippocampus (Ben-Ari et al., 1981), hippocampus itself seems to be a structure of
FIGURE 3 Emprosthotonus in PN12 rat induced by injection of Nmethyl-d-aspartate (NMDA).
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Choice of Administration Route
origin of pilocarpine-induced seizures (Cavalheiro et al., 1987). Automatisms dominate the initial phase following pilocarpine injection. Animals become akinetic and ataxic with gustatory automatisms, scratching, and head tremor. The behavior progresses into forelimb clonus with rearing and intense masticatory movements. Tonic–clonic seizures occur following higher doses and are often observed when lithium pretreatment is used. During development, initial hyperactivity is followed by hypoactivity, then body tremor and automatisms, including vigorous scratching, can be observed. Motor seizures can be demonstrated from the second postnatal week and include forelimb clonus with rearing and falling (Cavalheiro et al., 1987).
Hypoglycemic Seizures Hypoglycemic seizures have been reliably demonstrated in adult rats by injection of insulin (Gastaut et al., 1968). The mechanism of hypoglycemic is unknown, but the seizures seem to originate in the deep brainstem structures. The hypoglycemic seizures have very characteristic behavioral expression. Twitches, the first sign, consist of strong jerks of the body with jumps. Barrel rotations are typical for hypoglycemic seizures. The rat is fast rolling without the righting reflex (Figure 4). Clonic seizures involve all four limbs and look like uncoordinated swimming movements.
choose the right dose for the hypothesis to be tested. A low dose of a convulsant drug may not lead to more advanced stages, such as occurrence of tonic–clonic seizures. High doses, on the other hand, can induce rapid spread of seizures and individual stages can be too short or even masked by more advanced seizure stages (Velísˇková and Velísˇek, 1992). A pilot experiment is highly recommended to determine the dose of a convulsant drug. This is because of differences in strain sensitivity (Loscher et al., 1998; Schauwecker, 2002; Brandt et al., 2003) and also within the same strain because of the housing conditions or diet. It has been shown that environmental enrichment among farms or institutions (e.g., cage size or handling) has profound influence on seizures (Young et al., 1999). A different dose of a convulsant drug may represent a model for different types of seizures. Pentylenetetrazol- or bicuculline-induced seizures are an example of such situation (Schickerová et al., 1984; de Feo et al., 1985; Velísˇek et al., 1993). Low doses of PTZ (40 mg/kg) produce short episodes of loss of consciousness with staring, which is characteristic for absence seizures (Velísˇek et al., 1993), and EEG recordings show typical spike-and-wave rhythmic activity (Schickerová et al., 1984; de Feo et al., 1985). Higher doses of PTZ (100 mg/kg) produce generalized clonic seizures with progression to tonic–clonic seizures.
CHOICE OF ADMINISTRATION ROUTE CHOICE OF THE RIGHT DOSE Development of distinct phases of behavioral seizures depends on the dose of a convulsant. It is important to
Modeling of distinct seizure types may not depend only on the drug used but also on the choice of administration route. Topical application of PTZ on the neocortex is a model for simple partial seizures, whereas systemic administration of PTZ serves as a model of absence seizures (using low doses) or generalized clonic and tonic–clonic seizures (higher doses) (Velísˇek et al., 1992; Velísˇek et al., 1993).
Focal Administration
FIGURE 4 Barrel rotation in adult rat during insulin-induced hypoglycemic seizures.
Besides the example mentioned, focal drug application intracerebroventricularly might be chosen for better survival rate following seizures (homocysteine acid, kainic acid) (Nadler et al., 1978; Folbergrová et al., 2000). Drugs can also be applied directly into the brain structures (i.e., area tempestas [Maggio and Gale, 1989]; hippocampus [Lee et al., 1995]) or on the surface of the cortex (Soukupová et al., 1993; Chang et al., 2004). Following focal drug application, the seizure behavior can occur acutely or with a delay. The first sign of seizures depends on the application site of the drug. With generalization, other seizure behavior occurs, depending on the networks involved.
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Systemic Administration Behavioral expression of seizures following systemic administration of the drug depends on many factors. One of the important factors is permeability of the BBB and the other drug penetration. One of the interesting examples is the atypical features of seizure behavioral following bicuculline methiodide. As described above bicuculline, an antagonist at GABAA receptors, easily penetrates the BBB and induces generalized seizures with behavioral arrest, myoclonic twitches progressing into clonic seizures, and finally into tonic–clonic seizures. In contrast, bicuculline methiodide, which does not crosses the mature BBB, elicits automatisms consisting of sniffing, rearing, chewing, washing, and in adult rats WDS and scratching (Maresˇ et al., 2000), which are typical for limbic seizures rather than primary generalized seizures and are never observed following bicuculline or other GABAA receptor antagonists. Moreover, up to age P18, automatisms progressed into clonic seizures and later to tonic–clonic seizures, although the tonic phase was never observed in this model (Maresˇ et al., 2000). This unusual seizure behavior, especially the presence of automatisms following bicuculline methiodide, may be explained by the path of its penetration into the brain, because these automatisms suggest hippocampal involvement. Thus, bicuculline methiodide probably first diffuses into the cerebrospinal fluid and, thus, the hippocampus represents the first brain structure in its way of tissue penetration. Seizures then spread into the motor cortex and clonic seizures occur. Another reason for choosing the right route of drug administration is the progression of individual seizure stages, which can develop too fast. Such an example is PTZinduced seizures. Subcutaneous (s.c.) administration results in slower drug absorption and produces reliably all seizure stages with dose necessary for occurrence of tonic–clonic seizures compared with intraperitoneal application, which leads to fast progression of individual seizure stages, whereas lower doses do not lead to development of tonic–clonic seizures. Moreover, s.c. application is less stressful for the animal compared with intravenous injection or through intraperitoneal microinjection cannula, which are other ways to ensure gradual development of individual seizure stages (Zouhar et al., 1980).
CHOICE OF AGE Translation of human developmental stages in neural events of rodent developing brain is important in developmental studies (Holmes, 1986; Moshé, 1987). Comparative ontogeny: Factors that need to be considered in comparisons of the development of the rat’s brain to human development are brain growth, rate of protein synthesis, myelinization,
structural connectivity, synaptic density, levels of neurotransmitters, or cerebral glucose utilization rate. Based on these confounding factors, rats at PN8 through PN10 roughly correspond to full-term human newborn (Gottlieb et al., 1977; Dobbing and Sands, 1979; Moshé, 1987; Nehlig, 1997; Avishai-Eliner et al., 2002; Herlenius and Lagercrantz, 2004). The 2- to 3-week-old rats seem to be equivalent to human infant-toddler (Nehlig, 1997; Velísˇek and Moshé, 2002). Rats 3- to 5-weeks-old correspond to a prepubescent child (Nehlig, 1997; Velísˇek and Moshé, 2002); puberty in rats starts around P32 to P38 and the maturity in the rat is achieved after P55 (Ojeda and Urbanski, 1994; Velísˇek and Moshé, 2002). An important factor is regional differences in maturation, and experiments targeting a specific brain structure should reflect selective regional maturation state (Avishai-Eliner et al., 2002). The sensitivity to epileptogenic stimuli and seizure severity is highest during early development, on the other hand mortality and occurrence of seizure-induced damage is usually lower compared with adult rats (Wasterlain, 1978; Moshé and Albala, 1982; Moshé et al., 1983; Cavalheiro et al., 1987; Velísˇková et al., 1988; Maresˇ and Velísˇek, 1992; Haut et al., 2004). Status epilepticus involving tonic–clonic seizures lasting for up to 2 hours can be induced by flurothyl in rats during the first 2 weeks of life (Giorgi et al., in press). Adult rats, however, die within 30 minutes of tonic–clonic seizure onset (Sperber et al., 1999). Finally, certain seizures occur as a function of age. There is high sensitivity to febrile- or corticotropin-releasing hormone-induced seizures in developing rats during a discrete developmental window (Baram and Schultz, 1991; Baram et al., 1997). Development of kindling is another seizure model with age-dependent features (Moshé, 1981). Although kindling in adult rats takes several weeks, the immature animals can be kindled within 2 days. On the other hand, certain seizure phenomena cannot be demonstrated in immature brain. Typical features of absence seizures with EEG spike-and-wave discharges can be observed in adult rats but not in developing rats (Marescaux et al., 1984; Schickerová et al., 1984; de Feo et al., 1985; Maresˇ et al., 1997a). In conclusion, seizure expression in the laboratory animals often closely models the variety of seizure behavior in humans. Any seizure behavior must be first confirmed and correlated with electrographic discharges. This is important, especially in developing animals, in which certain manifestations of normal behavior can resemble a seizure or in models characterized by atypical behavioral pattern (e.g., absence or limbic seizures). Many variables (e.g., age, sex, strain, or housing conditions) can influence the seizure behavior or seizure threshold. Different seizure types have distinct substrates, thus the choice of appropriate model for the question asked is important.
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49 Behavioral and Cognitive Testing Procedures in Animal Models of Epilepsy CARL E. STAFSTROM
the behavioral and cognitive deficits. Such confounders include the underlying etiology and brain pathology, present and past anticonvulsant treatments, degree of seizure control, and developmental status of the patient. Animal models can alleviate some of those variables and provide insights into seizure effects that can be applied to the human patient. In animal models, the investigator has control over the seizure type, duration, frequency, and pharmacologic and other interventions, permitting correlation of behavioral outcomes with physiologic and histologic data. Accumulating evidence suggests that, in models of experimental epilepsy, both brief and prolonged seizures lead to deficits in cognition and behavior, even in the absence of overt structural neuronal damage. These deficits are dependent on the age at which seizures occur (less severe deficits at younger ages), seizure frequency, and seizure severity. Seizure-related cognitive deficits appear to be less strongly dependent on seizure etiology, as they occur as a consequence of a wide variety of seizure induction methods, including kindling, flurothyl exposure, and numerous chemical convulsants, as well as in genetic epilepsy models. With appropriate caveats, data derived from animal models can be extrapolated to the human, in whom such comprehensive studies cannot be performed. The goals of using animal models to study experimental epilepsy are to understand how seizures alter cognitive function, which aspect(s) of cognition are most at risk for impairment, and how, if at all, some of the cognitive deficits can be compensated for or restored. It would be informative to know which brain region or system subserves various aspects of learning, memory, behavior, and cognition, to determine which brain function is altered by seizures. Additional important considerations are whether (1) seizure-induced
EFFECTS OF SEIZURES ON BEHAVIOR AND COGNITION: DEFINITIONS AND PERSPECTIVES The consequences of seizures and epilepsy can be assessed in several ways: structural or histologic changes (both cell death and compensatory processes) at the levels of single cells or neuronal networks; electrophysiologic changes in membrane or circuit properties; or behavioral measures, including learning, memory, and other cognitive processes. The testing of behavioral and cognitive function is a critical aspect of the characterization of an animal epilepsy model. The degree to which seizures result in long-term deficits in cognition and behavior remains an unresolved clinical question. Behavioral and cognitive deficits, which may not become obvious until long after the onset of epilepsy, can be equal or more detrimental to an individual’s overall function than the seizures themselves. Accumulating evidence indicates that epilepsy is a progressive disorder (Pitkanen and Sutula, 2002), as judged by neuroimaging, pathological, and neuropsychological studies, with recurrent seizures resulting in long-term adverse behavioral and neurocognitive consequences. In humans, a large literature strongly suggests that repeated seizures are associated with poorer cognitive skills, memory impairment, and a wide variety of behavioral and psychological disorders (Elwes et al., 1988; Beckung and Uvebrant, 1997; Bourgeois, 1998; Schoenfeld et al., 1999; Ploner et al., 1999; Austin and Dunn, 2002; Helmstaedter et al., 2003; Sillanpaa, 2004; Elger et al., 2004). Several confounding variables in the clinical setting, however, preclude direct demonstration that seizures themselves are responsible for
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central nervous system to acquire, process, store, and act on information from the environment (Pickens and Holland, 2004). “Learning” is the process of acquiring a new behavioral repertoire or information. “Memory” is defined as the retention of such learned information, as evidenced by its use at a later time. “Behavior” comprises the animal’s actions that are observable and quantifiable. Obviously, these terms overlap considerably, yet they must be defined separately to analyze the complexities of animal behavior. Behavior and cognition are used together in this chapter to denote external and internal states, respectively. Because animals are nonverbal, cognition must be measured indirectly using behavioral tests. Overall, the ability of an animal to function optimally in its environment depends on several interacting neural systems, mediating cognition, learning, memory, and behavior. Many ways exist to conceptualize these cognitive variables. Figure 1, based on the pioneering work of the cognitive neuroscientist E. Tulving (1983), attempts to synthesize and conceptualize memory into units that can be tested specifically. The brain substrates (sometimes, but not
brain damage is permanent, (2) histologic evidence of damage necessarily implies functional impairment, (3) behavioral impairment can exist in the absence of structural damage, and (4) the brain possesses age-dependent repair mechanisms. This chapter critically reviews behavioral and cognitive methods that are used to assess the consequences of seizures in animal models of epilepsy. The investigator must choose carefully among the various tests of behavior, learning, memory, and cognition, because the existence or extent of deficits depends on which test is selected, how the test is performed, and how the data are analyzed. Details of specific behavioral and cognitive effects of seizures, a vast topic of obvious relevance here, are mentioned but not discussed extensively in this chapter. Recent comprehensive reviews cover this topic in detail (Stafstrom et al., 2000; Stafstrom 2002; Majak and Pitkanen, 2004; Holmes, 2004). Here, the primary intent is to describe and discuss the tests themselves. First, it is essential to define certain concepts and terms. For this chapter, “cognition” is defined as the ability of the
MEMORY LONG TERM (reference)
SHORT TERM (working) ∑
DECLARATIVE (explicit)
EPISODIC (events)
∑ ∑ ∑
PREFRONTAL CORTEX
NONDECLARATIVE (implicit)
SEMANTIC (facts)
PROCEDURAL
MEDIAL TEMPORAL LOBE DIENCEPHALON NEOCORTEX, ESPECIALLY PREFRONTAL
PERCEPTUAL REPRESENTATIONAL
SKILLS (MOTOR AND COGNITIVE)
∑ ∑
BASAL GANGLIA, CEREBELLUM NOT HIPPOCAMPALDEPENDENT
CLASSICAL CONDITIONING
PERCEPTUAL PRIMING
∑
∑
PERCEPTUAL AND ASSOCIATION NEOCORTEX (occipital, parietal, frontal) NOT HIPPOCAMPALDEPENDENT
CONDITIONED RESPONSES BETWEEN 2 STIMULI ∑ ∑ ∑
SKELETAL MUSCLE (cerebellum) EMOTIONAL (amygdala) NOT HIPPOCAMPALDEPENDENT
NONASSOCIATIVE CONDITIONING
HABITUATION SENSITIZATION
∑ ∑
FIGURE 1 Taxonomy of human memory systems (boxes), with presumed brain substrates mediating the various memory subtypes indicated below in bold letters. This schematic is based on work of Tulving, Squire, and others (Tulving, 1983) (Squire and Zola, 1996). The relationship between human and non-human animal memory systems is the subject of active research (see text).
REFLEX PATHWAYS NOT HIPPOCAMPALDEPENDENT
Behavioral Testing of Laboratory Animals: General Principles
always equivalent to specific brain structures) that participate in the mediation of each component are also indicated. This information can be used to design experiments to test specific aspects of seizure-induced cognitive deficits. Notably, this taxonomy is derived from human studies, so it is unclear how well it fits nonhuman animals; nevertheless, it serves as a conceptual framework. Other aspects of psychological function (e.g., anxiety and stress) also affect cognition, behavior, and performance on tests. Memory is a major measurable aspect of cognition, often addressed by behavioral tests. The two major types of memory are short-term (working) memory and long-term (reference) memory. Short-term memory implies a limited capacity for the temporary storage of information, whereas long-term memories can be stored indefinitely and represent learned information. Most cognitive tests in animals assess long-term memory (see Figure 1). In humans, short-term memory is considered to last less than 1 minute, and usually less than 30 seconds. An example is memorizing a telephone number, which is forgotten soon after the number is dialed. A direct analogy to working memory duration in nonhuman animals is not available (Dudchenko, 2004), but can be defined operationally as memory required for one trial of an experiment, but not for subsequent trials (Dalley et al., 2004). Tests to evaluate the integrity of short-term memory usually involve a delayed response contingency, such as spatial delayed alternation and delayed nonmatching to sample. The prefrontal cortex is strongly implicated in the mediation of working memory. Rats with lesions of the prefrontal cortex have impairments in performing sequential behavioral tasks dependent on short-term memory. Similarly, short-term memory is altered by antiepileptic drugs (Shannon and Love, 2004). Long-term (reference) memory has many subdivisions (see Figure 1). The first division is into declarative (explicit) memory and nondeclarative (implicit) memory. Subdivisions of declarative memory are episodic (for events) and semantic (for facts and general knowledge about the world). Declarative memory in humans is considered to be roughly equivalent to spatial memory in rodents, justifying the widespread use of water maze and other tests of spatial learning and memory in epilepsy research. Nondeclarative memory, to which an individual has no conscious access, is not dependent on the hippocampus (Squire and Zola, 1996). Nondeclarative memory also includes several subcategories. Procedural memory relies on learned motor and cognitive skills. An example would be our ability to ride a bicycle or a rat’s ability to swim. Perceptual memory is a form of memory in which a priming stimulus initiates the memory. Finally, classical conditioning paradigms and habituation are forms of nondeclarative memory (Bouton and Moody, 2004). The correlation between memory subcategories and brain substrate, although inexact, is becoming increasingly better
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defined. Much of memory is distributed among several brain regions or pathways, rather than mediated by a single structure or nucleus (Maviel et al., 2004; Jeffrey et al., 2004; Miyashita, 2004). Nevertheless, lesions or pathology of certain brain regions (e.g., the hippocampus for episodic memory) suggest that partial seizures can affect some parts of the brain more than others. An important caveat is to avoid rigid adherence to the notion that, if pathology that exists in a certain brain structure is associated with a specific cognitive deficit, that function is mediated (only) in that structure. For example, if hippocampectomy or seizureinduced hippocampal pathology is associated with spatial learning dysfunction, this does not mean that spatial learning is subserved solely by the hippocampus. This observation does not prove that other brain regions do not play a role in spatial learning, or that spatial learning is the only hippocampus-mediated cognitive deficit. The scheme of Figure 1 was constructed for humans; whether these categories are directly applicable to rodents is uncertain (Miyashita, 2004). Controversy exists to whether nonhuman animals can truly “travel back in time” and represent past experiences (episodic memory) or whether this ability is unique to humans. Recent work provides evidence that, with appropriately designed experiments, animals can exhibit episodic memory (Clayton et al., 2003). Our task is to design tests to assess the involvement of various brain regions affected by seizures, and to see whether we can detect and perhaps intervene to prevent seizure-related damage and cognitive deficits. For example, in rats experiencing pilocarpine-induced status epilepticus, subsequent cognitive impairment on water maze tasks was reduced if the rats were housed in an enriched environment (a large area with toys, a treadmill for exercise, and piped in Mozart music) (Rutten et al., 2002).
BEHAVIORAL TESTING OF LABORATORY ANIMALS: GENERAL PRINCIPLES Several general principles must be taken into account when using animal models to provide insights into human epilepsy. First, species and strain differences are critical. Obviously, human language and behavior differs qualitatively and quantitatively from that of laboratory animals, yet mammalian brains share many structural and physiologic features that permit comparison between species. Ideally, a specific behavioral test in an animal will have a correlate in human neuropsychology; however, such correlation is rarely possible. Only by extrapolation, and with appropriate caution, can we attempt to compare seizure-induced cognitive changes across species. This chapter concentrates on rodents, the most prevalent animal models of seizures and epilepsy. Even among rodents, profound differences exist in performance on behavioral tests (Voikar et al., 2001;
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Schimanski and Nguyen, 2004). For example, in spatial navigation tasks in water mazes, rats out-perform mice, whereas in spatial navigation tasks on dry land, rats and mice perform similarly (Whishaw and Tomie, 1996). Gender differences must also be considered when testing seizure susceptibility (Mejias-Aponte et al., 2002; Galanopoulou et al., 2003) or cognitive function (Voikar et al., 2001; Gresack and Frick, 2003). The second important caveat is the laboratory setting. Any behavioral test performed in the laboratory is unnatural, in the sense that the laboratory is not the usual environment in which the animal must learn to cope with evolutionary stressors and compensate for functional deficiencies. An ethological perspective urges that an animal’s cognition and behavior be tested in an environment as “natural” as possible (Gerlai and Clayton, 1999). Laboratory animals are usually bred in captivity and live their entire lives in the confines of a laboratory cage. The “challenges” of life in a laboratory cage are radically different from those facing animals living in the wild (e.g., how often does a rat in the wild need to choose which arm of an enclosed chamber to navigate in order to obtain food?). The main point is not necessarily that we perform neurobehavioral experiments in an animal’s natural setting, but rather that we interpret behavioral data with a knowledge of speciesspecific behaviors (Crabbe and Morris, 2004). What is a “normal” behavior for a rodent in one environment may be quite different in another. These issues relate to the validity of a given test. A third crucial feature is reproducibility of test results related to the impact of experimenter handling and characteristics of the housing and testing environment. Despite efforts to ensure that behavioral experiments are performed identically from day to day and from animal to animal, inevitable variations in laboratory parameters affect outcomes. The list of laboratory variables that must be accounted for is lengthy (Table 1). Unless the time since the last seizure (e.g., in models with spontaneous seizures) is known, behavioral testing results can be misleading (Leung et al., 1994; Hort et al., 2000). Numerous specific examples attest to how behavioral results vary because of different handling and other aspects of laboratory practice (Arndt and Surjo, 2001; Pryce et al., 2001; Chesler et al., 2002). Efforts should be made to minimize the stress induced by a testing procedure, by acclimating animals to handling and to the testing apparatus, and providing sufficient rest between trials and a consistent home cage environment. Because each trial, each laboratory setting, and each experimenter is different, it is a major challenge to maximize the reliability of results. Fourth, it is likely that multiple brain regions mediate common behaviors and cognition. It is crucial that the experimenter have a specific hypothesis in mind when choosing and performing a behavioral test. For example, it is not suf-
TABLE 1 Some Laboratory and Animal Factors That Can Affect Behavioral Testing Results Animal specifications • Breeding conditions • Species or strain • Age • Sex • Nutrition • Maternal care (in young animals) Laboratory environment • Housing parameters (e.g., number of animals per cage, cage size) • Animal care • Acoustic background • Light/dark cycle Testing conditions • Test order • Time of day • Temperature and humidity • Experimenter handling • Testing apparatus Experimental procedures • Method of seizure induction • Age at time of seizure • Seizure frequency • Time elapsed since last seizure • Environmental enrichment • History of therapies or interventions
ficient to select the water maze as a measure of spatial learning, and then consider this test to be a reflection of overall cognition, as has been done frequently, especially in gene targeting studies. As discussed below in the section on water maze, the complexities of even this test, often considered a relatively “pure” measure of hippocampal integrity, can confound even the most carefully designed experiment. Lastly, in epilepsy and related research, genetic mutants are being increasingly used, and for good reason. Animals with particular genes knocked in or out provide an exceptional opportunity to test the effect of a specific mutation on both epilepsy predisposition and behavior. In such experiments, it is especially critical to be aware of confounders of behavior and cognition, to choose appropriate controls, and to avoid overgeneralization of results (Crawley, 1999; Voikar et al., 2001). With those caveats in mind, some selected tests of behavior and cognition are described that are often used in experimental epilepsy models.
BEHAVIORAL TESTS TO ASSESS THE EFFECTS OF SEIZURES In animal models of epilepsy, variables (e.g., seizure etiology, severity, frequency, and duration) can be controlled.
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Therefore, animal models have been used to explore these questions (Stafstrom and Holmes, 2002). The use behavioral data as a determinant of brain damage in seizure models is increasing, but the tests themselves have rarely been subject to critical scrutiny. Analysis of the validity and potential shortcomings of each behavioral test is needed before concluding that seizures damage the brain. Here, various behavioral tests used to assess seizure-induced cognitive damage in animals are reviewed, focusing on how each test is performed and analyzed, what neurologic function each test subserves, and whether a specific brain area mediates that neurologic function. Many variations of each test have been described in the literature, and only the most commonly used protocols are discussed here. Unless indicated, the tests are restricted to those used in rats and mice. Analogous tests in humans are discussed when appropriate. Finally, some examples of the use of each test in epilepsy models are provided; further details can be found in other chapters of this volume that cover specific models.
Tests of Sensorimotor Function and Reflexes Behavioral tests usually assess motor responses to sensory stimuli. Therefore, before concluding that seizures produce cognitive dysfunction, it must be shown that sensory and motor deficits are not responsible. In addition to primary deficits in an animal’s ability to perceive sensory stimuli and exhibit normal motor behavior, several factors can influence sensorimotor function, including general health, time of day, level of arousal, level of anxiety, age (Fox, 1965; Altman and Sudarshan, 1975), and genetic background (Crawley, 1999; Fox et al., 2001). The most popular test of gross motor function is the rotarod (Figure 2A), which assesses the ability of an animal to maintain balance on a rod that rotates at increasing speeds. The latency to falling off the rod is considered a measure of gross motor skill and coordination. The rotarod task challenges the integration of the animal’s sensory, motor, and coordination systems, and is thought to be mediated primarily by the brainstem and spinal cord (Ba and Seri, 1995), although there is undoubtedly a cortical component as well. Fine motor and vestibulomotor abilities can be assessed by having the animal walk on a narrow balance beam (Fox et al., 2001; Fujimoto et al., 2004). The animal grasps each side of the beam with its paws, and an error is defined as a misstep or foot slip (the animal’s grasp on the beam is lost). Another outcome measure is the latency to falling off the beam onto a soft pad below, usually scored on a 0 to 60-sec scale. A variation, the beam walk test, involves the animal walking across a beam toward a darkened box (quantifying the foot slips and latency to the box); this test is considered a measure of motor and cognitive function (Fujimoto et al., 2004).
FIGURE 2 Tests of sensorimotor function and exploration. A: Rodent undergoing rotarod testing. The animal attempts to maintain balance on the rotating rod (right). Gross motor function is assessed by the latency to falling off the rod (right; animal on floor). The rotarod test assesses sensorimotor function and balance. B: Rodent in open field test, which assesses exploratory activity in a novel environment. Recorded measures include the number of squares crossed and the time spent examining the novel objects in the middle of the field (here, spinning top and small ball). See text for further details.
Rodents exhibit numerous reflex behaviors that measure various levels of neurologic function, from spinal cord to brainstem to cortex. Examples include the righting reflex (pons and mesencephalon), grasp reflex (cerebral cortex), placing reflex (cerebral cortex), auditory startle (medulla), and corneal and pupillary reflexes (pons, mesencephalon, medulla, and upper cervical cord) (Tupper and Wallace, 1980). The righting reflex can be used as a measure of postictal recovery. Forelimb and hind limb reflexes have been used extensively in tests of cognition in experimentally brain-injured rodents (Fujimoto et al., 2004), because in that model, lateralized cerebral damage can profoundly affect cognitive results. Although it is probably not necessary to test all reflexes exhaustively, these could become important when searching for subtle phenotypic differences between groups of rodents (e.g., in transgenic or knockout [Crawley and Paylor, 1997]). In epilepsy research, the main goal is to ensure that motor, sensory, or reflex differences do not explain performance differences on behavioral or cognitive tests. Several detailed descriptions are available on how
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to perform a rodent neurologic examination (Fox, 1965; Tupper and Wallace, 1980; Tremml et al., 1998; Moser, 2000). When interpreting the above tests, the investigator must be sure that no “learning” has occurred, and that cognitive improvement has not been masked by motor deficits (and vice versa). These tests are scored either on all-or-none or graded scales. Graded scales have better sensitivity but introduce some subjectivity into the scoring; all-or-none dichotomous scales are easier to score but might overlook subtle differences in performance. Nonparametric statistical tests are ordinarily used to analyze these tests. In most models, no significant differences are seen in sensorimotor function between animals with epilepsy and controls, thus allowing the investigator to compare the groups on more specific cognitive tests (Suchomelova et al., 2002). Motor differences are found occasionally, however. For example, in a lithium-pilocarpine model of nonconvulsive status epilepticus, experimental rats had impaired performance on the rotarod test when tested 2 months after status epilepticus (Krsek et al., 2004). This result suggests that sensorimotor tests need to be performed to rule out such a confounding factor.
Tests of Locomotion and Exploration Spontaneous locomotor activity and exploration are aspects of motor function that can be used to compare animals having experienced seizures from controls. The open field test is the most popular test of these behaviors (Walsh and Cummins, 1976; Fox et al., 2001). The open field test evaluates the conflict between exploration of a novel environment and a rat’s inherent aversion to open spaces. Typically, rodents actively explore a new environment but become less active on subsequent exposures to the environment (habituation). When novel objects are introduced into the open field, rats actively investigate the objects. In the open field, the general activity level is dissociable from exploratory behavior (Whimbey and Denenberg, 1967; Corman and Shafer, 1968). Of note, exposure to an open field represents a form of stress for a rodent (see section, Tests of Anxiety and Stress). An animal is placed into a square or circular arena (open field), the floor of which is divided into identically sized areas. The degree of activity in the open field is measured by the number of times the animal moves into the various marked off areas (Figure 2B). The amount of time the animals spend exploring novel objects introduced into the field (e.g., a small toy, bottle cap, and so forth) or exploring holes in the field by dipping their noses into the holes are measures of each animal’s willingness to examine a novel environment. One variation of the standard open field test is the “playground maze,” exploration of which is decreased in rats with epilepsy induced by hippocampal tetanus toxin injection (Mellanby et al.,
1999). Other quantifiable parameters in the open field test include grooming behaviors, number of rearings, and stereotypical behaviors such as biting or head weavings. Some investigators have used the number of fecal boli that the animal produces as an outcome measure of anxiety, but this number can be unreliable, because defecation is dependent on time since the last meal and other confounders (Fox et al., 2001). Outcome measures are typically compared using analyses of variance (ANOVA). The open field test is simple to design and perform. Measurements can be made in an automated fashion (e.g., with infrared beam crossings) or by observation. The field must be cleaned from trial to trial, because rodent behavior on this task varies markedly as a function of odor trails (Walsh and Cummins, 1976). Rodent strain differences must also be considered (Crusio, 2001). It is still unclear what brain region(s) mediates each specific open field behavior; it is likely that multiple levels of the central nervous system are involved, including olfactory cortex, limbic areas (emotionality, fear), and higher neocortical regions (exploration, motor activity). Therefore, if differences in open field behaviors are detected between animals with and without epilepsy, the most conservative interpretation would be that some nonspecific aspect of emotionality or locomotion is affected, rather than a specific brain region. Interestingly, kindling, especially of the dorsal hippocampus, increases exploratory behavior (Hannesson et al., 2001). In the kainic acid epilepsy model, open field activity and exploration increase in an age-dependent manner (Stafstrom et al., 1993). Multiple episodes of pilocarpine-induced status epilepticus cause altered exploration (Santos et al., 2000), but not all epilepsy models cause persistent alterations in open field behavior (Erdogan et al., 2004).
Tests of Visual-Spatial Learning and Memory Water Maze The Morris water maze (WM) or its variations have become the most popular tests of “cognitive function” in recent decades (Morris et al., 1982; Brandeis et al., 1989). More than 3,500 studies employing the water maze have been published. A measure of declarative or episodic memory, the WM has been widely employed to examine rodent navigation (McDonald et al., 2004; Sutherland and Hamilton 2004), assess the effects of seizures on learning and memory in animals of different ages (Stafstrom et al., 1993; Lynch et al., 2000; Sayin et al., 2004), and define the behavioral phenotype of mice transgenic for a huge diversity of genes (Crawley, 1999; Schimanski and Nguyen, 2004). The WM is a powerful test of spatial (“place”) learning and memory, requiring the integrity of the hippocampus for optimal performance. The water maze, however, is often
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considered synonymous with “learning and memory.” In reality, learning and memory are complex and multidimensional (Willingham, 1997; Kessels et al., 2001) and care must be taken to define the relevant subcomponents of learning and memory, especially when using these functions to assess the consequences of seizures (see Figure 1). To use the WM as the sole test of learning (e.g., Brown-Croyts et al., 2000; Bo et al., 2004) might overlook some important aspects of cognition (Thorpe et al., 2004). In the WM, an animal uses visual cues outside of the pool, but within its field of vision, to learn the location of a platform slightly submerged under water in a swimming tank (Figure 3A). The latency (swimming time) for the animal to locate and climb onto the platform is used as the primary outcome measure. Other outcome measures include directionality (whether the animal is heading in the direction of the platform) and the length of the swim path. Although rats are proficient swimmers, they prefer to be out of water; therefore, the WM utilizes negative reinforcement (water immersion) to encourage the animal to learn and remember the platform location. The WM dissociates memory deficits from deficits in sensory function, motor function, motivation, and retrieval processes (McNamara and Skelton, 1993). The WM is a deceptively simple test. An animal is placed into the tank at various positions around its perimeter. It then swims around and first encounters the platform by chance. Alternatively, the animal is initially trained with the platform visible above water (“cued” version). On subsequent training trials (usually four to six per day), the animal uses extramaze visual cues to relocate and escape onto the platform. Performance typically improves in daily trials. Once plat-
form location is learned, a variety of methods are used to test memory for its location (known variously as retrieval memory, “spatial bias,” “probe” test, or “transfer” test). In the probe test (Figure 3B), the platform is removed from the water, and time the animal spends swimming in each quadrant (presumably searching for the platform) is recorded. It is assumed that the normal strategy is for the rat to spend greater time searching for the platform in its previous location. Eventually, however, a more adaptive strategy would be to abandon the search in the vacated quadrant and search elsewhere for the platform, an observation that has been verified in experiments employing probe trials longer than the usual 60 seconds (Blokland et al., 2004). Specific protocols for the WM test and its modifications are available (Morris et al., 1982; Hollup et al., 2001; Terry, 2001; McDonald et al., 2004). There is no one “right way” to perform the water maze task. Numerous variations on the standard water maze can be selected to address specific questions. Some examples are listed in Table 2. Investigators often preface the WM by using some “cued” (visible platform) trials, to ensure that the animal learns the motor aspects of the task; the cued version is especially used with mice, which learn the task slower than rats. The collapsible platform (“on-demand”) version of the WM prevents chance encounter of the platform (Buresova et al., 1985); in this version of the test, the platform rises to support the animal when it the subject has spent a criterion amount of time over the target area. This predetermined time can be varied; the longer the time required for the rat to remain over the target area, the slower the learning curve. Working memory versions of the WM have also been devised, in which short-term, all-or-none learning is
TABLE 2 Water Maze Test: Selected Variations How test is performed
Outcome measures
Aspect of cognition assessed
Visible platform (“cued” version)
Animal placed in pool and swims until escape onto the visible platform
Latency to escape onto visible platform
Motor ability, visual acuity
ANOVA
Standard: hidden platform (“noncued” version)
Animal placed in pool at different entry points and swims until escape onto the submerged platform
Latency to escape onto hidden platform
Acquisition learning of spatial information
ANOVA with repeated measures
Probe test (also called “transfer test”)
Remove platform; allow animal free swim
Time spent swimming in prior target quadrant
Retention or recall of spatial information
ANOVA
Reversal test
After learning one position, platform is moved to a new position
Latency to escape onto hidden platform
Ability to learn new platform location
ANOVA with repeated measures
Collapsible platform (“on-demand” task)
A platform automatically rises under an animal if it remains in a target position for a criterion time
Time to criterion over target area (platform)
Prevents finding the platform by chance
ANOVA
Working memory version
Platform is moved to a new position each day; 2 training trials per day
Latency to escape onto hidden platform
Single trial (allor-none) learning
ANOVA
Test variation
ANOVA, analysis of variance.
Method of analysis
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FIGURE 3 Tests of spatial learning and memory. A: Water maze acquisition test. Rodent is placed into the water maze tank facing the wall; the latency to finding and escaping onto the slightly submerged platform (indicated by square in upper left quadrant) is recorded over several daily trials. The graph shows hypothetical data comparing the learning curves of a control and experimental animal. Both groups learn to find the platform over successive testing days, but the experimental group’s performance is significantly impaired compared to the controls, suggesting a spatial learning deficit. B: Probe test. After multiple days of acquisition training (A), the rats’ swim pattern is recorded with the platform removed. The graph shows hypothetical data plotted for quadrant swim times for control and experimental groups. The controls spend a significant percentage of the time swimming in the quadrant where the platform was previously located, while the experimental group spends similar time in each quadrant, suggesting poorer memory for the platform’s location. C: Radial arm maze. A mildly food-deprived rodent is placed in the center of the maze and is allowed to explore the 8 arms. Entries into baited (e.g., raisin at the end of some arms, indicated by black dots) or unbaited arms are recorded. Working and reference errors can be assessed. See text for details. The graph shows hypothetical data of number of reference memory errors (entries into arms that have never contained bait) for control and experimental groups. In this example, experimental animals have impaired long-term spatial (reference) memory compared to controls, which becomes increasingly apparent over the 5 trials.
Behavioral Tests to Assess the Effects of Seizures
assessed (Dudchenko, 2004). This version uses two trials per day. The animal is required to learn the platform location on the first trial, and exhibit memory for the platform location on the very next trial. The platform is moved to a different location on each subsequent testing day. Rodents with hippocampal lesions are impaired in acquiring this short-term memory task, whereas intrahippocampal N-methyl-daspartate (NMDA) receptor blockade does not affect working memory (Steele and Morris, 1999). Optimal performance in the WM depends on intact hippocampal function (Morris et al., 1982; Florian and Roullet, 2004), partly accounting for its popularity in epilepsy models. Several other brain systems, however, modify WM performance, including neocortex, retrosplenial cortex, striatum, basal forebrain, and cerebellum (D’Hooge and Deyn, 2001; Gerlai et al., 2002; Hoh et al., 2003; Harker and Whishaw, 2004). WM performance requires intact cholinergic and glutaminergic function; blocking those receptors decreases spatial learning but not recall (McNamara and Skelton, 1993). Cholinergic pathways may be a potential target for improving spatial memory impairment after seizures (Holmes et al., 2002). An exciting new application of the WM is in the study of hippocampal place cells. Place cells are hippocampal neurons that fire selectively when an animal is in a certain location (“firing field”) in its environment. Place cells may represent the physiologic basis of an animal’s “cognitive map” of its environment (O’Keefe and Nadel 1978). In a new environment, place cell fields form quickly, often in less than 5 minutes, and remain stable on repeated tests over days to weeks. Any given location is encoded by a population of simultaneously active cells, rather than by the activity of single neurons (Wilson and McNaughton, 1993). Hippocampal subregions, CA1, CA3, and entorhinal cortex may be responsible for different aspects of spatial memory encoding (Brun et al., 2002; Nakazawa et al., 2004). The effect of seizures on place cell function is of major interest in epilepsy research. A recent study compared place cell firing patterns with visuospatial memory in the WM in control rats versus rats that had experienced an episode of lithium—pilocarpine-induced status epilepticus or a single generalized flurothyl seizure (Liu et al., 2003). In rats with status epilepticus, place cell fields were less precise and less stable than those of control animals or those with a single flurothyl seizure. The degree of place cell field abnormalities corresponded to the greatest impairment of WM learning and memory and maximal neuropathologic changes. These experiments represent a novel approach to the cellular mechanisms of cognitive dysfunction in epilepsy. Many studies have examined the effect of seizures on WM performance. Although this literature is too broad to review in detail here, the consensus is that spatial learning is impaired by both brief and prolonged seizures, in several species or strains, and at many ages. The deficits are
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maximal in adult animals and in models with hippocampal pathology. Examples of seizure induction methods in which this paradigm has been tested include kindling (Gilbert et al., 2000), tetanus toxin (Lee et al., 2001), electroshock (Lukoyanov et al., 2004), kainic acid (Stafstrom et al., 1993), pilocarpine (Rice et al., 1998; Mohajeri et al., 2003), pentylenetetrazole (Huang et al., 2002b), and flurothyl (Huang et al., 1999). Numerous studies have also looked at the effects of pharmacologic and environmental interventions on WM performance after seizures, as well as correlations with neuronal damage. The reader is referred to comprehensive reviews of this topic (Stafstrom, 2002; Holmes, 2004; Majak and Pitkanen, 2004). Given the popularity of the WM, the question arises as to how carefully the test is used and interpreted. The WM is a test for a specific set of cognitive abilities—spatial learning and memory. Caution should be taken not to interpret the results too broadly (i.e., as a general measure of cognition). For example, the terms “learning and memory” are fully applicable only when the probe trial is included after the standard place learning acquisition phase; the probe test assesses memory for platform location, whereas the hidden platform test measures spatial learning. Other caveats involve the aforementioned sensorimotor deficits; if the motor aspects of swimming are not equivalent in the experimental and control animals, group differences may be a result of motor skills, not cognitive abilities. For rodents, water exposure is stressful, and this stress might affect one experimental group, species, or strain preferentially. Also, escape from the water might be an insufficient reward for the animal to seek the platform (Jaffard et al., 2001). Water maze experiments ordinarily utilize independent group designs. The proper statistic for analyzing acquisition trials of WM learning is the 2-way ANOVA with repeated measures. The probe test must be analyzed using 2-way ANOVA with quadrant and treatment as factors. The WM is a powerful, reproducible test of spatial learning and memory that can be adapted to assess both working and reference memory. Results should be interpreted cautiously with regard to potential confounding variables and with appropriate attention to the memory subsystem being evaluated. Radial Arm Maze The radial arm maze (RAM) is another popular test of spatial memory in rodents. Both spatial memory (place learning) and nonspatial memory (associative learning) can be assessed in the RAM, which exploits the natural tendency of rodents to explore, learn, and remember different spatial locations of a food reinforcement (Figure 3C) (Levin, 2001). As opposed to the WM, which uses negative reinforcement (water immersion), the RAM uses positive reinforcement (food bait) to test visual-spatial learning and memory. In the
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RAM, “localization” of the reward is not required, because the animal travels down defined corridors, compared with the WM, in which the animal must localize the platform position (Hodges, 1996). As in the WM, rodents use extramaze visual cues to navigate the RAM. Performance on the RAM depends on the integrity of the hippocampus, frontal cortex, and forebrain cholinergic pathways (Becker et al., 1980; McDonald and White, 1995). The procedure of RAM testing involves initial adaptation to handling by humans and to the maze environment. A “win-shift” acquisition protocol is then administered. Each arm is baited with a small bit of food at the end. The animal is reinforced only once (“win”) for entry into each arm of the maze, and then it must “shift” to another baited arm. The test concludes when the animal enters all arms or a time limit elapses. Both working and reference memory can be tested in the RAM. Some arms do not contain food bait; entry into a nonbaited arm is a reference (long-term) memory error. Reentries into a baited arm is regarded as a working (shortterm) memory error because the food is now gone and the status of the arm has changed from baited to not baited. Once an animal learns the protocol, its performance remains stable and the effects of treatments on memory can be tested (e.g., drugs, seizures, genetic alterations, and a variety of other manipulations). Variations of the method allow tremendous flexibility in assessing learning and memory. The task can be made very challenging (e.g., by increasing the number of arms) or simplified, depending on the degree of sensitivity desired. A version of the RAM has even been devised that incorporates a water maze inside a RAM, to test reference and working memory simultaneously (Shukitt-Hale et al., 2004). The experimenter, however, should be aware of the disadvantages of the RAM as well. Animals must be modestly food deprived (to increase motivation sufficiently for optimal performance). The adaptation period and learning process can be quite laborious. Several outcome measures can be assessed in the RAM, including the number of trials or days to achieve a criterion performance (e.g., consumption of all baits within a given number of arm entries), number of working errors, and number of reference errors. The RAM uses a repeated measures design, so ANOVA with repeated measures is usually necessary for statistical analysis. Seizures caused by a variety of agents (e.g., kainic acid) cause impaired learning and memory in RAM tasks, in both adult (Letty et al., 1995) and young (Lynch et al., 2000) (Sayin et al., 2004) rodents. The degree of memory deficit in the RAM is related to the number of kindled seizures (Kotloski et al., 2002). In a novel rat model of atypical absence seizures (possibly mimicking Lennox-Gastaut syndrome), induced by a cholesterol synthesis inhibitor, RAM learning was markedly impaired compared with controls (Chan et al., 2004). In that study and another study using
kainic acid (Lynch et al., 2000), RAM deficits correlated with impairment of long-term potentiation, suggesting a cellular basis for memory dysfunction. Spatial Learning in Humans Spatial learning in humans is a form of declarative memory (Burgess et al., 2002). Although it is likely that no person has ever been tested in a Morris WM tank, a number of studies have used “virtual” water mazes and similar navigational tasks to assess human spatial learning and memory in both children and adults (O’Connor and Glassman, 1993; Overman et al., 1996; Abrahams et al., 1997; Lehnung et al., 1998; Hamilton et al., 2002). Some virtual experiments involve computer-generated tasks (similar to a video game), in which a person must learn to navigate along various paths and learn the location of certain targets. Among normal individuals, males and females differ significantly in their use of spatial information to master the task; males generally learn the task quicker and utilize both landmark and geometrical cues to navigate to the target, whereas females primarily use landmark cues (Astur et al., 1998; Sandstrom et al., 1998). The reasons for gender-related performance differences remain to be explained, and offer a caveat when testing groups of animals that include both sexes (Roof and Stein, 1999). Performance on virtual WM tasks is impaired in persons with traumatic brain injury (Skelton et al., 2000) and hippocampal lesions, especially right-sided lesions (Bohbot et al., 1998). Virtual tests of spatial learning and memory could be a powerful method to study memory deficits in patients with various types of hippocampal dysfunction (Ploner et al., 1999). Such experimental paradigms are now being applied to investigate human place cell function (Ekstrom et al., 2003), and it is hoped that this approach will yield insights into the effects of seizures on human spatial learning and memory.
Tests of Anxiety and Stress Human epilepsy is associated with a wide variety of emotional disorders, including anxiety (Vazquez and Devinsky, 2003). Because anxiety is an emotional state, it must be evaluated indirectly (by observation) in animals. A variety of tests are available to assess anxiety in experimental animals. Each test exposes the animal to an unfamiliar, aversive environment (Belzung and Griebel, 2001). One of the most popular tests of anxiety in rodents is the elevated plus-maze (EPM), which consists of two elevated, open (brightly lit) arms perpendicular to two enclosed (dark) arms (Figure 4A) (Lister, 1987; File, 1993). Rodents prefer dark, enclosed spaces to brightly lit open ones, but they are also highly exploratory by nature. Rodents also have an innate fear of heights, so elevating the EPM off the floor adds to their anxiety level. The EPM is regarded as an unconditioned
Behavioral Tests to Assess the Effects of Seizures
FIGURE 4 Tests of anxiety and socialization. A: Rodent in elevated plus maze (EPM). Rodents prefer dark enclosed spaces (shaded) and tend to avoid brightly lit areas. The EPM assesses anxiety by comparing the time spent in enclosed arms vs. open arms. B: Rodent undergoing the social recognition test. When a test animal (here, the larger adult rodent) is exposed to a conspecific juvenile (smaller rodent), it is expected that the test animal will spend time “socializing” with the juvenile, by sniffing, grooming, etc. (left). When later exposed to the same juvenile (right), it is expected that less time will be spent engaging in such social behaviors, as the animal is no longer “novel.” See text for further details.
spontaneous behavioral conflict test (Wall and Messier, 2001) that uses an ethologically relevant situation to measure the conflict between exploration of a novel environment and avoidance of a brightly lit, open area (Crawley, 1999). In the EPM, the animal is placed in the center of the maze. Outcome measures include the latency to travel in the open arm, the number of entries into each arm, the ratio of open arm entries to total arm entries, and the time spent in each arm are recorded over a given time period, providing an indication of anxiety-related behavior. The lower the ratio of time spent in open arms to total time, the greater the animal’s anxiety. Results are typically analyzed using ANOVA. In using behavioral tests of anxiety such as the EPM, investigators should realize that fundamental differences may exist between anxiety in humans and animals. In humans, anxiety is an affective, emotional state that may or may not have an externally observable correlate. In testing anxiety in animals, we must rely on observable behaviors and assume that these reflect the internal emotional state (Wall and Messier, 2001).
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The EPM is a well-validated test that can be used to compare anxiety levels in experimental settings, after anxiolytic or anxiogenic drugs, or in transgenic animals (Pellow et al., 1985; Hogg, 1996; Voikar et al., 2001). Multiple brain areas are probably involved in the mediation of anxiety, especially gamma-aminobutyric acid (GABA) pathways of limbic structures and neocortex. Anxiety can be subdivided into (1) anticipatory anxiety, a passive avoidance-conditioned component caused by expectation in a stressful situation, and (2) innate fear, an active avoidance-unconditioned component in which animals actively learn to avoid a dangerous situation. The elevated T-maze (ETM), a variation of the EPM in which there are two open arms (forming the top of the T) and one closed arm; both anticipatory anxiety and innate fear can be assessed with the ETM, depending on whether the animal is placed in the closed arm (tests anticipatory anxiety) or at the end of one open arm (forcing the animal to actively learn to escape into the closed arm) (Erdogan et al., 2004). It is unknown whether epilepsy differentially affects an anxiety subtype. Other tests of stress are also used, some of which attempt to mimic ethologically appropriate conditions. For example, predator exposure is a reliably anxiogenic stimulus, but has only rarely been used in epilepsy research (Merali et al., 2001). Separation of a young animal from its mother is a profound stressor that has been shown to cause marked alteration of the stress control system (Penke et al., 2001) and subsequent behavioral and cognitive dysfunction (Frisone et al., 2002) that is exacerbated by seizures (Huang et al., 2002a). As discussed above, the open field test also represents a form of anxiety, presenting a conflict between a rodent’s tendency to explore of a new environment and its aversion to open spaces (escape being prevented by the surrounding wall). The effects of anxiety on seizure threshold and susceptibility (and vice versa) vary, depending on the strain, age, method of seizure induction, and molecular and neurobiological factors (Post, 2004). Prenatal stress lowers kindled seizure threshold in rat pups (Edwards et al., 2002). In many studies, kindled animals exhibit heightened anxiety (Adamec and McKay, 1993) and increased aggression toward other animals (Kalynchuk et al., 1999) (see section, Tests of Socialization below), although in some reports, kindled rats perform similarly to controls on the EPM (Hannesson et al., 2001). Part of the variability in such results could be related to the size of the arena; in a large open field, kindled rats tend to huddle together (Haimovici et al., 2001). Status epilepticus caused by pilocarpine or kainic acid results in increased anxiety in adult and immature rats (Santos et al., 2000) (Sayin et al., 2004), whereas anxiety is unchanged by pentylenetetrazole-induced status epilepticus (Erdogan et al., 2004).
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Tests of Socialization
CONCLUSIONS
Persons with epilepsy, especially temporal lobe epilepsy, are susceptible to a variety of behavioral, emotional, and social difficulties, observations that have been verified in laboratory animals (Griffith et al., 1987). Rodent tests to determine sociability and interaction include the handling test and a variety of assessments of socialization when two previously unfamiliar animals are exposed to each other. The handling test is a measure of emotional response to graded amounts of discomfort, elicited by nonstressful handling (rubbing the fur along its grain), stressful handling (rubbing the fur against its grain), and graded tail pinch with a hemostat. Specific responses are scored according to a validated ordinal scale from 1 to 4. Kainic acid-induced epileptic animals are notably more aggressive on the handling test (Holmes et al., 1988), a finding that is age-specific; rats experiencing kainic acid status as neonates and juveniles are not more aggressive than controls (Stafstrom et al., 1993). Tests of socialization include the home cage intruder test (HCIT, also known as the resident intruder test). A test animal is placed into the cage of an experimental animal, and aggressive, passive, and other interactive and noninteractive behaviors are recorded (Mellanby et al., 1981; Thurmond, 1975; Kaliste-Korhonen and Eskola, 2000). Animals with experimental epilepsy (e.g., induced by kainic acid) are significantly more aggressive, irritable, and difficult to handle by experimenters; however, these animals often display uncharacteristic passivity toward intruder animals (Mellanby et al., 1981; Holmes et al., 1988). Pentylenetetrazole-kindled rats display decreased offensive behaviors on the HCIT (Franke and Kittner, 2001). A variation of the HCIT is the social recognition task (SRT) (Figure 4B), in which an experimental rodent is repeatedly exposed to a normal conspecific animal, usually a juvenile. In this situation, it is expected that the two animals will explore each other actively on the initial encounter (with outcome measures such as sniffing, grooming, and so on), but with repeated exposures, such interactions will diminish (Dantzer and Bluthe, 1987). Abnormalities on the SRT have been documented after pilocarpine-induced, nonconvulsive status epilepticus (Krsek et al., 2004) and kainic acid-induced status epilepticus (Letty et al., 1995), but not after amygdala kindling (Letty et al., 1995). The significance of many of the above findings remains uncertain. Given the immense complexity of emotional behavior, and the wide spectrum of possible effects of different types, durations, and ontogenetic features of epilepsy, caution should be exercised when trying to correlate animal and human behaviors with specific brain lesions or insults.
The behavioral and cognitive tests described above represent only a small sample of available methods to assess the effects of seizures. This review has concentrated on tests of sensorimotor function, locomotion or exploration, learning and memory, and anxiety or social interaction. Important forms of cognition and behavior (e.g., perceptual priming, classical conditioning, and nonassociative conditioning) were not discussed here, but have been addressed in some epilepsy models (Peele and Gilbert, 1992; Persinger et al., 1994; Neill et al., 1996; Santos et al., 2000; Sakamoto and Niki, 2001; Majak and Pitkanen, 2004). Although it would be imprudent to recommend a rodent “neuropsychological battery” that is applicable to all animal epilepsy models, the sample battery listed in Table 3 is fairly comprehensive, and can be modified according to the experimenter’s goals and questions. It is unwise to depend on a single test as a measure of behavior or cognition. Abnormalities of behavior and cognition in animal models of epilepsy are discussed throughout this volume. In spontaneous and induced genetic mutants, or in response to numerous chemical convulsants, electrical stimulation or kindling, or cortical dysplasias, deficits can be demonstrated across a wide range of behavioral and cognitive domains. Before concluding that seizures lead to impairment in behavior or cognition, one must ensure that elementary sensorimotor function (as well as sensory capabilities such as vision and hearing) is intact. When designing experiments to quantify the effects of seizures on behavior and cognition, the investigator should select tests to examine specific
TABLE 3 Selected Tests to Assess Seizure-Induced Behavioral and Cognitive Impairment Test
Function tested
Brain substrate*
Rotarod test
Motor coordination
Brainstem, spinal cord
Open field test
Locomotor activity, exploratory behavior, anxiety
Multiple
Water maze
Spatial learning and memory
Hippocampus
Radial arm maze
Spatial learning and memory
Hippocampus
Elevated plus maze
Anxiety
Multiple
Handling test
Response to graded discomfort
Multiple
Home cage intruder test
Socialization, aggression
Multiple
*Presumed brain regions involved in mediating the function.
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50 Morphologic Approaches to the Characterization of Epilepsy Models H. JÜRGEN WENZEL AND PHILIP A. SCHWARTZKROIN
ined. These types of experiments are difficult to carry out in human patients. For example, hilar cell loss (e.g., after status epilepticus) is often correlated with epileptogenesis, but it is virtually impossible to determine the cause—effect relationship between such damage and seizure development in patients. Similarly, granule cell dispersion and mossy fiber sprouting is often found in association with mesial temporal sclerosis and temporal lobe epilepsy (Houser, 1990). It is only within the context of animal models that we can assess the causal relationship between such morphologic abnormalities and epileptogenesis or the epileptic state. For purposes of illustration, this chapter focuses attention particularly on morphologic changes often seen in models of cortical dysplasia (defined by structural abnormality) and temporal lobe epilepsy (TLE). The pathologic characteristics of TLE, particularly mesial temporal lobe sclerosis (selective neuronal cell loss, gliosis, mossy fiber sprouting, and synaptic reorganization in hippocampus and related structures) were perhaps the first morphologic links to an epileptic state. Since the early study of mesial temporal sclerosis (Falconer et al., 1964), TLE-related pathology has been a frequent subject for neuropathologists (Armstrong, 1993). Whereas the neuropathologic features of both cortical dysplasia and Ammon’s horn sclerosis have been extensively studied in detail, the causes and processes involved in their development are poorly understood and the causal relationships between these anatomic abnormalities and seizure function remain controversial (Mathern et al., 2002). Given the difficulties in studying such questions in human patients, a real need exists for appropriate animal models in which key characteristics of the neurologic disorder can be manipulated. Advances in neuroanatomic techniques to assess
INTRODUCTION The pathogenesis of epilepsy is poorly understood. Cellular and molecular changes in various neurons and glial cells have been hypothesized as critical factors (determinants) for the development of spontaneous seizures (Honavar and Meldrum, 2002). In studying such pathogenetic processes, it is important to identify those factors that link an initial insult (or a genetic defect) to the occurrence of spontaneous seizures (epilepsy). In humans, these critical processes can occur over the course of decades. In experimental animal models of epilepsy, however, these changes are condensed into a shorter time-span (e.g., within a few weeks in the case of the pilocarpine model). The processes that are involved in epileptogenesis, or underlying an epileptic state, are dynamic. Morphologic contributions to understanding epileptogenesis or epilepsy tend to be static “snapshots,” taken at a given point during these processes. Thus, it is important to understand that initial morphologic changes may provide the bases for different (perhaps, more “permanent”) changes in structure, and that the initial pathology may be a critical precursor to the “plasticity” of neuronal function associated with seizure activity. A morphologic change (of neurons or glial cells) found in association with an epileptic syndrome may be causally related to the abnormal discharge (i.e., represent a critical change that contributes to spontaneous seizures or to the process of epileptogenesis). These changes, however, may be the consequences of abnormal activity (i.e., not a cause at all, but rather a result). It is helpful, therefore, to study the time course of such changes and to devise experiments in which “dissociation” of structure and function are exam-
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features at the circuitry, cellular, membrane, and molecular levels can help us address the key issues regarding structure—function relationships. The first part of this chapter focuses on structural cellular features, demonstrated via anatomic techniques, which appear to be underlying (or reflect) changes in cellular function that are related to chronic or repeated seizure occurrence. The second part of this chapter provides an overview of the neuroanatomic techniques that have been used to demonstrate such morphologic (pathologic) features of epileptic brain tissue. Within this context, we discuss issues related both to morphologic analyses and to the interpretation of anatomic (structural) data linked to seizures and epileptogenesis.
WHAT CELLULAR FEATURES (DEMONSTRABLE VIA ANATOMIC TECHNIQUES) MIGHT REFLECT EPILEPTOGENICITY? Different models of seizures and epilepsy are associated with a broad range of morphologic changes that are correlated with seizure occurrence. In characterizing animal models, as when describing clinical pathology in human patients, it is helpful to have as complete a description as possible of these features. Some features can serve as structural underpinnings of seizure development (i.e., provide clues about underlying causes). Other features may simply provide useful markers, which, while not necessarily causally related to the seizure activity, are consistent indicators of seizure activity (severity, location, and so on). The following paragraphs deal with only a select set of morphologic features of epileptic brain (in both animal models and in human tissue samples) that are useful examples of how epileptic tissue can be characterized.
Developmental Abnormalities (Cortical Dysplasia) Disorders of cortical development, often termed “cortical dysplasia”, are caused by deviations from the normal pattern of brain development (Palmini, 2000) and have been widely recognized as potential causes for functional impairment (e.g., mental retardation). Cortical dysplasia refers to a variety of structural abnormalities (e.g., lissencephaly, microgyria, heterotopia) that are associated with a large number of epilepsy syndromes (Crino et al., 2002; Gleeson and Walsh, 2000). Cortical dysplasias have been classified based on both anatomic (histopathological) criteria and on the basis of radiographic findings, underlying genetic abnormalities (Gaitanis and Walsh, 2004), or both. At least three major mechanisms involved in cortical development have been implicated: (1) aberrant neuronal and glial prolifera-
tion and differentiation (e.g., Taylor-type focal cortical dysplasia, hemimegalencephaly, tuberous sclerosis); (2) abnormalities in migration (e.g., periventricular or subcortical nodular and band heterotopia, lissencephalies); and (3) abnormalities in cortical organization (e.g., schizencephaly, polymicrogyria, microdysgenesis) (Barkovich et al., 1996; Schwartzkroin et al., 2004). The major histologic features of cortical dysplasia have been described as loss of lamination, loss of spatial orientation of neurons, abnormalities in neuronal morphology, and abnormalities of cellular commitment. Enlarged, dysmorphic neurons and so-called “balloon cells” have also been the focus of a number of pathologic studies (Cepeda et al., 2003; Mischel et al., 1995; Taylor et al., 1971). A classification scheme based on pathohistologic features of cortical dysplasia provides a framework within which we can obtain a clear view of the cellular constituents of the dysplastic cortex. Mischel et al. (1995) proposed a classification of cortical dysplasia based on histologic findings, as related to the presumed time-points of early, intermediate, and late developmental lesions. These authors hypothesized that earlier lesions would result in more severe clinical phenotypes and later lesions would produce milder symptoms. They classified histologic changes as severe (balloon cells, neuronal cytomegaly), moderate (polymicrogyria, white matter heterotopia), or mild (absence of the above pathologies), and found a correlation between histologic grade and seizure frequency (i.e., more severe grade was associated with higher seizure frequency) (Mischel et al., 1995). The cause–effect relationships in these studies (i.e., between the developmental defects and seizures) remain to be investigated. These relationships can be assessed in the numerous animal (mostly rat and mouse) models of cortical dysplasia, where comparisons of animal models with the human disease are based on similarities in histologic features and abnormalities in common stages of cortical development (Schwartzkroin et al., 2004). A few examples of relevant models are described below with respect to morphological features of interest. The p35 Knockout Mouse: A Model for Disruption in Later Neuronal Migration Stages Gene mutations, as described in other chapters in this volume, have resulted in a number of animal models of seizures and epilepsy. Cyclin-dependent kinase 5 (cdk5) and its neuronal regulators (e.g., p35) play important roles in normal cortical development, and mutation or deletion of these genes results in a profound disruption in migration (Chae et al., 1997). In mice, mutations of both cdk5 and p35 result in morphologic phenotypes with an inverted neocortex (Fig. 1A,B). In p35 mutant mice (see Chapter 19), the subplate lies beneath the cortical plate, and neurons cannot migrate through the cortical plate. Structural abnormalities
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FIGURE 1 Morphologic abnormalities in the cortex of p35 knockout mice. A–D: Coronal brain sections from wildtype (A,C) and p35 knockout (B,D) mice, immunoreacted for NeuN. Neocortex (Ctx) of the p35 knockout mouse shows absence of the normal neocortical lamination patterns (cf. A & B). Higher magnification of the hippocampal (H) dentate gyrus (DG) illustrates granule cell dispersion (arrows) into molecular layer (ml) and hilus (hi) (cf. C & D). E,F: Immunocytochemistry for glial fibrillary acidic protein (GFAP) demonstrates the characteristic radial glial fiber orientation and astrocyte cell body localization in the granule cell layer (gcl) of wildtype mice (E). These astrocytes are absent in p35 knockout mice (F). G,H: Parvalbumin-positive interneurons are localized to the gcl-hilar border or molecular layer in wildtype mice (e.g., arrow in G shows cell at gcl/ml border). In p35 knockout mice there is an abnormal distribution of parvalbumin-containing interneurons across the dentate layers (arrows in H). I: Camera-lucida drawing and reconstruction of a heterotypically localized (to the hilus) basket cell (1) and a granule cell (2), intracellularly filled with biocytin, from a p35 knockout mouse. The axonal plexus (ap) of the basket cell is abnormally distributed into the molecular layer (ml) and hilus (hi). (See color insert.) CA1-CA3, hippocampal subfields; gcl, granule cell layer; gcl, granule cell layer; mf, mossy fiber. Scale bars: A,B: 200 mm; C,D,I: 100 mm; E,F: 20 mm; G,H: 40 mm.
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in the hippocampal formation have also been identified in the p35 mutant mouse, particularly in the dentate gyrus (Fig. 1C,D). Histologic investigations (Wenzel et al., 2001) have revealed a dysplastic hippocampus that includes heterotopic pyramidal cells in strata oriens and pyramidale of CA3 and CA1 subfields, and dispersion of granule cells into the molecular layer and hilus. The dispersed granule cells exhibit abnormal axonal and dendritic orientation as well as mossy fiber sprouting into the granule cell and molecular layers. Immunocytochemical techniques have also revealed abnormalities in astrocyte localization (Fig. 1E,F) and inhibitory interneuron organization (Fig. 1G–I). In the immature p35 knockout mouse, structural abnormalities (e.g., granule cell dispersion, mossy fiber sprouting, and dislocation of interneurons) appear to develop early in pre- or postnatal corticogenesis (i.e., before seizure onset). Because similar abnormal structural features have been described in human mesial TLE, it is tempting to hypothesize that these features provide an initial substrate for epileptogenesis. Prenatal Insult with Methylazoxymethanol: A Model for Disruption of Neuronal Proliferation Early prenatal insults are hypothesized to play a crucial role in the pathogenesis of cortical dysplasia (Palmini et al., 1994). In utero exposure to methylazoxymethanol (MAM), a DNA methylating agent, has been investigated in the context of experimentally induced cortical dysplasia (Baraban et al., 2000; Singh, 1977). When administered to pregnant rats at E15, the treated offspring exhibit abnormalities that include microcephaly, cortical and hippocampal laminar disorganization, and periventricular and hippocampal heterotopia (neurons with variable morphology displaced across the hippocampal layers) (Fig. 2A) (see Chapter 23, this volume). The histologic features of disorganized and heterotopic neurons are similar to those described in children with acquired cortical dysplasia (Marin-Padilla et al., 2002). The in utero-exposed MAM rats show increased susceptibility to convulsant agents such as flurothyl (Baraban and Schwartzkroin, 1996), kainic acid (Germano and Sperber, 1997), and kindling (Chevassus-AuLouis et al., 1998), and exhibit a number of cellular or molecular functional abnormalities that might result in cellular hyperexcitability (Baraban et al., 2000; Castro et al., 2001). Morphologic techniques (e.g., anterograde and retrograde axon transport of dyes such as biotinylated dextran amine [BDA]) [Fig. 2B]) have been used to study the connectivity between heterotopic neurons and “normotopic” brain regions (Colacitti et al., 1998). Birth-dating and neurochemical profiling have also indicated that heterotopic neurons in the hippocampus have features similar to those of neocortical lamina II/III neurons. These results, together with physiologic studies, have led investigators to hypothesize that heterotopic neurons are integrated into both the
hippocampal and neocortical circuitry, providing a bridge for seizure propagation between the two regions (Baraban et al., 2000; Chevassus-Au-Louis et al., 1998). Cytologic Abnormalities in the Eker (TSC2 +/-) Rat: Incomplete Cellular Differentiation in a Model of Tuberous Sclerosis A key feature of some forms of cortical dysplasia (e.g., Taylor-type, tubers of tuberous sclerosis) is the occurrence of cells with unusual cytologic features. The description of “giant” and “balloon” cells, and of cells with mixed markers for both neurons and glia, have been frequently described by neuropathologists (Armstrong, 1993; Crino et al., 2002), but the occurrence of such cytologic abnormalities in animal models has been rare. Giant cells have been described in the flathead rat or Citron K mouse (see Chapter 19, this volume), and also in a rat model in which the TSC2 gene (one of two genes implicated in tuberous sclerosis) is mutated. Although tubers have not been found in animal models involving TSC1 or TSC2 mutation or deletion, the TSC2 +/- rat exhibits a number of relevant cell phenotypes, including cytomegalic neurons (Fig. 2C,E), giant astrocytes (Fig. 2D), and hamartoma-like structures in which cells sometimes exhibit immunoreactivity for both neuronal and glial markers (Fig. 2F) (Wenzel et al., 2004). The functional consequences of such cellular abnormalities are unclear.
Altered Cell Structure Early studies that focused on the neuropathologic substrates of intractable TLE described both dendritic spine loss and nodular swellings of dendrites of hippocampal pyramidal neurons and dentate granule cells (Scheibel et al., 1974). In more severe cases of TLE, spine loss and dendritic swellings were associated with a marked distortion of the dendritic structure and shrinkage of the dendritic tree. Similar spine pathology has been observed in neocortical pyramidal neurons in patients with TLE, as well as in Lennox-Gastaut syndrome (Du et al., 1993). In contrast, Isokawa, (2000) observed higher spine densities on dentate granule cells (cells that generate mossy fiber sprouting) of human epileptic hippocampi, suggesting that dendritic spines of epileptic neurons may respond over time in a plastic manner. Blumcke et al. (1999) also reported increased dendritic ramifications of pyramidal cells and interneurons in the dentate hilus in cases of Ammon’s horn sclerosis, associated mesial TLE. In addition, their electron microscopic analysis revealed altered cellular morphology, with accumulations of cytoskeleton filaments and increased number of mitochondria. Studies in animal models have also demonstrated that cellular pathologic features develop over time (Fiala et al., 2002; Isokawa, 2000). Immediately after induced seizures, dendritic spine loss and dendritic
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F FIGURE 2 Morphologic abnormalities in methylazoxymethanol (MAM)-induced CA1 heterotopia, and cellular features of incomplete differentiation in the TSC2 +/- (Eker) rat. A: Coronal section of the dorsal hippocampus (H) and overlying neocortex (Ctx) from a MAM-exposed (in utero) rat, showing a CA1 heterotopia (large arrow) and a periventricular nodular heterotopia (small arrow). Cresyl violet staining. B: Photomicrograph of stained cells in an intrahippocampal CA1 heterotopia, following injection of biotinylated dextran amine (BDA) into a normal region of the hippocampus. Retrograde dye transport labels cells (arrows) that project out of the heterotopia; dye-stained fibers (arrowheads) show axons entering the heterotopia. Neutral red counter-staining. C: A large dysmorphic neuron (arrow), located in the neocortex (lamina II/III) of an Eker (TSC2 +/-) rat, shows immunoreactivity for neurofilament in the cell body and irregularly-oriented “dendrites.” D: Photomicrograph of a giant astrocyte (large white arrow) in the neocortex of an Eker (TSC2 +/-) rat, showing glial fibrillary acidic protein (GFAP) immunoreactivity in its irregularly-oriented and extended processes. Small dark arrow indicates a normal GFAP-positive astrocyte for comparison. E,F: Photomicrographs show giant, balloonlike cells (arrows) in hamartomatous lesions of Eker (TSC2 +/-) rats. These cells exhibit eosinophilic cytoplasm in hematoxylin and eosin (H&E) staining (E) and immunoreactivity for both GAD67 (F) and GFAP (not shown). Scale bars: A: 200 mm; B: 20 mm; C–F: 10 mm. CA1, CA3, hippocampal subfields; DG, dentate gyrus. (See color insert.)
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swellings (beading) (as well as excitotoxic cell death) are evident. Initial pathology (e.g., spine reduction in the dentate gyrus) may recover at later time points (e.g., when mossy fiber sprouting is seen) (Isokawa, 2000), suggesting that long-term spine alterations, found in the chronic phase of epilepsy, may result from a cause other than the initial acute insult (e.g., to the occurrence of chronic seizure activity itself). The effects of seizure activity on dendritic morphology have been studied in the hippocampal slice culture preparation (Gahwiler et al., 1997) (see Chapter 5, this volume). Somatic and dendritic alterations (e.g., cytoplasmatic vacuolization, dendritic swelling) and reduction in dendritic spine density (hippocampal CA3 pyramidal neurons) have been reported (Drakew et al., 1996; Gahwiler et al., 1997). Importantly, the spine loss was reversible when cultures were returned to normal medium conditions for 1 week. Further, co-treatment with tetrodotoxin (TTX) (to block seizure activity induced by epileptogenic agents) prevented spine loss (N-methyl-d-aspartate [NMDA] or a-amino-3hydroxy-5-methyl-4 isoxazide proprionate (AMPA) receptor antagonist treatment only partially reduced spine loss) (Thompson et al., 1996). Recently, spiny basal dendrites were observed on many dentate granule cells in epileptic rats (Spigelman et al., 1998). Such basal dendrites also occur in a small percentage of normal rats, and are consistently observed in the primate dentate gyrus. Basal dendrites of dentate granule cells from epileptic rats were found to occur within the hilus, and also to extend into the molecular layer (Spigelman et al., 1998). In the rat, electron microscopic examination of retrogradely labeled granule cells revealed that new basal dendrites were innervated by sprouting mossy fibers (Ribak et al., 2000), and that they received numerous synapses in a pattern similar to that seen on normal apical dendrites. Similar basal dendritic morphologic abnormalities have been described in the aberrant circuitry of p35 mutant mouse
dentate gyrus (Patel et al., 2004). This type of altered connectivity may contribute to additional recurrent excitatory circuitry via sprouted mossy fibers in the epileptic brain (Patel et al., 2004). A number of studies have also examined seizure-related plasticity of axons, particularly with respect to the concept of activity-dependent “pruning” in the maturing brain. In normal development, there is often a simplification of axon collateralization, as has been shown for hippocampal CA3 pyramidal cells (Gomez-Di Cesare et al., 1997). Using intracellular labeling techniques, investigators showed that induction of early seizure activity did not appear to alter the extent or pattern of these axons; however, the density and size of axonal varicosities were increased, suggesting that these axons participate in more extensive excitatory recurrent collateral circuitry (Galvan et al., 2003).
Neuronal Cell Loss Neuronal cell loss is one of the morphologic features mostly frequently described in human TLE (Houser, 1999), as well as in animal models. It remains unclear, however, whether neuronal loss contributes to the causes of epileptogenesis or whether it is simply a result of recurring or prolonged seizures. The magnitude and consequences of neuronal cell loss in the epileptic brain are likely determined by a number of complex, interacting factors such as: (1) the brain region in which seizures (or initiating insult) occur (e.g., amygdala, entorhinal cortex, hippocampal formation); (2) the times at which the seizures (or initiating insult) occur; (3) the cell type primarily affected; and (4) the maturational stage of the animal (or patient). Studies on animal models of status epilepticus have shown, for example, that certain cell types within the hippocampus are particularly vulnerable to seizure-induced loss; the excitatory principal cells in CA1 and CA3 subfields (Fig. 3A–C), and the hilar neurons
FIGURE 3 Pathohistologic features of seizure-induced damage in neurons and glial cells in Kv1.1 knockout mice. A: Photomicrograph of the hippocampus of a Kv1.1 knockout mouse shows neuronal cell loss in the pyramidal cell layer (pcl) of the CA3 region (arrow) and within the hilus (hi). Principal neurons in CA1 and dentate gyrus (DG) show relatively little damage. Cresyl violet staining. B: Higher magnification of the CA3 region (pcl) (from A), showing cell loss and accompanying gliosis (arrows). Arrowhead indicates normal pyramidal cells. C,D: Photomicrographs show severe neuronal damage, including cell body and dendritic and axonal terminal degeneration, in the CA1 region of a Kv1.1 knockout mouse following chronic seizures. Fink—Heimer degeneration staining labels degenerated cell bodies (e.g., black cell bodies in CA1 pyramidal cell layer (pcl; white arrows in C) and hilar cells (hi; white arrows in D). In addition, hilar cell death induces terminal degeneration in the inner molecular layer (iml; white arrow in iml (D) indicates terminal projection of mossy cells) and outer molecular layer (oml; white arrow in oml (D) indicates terminal projection of a subpopulation of hilar interneurons). E,F: Photomicrographs of the dentate gyrus of a Kv1.1 knockout mouse, showing seizure-induced astrogliosis in the molecular layer (ml) and hilus (hi) (E). Higher magnification (F) shows hypertrophic (reactive) astrocytes (arrows). Glial fibrillary acidic protein immunocytochemistry, counter-stained with cresyl violet (F). G,H: Timm staining in coronal sections of the hippocampus from a Kv1.1 knockout mouse, demonstrating seizure-induced mossy fiber sprouting into the molecular layer (iml; arrows), granule cell layer (gcl), and CA3 (strata pyramidale and oriens; arrow). For comparison, see normal Timm staining patterns in the dentate gyrus of a wildtype mouse (inset g in G). (See color insert.) Scale bars: A,G: 200 mm; B: 20 mm; C, inset g, E,H: 100 mm; D: 40 mm; F: 20 mm. rad, s.radiatum; ml, molecular layer; gcl, granule cell layer.
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in the dentate gyrus (Fig. 3D), are relatively sensitive, whereas dentate granule cells and some inhibitory interneurons are relatively resistant to damage (Franck, 1993; Morimoto et al., 2004; Sloviter, 1987). In humans, severe seizures and status epilepticus induce neuronal cell death in the hippocampus within hours of seizure onset (Hauser, 1983); degenerating neurons can be seen in both the CA1 region and dentate gyrus (Dam, 1980). These patterns of cell loss in the hippocampal formation clearly suggest a selective vulnerability in the human brain. The pattern of neuronal cell loss, however, varies dramatically in human TLE; for example, end-folium sclerosis involves cell loss principally in the hilar region, whereas Ammon’s horn sclerosis involves a more general cell loss in most of the hippocampal subregions. The pattern of cell loss also varies significantly across different animal models (e.g., Fig. 4A,B). It is clear that this variability results from differences in both obvious variables (e.g., modes of seizure induction, and severity and duration of seizure activity) and such factors as the species or strain, sex, and age of the animal (Houser, 1999). For example, studies on status epilepticus induced by pilocarpine demonstrated widespread neuronal loss, particularly in the CA1 subfield, dentate gyrus, and piriform cortex; neuronal apoptotic cell death was also seen in the dentate granule cell layer (Covolan et al., 2000). In contrast, in the rat kainate model of TLE, the cell loss was primarily restricted to the hilus and CA3 region (and less extensive in CA1, a characteristic feature in humans) (Dam, 1980; Houser, 1999; Mathern et al., 2002). Because both animal models develop spontaneous recurrent seizures, it appears that substantial cell loss in CA1 is not essential for the development of recurrent seizure—at least in rats (Houser, 1999). Recent studies on the kainate and pilocarpine models of TLE have shown that different patterns of pyramidal cell damage in CA1 and CA3 are characteristic of different rat and mouse strains (Bouilleret et al., 1999; McKhann et al., 2003). For example, in Sprague-Dawley rats, CA3a neurons are most vulnerable, whereas in Wistar rats, the most vulnerable neurons are CA1 and CA3c pyramidal cells. Kainate administration did not produce damage to dentate granule cells, whereas pilocarpine induced extensive damage (including ultrastructural evidence of apoptosis) (Covolan et al., 2000; Fujikawa et al., 2000). Long-Evans rats appear to be more resistant than other rat strains to pilocarpineinduced seizure consequences on most morphologic measures (e.g., cell loss, mossy fiber sprouting); within the hippocampus, they show relatively less damage in the hilus, but more damage in CA1 and CA3 subfields (Xu et al., 2004). Similar observations have been made in experiments using different mouse strains (McKhann et al., 2003; Schauwecker, 2002). For example, mice from C3HeB/FeJ and C57BL/6J strains, exhibiting severe kainate-induced seizures, were resistant to cell death and synaptic reorgani-
zation; in contrast, 129/SvEms mice developed marked pyramidal cell loss in CA1 and CA3, dentate hilar cell loss, and mossy fiber reorganization, despite limited seizure activity (McKhann et al., 2003). Hilar mossy cells are one of the most consistently and severely affected neuronal cell types in human TLE, and their loss represents a pathologic hallmark of this disorder (Dam, 1980; Houser, 1999). The mossy cells are also vulnerable in experimental animal models of epilepsy (Nadler et al., 1980; Sloviter, 1987) and other brain injuries (e.g., head trauma) (Hicks et al., 1996; Lowenstein et al., 1992). Mossy cells provide some excitatory input to dentate inhibitory basket cells, but particularly to spines of granule cell dendrites (Buckmaster et al., 1996). Mossy cell death has been hypothesized to play a role in synaptic reorganization of granule cell axons by triggering or promoting sprouting of mossy fiber growth into the inner molecular layer (Buckmaster et al., 1996). Recent experimental studies have shown that some mossy cells survive pilocarpineinduced status epilepticus (Scharfman et al., 2001); in contrast to normal mossy cells, however, these surviving cells generate spontaneous and evoked epileptiform burst discharges. Ablation of mossy cells in hippocampal slice preparations has not supported a role for mossy cells in dentate hyperexcitability (Ratzliff et al., 2004). If and how loss of mossy cells might contribute to increased epileptogenicity remains to be clarified. The potential consequences of g-aminobutyric acid (GABA)ergic interneuron cell loss appear to be more obvious. Hippocampal interneurons control the excitability of principal cell networks (Freund and Buzsaki, 1996), and loss of the inhibitory neurons (or impairment of GABAergic neurotransmission) has long been suggested to be causally related to epileptiform discharge (Schwartzkroin and Prince, 1977). Early studies on hippocampi obtained from resections from patients with TLE, as well as on the kainate animal model of TLE, suggested that GABAergic neurons are relatively resistant to excitotoxic damage (Babb et al., 1989; Sloviter et al., 2003). More recent studies in various animal models of epilepsy, however, have shown that subpopulations of GABAergic neurons are selectively lost in the hippocampus following prolonged seizures or status epilepticus (Houser, 1999). For example, quantitative analysis in the rat pilocarpine model revealed an approximately 40% decrease in glutamic acid decarboxylase (GAD) mRNA-labeled neurons in the hilus, but preservation of other GAD mRNA-labeled neurons (Houser, 1999). GABAergic neurons were also lost in the hippocampal stratum oriens. Using the rat kainate model, Buckmaster and Dudek (1997) found a substantial loss of somatostatin-immunoreactive neurons, whereas the numbers of parvalbumin- and CCK-immunoreactive neurons were relatively unaffected. Kobayashi and Buckmaster (2003) showed, using in situ hybridization for
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F FIGURE 4 Seizure-induced hippocampal pathology in an infant monkey model of temporal lob epilepsy (TLE), and in the p53 knockout mouse following kainic acid-induced seizures. A,B: Coronal sections from monkey hippocampus, contralateral (A) and ipsilateral (B) to a unilateral bicuculline infusion (1.3 ml) into the left entorhinal cortex (1 hour of focal status epilepticus, 4-month survival). Immunocytochemistry for NeuN (counter-stained with cresyl violet) demonstrates a normal pattern of hippocampal cytoarchitecture in contralateral hippocampus (A), compared with the marked pathology of the ipsilateral hippocampus (B)—severe shrinkage, neuronal loss (in the dentate hilus [Hi], CA3 and CA1 subfields), and gliosis. Note the survival of CA2 neurons, which are less vulnerable to seizure-induced damage, and the dispersion of surviving granule cells (arrows) into the molecular layer. C,D: Timm staining of the mossy fiber system in the hippocampus, contralateral (C) and ipsilateral (D) to the injection site. Seizure-induced mossy fiber sprouting is evident within the inner molecular layer (iml; arrows) of the ipsilateral sclerotic hippocampus (D). E,F: Photomicrographs of a coronal section from a p53 knockout mouse following kainate-induced seizures, showing delayed neuronal cell loss (apoptosis) in the CA1 region (arrows point to black, TUNEL-positive neurons in E). Higher magnification (F) demonstrates that most cells in this section of the CA1 pyramidal cell layer are TUNEL-positive (pcl; arrows). Scale bars: A–D: 400 mm; E: 100 mm; F: 20 mm. DG, dentate gyrus; Sub, subiculum; Hi, hilus; gcl, granule cell layer; luc, s.lucidum.
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GAD65 mRNA, that severe loss of inhibitory interneurons in the dentate gyrus represents a characteristic pathologic feature in epileptic pilocarpine-treated rats that experienced status epilepticus. The profound morphologic changes specific to subpopulations (e.g., cells that have different axonal projection territories and different axonal arbors) suggest that the functional consequences of cell loss can be understood only within the context of detailed knowledge about network connectivity and about the properties (vulnerability, synaptic influences, and so on) of chemically distinct interneuron populations.
Circuitry Reorganization Reorganization of Excitatory Circuitry One of the key features shared by human TLE and animal models of TLE is the striking reorganization of the neurons and circuits that appears in association with neuronal cell loss and hippocampal sclerosis (Franck, 1993; Houser, 1999; Nadler, 2003). Sprouting of the granule cell axon (i.e., the mossy fiber) has been observed in human patients with mesial TLE and in subhuman primate models (Fig. 4D) (Franck et al., 1995; Houser, 1999; Ribak et al., 1998; Sutula, et al., 1989; Wenzel et al., 2000). Similar observations have been made in several rodent models of TLE: following kainate administration (Fig. 3G,H) (Buckmaster and Dudek, 1997; Wenzel et al., 2000), following pilocarpine treatment (Cavalheiro et al., 1996; Okazaki et al., 1995), and as a feature of kindling (Morimoto et al., 2004). Timm staining, which histochemically labels zinc-containing mossy fiber boutons, has shown that sprouting mossy fiber axons and their terminals target the inner molecular layer of the dentate, the hilus, and stratum oriens of the CA3 subfield. These observations indicate that remodeling of the synaptic pathways of the granule cell axon involves both the dentate granule cells and the circuits in the hilus (Sutula, 1998) and other hippocampal regions: for example, CA1 region (Cavazos et al., 2004); CA3 s.oriens (Represa and Ben-Ari, 1992), including other surviving neuronal populations (e.g., interneurons (Kobayashi and Buckmaster, 2003). Within the dentate molecular layer, recurrent mossy fibers form synapses preferentially with granule cells (Buckmaster and Dudek, 1997; Okazaki et al., 1995; Sutula, 1998; Wenzel et al., 2000), creating monosynaptic recurrent excitatory circuitry in a region where such circuitry is normally not present (Buckmaster et al., 2002; Dudek and Spitz, 1997; Nadler, 2003). Recent light and electron microscopic studies, using intracellular labeling and immunocytochemical techniques, have characterized details of seizure-induced morphopathologic alterations and reorganization in the dentate (Buckmaster et al., 2002; Okazaki et al., 1995; Wenzel et al., 2000), and confirmed reports using biocytin-labeling in
hippocampal slice preparations of epileptic human tissue (Franck et al., 1995) and retrograde-labeling (with biocytin) in pilocarpine-treated tissue (Okazaki et al., 1995). At the electron microscopic level, biocytin-labeled mossy fiber terminals in the inner molecular layer (Fig. 5A–C) exhibit ultrastructural features similar to molecular layer synapses seen in normal rats; no evidence was seen for giant mossy fiber boutons characteristic of the hilar mossy fiber axon (Wenzel, unpublished observation). Newly formed mossy fiber boutons in the inner molecular layer make synaptic contacts of the asymmetric, excitatory type with dendritic spines, dendritic shafts, and somata of granule cells, including contacts on dendrites of the same cell (i.e., autapses) (see also Buckmaster et al., 2002). Reorganization of excitatory circuitry in the hippocampus is not restricted to the granule cells. Studies with kainate and pilocarpine models have characterized the axonal branching patterns of individual biocytin-labeled CA1pyramidal neurons. CA1 pyramidal neurons have been shown to “sprout,” exhibiting an increased branching of the proximal axon, within the stratum oriens and into the stratum radiatum (layers that are normally not innervated by axonal collaterals of CA1 pyramidal cells) (Esclapez et al., 1999; Perez et al., 1996). Because activation of the reorganized CA1-associational pathway evokes epileptiform bursts in CA1 pyramidal neurons, the morphologic data have been interpreted to reflect the formation of novel recurrent excitatory circuits in the CA1 pyramidal cell population. In addition, a recent study revealed remodeling of the CA1 projections to the subiculum (Cavazos et al., 2004) following intense seizure activity, similar to the CA1subiculum pathway reorganization observed in human TLE (Lehmann et al., 2000). Iontophoretic injection of a retrograde tracer (sodium selenite) was used to label sprouted axon collaterals. Timm histochemistry and electron microscopy revealed sprouting of zinc-containing terminals of the CA1 pyramidal cells across hippocampal lamellae, suggesting a translaminar spread of hyperexcitability and enhancement of epileptic discharges. Reorganization of Inhibitory Circuitry Increasing evidence suggests that circuitry reorganization is not limited to the excitatory neurons and pathways. Earlier studies reported reactive sprouting of cholinergic and GABAergic septohippocampal fibers following intrahippocampal kainate injections (Nadler et al., 1980; Suzuki et al., 1997), and studies with experimental lesions of the entorhinal cortex confirmed the sprouting of GABAergic commissural axons (arising from hilar interneurons) into the dentate molecular layer (Frotscher et al., 1997). In the kainate and pilocarpine models of epilepsy, GAD immunoreactivity has been found to increase in the dentate molecular layer after treatment (Andre et al., 2001); a
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similar change has also been observed in the dentate molecular layer of human patients with TLE (Babb et al., 1989). The results of these initial studies suggest that surviving GABAergic interneurons might sprout to restore some of the lost inhibitory synaptic input to granule cells (Kobayashi and Buckmaster, 2003). Biocytin labeling of dentate basket cells in the kainate-induced epileptic rat showed abnormally extensive axonal sprouting into the entire molecular layer (Wenzel et al., 2000); electron microscopy or GABA immunocytochemistry showed that the sprouted axon collaterals of inhibitory GABAergic cells made synaptic contacts onto granule cell dendrites. In addition, dendrites of the GABAergic cells received synaptic contacts from recurrent mossy fiber axon collaterals within the granule cell layer. Recent observations on epileptic rats and mice revealed that, after an initial loss of somatostatin immunoreactivity, there was an increase in somatostatin-immunoreactive fibers in the dentate molecular layer (Houser et al., 2004; Kobayashi and Buckmaster, 2003). In pilocarpine-treated C57BL/6 mice, reorganization of the dentate somatostatin axonal plexus appeared to result from sprouting of axonal collaterals from CA1 stratum moleculare interneurons which crossed the hippocampal fissure.
Molecular Plasticity: Changes in Receptors and Channels Epilepsy-associated alteration in the expression patterns of various neurotransmitter receptors and ion channels has been studied in both human tissue and in animal models. The results of such studies suggest that seizure activity is associated with an impressive plasticity of molecular structure and that at least some of these changes can contribute to the development of a hyperexcitable or epileptic state associated with recurrent seizure generation (Coulter, 2001; Mody, 1998; Morimoto et al., 2004; Sperk et al., 2004). Among the best-studied molecular changes are those discussed below. Glutamate Receptors Glutamate is the major excitatory neurotransmitter in the brain, and its excitatory effects are mediated by ionotropic glutamate receptors (the NMDA, kainate, and AMPA receptors), and G-protein—coupled metabotropic receptors (and associated second messengers). Ionotropic glutamate receptors are assembled from distinct subunits in various combinations, and are localized to different subcellular regions (synaptic and nonsynaptic) of the neuronal membranes. For example, activation of GluR6 subunit-containing receptors on CA3 pyramidal cells (postsynaptic to mossy fiber terminals) is key to kainite-induced seizures. In contrast, kainate receptors containing the GluR5 subunit, which are localized to glutamatergic synapses onto GABAergic interneurons
(Ben-Ari and Cossart, 2000), are thought to mediate seizure suppression effects. Studies in the kindling model have shown transient but inconsistent changes in NDMA subunit receptor expression, so the role of these changes in kindling remains unclear. Studies in the human epileptic hippocampus (using in situ hybridization techniques) revealed increased levels of GluR1 and NR2 expression, but decreased NR1subunit expression. Reductions in GluR2 mRNA and protein were apparent in regions of cell loss (Blumcke et al., 1996; Mathern et al., 1997). GluR2 has been shown to be a determinant of Ca++ permeability of AMPA receptors (Pellegrini-Giampietro et al., 1997) and to play a key role in epileptogenesis; mice with a mutant GluR2 allele are epileptic (Friedman and Veliskova, 1998). In the kainate model, the expression of the GluR2 subunit was found decreased in the CA3 region. The expression of metabotropic receptor subunits mGluR1-8 has been studied in detail, and specific subunitcontaining receptors localized to the neuronal membrane of various neuronal populations; for example, mGluR1 and mGluR5 are perisynaptically localized on dendritic spines and dendrites; mGluR7 and mGluR8 subunits are found presynaptically (close to the synapse); and mGluR2 and mGluR3 appear to be axonal, on the initial segment (Lujan et al., 1997). In different animals models of epilepsy (e.g., kindling, kainate treatment), transient as well as long-lasting alterations of mGluR subunit expression were observed. For example, in amygdala-kindled rats, in situ hybridization showed transiently enhanced mRNA for mGluR1a and transiently decreased mGluR5 expression; mGluR1a was persistently increased in fully kindled rats and also in hippocampal slice preparations from these animals (Wong et al., 2004), suggesting that changes in mGluR may be associated with kindling-induced epileptogenesis. GABA Receptors g-aminobutyric acid is the principal inhibitory neurotransmitter in the central nervous system, and its inhibitory effects are mediated by two classes of receptors: GABAA receptors (which are ligand-operated ion channels) and the G-protein-coupled metabotropic GABAB receptors. GABAA receptors consist of five subunits, which together form a complex binding molecule (for GABA and modulatory agents) and the associated chloride channel. Various subunits (a1–a6, b1–b3, g1–g3, d, e, y) have been identified as components of different GABAA receptor complexes (Sperk et al., 2004), and alterations of the GABAA receptor compositions have been linked to genetic epilepsy syndromes (e.g., mutations of the g2 subunit in a GDFS+-like syndrome). Initial studies on hippocampi from epileptic patients, on kindled animals, and on animals following pilocarpine- or kainate-induced status epilepticus showed an overall increase in GABAA receptor binding sites (Morimoto
What Morphologic Techniques Can be Used to Characterize Tissue from Models of Epilepsy?
et al., 2004). Alterations in the expression of GABAA receptor subunits were found in the kainate seizure model in rats, showing lasting increases in subunits a1, a2, a4, and b1–3 (both mRNA level and immunoreactivity) (Schwarzer et al., 1997). Consistent with these findings are studies in which postembedding immunocytochemistry at the ultrastructural level was used to track localization of the b2 and b3 subunits of GABAA receptors in kindled rats; results indicated a 75% increase in GABAA receptor numbers at inhibitory synapses on somata and initial axon segments, and an increase in receptor density (34% to 40%) (Nusser et al., 1998). Increased levels of mRNA encoding subunits a3, a4, b3, d, and e, and decreased levels of a1 and b1 mRNA, were found in granule cells in the pilocarpine model of TLE weeks before the animals demonstrated spontaneous seizures (Brooks-Kayal et al., 1998), suggesting that these changes were related to the evolution of the chronic epileptic state. These changes were also associated with altered sensitivity of GABAA receptors to zinc and benzodiazepine ligands (Brooks-Kayal et al., 1998). Results from other studies, showing a loss of GABAergic interneurons and mossy fiber sprouting (Andre et al., 2001; Bouilleret et al., 2000), support the conclusion that the regulation of GABAA receptor expression might be related to chronic recurrent seizures. Neurodegeneration-induced loss in receptors is accompanied by altered expression of GABAA receptor subunits in remaining cells of the dentate gyrus and other hippocampal regions, suggesting altered physiologic and pharmacologic properties of these neurons (Sperk et al., 2004). GABAB receptors (R1a and 1b, and R2 subunits) mediate presynaptic control of transmitter release and postsynaptic inhibition through G-protein—mediated modulation of K+ and Ca++ channels. Both subunits are localized in the hippocampal formation and present in both GABAergic and glutamatergic terminals (e.g., the R1a subunit is most abundant on CA1 pyramidal neurons and dentate granule cells; subunit R1b on CA3 pyramidal cells and distal dendrites of CA1 pyramidal neurons) (Andre et al., 2001). Alterations of the GABAB receptor subunits are thought to be associated with neuronal excitability and development of seizures in animal models (Prosser et al., 2001). Strong evidence points to an involvement of GABAB receptor-mediated neurotransmission, particularly in models of absence seizures where mice lacking the GABAB R1 subunit experience seizures (Prosser et al., 2001). Straessle et al. (2003) investigated alterations in regional and cellular expression of GABAB receptor subunits (R1 and R2) with immunocytochemical techniques in a mouse model (unilateral kainate injections into the dorsal hippocampus). One day after kainate treatment, immunoreactivity for both GABAB R1 and R2 subunits was profoundly reduced in hippocampal CA1, CA3c, and dentate hilar regions, and no immunoreactivity was evident in interneurons. This GABAB receptor loss
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persisted at 3 months in CA1 and CA3, but recovered gradually in the dentate gyrus, including in neuropeptide Ypositive interneurons in hilus and the CA3 subfield. These data suggest that loss of GABAB receptors might contribute to epileptogenesis (caused by presynaptic control of neurotransmitter release or postsynaptic inhibition). Long-term changes may represent a compensatory response to recurrent seizures. Voltage-gated Ion Channels As is the case for receptors, many human epilepsy disorders have been attributed to mutations in genes encoding subunits of voltage-gated channels “channelopathies”. These discoveries have provided impetus to studies on the normal localization of specific ion channels and to the possibility that alterations in channel number and location (as a result of seizures or other initiating event/stimulus) give rise to “acquired” channelopathies. Many studies have provided electrophysiologic and/or biochemical evidence of such plasticity (Castro et al., 2001; Bernard et al., 2004; Jiang et al., 2004). Recent studies employing morphologic (immunocytochemical) tools have now provided interesting examples of activity- or seizure-related changes. For example, the voltage-gated potassium channel, Kv2.1, shows a dramatic change in pattern of expression associated with activity and dephosphorylation (Misonou et al., 2004). Investigators studying a model of febrile seizures found an augmented hyperpolarization- (and cyclic nucleotide)-gated channel (HCN1) (Bender et al., 2003).
WHAT MORPHOLOGIC TECHNIQUES CAN BE USED TO CHARACTERIZE TISSUE FROM MODELS OF EPILEPSY? General Histology and Quantitative Methods for Assessing Cell Loss A general histologic overview of the brain regions (or of tissue slices or cultures) of interest is an essential starting point for both qualitative and quantitative morphologic assessments. Such an overview requires an appropriate fixation protocol, the details of which depend on the techniques to be used (e.g., if electron microscopy [EM] procedures are required, then the fixative should include glutaraldehyde [Peters, 1970]) and the source of the tissue (e.g., different procedures are needed to fix cultured material [Phillips, 1998] vs. intact brain). Once tissue has been fixed, the investigator may choose to embed the tissue, often useful when dealing with culture material or delicate immature specimens (Phillips, 1998), or section it on a cryostat, often useful for thin sections (Bancroft and Gamble, 2002), or on a freezing microtome, probably the most “flexible” approach with
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respect to subsequent processing and analyses (Buckmaster and Dudek, 1997; Williams et al., 2003). If nonbiased stereologic techniques are to be used, the requirements of the analysis procedure (e.g., including the entire structure of interest in the sectioned material) must be addressed at the outset (West, 1999). In addition, it is important to include the appropriate controls (e.g., nontreated, age- and sexmatched animals or tissue) in parallel processing. This latter requirement is particularly difficult to achieve in analyses of human tissue from surgical resections. Once appropriate sections have been cut, a simple cell stain (e.g., a Nissl stain, such as cresyl violet) is useful for detailing the qualitative, light-microscopic features of the tissue. Well-stained material can give the investigator a good sense of cell loss, the general features of dysplastic lesions, or even the magnitude of reactive gliosis (Fig. 2A; Fig. 3A,B). Such cell-stained material also provides a potential basis for quantitative assessments (i.e., volume changes of a structure of interest, cell counting). Because no widely accepted protocols exist for making quantitative measures of many obvious qualitative changes, investigators may need to develop situation-specific approaches. For example, quantifying “cell dispersion” in our studies of p35 -/- mice required a semiquantitative approach that allowed us to estimate the degree of dispersion in comparing wildtype vs. knockout animals (Wenzel et al., 2001). Especially when using such “home-grown” procedures, the tissue analysis should be carried out in a blinded fashion whenever possible (i.e., with the investigator blind to the origin or treatment of the tissue). In addition to descriptive and semiquantitative measures, basic histology or cell staining has often been used to analyze cell loss; for example, a Nissl stain allows one to identify pyknotic cells. A host of additional procedures exist for making assessments of cell damage or loss; they can be grouped according to two major approaches: (1) procedures that reveal damaged or dying cells and (2) procedures that provide a measure of the cells that remain following an insult or treatment. The first category includes the classical silver stains that have been developed to identify “dying” cells (Gallyas et al., 1993; Guillery, 1970). Silver-staining procedures require a significant degree of artistry, and can be extremely useful, especially because some variations identify dying cell bodies as well as processes and terminals (Fig. 3C,D) (Heimer, 1970). In general, silver-stained material does not differentiate between necrotic vs. apoptotic cell death (an issue that we will not deal with here, but see Northington et al., 2001). Other procedures that have been designed particularly to highlight apoptosis include FluoroJade histofluorescence (Schmued and Hopkins, 2000) and various procedures to identify cells with injured DNA (e.g., TUNEL labeling; Fig. 4E,F) (Fujikawa et al., 2000). Although these procedures provide an index of cell damage, it remains controversial whether silver or Fluoro-Jade—or
even TUNEL—staining provides an unequivocal measure of cell death. Thus, it is important to consider the use of a complementary procedure, for counting the number of cells that remain after the insult (i.e., not what is injured, but what survives) (Muller et al., 2004). A number of different strategies exist for cell counting, whether of injured cells or of healthy surviving cells. The gold standard for quantitative assessment is nonbiased stereology, which provides a statistically significant determination of the entire number of cells of interest within a region of interest. A number of strict criteria must be met to apply this technique: (1) the use of systematic random sampling; (2) the calculation of total numbers rather than densities; (3) the counting of cells rather than profiles; and (4) the partitioning of the variance to determine the sampling precision (Long et al., 1999; West, 1999). In some cases (e.g., in making counts on brain slices), these criteria cannot be met and, therefore, alternative statistically weaker techniques (e.g., measures of cell density within a limited subregion) must be used (Houser, 1999; Mathern et al., 2002). Because Nissl stains are not specific for cell types, quantitative counts of neurons (or glial cells) are best carried out on tissue that has been stained with a cell-specific indicator (e.g., NeuN immunocytochemistry (ICC) for neurons; glial fibrillary acidic protein (GFAP) ICC for glia; GAD67 for inhibitory interneurons).
Single-Cell Labeling and Assessment of Cell Structure (Dendritic Tree, Axon Arbor) Whereas a general description, and even quantitative analysis, of an affected cell population can be extremely valuable, some forms of information require description at the single-cell level. Given this need, two basic technical issues arise: (1) how can one visualize the cell (i.e., the whole cell, or the part(s) of interest)? and (2) How can one best describe/quantify the cell features of interest? Investigators have addressed Question 1 in a variety of ways, starting with the early Golgi impregnation approaches (that give a complete view of the soma and dendritic tree of a randomly(?) selected group of cells (Valverde, 1970), and including intracellular labeling with dyes (fluorescent dyes such as Lucifer yellow; electron-dense dyes such as biocytin) that are transported into both dendrites and axons (Wilson and Sachdev, 2004). In addition, a number of ICC procedures now provide relatively complete visualization of cells of interest. The choice of which approach to use is determined by the goal of the experiment (and the complementary techniques to be used). For example, as indicated, Golgi techniques do (in theory) provide a randomly stained set of cells for analysis. Because the investigator has poor control over which cells are stained, this approach has obvious drawbacks (and advantages—lack of experimenter
What Morphologic Techniques Can Be Used to Characterize Tissue from Models of Epilepsy?
bias). A major disadvantage of this technique is that tissue fixation for Golgi is often incompatible or difficult to combine with other morphologic approaches, such as immunocytochemistry (Freund, 1993). In recent years, investigators have relied less and less on Golgi staining, in part, because effective use of this technique is an art form that few investigators have bothered to master. In contrast, because intracellular dye labeling can be combined effectively with electrophysiologic measurements, investigators have tried to correlate electrical features of a cell with its morphology. This approach can yield detailed analysis of a given, identified cell, including information about its dendritic tree and axons, as well as its synaptic targets (Buckmaster et al., 1996; Franck et al., 1995; Wenzel et al., 2000). Gathering sufficient numbers of cells for meaningful group comparisons is a painstaking process, however, especially when the cell type of interest is relatively rare (e.g., interneurons) and the intracellular electrophysiology difficult (Freund and Buzsaki, 1996; Scharfman, 1995). Perhaps the most contentious issue involved in assessing labeled cells is whether a given technique provides complete labeling. This difficulty has long been acknowledged for Golgi-stained material, and is also true for intracellular dye injection. Clearly, when intracellular staining is carried out in an acute slice preparation, it is very likely that the cell has been truncated and, thus, the dye cannot give a complete picture of the cell. Comparisons with cells stained in the intact brain are useful for evaluating such in vitro data (Buckmaster et al., 1996; Li et al., 1994; Sik et al., 1995), but are no means guaranteed to provide a picture of the complete neuron (or astrocyte). Other labeling approaches provide information about larger cell populations. For example, GFAP staining can provide informative staining of astrocytes (Fig. 1E,F; Fig. 2D; Fig. 3E,F), and can be used to determine whether astrocytes are lost or missing (Schmidt-Kastner and Ingvar, 1994) or whether they are reactive or hypertrophied (Blumcke et al., 2002). Similarly, neuron-specific ICC (e.g., with NeuN or neurofilament antibodies) (Fig. 1A–D; Fig. 2C) can provide pictures of cell somata (hypertrophic) and dendrites (Wenzel et al., 2004). With many of these general population markers, however, the number of stained cells in a field may be so large as to preclude analysis of individual cells (it becomes unclear which processes belong to which cells). In some types of analysis, this issue is not a problem. For example, Timm staining (for heavy metals) has been widely used to examine mossy fiber sprouting in hippocampus (Houser, 1999; Nadler, 2003; Sutula, et al., 1989). The technique labels bundles of axons and terminals (particularly of dentate granule cells), which can thus be easily followed with standard light microscopic procedures. Other tracing procedures, to determine which cells project to what parts of the brain and to what cell targets, include anterograde and retrograde techniques (e.g., with biotinylated dextran
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amine [BDA], and so on) (Vercelli et al., 2000) (Fig. 2B). These approaches provide additional procedures for examining axonal plasticity as a function of brain abnormality or repeated seizure discharge (Lehmann et al., 2000). Once an investigator has chosen and applied a cell labeling procedure, the more difficult task of deciding how to describe and analyze the data exists. In some cases, a descriptive measure may suffice (e.g., noting the appearance of basal dendrites on granule cells of epileptic mice or rats) (Patel et al., 2004; Ribak et al., 2000). Such simple descriptions are most useful when the feature in question is never (or rarely) seen in controls (e.g., balloon cells in dysplastic neocortex) (Fig. 2E). In most cases, however, even these features deserve some efforts toward quantification (e.g., the percentage of granule cells in which basal dendrites occur; the actual size (diameter) of balloon cells as compared with normal neurons). Investigators may choose to follow up qualitative descriptions regarding cell size or process abnormalities with specific ICC procedures for characterizing related features. Often, the most interesting feature of a labeled abnormal cell lies in the architecture of its dendrites and axons. A number of procedures have been developed to quantify cell features (e.g., dendritic branching, Sholl analysis (Sholl, 1955) and axon length and branching patterns (Drakew et al., 1996; Gomez-Di Cesare et al., 1997; Jiang et al., 1998). Many laboratories now employ a semiquantitative approach to describing mossy fiber sprouting (as seen with Timm staining), using a four-point descriptive categorization of the degree (intensity, location) of aberrant Timmpositive granules (Mathern et al., 1995; Tauck and Nadler, 1985).
Immunocytochemistry and Description of Cell Subpopulations (Presence and Location of Proteins of Interest) Although immunocytochemical analysis can assist in some of the cell-descriptive procedures mentioned above, they are perhaps most useful in providing a window to particular subpopulations of cells (their location, loss with seizure-related stimulation, and so on) and their expression of functionally important molecules (e.g., neuropeptides, receptors, and so on). Although neurochemical approaches (e.g., Western blotting) probably gives a more sensitive measure of the presence or absence of proteins, ICC provides invaluable information about which cells contain what substances and where. Antibodies to molecules of interest are offered in dizzying profusion. Investigators are faced with the rather formidable task of determining how specific an antibody is for the protein of interest within their preparations (e.g., will a protein characterized for a rat work equally well in a mouse?). Technical tricks (freezing, deter-
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gent treatment, microwaving) are often needed to permeabilize cell membranes so that the antibody has access to intracellular sites (Boenisch, 2004; Fritschy et al., 1998). Critically, results with specific antibodies, even antibodies that normally characterize given cell populations, must be interpreted with caution. For example, loss of neuropeptide staining in a subpopulation of interneurons (Scotti et al., 1997) may or may not mean that those cells have been killed by an epileptogenic treatment or epileptic condition. The cells may survive, but no longer make the protein of interest. Or they may make that protein, but the protein’s structure has been modified by the treatment, so that the antibody no longer recognizes the epitope against which it was generated. Because of these caveats, ICC analysis is often carried out using multiple antibodies that provide complementary information. Approaches using ICC have been particularly valuable in epilepsy studies to identify changes in receptors (Coulter, 2001; Mody, 1998; Sperk et al., 2004), interneuron subpopulations (using GABA-related antibodies as well as neuropeptide antibodies that label different subpopulations) (Bouilleret et al., 2000; Buckmaster and Dudek, 1997), and molecules that have age-specific expression (e.g., NeuroD2 in granule cells) (Pleasure et al., 2000). ICC-stained tissue can be analyzed using cell counting procedures (e.g., nonbiased stereology), although quantitative ICC measurements have been notoriously difficult to generate. The difficulty and limitation of ICC arise from, among other issues, the variable staining intensity seen with different primary antibody concentrations and times of exposure, the variable visibility associated with choice of secondary antibody, and general sensitivity of the procedure to often undetermined or uncontrolled factors (e.g., methods of sectioning, degree of fixation, and so on). Thus, to carry out quantitative measures on ICC-stained cells, it is critical to incorporate within each protocol the following measures: first, control (nontreated? nonepileptic?) tissue, is processed identically to the experimental tissue (i.e., with the same reagents, for the same amount of time, and so on). Ideally, one would like to use a control cell population within the same tissue section (e.g., the contralateral homotopic cell group), although for most studies, this type of control is not possible. Second, in controls in which the primary antibody is omitted (or preabsorbed), be sure the antibody reaction is specific to the antigen of interest. Third, because different antibody dilutions are available, identify an appropriate antibody concentration that minimizes nonspecific background staining. Even given these procedural cautions, one is then faced with the issue of deciding what level of staining (i.e., what threshold) constitutes immunopositivity. Subjective determinations may be acceptable in cases of a clear positivity—negativity dichotomy. Computer programs have now been developed for more objective determination of threshold for ICC positivity; these programs provide a
basis for making a quantitative densitometric measure of immunostained tissue (Finkelstein et al., 2004). One last caveat is warranted in considering the vagaries of ICC approaches to characterizing epileptic tissue. As indicated, ICC procedures provide information about if and where a molecule is expressed. An experimental focus entirely on neuronal somata, therefore, may give negative data for proteins that have exclusive dendritic or axonal localization. Although most proteins are manufactured in the cell body and shipped to appropriate target sites, slow cycling and, therefore, low levels in the soma, may make the protein undetectable at the cell body (Uhl, 1993). For that reason, it is often useful to have complementary in situ hybridization data for a given molecule of interest (Young and Mezey, 2004). In situ approaches clearly identify which cells are making the mRNA that is required to manufacture a given protein. Unlike the ICC data, in situ results are all focused on the cell body and, thus, provide no information about where a protein is (should be) localized for appropriate function. These results, however, do provide easily quantifiable data (again, using densitometric techniques) (Le Moine, 2003) regarding which cells are able to make a given protein, and how much. Thus, in situ hybridization procedures are useful means of assessing gene expression.
Electron Microscopy (and ICC) for Assessment of Synaptic Connectivity Electron microscopic analyses have traditionally been used to provide ultrastructural details of cells of interest. Within the context of epilepsy research, the real advantage of EM procedures is the assessment of synaptic connectivity. The difficulty of interpreting morphologic data in terms of function, although not completely resolved by such EM studies, takes on a different perspective once the investigator learns that a given cell (or cell population) makes synaptic contact with a given prospective target (i.e., establishes connections that include specializations associated with synaptic function). This approach has been particularly powerful when used in conjunction with individual cells that have been labeled with an electron dense marker (e.g., biocytin) (Buckmaster et al., 1996; Patel et al., 2004). The synaptic contacts of these cells can be visualized and described (Fig. 5A–C) (synaptic vesicles—round or flattened, symmetric vs. asymmetric synaptic densities, densecore vesicles, gap junctions, and so on) using features that have previously been associated with specific functional properties (Peters et al., 1991). When used in combination with ICC approaches (including immuno-gold labeling) (Fig. 5D–F), features of both the pre- and postsynaptic elements can be described. For example, mossy fiber connectivity to GABAergic vs. glutamatergic postsynaptic elements has been examined with such techniques (Buckmaster et al., 2002; Wenzel et al., 2000).
Dangers and Difficulties in Interpreting Morphologic Data
Electron microscopic data can also be used to help interpret light microscopic features. For example, light microscopic visualization of axonal varicosities has often been used to support the view of functional connectivity (GomezDi Cesare et al., 1997; Sik et al., 1995). Further, the number of varicosities in a labeled axon is commonly correlated with the number of synapses made by the presynaptic neuron. EM data have now shown, at least for some axonal systems, that axonal varicosities correlate imperfectly with synaptic connectivity, based on the presence or absence of synaptic specializations (Buckmaster et al., 1996; Buckmaster et al., 2002). Thus, the observation of increased numbers of varicosities following an epileptogenic treatment may not mean that a given axonal system is making more functional synaptic connections (Benke and Swann, 2004; Galvan et al., 2003). Finally, it should be noted that EM analysis is not a technique for every laboratory. EM is an exacting technique, requiring expert instruction, technical experience, and sophisticated interpretation. Although the information available from EM studies is important, mistakes are easily made and misinformation is readily propagated.
DANGERS AND DIFFICULTIES IN INTERPRETING MORPHOLOGIC DATA
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function. Causation requires that the hypothesized structural change occurs previous to (or, at the very least, simultaneous with) the functional change. Thus, it is important to carry out studies that investigate the time relationships between a morphologic feature of interest and the development of seizure activity. To be considered causal, a structural change cannot first appear after the epileptiform state has occurred. Thus, it is unlikely that mossy fiber sprouting is always a reasonable prospect for the cause of seizure activity, because sprouting often is not seen until after the occurrence of multiple seizures (Dudek and Spitz, 1997; Nadler, 2003). Certainly, one can argue that the critical threshold of sprouting that underlies the epileptogenic process is not detectable by current methods. As indicated, this possibility then requires a careful quantitative assessment. Morphologic changes associated with epileptiform activity, even if not causally related, can still provide a useful set of markers for identifying where in the brain seizure activity is initiated or generated. Such markers are likely to have different degrees of fidelity in identifying key brain structures, with variability depending on quantitative factors, timing issues, and the type of epilepsy in question. It is important to note that just because morphologic plasticity cannot be implicated as a causal factor, that does not mean that it is not interesting and important for studying models of epilepsy.
Quantitative Versus Descriptive Morphologic data have often been presented as descriptive. As we become more aware of the continuum of changes potentially involved in epileptogenesis, the need to couch even structural changes in quantifiable terms becomes more obvious and important. Is there a critical threshold of cell loss (e.g., in a given interneuron sub-population) that is precursor to epileptiform function? Is there a threshold degree of mossy fiber sprouting (or sprouting in other systems) that is correlated with hyperexcitability? Is there a threshold change in receptor subunit composition that is critical for epileptogenesis? These types of questions can only be answered when we report morphologic data in quantitative terms, terms that can be compared across experiments, preparations, and laboratories. Although descriptive data provide an essential starting point for many studies, investigators should be encouraged to plan their morphologic analyses so that they can take advantage of the many quantitative tools now available.
Cause or Effect (Timing, Marker, Causation) As indicated in earlier paragraphs, most of the morphologic correlates of epileptiform activity are just that— correlates. Clearly, one of the important goals of morphologic studies on epilepsy models is to identify changes (reorganization) that may be causally related to changes in
Cell, Age, Sex, Species (Strain) Specificity Although everyone seems to know that morphologic features can vary significantly with cell type, an animal’s age and sex, and the species or strain under investigation, considerable carelessness is found in reporting these variables. It is well-accepted, for example, that immature animals are less likely to show cell loss or damage following status epilepticus, although this reduced susceptibility is clearly not absolute (Haas et al., 2001). Relatively few studies, however, have explored the differences between male and female subjects with respect to seizure-related morphologic changes, although it seems likely that such differences exist (Galanopoulou et al., 2003). The cell types affected in various seizure and epilepsy models may be dramatically different, even in species as similar as rat and mouse (McKhann et al., 2003; Xu et al., 2004). For example, the hilar mossy cells, so susceptible to seizure-induced loss in rat models of status epilepticus, are relatively well-preserved in the mouse (Bouilleret et al., 1999; Silva and Mello, 2000). Identifying the features that underlie these differences constitutes a very important area of investigation, for example, current research on the bases for mouse strainspecific differences in seizure-related cell vulnerability (McKhann et al., 2003; Schauwecker, 2002). We do not argue here that there is a correct age, sex, species, or strain to use in epilepsy experiments. We do urge investigators,
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however, to be careful in reporting these variables as important factors that may influence the morphologic findings on different model systems.
Static vs. Dynamic (Multiple Time Points) Epileptogenesis is almost certainly a dynamic process. Maintenance of the epileptic state may be dynamic as well. This dynamic quality presents a considerable experimental hurdle for morphological investigation, since anatomy has traditionally provided only a one-point-in-time snapshot of the system of interest. The “static” quality of morphological study is one reason why structural analysis on human epileptic tissue (whether from autopsy material or from surgical resections) has been so frustrating. We see this tissue at only one point in time. Animal models provide an opportunity for multiple snapshots — perhaps even “movies” — that better capture the dynamic nature of the system. It is up to the investigator to take advantage of this potential, to devise experiments (and techniques) that allow us to see the changing nature of the epileptic (and epileptogenic) system.
Functional Significance of Structural Data Clearly, an ultimate goal of morphologic description is to provide data that reflect functional alterations. This is a tricky business. Structure is not function. Any attempt to relate structure to function requires continuing efforts at dissociation. If experimental results do not support dissociation, then there is a chance that the structural feature of interest is tightly linked to (causally related to) the function of interest (e.g., epileptogenicity). For example, mossy fiber sprouting has long been recognized as a frequent pathologic feature of the epileptic hippocampus (Franck et al., 1995; Houser, 1999; Sutula, et al., 1989). Authors continue, however, to argue about whether sprouting is causally related to functional epileptiform alterations of the dentate circuitry. One study on rats treated with cycloheximide (a potent inhibitor of protein synthesis) before intrahippocampal kainate or systemic pilocarpine administration, showed that these animals did not develop mossy fiber sprouting but did develop spontaneous recurrent seizures (Longo and Mello, 1997), a clear dissociation between mossy fiber sprouting and epileptogenesis (Pitkanen et al., 2002). In a study by another laboratory to reassess the effects of cycloheximide, pretreatment with this inhibitor did not affect mossy fiber sprouting into the molecular layer (Williams, et al., 2002). Even in this study, however, a small number of animals developed spontaneous seizures without morphologic evidence of sprouting, a finding reminiscent of observations in some cases of human mesial TLE in which no mossy fiber sprouting is evident (Gabriel et al., 2004; Pitkanen et al., 2002; Proper et al., 2001). Such issues require careful, model-by-model consideration if investigators hope
to offer generalizations about the functional significance of morphologic changes.
FUTURE OPPORTUNITIES FOR MORPHOLOGIC INVESTIGATION Epileptic activity in animal models of epilepsy is associated with profound alterations in the structural organization of the brain. Each model system is characterized by a unique set of morphologic changes, just as each form of human epilepsy has seizure or syndrome-specific histopathologic features. Morphologic investigation, therefore, has been an invaluable tool for providing a full description of a given model and for helping to relate a given model to a set of potentially relevant clinical epilepsies. In the present chapter, we have described some of the conventional morphologic approaches that can be used to characterize key structural features that have been linked to epileptic function. These tools can be particularly valuable when describing new models, to generate a description of the brain pathology that constitutes an essential aspect of any attempt to generate a meaningful phenotype. In the world of modern neuroscience, however, morphologic techniques are capable of doing more than provide a static picture of brain abnormalities associated with a given clinical pathologic condition. In the future, morphologic techniques, in conjunction with new imaging, genetic, molecular biological, and electrophysiologic tools, must help us to identify causal mechanisms and dynamic processes as they relate to seizures and epileptogenesis, and, thus, identify new therapeutic targets. For example, imaging modalities (see Chapter 47, this volume) both provide useful anatomic descriptions and generate data that can be related (in the intact brain) to functional and metabolic abnormalities. As the level of resolution of such imaging improves, we may soon have a means for simultaneous chronic monitoring of structural and functional changes in small populations of neurons. Cellular molecular techniques (e.g., polymerase chain reaction [PCR]) can now be carried out on single, morphologically identifiable neurons using either laser microdissection of fixed tissue, or pipette capture of the intracellular contents of a living cell. This technique provides us with a means of directly relating morphologic abnormalities to genetic mutations and then, it is hoped, to epilepsy-relevant function. Using new labeling approaches (e.g., introducing into the genome of a mouse a fluorescent tag that labels a specific cell subpopulation, perhaps at a given period of maturation), the investigator can now describe the development of histopathologic features that may be linked to epileptogenesis (i.e., produce a dynamic view of developing pathology as it relates to epileptic function). Whereas such live imaging “movies” are currently limited to culture systems (see
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Chapter 5, this volume), these labeling approaches may soon make analyses possible from intact brains of animal models. Finally, the application of many of these modern techniques to studies of epileptic human tissue—analyses of genetic mutations in identified neurons or glia, highresolution structural and functional imaging—will undoubtedly yield important hypotheses to be tested in animal models. Among the most important of these hypotheses are those that relate to the role of normal brain compensatory processes during epileptogenesis or in response to the epileptic state (e.g., axonal reorganization, synaptogenesis, neurogenesis). A better understanding of these processes, as viewed structurally, but understood molecularly and functionally, is likely to reveal targets for new treatments to stop seizures (antiepileptic) and to prevent the development of epilepsy (antiepileptogenesis). Clearly, the future—and power—of morphologic description lies in its integration with other investigative tools. Whereas morphologic techniques can (and do) provide important information on their own. There are exciting new opportunities for collaboration. Laboratories that can use morphology for studying dynamic, functional issues, especially with respect to specific hypotheses addressable in appropriate animal models, will lead the way toward clinically useful insights into seizure and epilepsy mechanisms.
Acknowledgments Research in the Wenzel/Schwartzkroin laboratory has been supported by grants from NIH/NINDS (NS 18895).
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Represa, A., and Ben-Ari, Y. 1992. Kindling is associated with the formation of novel mossy fibre synapses in the CA3 region. Exp Brain Res 92: 69–78. Ribak, C.E., Tran, P.H., Spigelman, I., Okazaki, M.M., and Nadler, J.V. 2000. Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry. J Comp Neurol 428: 240–253. Ribak, C.E., Seress, L., Weber, P., Epstein, C.M., Henry, T.R., and Bakay, R.A. 1998. Alumina gel injections into the temporal lobe of rhesus monkeys cause complex partial seizures and morphological changes found in human temporal lobe epilepsy. J Comp Neurol 401: 266–290. Scharfman, H.E. 1995. Electrophysiological evidence that dentate hilar mossy cells are excitatory and innervate both granule cells and interneurons. J Neurophysiol 74: 179–194. Scharfman, H.E., Smith, K.L., Goodman, J.H., and Sollas, A.L. 2001. Survival of dentate hilar mossy cells after pilocarpine-induced seizures and their synchronized burst discharges with area CA3 pyramidal cells. Neuroscience 104: 741–759. Schauwecker, P.E. 2002. Complications associated with genetic background effects in models of experimental epilepsy. Prog Brain Res 135: 139–148. Scheibel, M.E., Crandall, P.H., and Scheibel, A.B. 1974. The hippocampaldentate complex in temporal lobe epilepsy. A Golgi study. Epilepsia 15: 55–80. Schmidt-Kastner, R., and Ingvar, M. 1994. Loss of immunoreactivity for glial fibrillary acidic protein (GFAP) in astrocytes as a marker for profound tissue damage in substantia nigra and basal cortical areas after status epilepticus induced by pilocarpine in rat. Glia 12: 165–172. Schmued, L.C., and Hopkins, K.J. 2000. Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874: 123–130. Schwartzkroin, P.A., and Prince, D.A. 1977. Penicillin-induced epileptiform activity in the hippocampal in vitro prepatation. Ann Neurol 1: 463–469. Schwartzkroin, P.A., Roper, S.N., and Wenzel, H.J. 2004. Cortical dysplasia and epilepsy: animal models. Adv Exp Med Biol 548: 145–174. Schwarzer, C., and Sperk, G. 1995. Hippocampal granule cells express glutamic acid decarboxylase-67 after limbic seizures in the rat. Neuroscience 69: 705–709. Schwarzer, C., Tsunashima, K., Wanzenbock, C., Fuchs, K., Sieghart, W., and Sperk, G. 1997. GABA(A) receptor subunits in the rat hippocampus II: altered distribution in kainic acid-induced temporal lobe epilepsy. Neuroscience 80: 1001–1017. Scotti, A.L., Bollag, O., Kalt, G., and Nitsch, C. 1997. Loss of perikaryal parvalbumin immunoreactivity from surviving GABAergic neurons in the CA1 field of epileptic gerbils. Hippocampus 7: 524–535. Sholl, D.A. 1955. The organization of the visual cortex in the cat. J Anat 89: 33–46. Sik, A., Penttonen, M., Ylinen, A., and Buzsaki, G. 1995. Hippocampal CA1 interneurons: an in vivo intracellular labeling study. J Neurosci 15: 6651–6665. Silva, J.G., and Mello, L.E. 2000. The role of mossy cell death and activation of protein synthesis in the sprouting of dentate mossy fibers: evidence from calretinin and neo-timm staining in pilocarpine-epileptic mice. Epilepsia 41 Suppl 6: S18–23. Singh, S.C. 1977. Ectopic neurones in the hippocampus of the postnatal rat exposed to methylazoxymethanol during foetal development. Acta Neuropathol (Berl) 40: 111–116. Sloviter, R.S. 1987. Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy. Science 235: 73–76. Sloviter, R.S., Zappone, C.A., Harvey, B.D., Bumanglag, A.V., Bender, R.A., and Frotscher, M. 2003. “Dormant basket cell” hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory
interneurons after hippocampal status epilepticus in the rat. J Comp Neurol 459: 44–76. Sperk, G., Furtinger, S., Schwarzer, C., and Pirker, S. 2004. GABA and its receptors in epilepsy. Adv Exp Med Biol 548: 92–103. Spigelman, I., Yan, X.X., Obenaus, A., Lee, E.Y., Wasterlain, C.G., and Ribak C.E. 1998. Dentate granule cells form novel basal dendrites in a rat model of temporal lobe epilepsy. Neuroscience 86: 109–120. Straessle, A., Loup, F., Arabadzisz, D., Ohning, G.V., and Fritschy, J.M. 2003. Rapid and long-term alterations of hippocampal GABAB receptors in a mouse model of temporal lobe epilepsy. Eur.J.Neurosci. 18: 2213–2226. Sutula, T., Cascino, G., Cavazos, J., Parada, I., and Ramirez, L. 1989. Mossy fiber synaptic reorganization in the epileptic human temporal lobe. Ann Neurol 26: 321–330. Sutula, T.P. 1998. A glimpse into abnormal cortical development and epileptogenesis at epilepsy surgery. Neurology 50: 8–10. Suzuki, F., Makiura, Y., Guilhem, D., Sorensen, J.C., and Onteniente, B. 1997. Correlated axonal sprouting and dendritic spine formation during kainate-induced neuronal morphogenesis in the dentate gyrus of adult mice. Exp Neurol 145: 203–213. Tauck, D.L., and Nadler, J.V. 1985. Evidence of functional mossy fiber sprouting in hippocampal formation of kainic acid-treated rats. J Neurosci 5: 1016–1022. Taylor, D.C., Falconer, M.A., Bruton, C.J., and Corsellis, J.A. 1971. Focal dysplasia of the cerebral cortex in epilepsy. J Neurol Neurosurg Psychiatry 34: 369–387. Thompson, S.M., Fortunato, C., McKinney, R.A., Muller, M., and Gahwiler, B.H. 1996. Mechanisms underlying the neuropathological consequences of epileptic activity in the rat hippocampus in vitro. J Comp Neurol 372: 515–528. Uhl, G.R. 1993. A review of in situ hybridization technique: relevance to combined immunocytochemical studies. In Immunocytochemistry II Ed. A.C. Cuello. pp. 281–300. New York: John Wiley & Sons. Valverde, F. 1970. The Golgi method. A tool for comparative structural analyses. In Contemporary Research Methods in Neuroanatomy Ed. W.J. Nauta, and S.O. Ebbesson. pp. 12–31. New York: SpringerVerlag. Vercelli, A., Repici, M., Garbossa, D., and Grimaldi, A. 2000. Recent techniques for tracing pathways in the central nervous system of developing and adult mammals. Brain Res Bull 51: 11–28. Wenzel, H.J., Born, D.E., Dubach, M.F., Gunderson, V.M., Maravilla, K.R., Robbins, C.A., Szot, P. et al. 2000. Morphological plasticity in an infant monkey model of temporal lobe epilepsy. Epilepsia 41 Suppl 6: S70–75. Wenzel, H.J., Woolley, C.S., Robbins, C.A., and Schwartzkroin, P.A. 2000. Kainic acid-induced mossy fiber sprouting and synapse formation in the dentate gyrus of rats. Hippocampus 10: 244–260. Wenzel, H.J., Robbins, C.A., Tsai, L.H., and Schwartzkroin, P.A. 2001. Abnormal morphological and functional organization of the hippocampus in a p35 mutant model of cortical dysplasia associated with spontaneous seizures. J Neurosci 21: 983–998. Wenzel, H.J., Patel, L.S., Robbins, C.A., Emmi, A., Yeung, R.S., and Schwartzkroin, P.A. 2004. Morphology of cerebral lesions in the Eker rat model of tuberous sclerosis. Acta Neuropathol (Berl) 108: 97–108. West, M.J. 1999. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci 22: 51–61. Williams, P.A., Wuarin, J.P., Dou, P., Ferraro, D.J., and Dudek, F.E. 2002. Reassessment of the effects of cycloheximide on mossy fiber sprouting and epileptogenesis in the pilocarpine model of temporal lobe epilepsy. J Neurophysiol 88: 2075–2087. Williams, R.W., Bartheld, C.S., and Rosen, G.D. 2003. Counting Cells in Sectioned Material: a suite of techniques, tools, and tips. In: Current
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FIGURE 5 0 - 1 Morphologic abnormalities in the cortex of p35 knockout mice. A-D: Coronal brain sections from wildtype (A,C) and p35 knockout (B,D) mice, immunoreacted for NeuN. Neocortex (Ctx) of the p35 knockout mouse shows absence of the normal neocortical lamination patterns (cf. A & B). Higher magnification of the hippocampal (H) dentate gyrus (DG) illustrates granule cell dispersion (arrows) into molecular layer (ml) and hilus (hi) (cf. C & D). E,F: Immunocytochemistry for gliaI fibrillary acidic protein (GFAP) demonstrates the characteristic radial glial fiber orientation and astrocyte cell body localization in the granule cell layer (gcl) of wildtype mice (E). These astrocytes are absent in p35 knockout mice (F). G,H: Parvalbumin-positive interneurons are localized to the gcl-hilar border or molecular layer in wildtype mice (e.g., arrow in G shows cell at gcl/ml border). In p35 knockout mice, there is an abnormal distribution of parvalbumin-containing interneurons across the dentate layers (arrows in H). I: Camera-lucida drawing and reconstruction of a heterotypically localized (to the hilus) basket cell (1) and a granule cell (2), intracellularly filled with biocytin, from a p35 knockout mouse. The axonal plexus (ap; red fibers) of the basket cell is abnormally distributed into the molecular layer (ml) and hilus (hi). CAI-CA3, hippocampal subfields; gcl, granule cell layer; gcl, granule cell layer; mf, mossy fiber. Scale bars: A,B: 200~tm; C,D,I: 100~tm; E,F: 20~m; G,H: 40~m.
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FIGURE 5 0 - 2 Morphologic abnormalities in methylazoxymethanol (MAM)-induced CAI heterotopia, and cellular features of incomplete differentiation in the TSC2+/- (Eker) rat. A: Coronal section of the dorsal hippocampus (H) and overlying neocortex (Ctx) from a MAM-exposed (in utero) rat, showing a CA1 heterotopia (large arrow) and a periventricular nodular heterotopia (small arrow). Cresyl violet staining. B: Photomicrograph of stained cells in an intrahippocampal CAI heterotopia, following injection of biotinylated dextran amine (BDA) into a normal region of the hippocampus. Retrograde dye transport labels cells (arrows) that project out of the heterotopia; dye-stained fibers (arrowheads) show axons entering the heterotopia. Neutral red counter-staining. C: A large dysmorphic neuron (arrow), located in the neocortex (lamina II/III) of an Eker (TSC2+/-) rat, shows immunoreactivity for neurofilament in the cell body and irregularly-oriented "dendrites." D: Photomicrograph of a giant astrocyte (large white arrow') in the neocortex of an Eker (TSC2+/-) rat, showing glial fibrillary acidic protein (GFAP) immunoreactivity in its irregularly-oriented and extended processes. Small dark arrow indicates a normal GFAP-positive astrocyte for comparison. E,F: Photomicrographs show giant, balloonlike cells (arrows) in hamartomatous lesions of Eker (TSC2+/-) rats. These cells exhibit eosinophilic cytoplasm in hematoxylin and eosin (H&E) staining (E) and immunoreactivity for both GAD67 (F) and GFAP (not shown). Scale bars: A: 200 ~tm; B: 20 gm; C-F: 10 gm.CAl, CA3, hippocampal subfields; DG, dentate gyrus.
FIGURE 5 0 3 Pathohistologic features of seizure-induced damage in neurons and glial cells in K v l . l knockout mice. A: Photomicrograph of the hippocampus of a Kvl.1 knockout mouse shows neuronal cell loss in the pyramidal cell layer (pcl) of the CA3 region (arrow) and within the hilus (hi). Principal neurons in CA1 and dentate gyms (DG) show relatively little damage. Cresyl violet staining. B: Higher magnification of the CA3 region (pcl) (from A), showing cell loss and accompanying gliosis (arrows). Arrowhead indicates normal pyramidal cells. C,D: Photomicrographs show severe neuronal damage, including cell body and dendritic and axonal terminal degeneration, in the CA1 region of a Kvl. 1 knockout mouse following chronic seizures. F i n ~ H e i m e r degeneration staining labels degenerated cell bodies (e.g., black cell bodies in CAI pyramidal cell layer (pcl; white arrows in C) and hilar cells (hi; white arrows in D). In addition, hilar cell death induces terminal degeneration in the inner molecular layer (iml; white arrow in iml (D) indicates terminal projection of mossy cells) and outer molecular layer (oml; white arrow in oml (D) indicates terminal projection of a subpopulation of hilar interneurons). E,F: Photomicrographs of the dentate gyms of a Kvl. 1 knockout mouse, showing seizure-induced astrogliosis in the molecular layer (ml) and hilus (hi) (E). Higher magnification (F) shows hypertrophic (reactive) astrocytes (arrows). Glial fibrillary acidic protein immunocytochenfistry, counter-stained with cresyl violet (F). G,H: Timm staining in coronal sections of the hippocainpus from a Kvl.1 knockout mouse, demonstrating seizure-induced mossy fiber sprouting into the molecular layer (iml; arrows), granule cell layer (gcl), and CA3 (strata pyramidale and oriens; arrow). For comparison, see normal Timln staining patterns in the dentate gyrns of a wildtype mouse (inset g in G). Scale bars: A,G: 200 ~lm; B: 20 ~tm; C, inset g, E,H: 100 gm; D: 40 gm; F: 20 gtm. rad, s.radiatum; ml, molecular layer; gcl, granule cell layer.
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52 What Good Are Animal Models? PHILIP A. SCHWARTZKROIN AND JEROME ENGEL, JR.
constitutes a critical set of goals for modern research (Jacobs et al., 2001). Our research efforts, of course, are built on a major assumption that basic mechanistic insights, in fact, will assist us in the development of more effective clinical management of human diseases and medical disorders. It could be argued, although perhaps no longer very convincingly, that basic biomedical research in general, and neuroscience research in particular, has not really contributed much toward patient care beyond the introduction of antibiotics. In the current world of molecules and genes, however, that perspective is rapidly fading. Basic understanding can, and does, lead to new therapies. Perhaps the epilepsy field has been relatively slow in turning our ever-increasing understanding of epileptogenic and seizure mechanisms into effective therapies, but we still believe that this application is an inevitable consequence of our efforts. Why, then, has our progress in translating basic research advances into clinical application been so slow? Why has the laboratory investigation of animal models yielded, to date, so little of obvious clinical usefulness? The rationale most often offered in explanation is that epileptic disorders are too complicated to be adequately modeled in the laboratory. Even the clinicians are not really sure what factors to use as defining features (see introductory chapter). So it may be that one of the most valuable contributions we can make from our modeling work is to help define epilepsies more accurately. As should also be clear from the introductory chapter, most of us now recognize that realistic modeling must focus on critical aspects of epilepsy disorders. Because modeling requires us to be clear about what features we are trying to understand, we in turn must pressure the clinician to be clear about what is important to treat clinically. We
GENERAL GOALS OF ANIMAL MODELING As should be clear after perusing this volume, a staggering number and variety of animal models are potentially relevant to seizures and epilepsy. Why have we devoted so much time and energy (and money) to the generation, characterization, refinement, and critique of these models? Implicit in our efforts is the belief that these models are valuable, not just inherently valuable (although they are certainly interesting from a purely “basic science” perspective) (Delgado-Escueta et al., 1999), but valuable in our quest to understand and treat epilepsy more effectively at a clinical level. What, specifically, do we expect that these animal models will offer? How can we justify their use (as opposed, say, to computer models)? As indicated in the introductory chapter to this volume (What Should Be Modeled?), one key reason for using animal models—for studies that cannot be pursued in humans—is based on the belief that more extensive investigations will provide us with an understanding of basic, underlying mechanisms of seizures, epilepsy, epilepsy syndromes, and the consequences of seizures and epilepsy. Working toward such understanding is more than just an intellectual exercise. We assume that our research on basic mechanisms will yield improved approaches to diagnosis and better therapies for treating seizure disorders, which in turn can often be tested first with animal models. Insight into these mechanisms also will provide us with rationales and directions for devising antiepileptogenic therapeutic approaches to prevent epilepsy. Indeed, the development of preventive measures and of cures for epilepsy (which, except for surgical “cures” in some patients, are now completely absent from our medical epilepsy armamentarium)
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will, inevitably, end up addressing selected components of clinically important conditions. Another explanation offered for our slow progress has been that the brains of animals most often used for modeling (particularly the rat and mouse) are unlike the human brain structurally, developmentally, genetically, and so forth. This rationale reflects a real and important concern for modeling approaches. Brains and neural processes do differ significantly, even across subprimate species (e.g., between rats and cats). For the most part, however, investigators have ignored (or at least minimized the significance of) such differences in their attempts to draw inferences from animal model work to the human condition. Indeed, a very basic assumption in adopting animal modeling strategies is that the model brain (be it rat, cat, or monkey—or a slice from one of them) is similar to the human brain, at least with respect to key contributors to seizure generation. Perhaps a more compelling explanation for our frustrations with animal models is the recognition that most of our early modeling attempts were based on normal animals that were made to have seizures by applying exogenous stimuli, whereas the human epileptic brain is abnormal by definition, otherwise no spontaneous seizures would occur. Thus, it is unclear why maximal electroshock (MES) or Metrazol (pentylenetetrazol [PTZ]) models, involving generalized convulsive and absence seizures induced in a normal mouse brain, should provide adequate assessment of antiepileptic (i.e., anticonvulsant) drugs (AED) that are intended to be used to treat other seizure types associated with chronic conditions arising from an abnormal brain. Our present sophistication—or at least, our increased level of awareness with respect to these issues—offers hope for a more rational approach to developing animal models and for applying our insights at the clinical level.
HOW TO CHOOSE THE “RIGHT” MODEL Advantages of Current Models for Different Modeling Goals In the introductory chapter, we attempted to address the question of “What should we model?” Within the framework of that question, different modeling approaches have different advantages. Any categorization of available models, with respect to their optimal applications, is undoubtedly somewhat arbitrary. At the risk of that arbitrariness, and with apologies to the chapter authors who have already addressed this issue for the models included in their discussions, we thought it might be helpful to consider optimal model properties under the various “components of epilepsy” targets set forth in the introductory chapter.
Epileptogenesis Useful models require a clear “pre-epileptic” state and a reasonable time interval between the initiating event and the emergence of the epileptic state. In general, the “acquired epilepsy” models fall into this category. It is important to note that epileptogenesis involves a complex set of processes, and some models may reproduce only some aspects of a relevant cascade of mechanisms. Currently, the most intensely studied models include (1) the kainate, pilocarpine, and stimulation-induced status epilepticus models, in which chronic mesial temporal lobe epilepsy-like activity emerges after a “subclinical” latent interval; (2) kindling models, which lack an initial precipitating event (and usually do not progress to a spontaneous seizure state), but are useful for studying the progression of epileptic excitability and recruitment of nonstimulated brain structures; and (3) insult models (e.g., head trauma) in which the precipitating event can be clearly identified (D’Ambrosio et al., 2004; Dudek et al., 2002; Graber and Prince, 2004; Leite et al., 2002; Morimoto et al., 2004). A number of models in the immature animal, including hypoxia and febrile seizures, are also appropriate choices for these studies (Bender et al., 2004; Jensen, 1999). Other acquired epilepsy models may be more problematic for studying epileptogenesis if (1) the initiating event cannot be well-defined; (2) the interval between the initial insult and the emergence of the epileptic state is very short; (3) the latent interval is so unpredictable that one cannot reasonably time experimental analyses or interventions. The Interictal State Useful models must involve recurrent seizure events, with interseizure intervals characterized by relatively normal brain and behavioral activity. Given that epileptic seizures are episodic, and that the brain functions reasonably normally during the intervals between seizures, the most useful models should mimic this aspect of the epileptic state. Models that provide useful windows include both those in which an early status epilepticus event leads to later chronically recurring spontaneous seizures (e.g., the pilocarpine and kainate models) and those models of idiopathic epilepsy (e.g., the genetic absence epileptic rats from Strasbourg [GAERS]) (Marescaux and Vergnes, 1995; Hosford, 1995). Some of the in vitro models in which recurrent seizure activity has been generated are also useful for detailed investigations of the properties of the tissue between electrographic seizures. In studying such in vitro models, however, it is important to keep in mind the possibility that the acute interseizure state created in a dish may be different from the chronic interictal state and that such in vitro data provide a basis for hypothesis testing in the intact, chronically epileptic animal.
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Ictal Onset
Postictal Period
In studying seizure onset, it is necessary to have a model in which seizures occur spontaneously, reliably, and sufficiently frequently to be amenable to repeated analysis. These frequency and reliability features are often difficult to achieve in intact animal models, so that in vitro models (e.g., in slices) can serve an important role. Particularly in studying tissue from chronically epileptic animals in vitro, manipulation of the bathing medium can increase the frequency (and regularity) of occurrence of electrographic seizure-like events to a level that allows detailed study of underlying mechanisms; however, as with acute recurrent seizures created in a dish, in vitro studies of tissue from chronic models also may not accurately reproduce the in vivo condition. Given the current interest in seizure prediction in the human clinical condition (Lehnertz and Litt, 2005), it is likely that we will soon have a means of focusing more effectively on ictal onset mechanisms in intact animal models. An alternative approach for studying onset, of course, is to use a known stimulus to trigger the seizure event. If the interval between stimulus and seizure onset is sufficiently prolonged (e.g., with low dose PTZ), it is possible to examine the basis of seizure initiation, even in a nonepileptic brain. Of course, studies of ictal onset in which the seizure is induced by an external stimulus provide only hypotheses about seizure onset mechanisms; again, such hypotheses must be subsequently tested in models in which seizures occur spontaneously.
The ideal models for studying postictal phenomenology, as indicated, are those systems in which spontaneous seizures occur in the intact brain. An important role exists for using acutely induced seizure models (even those that are based on seizure induction in normal brain) to examine features of the postictal period. In such models, the investigator has control over seizure onset and, because seizures are usually quite brief, one can study both seizure termination (above) and the characteristics of the period after the seizure, in many cases corresponding to postictal depression. In vitro models in which electrographic seizures can be reliably induced also present an attractive environment for such studies. The caveat here is that the postictal period may be different in an epileptic brain compared with a normal brain (even a normal brain that has just had a seizure). Thus, it is also important to examine the period directly after seizure occurrence in models that generate spontaneous recurrent seizures.
Ictus and Seizure Termination In vitro models have a major advantage for studying these issues, because seizures can be triggered (or occur spontaneously) at fairly frequent intervals. Cellular recording tools can be applied effectively to study relatively brief events and processes. What most in vitro models do not provide, however, is a sense of whether or how extensive circuits and networks participate in seizure activity (Spencer, 2002), particularly as seizures go through different stages (e.g., changes from tonic to clonic discharge patterns). Acutely induced seizures in the intact brain provide an opportunity to examine such circuitry contributions and interactions. Importantly, perhaps especially with respect to seizure termination, the interaction of neural activity with circulatory and hormonal influences can also be examined (Binaschi et al., 2003). It is important not to assume that the mechanisms of ictus and seizure termination are the same for induced seizures as for spontaneously occurring seizures. Hypotheses generated in systems or models that rely on induced seizure activity need to be tested in models of spontaneous seizure occurrence.
Long-term Consequences The study of long-term consequences is quite complex, and different investigators will undoubtedly favor different strategies (Cilio et al., 2003; Holmes, 2004; Koh, et al., 1999; Helig et al., 2002). One approach is to examine the long-term consequences of seizures in a model in which there is only an initial episode (e.g., status epilepticus) of seizure activity. One can then examine relevant sensitivities (e.g., seizure threshold), behavioral abnormalities (e.g., performance in specific tasks), and cellular and structural changes (changes in receptors, axonal reorganization) in the absence of additional confounding activity. An alternative approach is to deal explicitly with the complexities of seizure number and inter-seizure interval. Long-term consequences can reflect a cumulative effect of recurrent seizures over long periods. Rapidly occurring seizures (with brief inter-seizure intervals) can have different consequences from seizures (even the same number) with longer interseizure intervals. Studies of long-term consequences are particularly important in following challenges to the immature brain (e.g., in immature models of repetitive seizures, kindling, febrile seizures), where clinical concerns include a broad range of potential consequences for the maturing organism (e.g. Nehlig et al., 2002). Pharmacologic (or Other Treatment) Efficacy The history of drug discovery has been based, in large measure, on assessing the ability of drugs to raise seizure threshold in models of acutely induced seizures in normal brain. Although such models remain important, perhaps mostly for their quick “throughput,”it is now clear that
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potential therapies need to be assessed at a variety of levels, requiring other types of models. Change in rate of seizure occurrence can be assessed only in models generating spontaneous recurrent seizures (both in vivo and in vitro). Antiepileptogenesis treatments require models, as described, in which one can intervene during a period corresponding to the epileptogenic process (Loscher, 2002; Pitkanen, 2002). Kindling has long been used for such investigation, but now other models exist in which the epileptogenic effect of the insult is sufficiently reliable to make treatment testing practical, although it is certainly time- and laborintensive. Multifactorial Influences Many current models do not present with reliable seizure activity, but rather with seizure “sensitivity” or “predisposition.” It is thus possible to examine the interaction of multiple, potentially epileptogenic influences, including genetics, early insult, developmental abnormalities, and adult traumas. A number of studies have shown that genetic background—even in the absence of obviously mutated or epilepsy genes—is a powerful determinant of whether a superimposed insult actually results in epilepsy. We, thus, have an opportunity to study the likely complex, multifactorial bases for most human epilepsies. A combination of model systems can be used for such analyses; for example, kindling may be applied to a genetically epilepsy susceptible animal (from rat to monkey), which is exposed to an environmental challenge.
CURRENT STATUS OF MODEL DEVELOPMENT WITH REGARD TO ESTABLISHED BENCHMARKS Following a landmark White House-initiated conference (“Curing Epilepsy: Focus on the Future”) in March 2000, a series of goals and benchmarks were generated in an attempt to identify optimal strategies for realizing critical goals in the field. In light of that discussion (Jacobs et al., 2001), and following a number of workshops that have attempted to implement those goals in more practical terms (Stables et al., 2002), it may be useful both to assess the potential contributions of current animals models toward achieving those benchmarks and to determine what additional models might be necessary for achieving those benchmarks.
Modeling Priorities The benchmarks paper (Jacobs et al., 2001) identified the following strategies as priorities for research investigation.
Understanding Mechanisms of Epileptogenesis A number of the acquired models (i.e., models that start with a normal brain and develop, over time, an epileptic state) have been critical in generating insights into mechanisms of epileptogenesis. Exciting findings have been reported from kindling studies, from investigations on chemoconvulsants, and from the development of new models of immature insult. Insights from Genetics A host of genetic models (in diverse animals, from fish to fly to mouse) are available for investigation, and many have provided important homologs to human genetic epilepsies (Meisler et al., 2001; Noebels, 2001). Among the beststudied of these models are animals with (1) channel gene mutations (giving rise to a new appreciation of some epilepsies as “channelopathies”); (2) mutations in neurotransmitter and receptor systems, from peptides to transporters; and (3) differences in genetic background (showing how normal background genetics influences seizure susceptibility, seizure phenotype, and even drug resistance) (Ferraro et al., 1999; Schauwecker, 2002). New models are now being developed to investigate the interaction among multiple genes, and between genes and environmental insults. Insights from Imaging and Electrophysiology Whereas the investigative emphasis for this priority is on techniques, identifiable model systems have been particularly amenable for such investigations. Models with clear structural lesions (where the structural abnormality appears to be a precursor of epilepsy, or where it is a result of the seizure activity) have offered investigators an avenue for linking particular structural abnormalities with a given seizure phenotype or for examining, longitudinally, how lesions develop following an epileptogenic insult. In vitro models (particularly variations of the slice preparation) have offered important technical advantages for cellular electrophysiology and imaging (e.g., with voltage-sensitive dyes) (Demir et al., 2000); recent developments include application of sophisticated electrophysiologic techniques to intact systems (Bragin et al., 1997). Insights from Developmental Biology Although there are significant difficulties in comparing brain development in rodent and human (or in culture systems), details about normal brain maturation can, and have, guided model generation, particularly for pediatric epilepsies (Liu et al., 2000). Models are emerging that are based on genes known to be involved in key steps of brain development (cell proliferation, migration, differentiation)
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and on the processes (and their timing) involved in cell, synapse, and circuitry development (e.g., receptor and transporter maturation). Developing New Therapies Simple model systems (e.g., in vitro slices, seizure induction in the normal brain) have offered a convenient context for initial evaluation of therapeutic approaches. In addition, now numerous intact models exist in which therapy-related hypotheses can be tested. Virtually all of the latter models are time- and labor-intensive.
MODELING NEEDS FOR ACHIEVING SPECIFIC BENCHMARKS
5.
Elucidate Basic Disease Mechanisms 1. Develop noninvasive dynamic imaging and physiology monitoring systems, to be used in conjunction with surrogate markers to diagnose epilepsy, localize the epileptogenic region, determine whether a drug or treatment is going to work (without waiting for additional seizures to occur), and predict who will develop epilepsy after an insult. Toward these ends, new animal models are needed both to test the usefulness of current putative surrogate markers and to identify additional surrogate markers. Animal models are needed to develop and test new imaging or monitoring approaches (particularly in coregistering functional and structural information). Finally, appropriate models are needed to test specific hypotheses regarding treatment efficacy. 2. Create a magnetic resonance imaging (MRI) database to coregister anatomic data with functional studies. Although this benchmark is initially in the realm of the clinical investigator, the long-term goal involves establishing a framework of structure—function correlations. Correlations between specific anatomic disturbances and specific functional abnormalities, as seen in animal models, provide hypotheses for these clinical studies. In turn, as the correlations are established at the clinical level, cause—effect relationships can be tested in model systems (e.g., to see if creation of a specific anatomic lesion leads to a predicted functional outcome). 3. Establish a gene chip collaborative network. Here, too, the findings (epilepsy-related genetic influences) at the clinical level will provide specific hypotheses to be tested in animal models. The model findings, in turn, will provide specific genetic targets to be sought in the clinical population. 4. Establish a national collaboration for identifying or cloning monogenic epilepsy syndrome genes. It is widely recognized that identifying a specific gene abnormality is only a first step in understanding how a gene defect leads
6.
7.
8.
to the epileptic state. Clinical descriptions of single gene mutations associated with epilepsies will continue to generate models (e.g., knock-ins and knockouts, conditional models, and so on) in which the mechanisms that follow from the gene mutation can be studied. Toward these ends, there is an increasingly urgent need is seen to develop model systems in which gene mutations can be rapidly generated (e.g., in flies, zebrafish, worms) and studied. Such models may also provide an initial system in which to examine both treatment effects (see section on Treatment below) and to develop information about genes that regulate response to pharmacotherapy. Establish a national consortium to identify epilepsy susceptibility genes. The needs here are perhaps even more acute than in identifying monogenetic epilepsy syndrome genes because the relevant clinical population is likely to be much larger. Animal models can be particularly useful in this effort, because genetic background can be carefully controlled (e.g., with inbred mouse strains), and epileptogenic challenges can be superimposed on those well-characterized backgrounds. Validate models of epileptogenesis. As discussed here, there are currently a number of mesial temporal lobe epilepsy (MTLE) animal models of epileptogenesis. Models of epileptogenesis, relevant to other epilepsies or syndromes, are needed. In addition, there is a critical requirement for basic laboratory scientists to work closely with clinical investigators in order to agree on the criteria for model validation. Develop new models of epilepsy (particularly therapyresistant epilepsies) and epileptogenesis in the immature brain. Although we have developed many animal models of symptomatic partial epilepsies, there is a marked paucity of models of catastrophic symptomatic generalized epilepsies that arise in the immature central nervous system (CNS). Many of these epilepsies and syndromes are particularly treatment-resistant (e.g., infantile spasms, Lennox–Gastaut syndrome), and many of them have long-term consequences, not only for seizure activity in the adult, but also for neurologic and cognitive development. Thus, generation of animal models of catastrophic pediatric epilepsies—or for critical components of these disorders—is an area of special need. Identify and characterize potential surrogate markers of epilepsy and epileptogenesis. See Elucidate Basic Disease Mechanisms, paragraph 1.
Prevention 1. Use surrogate markers to identify potential epileptogenic brain regions and to direct therapies. See Elucidate Basic Disease Mechanisms, paragraph 1.
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2. Establish multicenter trials to test effectiveness of potential neuroprotective or antiepileptogenic compounds or treatments. Ideally, initial screening of potential neuroprotective and antiepileptogenic compounds would occur in appropriate animal models. Although there are current models in which such screening could occur, few of them have been characterized explicitly for studying antiepileptogenesis or neuroprotection. Additional models for these purposes will be necessary. Perhaps even more importantly, however, is an investigative change of focus to test antiepileptogenic and neuroprotective effects of new treatments.
Treatment 1. Develop individualized treatments that are based on information about maturational and hormonal status. The need for developmental models has already been identified as critical. Model development that focuses on hormonal state is also sorely neglected. Treatment strategies that differ across sexes and take into account other hormonal issues (e.g., associated with stress) can and should be addressed at the animal model level, as a precursor to clinical treatment trials. 2. Develop genetic “fingerprint” diagnostic tests for epilepsy. See Elucidate Basic Disease Mechanisms, paragraph 3. 3. Investigate and define the extent of intrinsic diffuse, seizure-suppressing systems, and identify methods for activating these systems. Currently, relatively little work focuses on intrinsic seizure-suppression mechanisms. This goal requires the development of monitoring tools (recording, imaging) that provide a broad (systems-wide) view of seizure initiation and propagation. Such research also requires models that generate frequent spontaneous seizures, so that experimental procedures to study seizure-suppression mechanisms (e.g., brain or tissue stimulation) can be easily evaluated. 4. Achieve a complete cure for at least one genetic form of epilepsy. Specific genetic epilepsies must be modeled so that interventions can be tested. Novel intervention strategies need to be developed within this context. 5. Develop a device that reliably anticipates seizures and can apply targeted treatment to abort the seizure. As indicated, development and testing of new devices would be greatly facilitated by the availability of new models in which frequent spontaneous seizures occur. 6. Widen the use of epilepsy surgery to more patients for whom it is an effective treatment. Although this specific goal does not really involve a contribution from the animal model arena, the broader use of epilepsy surgery procedures would provide increased opportunity for
validating current animal models and perhaps for obtaining new insights that will help in developing new models. 7. Develop and apply a new therapeutic strategy to reduce seizure frequency in at least one form of epilepsy. Novel therapeutic strategies should be a major product of the animal laboratory. Such strategies can be tested on models already available (e.g., models of MTLE). A great need is seen, however, to develop models of other types of pharmacoresistent epilepsy syndromes and seizure types to test novel treatment strategies designed for disorders currently refractory to treatment.
GENERAL STRATEGIES FOR MODELING AND FOR GENERATING REALISTIC GOALS The successful development and use of an animal model depends, in large measure, on establishing realistic expectations. Given the potential complexity of the issue, we researchers have all adopted strategies—often implicit—for developing models of epilepsy. It is useful, however, to make those strategies explicit, so that we can develop realistic goals and assess our progress, even in the face of factors that might interfere with or confuse our efforts. Most of our work, indeed, most of the work in biomedical research involving animal models, can be seen in light of the following strategies for modeling a complex human disorder. 1. Imitate what we think is the etiologic insult (i.e., the event that initiates the pathologic process), often (usually) with the outcome undefined. This is a good strategy if one can identify the precipitating event or insult, not usually easy to do for epilepsy. A number of epilepsy models, however, use this approach. For example, our animal models of posttraumatic epilepsy are based on some type of injurious event that we think mimics the naturally occurring insult giving rise to this clinical condition. Other models that are based on this strategy are listed among the acquired models in this volume, including insults that occur in childhood (e.g., febrile seizures, status epilepticus) and events that can trigger the epileptogenic process in adulthood (head trauma, stroke, infection, tumors, immunologic reactions). As is becoming increasingly clear from our work with such models, however, there are significant complications in reproducing a relevant insult, perhaps especially so when we attempt to translate an event in the human to a small animal like a rat or mouse. What are the salient parameters that we need to mimic to produce an epileptogenic insult, and how can we reproduce them in a small animal model? For example, should traumatic brain insult focus on shear injury? On penetrating injuries? To what part of the brain? Do we really need to know (and do we really need to measure) the key features
General Strategies for Modeling and for Generating Realistic Goals
of the insult, or is it adequate to make an approximation of the clinically relevant event? Even assuming we can answer some of these questions, we are then faced with the need to validate the model by measuring outcome features that define posttraumatic epilepsy. What are those measures? 2. Study what we think is the key underlying mechanism that can be manipulated experimentally. In the epilepsy field, several examples of hypothesized mechanisms provide the impetus for our studies. This approach has been particularly popular among investigators using in vitro (e.g., slice) model systems, but one can also treat intact animals to manipulate a mechanism of interest and then examine the resultant output. For example, investigators have hypothesized that seizures result from a compromised g-aminobutyric acid (GABA)ergic inhibitory system, and have carried out experiments in which the level of GABAergic inhibition is manipulated with pharmacologic agents and correlated with seizure activity. Basic mechanism hypotheses are also being increasingly tested at the genetic level, where a mouse knockout, for example, can be generated to show that loss of a given protein can alter excitability levels in the brain. Within the context of this strategy, it is important to consider what should be measured to validate the model and to be clear about what the model-producing experimental manipulation will tell us. Many of these types of studies have confirmed that a compromised mechanism can lead to epileptiform activity. It has been much more difficult to determine from these experiments (e.g., in which we have blocked GABA receptors or channels) that the mechanism under investigation in the laboratory has any relevance to a clinical seizure disorder. Using this approach, an investigator runs the considerable risk of falling into a tautological trap wherein an epilepsy-like outcome is taken for confirmation that a particular mechanism plays a key role in human epileptic activity. That trap has certainly confused the research progress with respect to the GABA hypothesis and has led to years of apparently circular hypothesis testing. 3. Reproduce a genetic abnormality known to underlie the disorder of interest, but where the phenotype may be undefined or unknown at the level of the model. More and more, we are learning that clinical diseases can be caused by specific mutations in one or more genes. In the epilepsy field, a number of epilepsy syndromes are known to arise from single gene mutations. It would seem relatively straightforward, therefore, to reproduce that genetic malfunction in an animal, to produce a model system in which one could explore how the gene defect leads to the clinical abnormality. There are a number of conceptual problems, however, associated with the attempt to model single gene epilepsies using knockout
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or transgenic animal models: (1) Experimental manipulation of a gene can result in compensatory changes in other genes, in a pattern of unknown relationship to that seen in the clinical disorder. Because genetic mutations are often expressed during brain development, some compensatory changes can occur during early development and involve complex reorganization that we cannot knowingly reproduce in a mouse. Conversely, specific compensatory changes could occur uniquely in the mouse and result in a phenotype that is not relevant to the clinical syndrome. (2) Deletion (or overexpression) of a gene in a rat or mouse may lead to a phenotype that is only marginally related to that seen in the clinical disorder (most critically, no seizures may occur). This problem is obvious in a number of existing mouse models (e.g., associated with cortical dysplasia). (3) Genetic deficits in human disorders rarely involve gene deletion per se, but rather specific site mutations. Depending on the site of the mutation, there can be complete or incomplete functional inactivation of the gene. The precise nature of the mutation often determines its phenotype (e.g., mutations at different sites within a given gene can result in different seizure types, and mutations involving different genes may result in rather similar seizure phenotypes). 4. Establish models in which the goal is to mimic a clinical phenotype (behavior, neuropathology, EEG, and so forth). This approach is perhaps the most common basis for animal models of epilepsy. We generate, or would like to establish, animal models that exhibit a certain seizure type (e.g., limbic seizures) that show clinically relevant patterns of neuropathology (e.g., mossy fiber sprouting) and imaging abnormalities (e.g., areas of high or low intensity on MRI, hypometabolism on positron emission tomography [PET]), or that are characterized by an epileptic EEG pattern (e.g., spike and wave discharge). The pilocarpine and kainate models of MTLE fit into this category; they both are based on establishing an animal that generates spontaneous limbic seizures associated with hippocampal sclerosis and reorganization (i.e., clinical phenotypic characteristics). Many of the pediatric epilepsy models associated with cortical dysplasia (e.g., irradiation, freeze) are variations on this theme. And idiopathic models also fit into this category; for example, the GAERS model is based on characteristic generalized spike–wave electrical activity, behavioral absence, and 2-deoxyglucose autoradiography data resembling fluorodeoxyglucose (FDG)-PET in human absence epilepsy. In such models, the match between the animal and the human clinical features is often considered the basis for assessing the value of the model. 5. Identify and study markers that are associated with the clinical disorder of interest. An explicit focus on
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surrogate (or bio-) markers is a relatively new strategy for epilepsy research. Indeed, it is perhaps not obvious what constitutes such markers. Simply (and perhaps arbitrarily) defined, a surrogate marker is a true marker of epileptogenicity or epileptogenesis. That is, such markers provide clues to basic mechanisms of the epilepsy or the epileptic process under investigation. An electrophysiologic example of a marker that might satisfy this criterion is the “Fast Ripple” EEG pattern, thought to be associated with seizure initiation sites in MTLE (Bragin et al., 2000). Similarly, investigators have proposed that a-methyl-l-tryptophan (AMT) uptake, seen on PET, is linked to the epileptic process (Juhasz et al., 2003). These markers help us to know where and when to look for those processes involved in seizure initiation or epileptogenesis. Because these markers are mechanistically tied to the epileptic phenomenon of interest, if one creates models that consistently show these markers, those models will provide a framework for investigating the underlying seizure or epileptogenic processes. There are, in contrast, a set of less-specific diagnostic indicators that are helpful in identifying features of the brain (functional, structural, molecular) that are relatively consistently related to a defining feature of epilepsy (e.g., seizures), but are not connected in a mechanistic way. Such indicators are easier to identify, because one does not need to know that, or how, the marker is causally related to the epilepsy; they have only a correlational relationship with the epileptogenic (or seizure-genic) process of interest. For example, at a structural level, loss of GABA cells has for years been taken to be an indicator of a seizure-generating area; at the imaging level, PET hypometabolism has been often associated with mesial temporal lobe seizure sites; and at the electrophysiologic level, interictal EEG spikes have been associated with seizure onset.
MODEL VALIDATION For most of the history of research on animal models of epilepsy, investigators have not been too concerned about model validation. Certainly, as used as assays for evaluating potential AED, our models appeared to be relatively “blind constructs” with respect to the relevant clinical conditions. Some attention was given to the pattern of seizure activity against which a drug was (or was not) effective (e.g., generalized vs. focal spike-and-wave), but few other variables were addressed. Indeed, the most widely used testing procedures for drug identification had no scientific bases. MES and Metrazol were proposed as models of generalized tonic–clonic seizures and absence seizures, and were used to select drugs that were effective against generalized tonic–clonic seizures and absence. These models have con-
tinued to be used in this way (based on a number of important early empiric successes). Not surprisingly, however, the application of these models to AED assessment has resulted simply in finding more of the same type of drug. We are no longer satisfied with this level of model relevance and application (White, 2003). What do we mean, now, by “validating” a model? Indeed, how can a model be validated? Generally, when we speak of model validation, we are talking about how closely the model reproduces the clinical entity of interest. The apparently obvious response to the question of validation is to present a comparison of the properties of the model against the clinical condition that the model is supposed to mimic. It is important to note that this approach to model validation depends on the specific research questions to be addressed with the model. For example, methylazoxymethanol (MAM)-induced heterotopia mimics some of the features of epilepsy syndromes associated with cortical dysplasia (Schwartzkroin and Walsh, 2000) and, thus, may be useful in studying developmental dysplastic lesions often associated with early onset epilepsy. These animals, however, do not have (or rarely have) spontaneous seizures and, thus, are inappropriate for assessing treatment possibilities. In contrast, the pilocarpine model reproduces the spontaneous limbic seizure pattern and neuropathologic features of human MTLE and, thus, provides a useful model for drug screening. Ultimately, the model must involve seizures—or some phenomenon tightly linked to seizure activity—if we are to test the effects of an antiseizure treatment (drug, stimulation, and so on). Similarly, the model must exhibit an epileptogenic process (i.e., changing from a no-seizure background to a spontaneous seizure state) if we are to assess antiepileptogenic therapies. Other non-seizure model features may also be useful from the perspective of validation. Characteristics of interest include changes in excitability and synchronization and the molecular alterations that underlie such changes, as well as cell damage and reorganization (commonly seen in MTLE) thought to be associated with alterations in behavior. Models in which such changes occur, but in which no spontaneous seizures are seen, can help us to better understand and treat epilepsies. Model validation can be based on a broad range of features, and is necessary only for that aspect of epilepsy that the model is intended to reproduce. The ultimate approach to model validation is to test the results of manipulating some feature of the model on human tissue or, when ethically responsible, on human patients. This essential aspect of validation takes advantage of perhaps the main strength of laboratory research—the development and testing of hypotheses. Animal models offer us that opportunity—to test our ideas about what mechanisms may be critical to seizure development and expression and our hypotheses about what treatments may be effective. For example, current work on AED efficacy has focused atten-
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References
tion on the role of drug transport mechanisms. Investigators have hypothesized that particular, genetically determined but environmentally regulated mechanisms transport potentially efficacious drugs away from target cells; by limiting drug availability in the epileptogenic region, this multidrug resistance property reduces the usefulness of the drug for affecting cell discharge (Sisodiya, 2003). At least some of the key features of the mechanisms underlying multidrug resistance (e.g., the genetic basis for relevant transporters, the effects of extracellular environment on transport efficiency) do not necessarily need to be tested in a model system in which an animal has spontaneous seizures (at least not initially). Experimentally testable hypotheses can be evaluated in simple or artificial model systems, and those studies may lead to clinical predictions (e.g., under what conditions changes in transport mechanisms would result in blockade of drug efficacy). Animal models, even in vitro models, do give us a venue in which to test such hypotheses. From this perspective, studies in a model system simply provide a rational basis for pursuing ideas at the clinical level. It should be noted that because the results of animal model experiments do not guarantee the correctness of the hypothesis within the context of the human disorder, it is useful to debate the relevance of the system to the clinical disorder in which a hypothesis is tested. Although any experimental model can be used to test a hypothesis, the likelihood that the outcome has clinical applicability depends on how appropriate the model is to critical features of the disorder. For example, if transport is thought to be a function of vascular endothelial cells, then testing drug transport mechanisms in a pure neuronal culture system would provide little guidance in evaluating drug efficacy in a seizure disorder. Within this context of hypothesis testing, validation of a model means that it has been shown to be a good predictor of outcome in a more clinically relevant system. If a given hypothesis is supported by studies in model A, and also is confirmed when tested in the human epilepsy patient, then model A is validated with respect to that hypothesis. The more hypotheses that are tested and confirmed in both model A and in the human epilepsy patient, the more trustworthy model A becomes (i.e., the greater the degree of validation). Also, this hypothesis-testing approach may be used in comparing simple and complex models (e.g., in vitro vs. in vivo systems). For example, if the results of hypothesis testing in a brain slice are confirmed in the pilocarpine rat, then one might speak of validating the simpler system on the basis of its usefulness in predicting outcome in the more complex system. Finally, it is important to note that validation, at least with respect to an animal model, is not an “all-or-none” determination. A given model may be a good predictor for one hypothesis, but not for another. There is no perfect model of an epilepsy disorder. Indeed, one could argue with the adage that “the only model of a cat
is a cat—and preferably the same cat.” Even a given cat (or person or epilepsy disorder) changes over time. Exact reproduction is not an option. Having made that somewhat theoretic point, it is important to note that a perhaps neglected aspect of experimental model studies is to pay attention to and help define the key features of the disorder, so that we can reproduce them in our model systems. The fact that seizure disorders are quite heterogeneous, and that even a given disorder can present with considerable variability (even within the same patient), has always been viewed as one of the major obstacles to elucidating the underlying mechanisms of the disorder. If we recognize that epilepsy is complex and involves multiple variables, then this heterogeneity can eventually help us to identify the salient features of the disorder. Thus, although the rationale for using reductionist models in which all the variables can be defined and kept constant is certainly scientifically compelling, there is also an argument for accepting or studying those models that show considerable variability, just as do the human conditions. We have spent a lot of time talking about what we want to model and how to go about that process. Undoubtedly, at some point we must determine how our models are related to the human condition. Validation of a model, however, may not be simply an issue of determining how similar the model is to a human disorder. Validation really is an issue of whether and how that model can be used to understand and to develop better treatments for clinical epilepsies.
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Ferraro, T.N., Golden, G.T., Smith, G.G., St. Jean, P., Schork, N.J., Mulholland, N., Ballas, C. et al 1999. Mapping loci for pentylenetetrazolinduced seizure susceptibility in mice. J Neurosci 19: 6733–6739. Graber, D.K., and Prince, D.A. 2004. A critical period for prevention of posttraumatic neocortical hyperexcitability in rats. Ann Neurol 55: 860–870. Holmes, G.L. 2004. Effects of early seizures on later behavior and epleptogenicity. Ment Retard Dev Disabil Res Rev 10: 101–105. Hosford, D.A. 1995. Models of primary generalized epilepsy. Curr Opin Neurol 8: 121–125. Jacobs, M.P., Fischbach, G.D., Davis, M.R., Dichter, M.A., Dingledine, R., Lowenstein, D.H., Morrell, M.J. et al. 2001. Future directions for epilepsy research. Neurology 57: 1536–1542. Jensen, F.E. 1999. Acute and chronic effects of seizures in the developing brain: experimental models. Epilepsia 40(Suppl 1): 51–58. Juhasz, C., Chugani, D.C., Muzik, O., Shah, A., Asano, E., Mangner, T.J., Chakraborty, P.K. et al. 2003. Alpha-methyl-L-tryptophan PET detects epileptogenic cortex in children with intractable epilepsy. Neurology 60: 960–968. Koh, S., Storey, T.W., Santos, T.C., Milan, A.Y., and Cole, A.J. 1999. Earlylife seizures in rats increase susceptibility to seizure-induced brain injury in adulthood. Neurology 53: 915–921. Lehnertz, K., and Litt., B. Eds. 2005. The first international workshop on seizure prediction. Clin Neurol in press. Leite, J.P., Garcia-Cairasco, N., and Cavalheiro, E.A. 2002. New insights from the use of pilocarpine and kainate models. Epilepsy Res 50: 93–103. Liu, M., Pleasure, S.J., Collins, A.E., Noebels, J.L., Naya, F.J., Tsai, M.J., and Lowenstein, D.H. 2000. Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy. Proc Natl Acad Sci U S A 97: 865–870. Loscher, W. 2002. Animal models of epilepsy for the development of antiepielptogenic and disease-modifying drugs. A comparison of the
pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res 50: 105–123. Masescaux, C., and Vergnes, M. 1995. Genetic absence epilepsy in rats from Strasbourg (GAERS). Ital J Neurol Sci 16: 113–118. Meisler, M.H., Kearney, J., Ottman, R., and Escayg, A. 2001. Identification of epilepsy genes in human and mouse. Ann Rev Genet 35: 567–588. Morimoto, K., Fahnestock, M., and Racine, R.J. 2004. Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog Neurobiol 73: 1–60. Nehlig, A., Dube, C., and Koning, E. 2002. Status epilepticus induced by lithium-pilocarpine in the immature rat does not change the long-term susceptibility to seizures. Epilepsy Res 51: 189–197. Noebels, J.L. 2001. Modeling human epilepsies in mice. Epilepsia 42(Suppl 5): 11–15. Pitkanen, A. (2002). Drug-mediated neuroprotection and antiepileptogenesis: animal data. Neurology 59(Suppl 5): 27–33. Schauwecker, P.E. 2002. Complications associated with genetic background effects in models of experimental epilepsy. Prog Brain Res 135: 139–148. Schwartzkroin, P.A., and Walsh C.A. 2000. Cortical malformations and epilepsy. Ment Retard Dev Disabil Res Rev 6: 268–280. Sisodiya, S.M. 2003. Mechanisms of antiepileptic drug resistance. Curr Opin Neurol 16: 197–201. Spencer, S.S. 2002. Neural networks in human epilepsy: evidence of and implications for treatment. Epilepsia 43: 219–227. Stables, J.P., Bertram, E.H., White, H.S., Coulter, D.A., Dichter, M.A., Jacobs, M.P., Loscher, W. et al. 2002. Models for epilepsy and epileptogenesis: Report from the NIH workshop, Bethesda, Maryland. Epilepsia 43: 1410–1420. White, H.S. 2003. Preclinical development of antiepileptic drugs: past, present, and future directions. Epilepsia 44(Suppl 7): 2–8.
Index
Note: Page numbers followed by f indicate illustrations; those followed by t indicate tables.
A Absence seizures/epilepsy, 6, 6t, 8t, 68, 73–86, 229–230 acutely dissociated cell preparations, 18–19 atypical, 122–123 calcium channelopathy, 229–230 callosal model, 80–85, 82f, 83f characteristic features, 112t chemical kindling, 380–381 clinical presentation, 73 corpus callosum, 81–85 corticocortical models, 80–85, 82f, 83f EEG findings, 73, 74t, 75, 122 ferret dorsal lateral geniculate nucleus model, spike-wave discharges, 77–79, 78f, 79f genetic models, 73–75, 74t mouse, 229–230 rat, 73–77, 74t, 76f, 233–243 animal issues, 234 behavioral characteristics, 235–236 chromosome mapping, 238–239 drug screening, 243 EEG characteristics, 235, 235f, 238f genetic transmission, 238 imaging, 236–237 insights into human disorders, 239–243 limitations, 239 metabolic changes, 236 methodology, 234 monitoring, 234–235 neuropathology, 236 ontogeny, 236 response to anticonvulsants, 237–238, 237t response to proconvulsants, 237–238 sensorimotor cortex, 242–243 thalamic hypersynchronization, 239–242, 240f validity, 239 transgenic vs. spontaneous, 229–230 g-hydroxybutyrate model, 74t, 75 mechanisms, 122–123
overview, 73, 233–234 pathophysiology, 75–77 pharmacologic models, 74, 75, 111–123 age-related effects, 120 AY-9944, 111, 113t, 114, 115–116, 116f cell loss, 117 double-hit, 113t, 114–115, 116 drug screening, 120, 123 electrographic/EEG features, 117, 117t forebrain vs. hindbrain seizures, 117 future directions, 121–122 general description, 111–112, 112t genetics, 118–120 GHB, 111, 113, 113t, 115, 115f imaging, 118 limitations, 120–122 MAM-AY, 111, 113t, 114–115, 116 metabolic changes, 118 methods of generation, 112–115 modeling applications, 112 molecular changes, 118–120 mortality, 120 neuropathology, 117–118 penicillin, 111, 113t, 114 plasticity, 117–118 primary generalized epilepsies, 122 production, 120 PTZ, 111, 113–114, 113t, 115 reactive gliosis, 117 reproducibility, 120, 121t response to anticonvulsants, 120 in rodents, 111–123 seizure severity, 116 status epilepticus, 116 symptomatic generalized epilepsies, 122–123 THIP, 111, 112–113, 113t, 115 usefulness for treatment, 123 vs. clinical seizures, 111, 112t, 121t remission, 73, 74t, 233 spike-wave discharges, 73, 74t, 75–85, 122, 233. See also Spike-wave discharges, in absence epilepsy
669
spontaneous mutation models, 229–230 syndromes, 233 thalamic models, 75, 77–78, 78f, 79f, 85, 239–242, 240f thalamocortical models, 74t, 75–77, 76f, 79–80, 80f, 85, 240–243, 240f Acetylcholine-related substances, 137–139, 139t, 433 behavioral manifestations, 606–607 Acquired epilepsy. See Chronic epilepsy Action potentials acutely dissociated cell preparations, 17–19, 19f cell cultures, 26 Acute brain slices animal age, 64 epileptiform activity, 36–37 hippocampal, 59–69. See also Hippocampal slices imaging, 39–40 from normal vs. epileptic animals, 64 preparation, 38–39, 59–60 staining, 39–40. See also Staining in vitro seizure models, 36–43 Acutely dissociated cell preparations, 15–21. See also Cell culture models advantages, 15 animal characteristics, 16 applications, 18–20 burst electrogenesis, 18–20 disadvantages, 15–16 dissociation techniques, 16–17 electrophysiologic discharge patterns, 18, 19f generation of, 16–18 genetic models, 20 human tissue, 93 intrinsic neuronal behavior, 17–18 ionic current studies, 17–19, 19f modeling applications, 18–20 receptor properties, 18 technical aspects, 16–18 Acute seizure models, 2
670 Afterdischarges, 155–158. See also Electrically induced afterdischarges kindling and, 355, 371, 372–373, 373t, 374, 395, 397, 398 Age differences, 13, 608 absence seizures, 120 acute brain slices, 64 alcohol withdrawal seizures, 162 behavioral manifestations, 608 cerebral metabolism, 585 hypoxia-induced seizures/hypoxic encephalopathy model, 327–328, 328f, 329 kindling, 399 middle cerebral artery occlusion model, 508 morphologic changes, 645–646 protothrombosis model, 517 spontaneous seizures, 579–580 status epilepticus, 462, 585, 586–587 Agnosia, visual, in kindled cats, 367, 367t Air blowing, as Mongolian gerbil seizure trigger, 275–276, 283, 285, 288 AKv1.1a transgenic mouse model, 205–208, 206t Alcohol intoxication behavioral rating scale, 162t mechanisms, 162t Alcohol withdrawal seizures, 161–173 alcohol administration methods, 162–164, 163f alcohol dependence induction, 162–164 anticonvulsant efficacy, 169–170 audiogenic, 164, 164t, 165–167 behavioral features, 162t, 164t, 165, 165t blood alcohol concentration measurement, 164 blood sampling methods, 164 brain mechanisms, 165, 166–167, 171–173 cellular/molecular mechanisms, 169–170 electrophysiologic studies, 166–167 genetics, 168 handling-induced, 164, 165–166, 165t, 166f, 167 in humans, 161, 171–173 gender differences, 162 relevance of rodent model, 171–173 local cerebral glucose utilization, 168 metabolic changes, 168–169 model selection, 165–166 neuropathology, 167 neuroplasticity, 172 overview, 161–162 repeated withdrawal, 172 seizure monitoring, 164, 164t, 165t single-neuron firing, 166–167 species/strain/gender/age factors, 162 spontaneous, 164, 165 susceptibility to, 166, 168 Allylglycine model, 128t, 131–132, 132f Alternative therapies, 5 Alumina cream model, 143, 265–268 Alumina gel injection models, 179–186 sensorimotor cortex, 180–183 behavioral and clinical features, 180 biochemical findings, 181
Index EEG features, 180 generation methods, 179–180 insights into human disorders, 183 limitations, 182–183 neuropathology, 180–181, 181f response to anticonvulsants, 181–182 seizure rates, 183 temporal lobe, 183–186 behavioral and clinical features, 183–184, 184f EEG features, 184 generation methods, 183 insights into human disorders, 186 limitations, 186 neuropathology, 184–185 plasticity and mossy fiber sprouting, 185, 185f, 186f a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). See under AMPA Amino acids, excitatory behavioral manifestations, 605–606 seizure induction, 133–137, 133t Aminophylline model, 139 4-aminopyridine, epileptiform activity and, 42 in human brain slices, 96f, 97–98, 97f Ammon’s horn sclerosis, 636. See also Hippocampal sclerosis AMPA (a-amino-3-hydroxy-5-methyl-4isoxasole propionic acid) model, 133t, 135–136 AMPA receptors behavioral manifestations, 605–606, 606f callosal model of absence seizures, 81, 83, 84 excitatory postsynaptic potentials, 27 hypoxia-induced seizures/hypoxic encephalopathy model, 325–327, 326f, 330 Amygdala alcohol withdrawal seizures, 165 alumina gel injection, 183–186 electrically induced afterdischarges in, 158. See also Electrically induced afterdischarges electrical stimulation for status epilepticus induction, 456–458, 544–545, 544t kindling, 2, 356, 356t, 357f. See also Kindling in baboons, 264 in drug-refractory epilepsy, 552–557 in kittens and cats, 366–369 in rats, 373t, 374–375 Analog recording format, 570–571, 572–573 Anesthetics, in pilocarpine model, 437 Animal models. See also Epilepsy; specific models for acute seizures, 2 advantages, 660–662 age factors, 13, 608 acute brain slices, 64 behavioral manifestations, 12–13, 601–609 chronic epilepsy, 2–3 comparative ontogeny, 608 component parts of epilepsy, 10–12, 660–662
development of current status, 662–663 recommendations/future directions, 655t, 656–657 for devising new diagnostic approaches, 3–4 for drug development, 5 of EEG patterns, 12 of epileptogenesis, 10–11, 660 established benchmarks, 662–666 future directions, 653–657 gender differences, 13, 622, 645–646 general goals, 659–660 for generalized seizures, 6t, 7 genetic factors, 13 ideal, characteristics of, 540 limitations, 659–660 multifactorial, 3, 662 parameters to measure, 12–13 for partial seizures, 6–7, 6t proconvulsants in. See Convulsant agents purposes, 1–5 rationale, 659 recommendations and priorities, 655t, 656–657, 662–666 response to therapy, 13 for seizure prevention, 5 selection of, 12, 660–662 species and strain specificity, 13 of structural abnormalities, 13 for testing new therapies, 4–5 for understanding basic mechanisms, 1–5 utility, 1–5, 659–667 validation, 12, 666–667 in vivo vs. in vitro, 12 Antibiotics absence seizures, 114 epileptogenesis in isolated guinea pig brain, 104–105, 107f topical (focal) administration, 145 Antibodies, in immunocytochemistry, 643–644 Anticonvulsants for absence seizures, 243 for alcohol withdrawal in seizure prevention, 169–170, 170t in treatment, 170t, 172–173 discovery/development, 539–547. See also Drug screening efficacy, 661–662 mechanism of action, 556 in cell cultures, 29–30 refractory disease. See Drug-refractory epilepsy response to. See also Drug screening in alumina gel injection models, 181–182 chemical kindling, 386, 387t–388t, 389 in cortical freeze lesion model, 300, 301 in cortical malformation models, 256 in electrical stimulation models for status epilepticus, 454 in febrile seizure model, 338 in genetic absence seizure model in rats, 237–238, 237t in hypoxia-induced seizures/hypoxic encephalopathy model, 325
Index in kindled animals, 360, 361, 368, 375–376, 387t–388t, 389, 542–544, 546 in middle cerebral artery occlusion model, 507 in Mongolian gerbil model, 280–283, 280t–282t in pilocarpine model, 437, 562–564, 562f in tetanus toxin model, 411, 412 in tumor-associated epilepsy model, 528 therapeutic assays for, 539–547. See also Drug screening therapeutic index, 389 Antimony model, 143 Antiproliferative agents, in lesion models, 306–307 Antiseizure agents. See Anticonvulsants Anxiety tests, 622–623, 623f Apes genetic epilepsy, 261–265 spontaneous seizures, 262, 263 Apoptosis glutamate and, 50 in hippocampal sclerosis, 50 kindling-related, 400 Area tempestas, kindling in, 373, 373t ARX mutations, cortical malformations and, 257 Astatic seizures, 6, 6t, 8t Astrocytes kindling and, 401 in posttraumatic epilepsy, 474 Astrocytomas. See Tumor-associated epilepsy model Atonic seizures, 6, 6t, 8t ATPases, in pilocarpine model, 442 Audiogenic kindling, 266, 267 Audiogenic seizures alcohol withdrawal, 164, 164t, 165–167. See also Alcohol withdrawal seizures behavioral rating scale, 164t cerebral blood flow mapping, 587 Auditory evoked potentials, alcohol withdrawal seizures and, 171 Aura, 6 Autofluorescent staining, 39–40, 47, 48f, 642. See also Staining Autoimmune mechanisms, in Rasmussen’s encephalitis model, 537 Automatisms, 603–604. See also Behavioral manifestations Autoradiography, 583–589 advantages, 583, 597 age-related changes in cerebral metabolism, 585, 586f Alzheimer’s disease, 597 blood-brain barrier permeability, 588–589 catheterization, 584 cerebral blood flow/metabolism and brain damage, 587–588, 588f cerebral glucose use during seizures, 584–585 circuits in acute limbic seizures, 585–586 limitations, 583–584, 597 local cerebral blood flow measurement, 587
long-term metabolic changes from acute sustained seizures, 586–587 quantitative vs. qualitative approach, 584 sources of error, 584 technical aspects, 583–589 temporal resolution, 584 three-dimensional, 589, 589f 2DG method, 584 Axonal plasticity, 634. See also Neuroplasticity Axonal sprouting, 638. See also Mossy fiber sprouting; Neuroplasticity in kainate model, 424, 424f, 426–428 kindling-related, 359, 400–401 in lateral fluid-percussion brain injury model, 470, 471, 472f, 473, 474 posttraumatic, 52–54, 53f seizure-induced, 51 AY-9944 model, 111, 113t, 114, 115–116, 116f 5-Azacytidine, for lesion models, 306
B Baboons genetic epilepsy, 261–265 kindling in, 264 spontaneous seizures, 262, 263 Balance beam test, 617 Bang-sensitive Drosophila, 189 Barbiturates. See also Anticonvulsants and specific agents withdrawal seizures from, 170–171 Basal dendrites, 634 BCNU, for lesion models, 306–307 Behavioral/cognitive testing, 613–625. See also Cognition animal issues, 615–616, 616t anxiety tests, 622–623, 623f, 624t balance beam test, 617 concepts and terminology, 614–615 elevated plus-maze, 622–623, 623f, 624t elevated T-maze, 623 general principles, 615–616 genetic mutants, 616 handling test, 624, 624t home cage intruder test, 623f, 624, 624t laboratory variables, 616, 616t locomotion and exploration, 617f, 618, 624t memory, 614–615, 614f, 618–621, 619t, 620f, 624t open field test, 617f, 618, 623, 624t overview, 613–615 parameters, 604, 604t, 605t playground maze, 618 radial arm maze, 620f, 621–622, 624t reproducibility, 616 rotarod test, 617, 617f, 624t scoring systems, 604, 604t, 605t seizure effects, 616–622 seizure incidence, 604 seizure severity, 604 seizure threshold, 604 sensorimotor function and reflexes, 617–618, 617f, 624t socialization tests, 623f, 624, 624t
671 social recognition task, 623f, 624, 624t stress tests, 622–623, 623f, 624t visual-spatial learning, 618–621, 619t, 620f, 624t water maze, 618–621, 619t, 620f, 624t Behavioral manifestations, 601–608 age considerations, 608 atypical, 603–604 automatisms, 603–604 clonic seizures, 602, 603f, 606 convulsant dose, 607 convulsant route of administration, 607–608 developmental aspects, 608 emprosthotonos, 606, 606f evaluation of. See Behavioral/cognitive testing factors influencing, 601–602 kindling, 358 motor seizures, 602–603 neurotransmitters, 605–607 normal, 601–602 seizure induction methods, 607–608 tail twisting, 606, 606f tonic-clonic seizures, 603, 603f, 606 wet dog shakes, 134, 358, 420f, 606, 606f Benzodiazepines. See also Anticonvulsants and specific agents for alcohol withdrawal, 169–170, 170t, 172–173 response to, in alumina gel injection models, 182 withdrawal seizures from, 170–171 Beta-carboline model, 128t, 132–133 Bicuculline, 128t, 130–131, 130f behavioral manifestations, 608 in human brain slices, 95–97 in isolated guinea pig brain, 104–105, 106, 106f systemic administration, 130–131, 608 topical (focal) administration, 144t Blindness, psychic, in kindled cats, 367, 367t Blood-brain barrier permeability, autoradiographic measurement, 588–589 Blood flow autoradiographic measurement, 587–588 in epileptiform activity, 107 link with metabolism, brain damage and, 587–588, 588f mapping, 587 Bradykinin, in pilocarpine model, 444 Brain, structural abnormalities, 12–13. See also Cortical dysplasia rat models of, 249–258 Brain-derived neurotrophic factor, in pilocarpine model, 440, 444 Brain injury vasogenic, autoradiography and, 587–589 posttraumatic. See Posttraumatic epilepsy seizure-induced. See specific models testing for. See Behavioral/cognitive testing Brain metabolism. See Metabolism Brain slices acute. See Acute brain slices ex vivo, vs. organotypic slice cultures, 47
672
Index
Brain slices (continued) ferret dorsal lateral geniculate nucleus, spikewave discharges in, 77–79, 78f, 79f human, 89–99. See also Human brain slices organotypic. See Organotypic brain slice cultures Brainstem auditory pathway, alcohol withdrawal seizures and, 171 Brainstem seizures in baboons, 262 in genetically epilepsy-prone rats, 266–268 Brain tumors, 527–532. See also Tumorassociated epilepsy model Burst electrogenesis in acutely dissociated cell preparations, 18–20 in cell cultures model, 30 g-Butyrolactone model, of absence seizures, 75
C [14C]2DG method, 584–587. See also Autoradiography CA1 pyramidal cells, 638 Cables, recording system, 575–576, 575f, 576f, 579, 580 Cacna1a knockout mouse, 206t, 214 Cacna1g knockout mouse, 206t, 214 Caenorhabditis elegans, lissencephaly gene mutant, 189 Caffeine model, 139 Cages, video monitoring, 576–578, 577f Calbindin, in in utero irradiation model, 318–319 Calcium, decreased absence seizures, 229–230 epileptiform activity, 40–41 in hippocampal slices, 66, 67f Calcium ATPases, in pilocarpine model, 442 Calcium currents. See also ion channels/currents in absence seizures, 229–230 in acutely dissociated cell preparations, 17–19, 18–19, 19f anticonvulsant activity, 556 in cell cultures, 26 Callosal model, of absence seizures, 80–85, 82f, 83f Callosal slices, preparation of, 82–84, 82f, 83f Callosotomy, 81, 85 Calretinin, 283 Cameras, 570–571 Cancer. See Tumor-associated epilepsy model Cannabinoids, anticonvulsant properties, 439 Carbachol, 433 Carbamazepine for alcohol withdrawal, 169–170, 170t, 172 in pilocarpine model, 437 response to, in kindled rats, 553, 555t, 556, 564 tetanus toxin model, 411 b-Carboline model, 128t, 132–133 Cardiazol. See PTZ model Carmustine, for lesion models, 306–307
Catheterization, for autoradiography, 584 Cats, kindling in, 365–369 Cdk5, 630 Cell, nerve. See Neuron(s) Cell counting, 642 Cell culture models, 23–32 chronic cultures on microchips, 31 drug mechanisms of action, 29–30 excitatory synapses, 27 excitotoxicity, 29 focal epileptogenesis, 30–31 historical perspective, 23–24 inhibitory synapses, 26–27 ionic currents, 30 limitations, 31 methodology, 24–25, 25f modeling applications, 31–32 overview, 23–24 single-cell molecular profiling, 28–29 species used in, 24 synaptic interactions, 30 synaptic plasticity, 27–28 tissue used in, 24, 25f utility, 26–31 Cell dissociation techniques, 16–17. See also Acutely dissociated cell preparations Cell labeling, 642–643 Cell signaling, in pilocarpine model, 439–440 Central nervous system depressants alcohol withdrawal seizures and, 161–170, 171–173. See also Alcohol withdrawal seizures nonalcoholic withdrawal seizures and, 170–171 Central nervous system lymphoma. See Tumorassociated epilepsy model Cephalosporins, topical (focal) administration, 145 Cerebral blood flow autoradiographic measurement, 587–588 in epileptiform activity, 107 link with metabolism, brain damage and, 587–588 mapping, 587 Cerebral cortex in absence seizures, 74t, 75–77, 76f, 85, 240–243, 240f, 242–243 malformation of. See Cortical dysplasia; Cortical malformation models Cerebral glucose utilization absence seizures in rats, 236 alcohol withdrawal seizures, 168 autoradiographic measurement, 584–585 decreased. See Hypoglycemia spike-wave discharges, 236 Cerebral heterotopia, in MAM model, 308–309, 308f–310f, 312 Cerebrovascular accidents. See Stroke models C-fos in kindling imaging, 383–385 in zebrafish model, 194 Chamber, inhalation, 140, 140f, 141 Channelopathies. See Ion channels/currents Chemical kindling, 379–389. See also Kindling, chemical
Chemically induced acute seizures, 127–146 absence, 74, 75, 111–123. See also Absence seizures/epilepsy, pharmacologic models convulsant administration in. See also Convulsant agents by inhalation, 141–143, 546 by topical (focal) application, 143–146, 144f, 144t Chemoconvulsants. See Convulsant agents Childhood epilepsy absence, 73, 74t. See also absence seizures/epilepsy pathophysiology, 75–77 spike-wave discharges, 74t, 75–79, 78f, 79f drug screening, 545–546 model organisms for, 189. See also Zebrafish model Chlormethiazole, for alcohol withdrawal, 169–170, 170t, 172 Cholinergic system, in seizure induction, 137–139, 139t, 433–434. See also Pilocarpine model Chromosome mapping, absence seizures in rats and, 238 Chronic epilepsy, hyperexcitability in, 427–428, 427f, 430 Chronic epilepsy models, 2. See also Status epilepticus models defining features, 420–422 epileptogenic mechanisms, 429–430 isolated guinea pig brain, 107–108 kainate-induced status epilepticus, 415–430 latent period, 420–421, 423f pilocarpine, 138, 346–347, 433–444 progression, 420–421 seizure frequency, 420–421, 422f Chronic partial cortical isolation model, 477–490 anatomic/pathologic features, 478, 485–486, 486f animal issues, 480 antiepileptogenesis, 487 behavioral features, 484 cellular recordings, 483, 485 characteristic/defining features, 484 critical period for epileptogenesis, 487 deafferentiation, 478 ease of development, 488 EEG features, 479f electrode implantation, 481 electrophysiologic features, 484–486 field potential recordings, 482–483, 484–485 focal injury, 478–479 future challenges, 488–489 general description, 477–478 insights into human disorders, 489–490 latent period, 477 limitations, 487–488 methodology, 478f, 480–483 naturally occurring injury, 480 neuroplasticity, 486, 486f pathology modeled by, 478–480 reliability, 488 surgical technique, 478f, 480–481
673
Index tetrodotoxin effects, 487, 490 timing of lesions, 488 usefulness, 477–478, 488 video-EEG monitoring, 484 in vitro recordings, 477–478, 481–485, 482f, 483f Circle of Willis, in isolated guinea pig brain, 104, 105f Clcn2 knockout mouse, 206t, 214 Clonazepam in pilocarpine model, 437 response to, in alumina gel injection models, 182 Clonic bursting phases, 61 Clonic seizures, behavioral manifestations, 602, 603f, 606 Cobalt model, 143 Cognition. See also Learning; Memory brain substrates, 614–615, 615f components, 614–615 evaluation of, 613–625. See also Behavioral/cognitive testing kindling, 388–389, 401–402 Complex febrile seizures. See Febrile seizure model Complex partial seizures, 6, 6t, 8t, 421–422. See also Temporal lobe epilepsy duration, 421–422 with secondary generalization, kindling as model of, 381 vs. absence seizures, 421–422 Computed tomography, 596–597 advantages and limitations, 597 Conditional knockouts, 209, 212, 213f Connector board, 575, 576f Convulsant agents. See also Chemically induced acute seizures; Pharmacologic models and specific agents absence seizures, 237–238 acetylcholine-related, 137–139 in chemical kindling, 379–389. See also Kindling, chemical dose, 607 excitatory amino acid–related, 133–137, 133t first-pass effect, 127 GABA-related, 128–133, 128t hypoxia-induced seizures/hypoxic encephalopathy model, 327 inhalation of, 141–143 in drug screening, 546 insulin-induced hypoglycemia, 140–141, 140f, 607 for kindling, 379, 380t, 381–382. See also Kindling, chemical miscellaneous, 139–141 routes of administration, 607–608 strychnine, 139 systemic injection, 127–141, 608 technical aspects, 127 topical (focal) application, 143–146, 607 Corpus callosum, intracortical excitability and, 81–85 Corpus callosum model, of absence seizures, 81–85
Cortical dysplasia, 12–13. See also under Morphological acquired. See Cortical malformation models, injury-based classification, 630 cortical freeze lesion model, 295–301 cytologic abnormalities, 632–638 definition, 630 dendritic morphology, 632–634 GABA receptors, 640–641 genetic. See Cortical malformation models, genetic glutamate receptors, 640 ion channels, 641 MAM model, 305, 307–312 drug-refractory epilepsy, 564–565 mechanisms, 630 molecular plasticity, 640–641 neuronal cell loss, 634–638, 635f, 637f rat models, 249–258 species differences, 636 spontaneous seizures, 311, 312 synaptic reorganization, 638–640 in utero irradiation model, 315–321 Cortical focal epilepsy, alumina gel injection model, 179–183 Cortical freeze lesion model, 295–301 advantages, 300 animal issues, 296 behavioral features, 297–298 characteristics/defining features, 297–300 drug screening, 300, 301 EEG features, 298, 300–301 general description, 295 imaging, 299 insights into human disorders, 301 limitations, 300–301 metabolic changes, 299 methodology, 296, 297f molecular changes, 299–300 monitoring, 296–297 neuronal migration disorders, 295 neuropathology, 295–296, 298–299, 298f, 299f response to anticonvulsants, 300, 301 Cortical interneurons loss of. See also Neuron(s), loss of in alumina gel injection models, 180–181 in cortical freeze lesion model, 300 in flathead mutant rats, 253, 321 in genetic cortical malformation models, 253–255, 321 in humans, 321 in kainate model, 424, 424f, 426–428, 429 kindling-related, 359, 385, 400, 401–402 in Mongolian gerbil model, 283–284 in pilocarpine model, 437 in status epilepticus, 359 in in utero irradiation model, 318–319, 320–321 molecular profiling of, 28 Cortical malformation models, 295–321. See also under Cortical dysplasia; Morphological
genetic, 249–258, 630–632 behavioral features, 250t, 251–253 EEG findings, 251–253, 252f epileptogenic mechanisms, 257–258 future directions, 258 general clinical features, 249–251 insights into human disorders, 257–258 mechanisms of developmental disruption, 250t, 256–257 neuropathology, 250t, 253–255 neurophysiology, 255–256 response to anticonvulsants, 256 seizure rate, 250t seizure types, 250t, 251–253 injury-based antiproliferative agents, 306–312 5-azacytidine, 306 carmustine, 306–307 cortical freeze lesion, 295–301 MAM, 305, 307–312, 632 methyl-mercury, 306 nitrosoureas, 306 in utero irradiation, 315–321 Cortical protothrombosis. See Protothrombosis model Corticocortical models, of absence seizures, 80–85, 82f, 83f, 85 Cryptogenic epilepsy, 9 Cultures dissociated cell, 23–32. See also Cell culture models organotypic slice, 37–43. See also Organotypic brain slice cultures Currents. See Ion channels/currents Cycad plants, 307 Cyclin-dependent kinase 5, 630–632 Cyclosarin, 138–139, 139t Cysticercosis, 522–524
D Danio rerio. See Zebrafish model DAT protein, alcohol withdrawal seizures and, 168 DCX mutations, cortical malformations and, 257 Declarative memory, 614f, 615 Deep endopiriform nucleus, kindling in, 373, 373t Dendritic hyperexcitability, posttraumatic, 53, 54–55, 55f Dendritic plasticity, 634. See also Neuroplasticity Dendritic remodeling, posttraumatic, 474 Dendritic spines, loss of, 50–51, 632–634 Dendritic swelling, 632–634 Dentate gyrus, kindling-related alterations in, 402 Developmental abnormalities. See Cortical dysplasia; Cortical malformation models Diazepam for alcohol withdrawal, 169–170, 170t, 172 in pilocarpine model, 439 withdrawal from, 171
674 Diet, ketogenic, 5 Diffusion-weighted imaging, 594–595. See also Magnetic resonance imaging Digital recording systems, 573–574, 580. See also Seizure monitoring Diphenylhydantoin, in pilocarpine model, 437 Disease-modifying therapies, 546–547 Dissociated cell preparations. See Acutely dissociated cell preparations; Cell culture models Dizocilpine, for alcohol withdrawal, 169–170, 170t, 172 DMCM model, 128t, 132–133 DNA microarrays, for human brain slices, 94 Domoic acid model, 135, 416 Dopamine, in pilocarpine model, 438 Dopamine transporter (DAT) gene, alcohol withdrawal seizures and, 168 Dorsal hippocampus. See also Hippocampus kindling in, 356t, 358, 374 in rats, 373, 373t, 374 Double-hit model, 113t, 114–115, 116 Drop attacks, 6 Drosophila, bang sensitive, 189 Drug-refractory epilepsy associated syndromes, 655t classification, 655t epidemiology, 655–656, 656t etiology, 655–656 incidence and prevalence, 655–656, 655t Drug-refractory epilepsy, drug screening for, 546 Drug-refractory epilepsy models, 551–565 general description, 551–552 genetics, 555–556, 557 kindled rats, 552–557, 553f, 554f, 555t MAM, 564–565 mechanisms of pharmacoresistance, 552 post-status epileptic models of temporal lobe epilepsy, 559–564 PTZ, 564 6 Hz psychomotor seizure model, 557–559 terminology, 551–552 in vitro, 565 Drug screening, 4–5, 539–547. See also Anticonvulsants, response to alcohol withdrawal seizures, 169–170, 170t animal models in, 540t relevance to human disorders, 547 chemical kindling, 386, 387t–388t, 389 cortical freeze lesion model, 300, 301 current era of, 540–541 differentiation of anticonvulsant activity, 543–544 disease-modifying drugs, 546–547 drug-refractory epilepsy, 546 early identification of anticonvulsant activity, 540–541, 540–543, 542t electroshock seizures, 153–155, 154f, 541, 542t, 543, 543t febrile seizure model, 338 future directions, 544–547 genetic epilepsy models, 268, 545 ideal models, 540
Index kindling models, 360, 361, 368, 375–376, 387t–388t, 389, 542–544, 542t, 546 limitations, 544 low-frequency (6 Hz) seizure test, 542t, 543, 543t maximal electroshock model, 541 middle cerebral artery occlusion model, 507 Mongolian gerbil model, 280–283, 280t–282t National Institutes of Neurological Disorders and Stroke, 540 overview, 539–540 pediatric epilepsy models, 545–546 pharmacoresistant epilepsy models, 546 pilocarpine model, 437, 560–562 primary screens, 540–543, 542t PTZ model, 541 secondary screens, 543–544 spontaneous seizure models, 544–545, 544t tetanus toxin model, 411, 412 therapeutic index, 389 Woodbury criteria, 195 zebrafish model, 190–191, 195, 196
E Electrical kindling. See Kindling Electrically induced afterdischarges, 155–158 in amygdala, 158 in entorhinal cortex, 158 in hippocampus, 64–66, 65f, 157 in kindling, 355, 371, 372–373, 373t, 374, 395, 397, 398 in neocortex, 156–158, 157f in piriform cortex, 158 Electrical stimulation models, 153–158 afterdischarges, 155–158, 157f. See also Electrically induced afterdischarges in brain slices, 42 electroshock seizures, 153–155, 154f, 541 for recurrent seizures in immature brain, 347–349 in immature rats, 347–349 status epilepticus, 449–462 age factors, 462 amygdala stimulation, 456–458 continuous hippocampal stimulation, 460–462 drug-refractory epilepsy, 559, 562–564, 563f general features, 449–450 in immature rats, 347–349 insights into human status epilepticus, 462 model development, 449–450 outcome measures, 461–462 overview, 449 perforant path stimulation in adult rats, 450–455 in anesthetized rats, 458–460 in immature rats, 455–456 in mice, 456 spontaneous seizures, 544–545, 544t usefulness, 462
Electrodes implantation, 578–579. See also specific models placement, for kindling, 350–354 types, 579 Electroencephalography, 3–4, 12, 30. See also specific epilepsies and models afterdischarges, 155. See also Electrically induced afterdischarges analog systems, 572–573 digital systems, 573–574, 580 digitization frequency, 573–574 electrical artifacts, 578 fast ripples, 3 with functional MRI, 4 monitoring, 569–582. See also Seizure monitoring seizure prediction, 3 spike-wave discharges, in absence seizures, 74t, 75–77, 76f surrogate markers of epileptogenicity and epileptogeneis, 3, 12 Electrogenesis, burst in acutely dissociated cell preparations, 18–20 in cell cultures model, 30 Electromyography, in tetanus toxin model, 408 Electrophysiologic studies, 3–4, 13. See also specific types Electroshock seizures, 153–155, 154f, 541. See also Electrical stimulation models drug-refractory epilepsy, 557–559 drug screening, 541, 542t, 543, 543t recurrent seizures in immature brain, 347–349 Elevated plus-maze, 622–623, 623f, 624t Elevated T-maze, 623 Emprosthotonos, 136, 606, 606f EMX2 mutations, cortical malformations and, 257 Encephalitis, 521–525. See also Infectioninduced seizure models Encephalopathy, epileptic, 10 hypoxia-induced seizures/hypoxic encephalopathy model, 323–330 End-folium sclerosis, 636 Entorhinal cortex alumna gel injection in, 183–186 electrically induced afterdischarges in, 158 in temporal lobe epilepsy, 60–61 Entorhinal cortex–hippocampal slices. See Acute brain slices Enzymes, for cell dissociation, 16–17 Epidemiology, 653–654 Epilepsia partialis continua, 411, 412 Epilepsy. See also Seizure(s) associated syndromes, 653–654, 654t chronic phase, 68 classification, 7–10, 9t, 653–654 cryptogenic, 9 definition, 5 drug-refractory. See Drug-refractory epilepsy epidemiology, 653–654 epileptogenesis, 68. See also Epileptogenesis etiology, 653–654, 654t
675
Index familial, 9–10 functional consequences, 388–389 generalized, 7, 9t genetic, 2–3. See also under Genetic idiopathic, 7–9, 9t, 653–654 incidence, 653–654 latent period, 68 localization-related, 7, 9t mendelian inheritance of, 223–234 mesial temporal lobe. See Mesial temporal lobe epilepsy; Temporal lobe epilepsy posttraumatic, 564–590. See also Posttraumatic epilepsy prevalence, 653–654 progressive nature of, 361. See also Kindling risk factors, 653–654 symptomatic, 7–9, 9t Epileptic afterdischarges. See Afterdischarges Epileptic encephalopathy, 10 Epileptic focus, 7 Epileptic seizures. See Seizure(s) Epileptic spasms, 7 Epileptiform activity. See also Seizure(s) blood flow, 107 dendritic spine loss, 50–51 neuronal death, 50 neuronal injury, 51–52, 52f neurovascular-neuronal interactions, 107 potentiation of excitatory synaptic transmission, 52, 52f signal transduction, 439–440 Epileptogenesis, 10–11, 660 absence seizures, 111–123 cell cultures, 30–31 chemically induced, 127–143. See also Chemically induced acute seizures growth factors, 440 inflammatory mediators, 441–442 kindling. See Kindling seizure-induced cell injuries, 51–52, 52f surrogate markers, 3 temporal lobe, in isolated guinea pig brain, 105 in vitro isolated pig brain model, 103–108 Ethanol. See Alcohol withdrawal seizures Ethosuximide, in 6 Hz test, 545, 545t Ethylenediamine model, 139 N-Ethyl-N-nitrosurea, for lesion models, 306 Etiology, 645t, 653–654 Excessively bursting neurons, 310, 311f Excitatory amino acid–related substances behavioral manifestations, 605–606 seizure induction, 133–137, 133t Excitatory neurons inhibitory activity, 28 molecular profiling, 28 reorganization of, 638 Excitatory postsynaptic currents, in corpus callosum, 81 Excitatory postsynaptic potentials, in cell cultures, 27 Excitatory synapses, in cell cultures, 26–27 Excitatory synaptic transmission, potentiation of, epileptiform activity and, 52, 52f
Excitotoxicity, 29 mechanisms, 50 Exploratory behavior tests, 617f, 618 Expression profiling, 28–29 Extracellular ionic concentrations. See also Ion channels/currents in human brain slices, 93
F FADH staining, of brain slices, 40 Familial epilepsy syndromes, 9–10 Fast ripples, 3 Febrile seizure model, 333–339 animal issues, 334 behavioral features, 336 characteristics/defining features, 336–338 core/brain temperatures, 335, 335t, 336f drug screening, 338 EEG monitoring/features, 335–336, 336–337, 337f fever vs. hyperthermia, 334 general description, 333–334 genetics, 338 imaging, 338 insights into human disorders, 339 limitations, 338–339 methodology, 334–335, 335t molecular changes, 338 monitoring, 335–336 neuropathology, 337–338 prolonged seizures, 335 reproducibility, 339 response to anticonvulsants, 338 spontaneous seizures, 336 usefulness, 333–334 Felbamate, response to, in kindled rats, 553, 555t Ferret dorsal lateral geniculate nucleus slice, spike-wave discharges in, 77–79, 78f, 79f Ferrous chloride injection model, 143, 495–499 behavioral/clinical features, 496 EEG features, 496, 497f genetics, 497 histopathology, 496–497, 498f insights into human disorders, 498–499 methodology, 495–496 molecular changes, 497 rationale, 495 response to anticonvulsants, 497–498 spontaneous seizures, 544–545, 544t Field potential recordings in chronic partial cortical isolation model, 482–483, 484–485 in human brain slices, 91–92, 94–95, 95f, 97f First-pass effect, 127 Flathead mutant rat, cortical malformations and spontaneous seizures in, 249–250, 250t, 251, 252f, 253, 254f, 255, 256, 321. See also Cortical malformation models, genetic Flexion seizures, 136, 606, 606f
Flickering light–induced seizures, in baboons, 261–265 Flipase-frt site-specific recombinase (SSR) systems, 212, 213f FLMs (funny looking mice), 225 Floxed, 212 Fluid-percussion brain injury model. See Lateral fluid-percussion brain injury model Flumazenil withdrawal, 171 Fluorescence imaging, of brain slices, 39–40, 47, 48f Fluorescent markers, in tumor-associated epilepsy, 529–532, 530f–532f Fluorescent staining, 39–40, 47, 48f, 642 Fluorodeoxyglucose positron emission tomography (FDG-PET), 4 Flurothyl model, 141–143, 141f, 142f drug screening, 546 status epilepticus, 341–344, 342f, 344f Focal motor monkey model, 179–183 Focal seizures, 6t, 7, 8t alumina gel injection model, 179–183 in baboons, 262 in brain slices, 43 tetanus toxin model, 145–146, 407–412 Forebrain seizures in baboons, 262 in genetically epilepsy-prone rats, 266–268 triggers, 266–268 Free radicals, in pilocarpine model, 441, 443–444 Frontal cortex, kindling in, 356t, 357–358 Frt site-specific recombinase (SSR) systems, 212, 213f Functional magnetic resonance imaging, 4, 595–596. See also Magnetic resonance imaging in absence epilepsy rats, 236–237 in cortical freeze lesion model, 299 with EEG, 4 Functional neuroimaging, 4 Funny looking mice, 225
G GABA absence seizures, 237–238, 241–242 behavioral manifestations, 605 burst electrogenesis, 30–31 cell cultures, 26–27, 28, 30–31 cortical freeze lesion model, 299–300 intraictal synthesis, 28 postsynaptic inhibition, 26–27, 28 synthesis, 132, 132f withdrawal seizures, 171 GABAergic neurons alumina gel injection models, 180–181 changes in, 638–640 loss of, 636. See also Neuron(s), loss of in utero irradiation model, 318–319 GABAergic neurotransmission, 636–640 absence epilepsy, 237, 241–242 cortical freeze lesion model, 300, 301 disinhibition in hippocampal slices, 65f, 66
676 GABAergic neurotransmission (continued) kindling, 402 Mongolian gerbils, 278t–279t, 285–288, 286t pilocarpine model, 439 tetanus toxin model, 411, 412 Gabapentin for alcohol withdrawal, 169–170, 170t, 173 response to, in kindled rats, 553, 555t GABA-potentiating drugs, withdrawal seizures, 170–171 GABA receptor complex allosteric modulators, mechanism of action of, in cell cultures, 29 GABA receptors, 640–641 absence seizures, 76–77, 237, 241–242 alcohol withdrawal seizures, 169 cortical freeze lesion model, 299–300, 301 hypoxia-induced seizures/hypoxic encephalopathy model, 325 kindling, 388 structure, 640–641 GABA-related substances, seizure induction by, 128–133, 128t GABA uptake inhibitors, mechanism of action in cell cultures, 29 GABA withdrawal seizures, 171 Gabba1 knockout mouse, 206t, 214 Gabbr1 knockout mouse, 206t, 214 Gabrb3 knockout mouse, 206t, 214 Gabrg2 knockout mouse, 206t, 214 Gad2 knockout mouse, 206t, 215 GAERS model, 2–3, 74t, 75–77, 76f, 233–243 absence seizures in, 74t, 75–77, 76f in drug screening, 544, 545 Gamma-aminobutyric acid. See GABA GAP-43, in pilocarpine model, 440 Gender differences, 13 alcohol withdrawal seizures, 162 morphologic changes, 645–646 spatial learning, 622 Gene(s), susceptibility, 13 in baboons, 261–265 in genetically epilepsy-prone rats, 265–269 Gene expression profiling, 28–29 Gene knock-in, 212, 213f Gene knockout, 209–210 in mouse epilepsy model, 209–216. See also Mouse epilepsy models, gene replacement Generalized seizures, 6, 6t, 7, 8t convulsive, 6, 6t, 8t nonconvulsive, 6, 6t, 8t Gene replacement/deletion models, mouse, 209–216. See also Mouse epilepsy models, gene replacement Genetic Absence Epilepsy Rat from Strasburg (GAERS) model. See GAERS model Genetically epilepsy-prone rats, 261, 265–268 in drug screening, 544, 545 Genetic epilepsy, 2–3 models of. See Genetic models Genetic factors, 13 Genetic heterogeneity, 223–224
Index Genetic models absence epilepsy. See Absence seizures/epilepsy, genetic models acutely dissociated cell preparations in, 20 baboon, 261–265 cortical malformation, 249–258. See also Cortical malformation models, genetic in drug screening, 545 gene replacement, in mice, 209–216 genetically epilepsy-prone rats, 261, 265–268 in drug screening, 544, 545 Mongolian gerbils, 273–288 orphan, 225 sensory-evoked seizures, 261–268 in baboons, 261–265 in genetically epilepsy-prone rats, 265–268 spontaneous mutations in mice, 223–230 transgenic mouse, 202–209 Genetic mutations. See mutations Gene transfection, in organotypic slice cultures, 49 Geographic factors, 653–654 Gerbils. See Mongolian gerbil model GF (nerve agent), 138–139, 139t GHB model, of absence seizures, 113, 113t, 115, 115f Glial proliferation, kindling-related, 401 Glial scar, 441. See also Reactive gliosis Gliomas. See Tumor-associated epilepsy model Glucose utilization absence seizures in rats, 236 alcohol withdrawal seizures, 168 autoradiographic measurement, 584–585 decreased. See Hypoglycemia spike-wave discharges, 236 GluR3 protein, in Rasmussen’s encephalitis model, 535–537 Glutamate behavioral manifestations, 605 cell death from, 50 kindling, 388 in pilocarpine model, 439, 442, 443, 444 posttraumatic hypersensitivity to, 54–55, 55f Glutamate receptor antagonists, mechanism of action in cell cultures, 29–30 Glutamate receptors, 640 changes in, 640 excitatory postsynaptic potentials and, 27 in excitotoxicity, 29 in pilocarpine model, 437, 439 in zebrafish model, 194 Glutamic acid decarboxylase (GAD) in excitatory neurons, 28 in Mongolian gerbils, 277, 279t, 284, 285, 287 tetanus toxin model, 411 Glutamic acid decarboxylase (GAD) inhibitor model, 128t, 131–132, 132f Glutathione peroxidase, in pilocarpine model, 441 Glutathione S-transferase, in Rasmussen’s encephalitis model, 535–537 Glycosaminoglycans, in pilocarpine model, 442–443 Golgi staining, 642–643
Granule cell axonal sprouting, 638 as marker for prior seizures, 227 Green fluorescence protein, in tumor-associated epilepsy model, 529–532, 530f–532f Gria2 knockout mouse, 207t, 215 Growth factors, in pilocarpine model, 440, 444 Guinea pig brain. See Isolated guinea pig brain
H Hamartoma, hypothalamic, with gelastic features, 10 Handling-induced convulsions alcohol withdrawal, 164, 165–166, 165t, 166f, 167. See also Alcohol withdrawal seizures behavioral rating scale for, 165t mechanisms, 165 in Mongolian gerbils, 275–276, 283 Handling test, 624, 624t Headsets, 579 Head trauma. See Posttraumatic epilepsy Hemosiderin, in posttraumatic epilepsy, 466–467, 495–499. See also Ferrous chloride injection model Herpetic encephalitis, 524–525 Hilar mossy cells, loss of, 636. See also Neuron(s), loss of Hippocampal dissociated cell cultures, 23–32. See also Cell culture models Hippocampal sclerosis, 634–636 Ammon’s horn, 636 domic acid poisoning, 135, 416 end-folium, 636 in human brain slices, 98 in kainate model, 423–424, 424f in lateral fluid-percussion brain injury model, 471, 472f, 473 in mesial temporal lobe epilepsy, 2, 12, 416 in posttraumatic epilepsy, 466 in status epilepticus, 416, 423–424, 636. See also Status epilepticus studies of, using organotypic slice cultures, 50–52, 52f tetanus toxin model, 410 Hippocampal slices. See also Acute brain slices; Organotypic brain slice cultures from acute animal models, 64–67 blocking K+ channels in, 66 electrical stimulation–induced afterdischarges in, 64–66, 65f. See also Electrically induced afterdischarges elevated K+ in, 67 GABAergic disinhibition in, 64–66, 65f kainic acid–induced epileptiform activity in, 66 low extracellular Ca2+ in, 66, 67f removal of Mg2+ in, 66–67 advantages, 59–60, 67, 68 animal age, 64 from chronic animal models, 67–68 future directions, 68 general features, 59
Index limitations, 60, 68 modeling applications, 60–61, 63–68 network properties, 68 preparation, 61–63, 62f specific features, 59–60 in toto, 68 voltage-sensitive dyes, 68 vs. intact preparations, 59–60 Hippocampus alumina gel injection, 183–186 circuitry reorganization, 638–640 drug-refractory epilepsy, 556 electrical stimulation for status epilepticus induction, 460–462 kindling, 356t, 358, 402 in rats, 373, 373t, 374 place cells, 621 Histopathology, 634–645 Home cage intruder test, 624, 624t Homocysteic acid model, 133t, 137 Homocysteine model, 133t, 137 Homologous recombination, 209–210 5HT, in pilocarpine model, 438–439 Human brain slices, 89–99 analysis of channel and receptors, 94 electrophysiologic monitoring, 91–93 epileptiform activity in, vs. clinical seizure patterns, 89–90 evoked epileptiform discharges as models of epileptiform synchronization, 95–98 extracellular ionic concentration measurement, 93 field potential recordings, 91–92, 94–95, 95f, 97f hippocampal sclerosis, 98 histologic analysis, 94 intracellular recordings, 92–93 isolated neurons, 93 limitations, 89, 98–99 optical imaging, 93–94 pharmacologic manipulations, 95–98 preparation, 91 resting and passive properties of neurons, 92 spontaneous epileptiform activity, 94–95, 95f spontaneous synaptic activity, 92 synaptic activation by focal stimulation, 92–93 synaptic plasticity, 98 tissue maintenance, 91, 92f tissue procurement and handling, 90–91 tissue sources, 89, 90–91 g-Hydroxybutyrate model, of absence seizures, 74t, 75 Hyperexcitability chronic epilepsy, 427–428, 427f, 430 drug screening, 546 in kainate model, 427–428, 427f, 430 in pilocarpine model, 439 posttraumatic, 53, 54–55, 55f, 481–483, 489, 490 Hypoglycemia. See also Glucose utilization alcohol withdrawal seizures, 168 behavioral manifestations, 607
Hypoglycemic seizures, insulin-induced, 140–141, 140f, 607 Hypothalamic hamartoma with gelastic seizures, 10 Hypoxia-induced seizures/hypoxic encephalopathy model, 323–330, 544t, 546 advantages, 329 age-specificity, 327–328, 328f, 329 altered gene/protein expression, 325–327 altered network excitability, 325 animal issues, 323 behavioral features, 324 ease of development, 328 EEG features, 324, 325f, 326f excitotoxicity, 329–330 future developments, 329–330 glutamate, 330 hypoxia induction, 323–324 insights into human disorders, 330 late-onset seizures, 327–328, 329 limitations, 328–329 long-term effects, 327–328 metabolic changes, 325 methodology, 323–324 monitoring, 324 mortality, 328 neurobehavior, 327 neuropathology, 324–325 overview, 323 protective effects of pre-/post-treatment anticonvulsants, 328, 328f recurrent hypoxia, 329 reproducibility, 328 response to anticonvulsants, 325 spontaneous seizures, 544–545, 544t status epilepticus, 329–330 susceptibility to later neuronal injury, 327 susceptibility to seizures, 327 variants, 329–330
I Ictal onset, 11, 661 Ictogenesis, in cell cultures, 30–31 Ictus, 11, 660 Idiopathic epilepsy, 7–9, 9t, 653–654 Imaging. See Neuroimaging Immunization, tetanus toxin, 408 Immunocytochemistry, 643–645 Immunohistochemistry, for human brain slices, 94 Imprinting, of transgenes, 209 Incidence, 653–654 Indoklon model, 141–143, 141f, 142f Induced mutagenesis, 226–227 Inducible knockouts, 209, 212, 213f Infantile spasms, 7 Infection-induced seizure models, 521–525 general description, 521–522 herpes virus, 524–525 neurocysticercosis, 522–524 viral encephalitis, 521–522, 524–525
677 Inferior colliculus, in alcohol withdrawal seizures, 165, 166, 169, 171–172 Inflammatory mediators, in epileptogenesis, 441–442 Inhalation chambers, 141, 141f, 142 Inhalation convulsant substances, 141–143, 546 Inherited epilepsy. See under Genetic Inhibitory neurons. See also Cortical interneurons; Neuron(s) molecular profiling of, 28 Inhibitory postsynaptic potentials, in cell cultures, 26–27 Inhibitory synapses, in cell cultures, 26–27 Injury-based models. See also Cortical malformation models antiproliferative agents, 306–312 5-azacytidine, 306 carmustine, 306–307 cortical freeze lesion, 295–301 MAM, 305, 307–312, 632 methyl-mercury, 306 nitrosoureas, 306 in utero irradiation, 315–321 Inositol 1-4-5-triphosphate (IP3), in pilocarpine model, 440 Insertion site variability, 209 In situ hybridization, 644 Insulin-induced hypoglycemia model, 140–141, 140f, 607 Interictal state, 11, 660 Intermittent light–induced seizures, in baboons, 261–265 Interneurons. See Cortical interneurons; Neuron(s) Intracellular recordings, in human brain slices, 92–93 Intracellular signaling, in pilocarpine model, 439–440 Intracranial hemorrhage, hemosiderin deposition in, posttraumatic epilepsy and, 466–467, 495–499. See also Ferrous chloride injection model Intracranial tumors, 527–532. See also Tumorassociated epilepsy model Intractable partial epilepsy. See also Drugrefractory epilepsy neuronal migration and, 301 In utero irradiation model, 315–321 animal issues, 316–317 behavioral/clinical features, 317 cortical dysplasia modeled by, 315, 316, 319 cortical interneuron loss, 318–319, 320–321 EEG features, 317–318 EEG monitoring, 317 general description, 315–316 insights into human disorders, 319, 320–321 limitations, 319 methodology, 316–317 neuropathology, 318–319, 319f, 320f overview, 315 In vitro brain slices. See Brain slices
678 In vitro seizure models in acute brain slices, 36–43. See also Acute brain slices advantages, 35 epileptiform activity in 4-aminopyridine–induced, 42 elevated potassium–induced, 42 focal onset of, 43 low calcium–induced, 40–41 low magnesium–induced, 41–42 stimulation-induced, 42 imaging studies for, 39 limitations, 35 in organotypic brain slices, 37–43. See also Organotypic brain slice cultures seizure characteristics in, 35–36 seizure criteria in, 35–36 In vivo models, vs. in vitro models, 12. See also In vitro seizure models Ion channels/currents, 641 in acutely dissociated cell preparations, 17–19, 19f alcohol effects, 169 anticonvulsant activity, 556–557 in cell cultures, 26, 30 changes in, 641 in drug-refractory epilepsy, 556–557 excitatory postsynaptic, in corpus callosum, 81 gene targeting for, 199–219. See also Mouse epilepsy models in human brain slices, analysis of, 94 potassium, blockade of, in hippocampal slices, 66 Ionic concentrations, extracellular, in human brain slices, 93 Ionotropic receptors, in acutely dissociated cell preparations, 20 IP3, in pilocarpine model, 440 Iron deposition, posttraumatic, 466–467, 495–499 Iron model, 143 Iron salts. See Ferrous chloride injection model Irradiation, in utero. See In utero irradiation model Isolated guinea pig brain, 103–108 advantages, 106–107 chronic epilepsy, 107–108 general description, 103 generation of epileptiform activity, 104–106 limitations, 107–108 magnetic resonance imaging, 107, 108f modeling applications, 103–104 network interactions, 106–107 neurovascular-neuronal interactions, 107 pharmacologic manipulations, 104–106, 106f, 107–108, 107f preparation, 104 structural changes, 107 tetanic stimulation, 106 vascular anatomy, 104, 105f Isonicotinehydrazide model, 128t, 131–132, 132f
Index
J Juvenile epilepsy. See Childhood epilepsy
K Kainic acid/kainate behavioral manifestations, 605–606, 606f in hippocampal slices, 66 in hypoxia-induced seizures/hypoxic encephalopathy model, 327–328, 329–330 in Mongolian gerbil model, 283 spontaneous seizures, 544–545, 544t in status epilepticus model, 346–347, 415–430 advantages, 429 animal issues, 417–418 anticonvulsant rescue, 416 behavioral/clinical features, 134, 144, 420–422, 420f, 605–606, 606f blood-brain barrier permeability, 588–589 characteristics/defining features, 134–135, 144, 420–429 domoic acid poisoning, 416 dose titration, 418–419 drug screening, 428–429 ease of use, 429 EEG features, 134–135, 420, 420f, 428 electrophysiology, 426–428 enhancing survivability, 418 epileptogenic mechanisms, 426–428 general description, 415 genetics, 425–426 hippocampal sclerosis, 423–424, 424f hyperexcitability, 427–428, 427f, 430 imaging, 424–425 insights into human disorders, 429–430 intracranial injection, 143–145, 144f, 144t, 416–417 latent period, 420–421, 423f limitations, 135, 144–145, 429 low-dose protocol, 416–417, 418 mechanics, 429–430 mesial temporal lobe epilepsy with hippocampal sclerosis, 416 methodology, 133–134, 133t, 143–144, 416–419 molecular changes, 425–426 monitoring seizures, 418–419 monitoring status epilepticus, 419–420 mossy fiber sprouting, 424, 424f, 426–428 neuron loss, 423–424, 427–428 neuropathology, 423–426, 636 neurophysiology, 426–428 pathologies modeled by, 416 progression, 420–421 reactive gliosis, 423–424 reliability, 419 reproducibility, 429 response to anticonvulsants, 428–429 seizure duration/severity, 420–421 seizure frequency, 420–421, 422f
seizure types, 421 single intracranial injections, 416–417 stages, 415 status epilepticus definition, 419–420 systemic administration, 133–135, 133t, 134f, 134t, 135f, 416–417 topical (focal) administration, 143–145, 144f, 144t, 416–417 usefulness, 430 Kcna1 knockout mouse, 207t, 215 Kcnq2 knockout mouse, 207t, 215–216 Kcnq2 transgenic mouse, 207t, 208 Ketamine, in pilocarpine model, 437 Ketogenic diet, 5 Kindled rat model, 373t, 374–375 of drug-refractory epilepsy, 552–557 in drug screening, 542t, 543–544, 553f, 554f, 555t Kindling advanced-stage, 398 afterdischarges, 355, 371, 372–373, 373t, 374, 395, 397, 398 age, 399 amygdala, 356, 356t, 357f in kittens and cats, 366–369 in rats, 373t, 374–375 animal issues, 350 anxiety, 623 apoptosis, 400 astrocyte hypertrophy, 359 astrocyte proliferation, 401 audiogenic, 266, 267 axonal sprouting, 359, 400–401 in baboons, 264 brain changes, 388, 399–401, 402, 402f chemical, 379–389 absence seizures, 380–381 advantages, 385–386 behavioral/clinical features, 382–383, 383t, 388–389 brain changes, 388 chemoconvulsants for, 379, 380t, 381–382 criteria, 382 dose, 382–383 in drug screening, 386, 387t–388t, 389 EEG features, 383, 384f electrical kindling and, 386 epileptogenic mechanisms, 386 functional impairments, 388–389, 399–401 imaging, 383–385 induction, 379, 380t, 381–382, 397 insights into human disorders, 386–389 intracerebral infusions, 380t, 381 limitations, 385–386, 386 methodology, 381–382 mortality, 383 neuropathology, 385, 388 neurotoxin screening, 386 overview, 379 pesticides, 386 as primary generalized epilepsy model, 381 response to anticonvulsants, 386, 387t–388t, 389
Index spontaneous seizures, 383 systemic administration, 380t, 381–382 transfer effects, 386, 397 usefulness, 379–381 cognitive impairment, 388–389, 401–402 complex partial seizures with secondary generalization, 381 cross-species differences, 398 definition, 379 dentate gyrus, 360–361 developmental stage, 399 discovery, 395 in drug-refractory epilepsy, 552–557 drug screening, 360, 361, 368, 375–376, 386, 387t–388t, 389, 542–544, 546 electrical, 351–361 electrode placement, 350–354 forebrain seizures, 266–268 frontal cortex, 356t, 357–358 functional alterations, 399–401 functional impairments, 388–389, 399–401 gene expression, 399–400 genetic/molecular changes, 360 glial proliferation, 401 hippocampus, 356t, 358, 373, 373t, 374, 402 in humans, 361 imaging, 359–360 induction, 372–374, 396 insights into human disorders, 360–361 interstimulus intervals, 355–356, 398 in kittens and cats, 366–369 limbic, 2, 266, 396 in baboons, 264 long-term potentiation, 397–398, 403–404 massed simulation effects, 398, 399 memory deficits, 401–402 metabolic changes, 359–360 methodology, 351–354, 396–397 as model, 360, 368, 381, 396, 402–404 advantages, 385–386, 403 limitations, 360, 376, 402–403 in Mongolian gerbil model, 274 monitoring of, 356–360, 356t, 357f, 358f network synchronization, 395, 398, 404 neurogenesis, 400 neuron loss, 359, 400, 401–402 neuropathology, 359, 385, 388 neuroplasticity, 358, 395–396, 403–404 overview, 351, 395 perirhinal cortex, 356t, 357 permanent effects, 397–398, 403–404 persistence, 358, 395, 397–398 pilocarpine model, 434 positive transfer, 359, 379 procedures, 354–356 progression, 356–360, 356t, 357f, 358f, 361, 398, 399 Racine scale, 116, 356, 356t rapid, 398 in rats, 371–376, 373t, 374–375 afterdischarge threshold, 371, 372–373, 373t, 374 age-/site-specific protocols, 373, 373t animal issues, 371
behavioral/clinical features, 373t, 374 drug screening, 375–376 ease of development, 376 electrode implantation, 371–372, 372t genetics, 375 kindling antagonism, 374 kindling induction, 372–374 kindling persistence, 374–375 kindling rates, 374 limitations, 376 methodology, 371–374 molecular changes, 375 mortality, 376 neuroimaging, 375 neuropathology, 375 reliability, 376 response to anticonvulsants, 375–376 spontaneous seizures, 374 strain factors, 375 usefulness, 376 response to anticonvulsants, 360, 361, 368, 375–376, 387t–388t, 389, 542–544, 546 seizure phenotypes, 361 spontaneous seizures, 366–367, 369, 398, 402, 402f stereotaxic surgery, 350–351 stimulus parameters, 354–355 surgical procedures, 351 susceptibility to, 396–398, 402 synaptic transmission, 399–400 tonic-clonic seizures, 266 Kindling antagonism, 359 in rats, 374 Kindling transfer, 359, 379 Kinins, in pilocarpine model, 441–442, 444 Kittens, kindling in, 365–369 Kluver-Bucy syndrome, 368 Knock-in mice, 212, 213f. See also PTZ model Knockout mice. See also Mouse epilepsy models, gene replacement B1/B2, in pilocarpine model, 442 p35, 55, 56, 250t, 251, 253, 254, 630–632, 631f production of, 218–219
L Lamotrigine response to, in kindled rats, 553, 555t, 564 6 Hz test, 545, 545t tetanus toxin model, 411 Landau-Kleffner syndrome, 368, 369 Larval zebrafish. See Zebrafish model Latent period, 10 Lateral fluid-percussion brain injury model, 465–475 animal issues, 467 apnea, 468 behavioral features, 470–471 brain injury induction, 467–468 diurnal seizures, 471 EEG features, 470–471, 471f electrode implantation, 469
679 epilepsy occurrence, 470 follow-up, 469, 474 future challenges, 474–475 hippocampal pathology, 471, 472f, 473 histology, 470 immediate seizures, 468 interictal activity, 471 latency period, 470, 473 late seizures, 473 limitations, 474 methodology, 467–470, 468f, 469f mortality, 468, 474 mossy fiber sprouting, 470, 471, 472f, 473 neuropathology, 471–474 pathology modeled by, 473–474 remission, 471 seizure duration, 470–471 seizure frequency, 470 seizure type, 470, 471 spontaneous seizures, 469, 474, 544–545, 544t trauma severity, 473 video-EEG monitoring, 469, 474 Lateral geniculate nucleus, spike-wave discharges in, in absence seizures, 77–79, 78f, 79f Learning, 614. See also Cognition visual-spatial assessment, 618–622, 619f, 620f in humans, 622 Lennox-Gastaut syndrome, 257, 369 Leptazol. See PTZ model Lesion models. See Cortical malformation models Levetiracetam development of, 542 response to after status epilepticus, 560–562, 561f in kindled rats, 553–554, 555t, 557 in pilocarpine model, 560–562, 561f 6 Hz test, 545, 545t Limbic kindling, 2, 266, 396. See also Kindling in baboons, 264 Limbic system in alcohol withdrawal seizures, 165 seizure circuitry in, 585–586 Lindane, in chemical kindling, 383, 384f Lithium-pilocarpine model. See Pilocarpine model Local cerebral glucose utilization. See glucose utilization Locomotion tests, 617f, 618 Long-term memory, 614f, 615 Long-term potentiation kindling and, 397–398, 403–404 in organotypic slice cultures, 49 in tetanus toxin model, 411 Low-frequency (6 Hz) seizure model drug-refractory epilepsy, 557–559 drug screening, 542t, 543, 543t LoxP site-specific recombinase (SSR) systems, 212, 213f Lymphoma. See Tumor-associated epilepsy model
680
Index
M Magnesium, decreased, epileptiform activity and, 41–42 in hippocampal slices, 66–67 in human brain slices, 98 Magnetic resonance imaging, 4, 590–596 absence epilepsy rats, 236–237 animal handling, 592–593, 592f cortical freeze lesion model, 299 diffusion-weighted, 594–595 febrile seizure model, 338 functional, 4, 595–596 in absence epilepsy rats, 236–237 in cortical freeze lesion model, 299 with EEG, 4 image contrast, 593–595 instrumentation, 591–592, 591f, 592f isolated guinea pig brain, 107, 108f kainate model, 424–425 methodology, 590 software, 592 T1-weighted, 593 T2-weighted, 593 Magnetic resonance spectroscopy, 595 MAM-AY model, 113t, 114–115, 116 MAM model, 305, 307–312, 632 administration, 307–309 advantages, 307, 312 cerebral heterotopia, 308–309, 308f–310f, 312 drug-refractory epilepsy, 564–565 insights into human disorders, 310 limitations and pitfalls, 311 mechanism of action, 307 neurophysiologic effects, 309, 311f periventricular heterotopia, 312 single vs. double injection, 307–309, 308f spontaneous seizures, 311, 312 time of administration, 311 toxic effects, 307 Marijuana, anticonvulsant properties, 439 Massed stimulation effect, 398, 399 Matrix components, in pilocarpine model, 442–444, 443f Maximal electroshock seizures, 153–155, 154f, 541. See also Electroshock seizures Mazes. See also Behavioral/cognitive testing elevated plus, 622–623, 623f, 624t elevated T, 623 playground, 618 radial arm, 620f, 621–622, 624t water, 618–621, 619t, 620f, 624t Medical intractability. See Drug-refractory epilepsy Memory, 614. See also Cognition assessment, 615. See also Behavioral/cognitive testing brain substrate, 614f, 615 kindling-related deficits, 401–402 long-term, 614f, 615 nondeclarative, 614f, 615 short-term, 614f, 615 taxonomy, 614f, 615 tests for, 618–621, 619t, 620f
Mendelian inheritance, 223–234. See also under Genetic Meningiomas. See Tumor-associated epilepsy model 3-Mercaptopropionic acid model, 128t, 131–132, 132f Meriones unguiculatus. See Mongolian gerbil model Mesial temporal lobe epilepsy, 6, 8. See also Temporal lobe epilepsy chronic models of, 2–3 with hippocampal sclerosis, 2, 12, 416 kainate model, 416. See also Kainic acid/kainate neuropathology, 416 status epilepticus, 416. See also Status epilepticus Metabolic mapping, 588–589 autoradiographic, 584–585 Metabolism age-related changes, 585 autoradiography, 584–589 three-dimensional, 589, 589f brain damage and, 584–585, 587–588, 588f link with cerebral blood flow, 587–588, 588f long-term changes after seizures, 586–587 Metabotropic receptors, in acutely dissociated cell preparations, 20 Metalloproteinases, matrix, in pilocarpine model, 442–444, 443f Metal models, 143 of posttraumatic epilepsy, 466–467 Metastases. See Tumor-associated epilepsy model Methyl-6,7-dimethoxyl-4-ethyl-b-carboline (DMCM) model, 128t, 132–133 Methylazoxymethanol-acetate. See MAM model N-methyl-D-aspartic acid. See under NMDA Methyl-mercury, for lesion models, 306 Methyl-b-carboline-3-carboxylate model, 128t, 132–133 Metrazol model. See PTZ model Metrazol test, 195 Mice. See Mouse Microarrays, for human brain slices, 94 Microcephaly. See also Cortical dysplasia in in utero irradiation model, 318–319 Microchips, chronic cell cultures on, 31 MicroCT, 596–597 Microgyrus, in cortical freeze lesion model, 298–299, 299f MicroPET, 589–590, 596–597 MicroSPECT, 589–590, 596–597 Middle cerebral artery occlusion model, 501–509 age specificity, 508 animal issues, 502 behavioral features, 504–507, 504f–506f biochemical/biophysical changes, 507 drug screening, 507 ease of development, 507–508 EEG features, 503, 504f–506f, 507 electrode placement, 502–503 future challenges, 508
general description, 501–502 insights into human disorders, 508–509 latency period, 507 limitations, 507–508 methodology, 502–504 monitoring, 503 mortality, 508 neuropathology, 507 neuroplasticity, 507 pathology modeled by, 502 reliability, 503–504 remission, 504 reproducibility, 508 response to anticonvulsants, 507 seizure duration, 507 seizure frequency, 507 seizure severity, 504–507 spontaneous seizures, 504 transient focal ischemia induction, 502–503 treatment applications, 509 video-EEG recording, 503 Miller-Dieker lissencephaly, 257 Minimal electroshock seizure threshold model, 153–155, 154f, 557–559. See also Electroshock seizures drug-refractory epilepsy, 557–559 drug screening, 542t, 543, 543t Mitogen-activated protein kinases, in pilocarpine model, 440 MK-801 for alcohol withdrawal seizure prevention, 169–170, 170t in pilocarpine model, 437, 439 Molecular markers, for prior seizure activity, 227–228 Molecular techniques, for human brain slices, 94 Mongolian gerbil model, 273–288 animal issues, 273–274 anticonvulsant response, 280–283, 280t–282t brain abnormalities, 284 calretinin, 283 disinhibition hypothesis, 285–288 drug screening, 280–283, 280t–282t EEG recordings, 277–278 environmental triggers, 275–276, 283, 285, 288 GABAergic transmission, 278t–279t, 285–288, 286t genetics, 284 glutamic acid decarboxylase, 277, 279t, 284, 285, 287 handling-induced convulsions, 275–276, 283 immunocytochemistry, 283–284 insight into human disorders, 288 mechanisms, 284–288 neurotransmitters/neuromodulators, 278t–280t, 285–288, 286t–287t ontogeny of epilepsy, 274–275 overview, 273 parvalbumin, 283–284 as reflex epilepsy, 288 seizure behavior, 276–277, 278t seizure effects, 278, 278t–279t seizure incidence, 275
681
Index seizure mechanisms, 283–285 seizure-sensitive vs. seizure-resistant strains, 273–274, 284–288, 286t–287t seizure triggers, 275–276, 283, 285, 288 species-specific characteristics, 283–284 spontaneous seizures, 288 strain development, 273–274 Monkeys, alumina gel injection models in, 179–186. See also Alumina gel injection models Morphologic changes, 629–647. See also Cortical dysplasia; Cortical malformation models age/sex/strain-specific, 645–646 altered cell structure, 632–634 causal, 645, 646 cause-and-effect relation, 645 cell types, 645 circuitry reorganization, 638–640 dendritic, 632–634 developmental abnormalities, 630–632 functional significance, 645, 646 future investigations, 646–647 ion channels, 641 as markers, 645 molecular plasticity, 640–641 neuronal cell loss, 634–638, 635f, 637f receptors, 640–641 species differences, 636 static vs. dynamic, 646 synaptic reorganization, 638–640 Morphologic data cause-and-effect relation, 645 interpretation of, 645–646 quantitative vs. descriptive, 645 Morphologic techniques, 641–645 electron microscopy, 644–645 general histology and quantitative methods, 641–642 immunocytochemistry, 643–645 single-cell labeling, 642–643 Morris water maze, 618–621, 619t, 620f Mossy cells, loss of, 636. See also Neuron(s), loss of Mossy fiber sprouting, 638. See also Axonal sprouting; Neuroplasticity in alumina gel injection model, 185, 185f functional significance, 645 in kainate model, 424, 424f, 426–428 kindling-induced, 401 in lateral fluid-percussion brain injury model, 470, 471, 472f, 473 as marker for prior seizures, 228 in pilocarpine model, 438, 443 seizure-induced, 51 in tetanus toxin model, 411 Timm’s staining, 424, 425f, 426, 470 Motor seizures, behavioral manifestations, 602–603 Mouse age of, acute brain slices and, 64 alcohol withdrawal seizures in, 161–173 cortical freeze lesion model in, 295–301 cortical malformations in, spontaneous seizures and, 249–258
electrical stimulation models for status epilepticus in, 456–458 funny looking, 225 knock-in, 212, 213f knockout, 209–216, 630–632, 631f. See also Mouse epilepsy models, gene replacement in pilocarpine model, 442 production of, 218–219 mutant, development of, 225–226, 226t rocker, 226, 226t spike-wave discharges in, 234 tottering, 225, 226t, 234 transgenic, 202–209. See also Mouse epilepsy models, transgenic Mouse epilepsy models development, 199 gene replacement/deletion, 209–216 advantages, 209 Cacna1a model, 206t, 214 Cacna1g model, 206t, 214 Clcn2 model, 206t, 214 in current use, 206t–207t, 218 examples, 206t–207t, 212–216 future directions, 218–219 Gabbr1 model, 206t, 214–215 Gabra1 model, 206t, 214–215 Gabrb3 model, 206t, 214–215 Gabrg2 model, 206t, 214–215 Gad2 model, 206t, 215 gene knock-in, 212, 213f genome-environment interactions, 201–202, 202f genotype vs. phenotype modeling, 199–201 Gria2 model, 207t, 215 homologous recombination, 209–210 ineducable (conditional) knockouts, 209, 212, 213f Kcna1 model, 207t, 215 Kcnq2 model, 207t, 215 limitations and pitfalls, 215 methodology, 210–212, 210f, 213f model sources, 219 outsourced production, 218–219 overview, 209–210 p35, 55, 56, 250t, 251, 253, 254, 630–632, 631f phenotype analysis, 216–218 spontaneous vs. induced seizures, 217 variations, 212 gene- vs. phenotype-driven, 224 genome-environment interactions, 201–202, 202f genotype vs. phenotype modeling, 199–201 spontaneous mutation, 223–230 absence epilepsy phenotype, 229–230 analysis of pathogenic mechanisms, 229 EEG recordings, 227 gene-forward approach, 224, 229 gene identification strategies, 226–227 human counterparts, 225, 228–229 induced mutagenesis, 226–227 mendelian inheritance, 223–234 mutant mice for, 225–226, 226t
orthologous vs. orphan, 224–228 phenotype validation, 226–227 phenotypic variation, 228–229 strain origin, 226 surrogate markers for screening, 227–228 vs. engineered single-gene models, 224 vs. transgenic approach, 224–225 working backward from phenotype, 229 transgenic, 202–209 AKv1.1a model, 205–208, 206t basic concept, 202–203 chimeric founders, 208 examples, 205–208, 206t–207t future directions, 218–219 gender-dependent transgenes, 209 genome-environment interactions, 201–202, 202f genotype vs. phenotype modeling, 199–201 historical perspective, 199 insertional mutations, 208 insertion site and copy number variability, 209 Kcnq2 model, 207t, 208 limitations and pitfalls, 208–209 methodology, 203–205, 203f, 204f model sources, 219 multiple insertion sites, 209 outsourced production, 218–219 perforant path stimulation, 456–458 phenotype analysis, 216–218 Scn2a model, 207t, 208 spontaneous vs. induced seizures, 217 transgene instability, 209 transgene lethality, 208 vs. spontaneous mutations, 224–225 utility, 199 MPDZ protein, alcohol withdrawal seizures and, 168 Multifactorial models, 3, 662 Multiple drug resistance gene, 556 Muscarinic receptors, in pilocarpine model, 443 Mutagenesis, induced, 226–227 Mutant mice, development of, 225–226, 226t Mutations genetically engineered. See Mouse epilepsy models, gene replacement; Mouse epilepsy models, transgenic identification of, 226–227, 226t induced, 227 null, 224 point, 223–224 screening for, 227–228 spontaneous, in mice, 223–230. See also Mouse epilepsy models, spontaneous mutation surrogate markers for, 227–228 Myoclonic seizures, 6, 6t, 8t
N NAD(PH) autofluorescent staining, 40 Naloxone, 439 Narcotic receptors, 439
682 National Institutes of Neurological Disorders and Stroke, 540 NBQX, hypoxia-induced seizures/hypoxic encephalopathy model, 325, 328 Neocortex, electrically induced afterdischarges in, 156–158, 157f. See also Electrically induced afterdischarges Neonatal hyperthermia model, 544t, 546 Neonatal seizures hypoxia-induced seizures/hypoxic encephalopathy model, 323–330, 544t, 546. See also Hypoxia-induced seizures/hypoxic encephalopathy model rat model, 341–349. See also Recurrent seizures, in immature rats Nerve agents, 138–139, 139t Nerve cells. See Neuron(s) Neurocysticercosis, 522–524 Neurodegeneration, alcohol withdrawal seizures and, 167 Neurogenesis, kindling-related, 400 Neuroimaging, 3–4, 583–597 advantages and limitations, 596–597 autoradiography, 583–589. See also Autoradiography catheterization, 583–584 computed tomography, 596–597 functional, 4. See also Functional magnetic resonance imaging histologic correlates, 596–597 magnetic resonance imaging. See Magnetic resonance imaging microPET, 589–590 microSPECT, 589–590 Neuron(s). See also Cortical interneurons acutely dissociated single, 15–21 culture of, 23–32. See also Cell culture models death of glutamate and, 50 in hippocampal sclerosis, 50 kindling-related, 400 excessively bursting, 310, 311f excitatory inhibitory activity, 28 molecular profiling, 28 reorganization of, 638 GABAergic in alumina gel injection models, 180–181 changes in, 638–640 loss of, 636. See also Neuron(s), loss of in in utero irradiation model, 318–319 inhibitory, reorganization of, 638–640. See also Cortical interneurons isolated. See also Acutely dissociated cell preparations in human brain slices, 93 loss of, 634–638, 635f, 637f in chemical kindling, 385 in hippocampus. See Hippocampal sclerosis in kainate model, 423–424, 423f, 427–428, 429 kindling-related, 400, 401–402 in lateral fluid-percussion brain injury model, 471, 472f
Index in middle cerebral artery occlusion model, 507, 507f in pilocarpine model, 437 in protothrombosis model, 514 quantitative assessment, 641–642 on microchips, 31 quantitative analysis, 642–644 reticular thalamic, spike-wave discharges in, in absence seizures, 76–77, 76f, 241 single acutely dissociated, 15–21. See also Acutely dissociated cell preparations vs. intact-brain neurons, 31 Neuronal growth factor, in pilocarpine model, 440, 444 Neuronal migration disorders, cortical freeze lesion model for, 295–301 Neuropathology. See specific models Neuroplasticity. See also specific models axonal, 634. See also Axonal sprouting dendritic, 634 kainate model, 424 kindling and, 358 long-term potentiation kindling and, 397–398, 403–404 in organotypic slice cultures, 49 in tetanus toxin model, 411 mossy fiber sprouting, 638. See also Mossy fiber sprouting Neurosyphilis, 521–522 Neurotoxins, screening for, chemical kindling in, 386 Neurotransmitters/neuromodulators. See also specific types behavioral manifestations, 605–607 changes in, 640–641 in Mongolian gerbil model, 278t–280t, 285–288, 286t–287t in pilocarpine model, 438–439, 443–444, 443f Neurovascular-neuronal interactions, in isolated guinea pig brain, 107 Nitrous oxide withdrawal, 171 NMDA (N-methyl-D-aspartic acid) model, 133t, 136–137, 137f, 138f NMDA receptor antagonists, in pilocarpine model, 437 NMDA receptors alcohol withdrawal seizures, 167, 169 behavioral manifestations, 605–606, 606f callosal model of absence seizures, 167 excitatory postsynaptic potentials, 27 kindling, 399–400 in pilocarpine model, 437, 439, 444 Nonbiased stereology, 642 Nondeclarative memory, 614f, 615 Nonhuman primates. See Baboons; Monkeys Norepinephrine, in pilocarpine model, 438, 443 Null mutations, 224
O Ohtahara syndrome, 257 Oligodendrogliomas. See Tumor-associated epilepsy model
Open field test, 617f, 618, 623, 624t Opioid receptors, 439 Optical imaging, of human brain slices, 93–94 Organophosphorus compounds, weapons-grade, 138–139, 139t Organotypic brain slice cultures, 37–43, 45–55 advantages, 47 brain injury consequences, 52–55 cell death, 50 cocultures, 47–48 dendritic spine loss, 50–51 epileptiform activity, 37–38 epileptogenicity of chronic epileptiform activity, 51–52, 52f gene transfection, 49 hippocampal sclerosis, 51–52 imaging, 39–40, 47, 48f limitations, 50 long-term potentiation, 49 modeling applications, 49–55 plasticity, 49 posttraumatic epilepsy, 52–55, 53f, 55f preparation, 39, 45–46, 46f properties, 46–47, 47f staining, 39–40 survival, 47 thickness, 47 from transgenic animals, 48–49 in vitro seizure models, 37–43 vs. ex vivo slices, 47 Orphan models, 225 Otx-1-/- mouse, cortical malformations and spontaneous seizures in, 250–251, 250t, 255–256. See also Cortical malformation models, genetic
P P35-/- mouse, cortical malformations and spontaneous seizures in, 55, 56, 250t, 251, 253–254, 630–632, 631f. See also Cortical malformation models, genetic Pacemaker cells, in acutely dissociated cell preparations, 18 Papio papio, genetic epilepsy in, 261–265 Parasitic infections, 522–524 Partial cortical isolation model. See Chronic partial cortical isolation model Partial epilepsy, intractable. See also Drugrefractory epilepsy neuronal migration and, 301 Partial seizures, 6–7, 6t, 8t Parvalbumin in cortical freeze lesion model, 300 in Mongolian gerbil model, 283–284 in in utero irradiation model, 318–319 PCR (polymerase chain reaction), for human brain slices, 94 Pediatric epilepsy. See Childhood epilepsy Penicillin model, 74t, 75 absence seizures, 114 in isolated guinea pig brain, 104–105, 107f topical (focal) administration, 145 Pentamethylenetetrazol. See PTZ model
Index Pentazol. See PTZ model Pentetrazol. See PTZ model Pentobarbital withdrawal, 171 Pentylenetetrazol model. See PTZ model Perceptual memory, 614f, 615 Perforant path stimulation models, 449–462, 544t. See also Electrical stimulation models, status epilepticus Periaqueductal gray, in alcohol withdrawal seizures, 165, 167 Perigeniculate nucleus, spike-wave discharges and, 77 Perirhinal cortex alumina gel injection in, 183–186 kindling in, 356t, 357 Periventricular heterotopia, in MAM models, 312 Pesticide testing, chemical kindling in, 386 P-glycoprotein, in drug-refractory epilepsy, 557 Pharmacologic models. See also Convulsant agents and specific compounds of absence seizures, 74, 75, 111–123. See also Absence seizures, pharmacologic models of acute seizures, 127–143. See also Chemically induced acute seizures Pharmacoresistance, 551–565 animal models, 552–565. See also Drugrefractory epilepsy models causes, 552 definition, 552 Phenobarbital in pilocarpine model, 437 response to in alumina gel injection models, 181 in kindled rats, 553, 555t Phenytoin for alcohol withdrawal, 169–170, 170t, 172 in pilocarpine model, 437 response to in alumina gel injection models, 181 in kindled rats, 552–557, 553f, 554f, 556t 6 Hz test, 545, 545t Phenytoin-resistant kindled rat, 542t, 543–544 Phospholipids, inositol-containing, in pilocarpine model, 440 Phosphotyrosine proteins, in pilocarpine model, 440 Photic-flickering–induced seizures, in baboons, 261–265 Physostigmine model, 433 Picrotoxin model isolated guinea pig brain, 104–105, 106 systemic administration, 128t, 131 topical (focal) administration, 143–145, 144t Pilocarpine model, 138, 433–444 acute period of, 433 animal issues, 435–436 applications, 138 ATPases, 442 behavioral/clinical features, 436–437, 606–607 biochemical pathways, 438–444, 443f blood-brain barrier permeability, 588–589 chemical alterations, 438–444
chronic period, 434 defining features, 138 dose titration, 435–436 drug-refractory epilepsy, 559–562, 561f drug screening, 437, 560–562 EEG features, 436–437 general description, 433 generation, 138 imaging, 588f–590f in immature rats, 346–347 inflammatory mediators, 441–442 in isolated guinea pig brain, 105, 107–108 kindling, 434 limitations, 138, 562 matrix components, 442–444 methodology, 434–436 mortality, 436 natural history, 433–434 neuropathology, 437–438, 636 neurotransmitters, 438–439, 443–444, 443f overview, 433 response to anticonvulsants, 437 seizure frequency, 436 seizure induction, 436 seizure monitoring, 436–437 signal transduction, 439–440 spontaneous seizures, 433, 544–545, 544t Pink eye dilution, 227 Piriform cortex electrically induced afterdischarges in, 158. See also Electrically induced afterdischarges kindling in, 373, 373t, 374 Place cells, hippocampal, 621 Plasticity. See Neuroplasticity Plateau potentials, 54 Playground maze, 618 Point mutations, 223–224 Polymerase chain reaction, for human brain slices, 94 Pontine reticular formation, in alcohol withdrawal seizures, 165, 167 Positive transfer, 359 Positron emission tomography (PET), 589–590, 596–597 with fluorodeoxyglucose, 4 Postictal period, 11, 661 Post-status epileptic models of temporal lobe epilepsy, 559–564 Poststroke epilepsy models, 501–518 middle cerebral artery occlusion, 501–509 protothrombosis, 509–518 Postsynaptic potentials, inhibitory, in cell cultures, 26–27 Posttraumatic epilepsy, 465–490 axonal sprouting, 52–54, 53f chronic partial cortical isolation model, 477–490. See also Chronic partial cortical isolation model early animal models, 466–467 hemosiderin, 466–467, 495–499. See also Ferrous chloride injection model hyperexcitability, 53, 54–55, 55f, 481–483, 489, 490 incidence, 465
683 latency, 465–466, 466t, 473, 477 lateral fluid-percussion brain injury model, 465–475. See also Lateral fluidpercussion brain injury model late seizures, 473 metal models, 466–467 natural history, 465–466 neuropathology, 465–466, 473–474, 489 overview, 465–466 pathogenesis, 52–55 pathophysiology, 489 postsynaptic changes, 54–55, 55f presynaptic changes, 52–54, 53f prevention, 466, 489–490 risk factors, 465, 466t spontaneous seizures, 467 subpial undercut model, 467, 477. See also Chronic partial cortical isolation model trauma severity, 465, 466t, 473 Potassium, elevated, epileptiform activity and, 42 in hippocampal slices, 67 Potassium currents. See also Ion channels/currents Potassium ion channel modulators, mechanism of action of, in cell cultures, 29–30 Potassium ion channels/currents in acutely dissociated cell preparations, 17–19 blockade of, in hippocampal slices, 66 Preamplifiers, 572 Prevalence, 653–654 Primary generalized epilepsy, kindling as model of, 381 Primates, nonhuman. See Baboons; Monkeys Probe test, 619, 619t, 620f Proconvulsants. See Convulsant agents Prostaglandin, in pilocarpine model, 441, 444 Proteoglycans, in pilocarpine model, 442–443, 444 Protothrombosis model, 509–518 age-specificity, 517 animal issues, 509 behavioral features, 510–511, 513f characteristics/defining features, 510–516 ease of development, 516–517 EEG features, 511f–516f, 512–513 electrode placement, 509–510, 510f focal rhythmic 1-Hz sharp- and slow wave discharges, 511, 512f focal spike-wave discharges, 512, 513f–515 future challenges, 517 general description, 509 generalized tonic-clonic seizures, 512 genetics, 516 imaging, 514–515 insights into human disorders, 517–518 ischemic penumbra, 514 latency period, 513–514 limitations, 516–517 metabolic changes, 514–515 methodology, 509–510 molecular changes, 516 monitoring, 510 mortality, 517
684
Index
Protothrombosis model (continued) neuropathology, 514 neuroplasticity, 514 pathology modeled by, 509 procedures, 509, 510f reactive gliosis, 514 remission, 510 reproducibility, 517 response to anticonvulsants, 516 seizure duration, 513 seizure frequency, 513–514 seizure severity, 511 spontaneous seizures, 510 Psychic blindness, in kindled cats, 367, 367t PTZ model absence seizures, 111, 113–114, 113t, 115 acute seizures, 128, 128t, 129–130, 129f drug-refractory epilepsy, 564 drug screening, 541–542, 542t kindling, 379–381, 564. See also Kindling, chemical status epilepticus, 129–130, 345–346 systemic administration, 128t, 129–130, 129f, 608 topical (focal) administration, 143–145, 144f, 144t, 607 zebrafish model, 191–196, 195f Pyramidal cells loss of. See also Neuron(s), loss of in status epilepticus, 636
Q Quisqualic acid model, 133t, 135–136 behavioral manifestations, 606
R Racine scale, 116, 356, 356t Radial arm maze, 620f, 621–622, 624t Radiation, in utero. See In utero irradiation model Radiotelemetry, 572 Rapid kindling, 398 Rasmussen’s encephalitis model, 535–537 Rat absence seizures in, 73–77, 74t, 76f, 233–243. See also Absence seizures/epilepsy, genetic models of, rat age of, acute brain slices and, 64 alcohol withdrawal seizures in, 161–173 cortical freeze lesion model in, 295–301 cortical malformations in, spontaneous seizures and, 249–258. See also Cortical malformation models electrical stimulation models for status epilepticus in, 450–456, 458–462 genetically epilepsy-prone, 261, 265–268 in drug screening, 544, 545 immature, recurrent seizures in, 341–349. See also Recurrent seizures, in immature brain
kindling in. See also Kindling chemical, 381 electrical, 371–376 lateral fluid-percussion brain injury model in, 465–475. See also Lateral fluidpercussion brain injury model seizure monitoring in, 569–581. See also Seizure monitoring; specific models Reactive gliosis in alumina gel injection models, 180, 181f, 184–185 in kainate model, 423–424, 423f in middle cerebral artery occlusion model, 507 in pilocarpine model, 441 in posttraumatic epilepsy, 474 in protothrombosis model, 514 Receptor(s) AMPA. See AMPA receptors GABA. See GABA receptors glutamate. See Glutamate receptors metabotropic, in acutely dissociated cell preparations, 20 morphologic changes in, 640–641 NMDA. See NMDA receptors opioid, 439 Receptor autoradiography, for human brain slices, 94 Receptor protein tyrosine phosphatase b, in pilocarpine model, 443 Recurrent seizures. See also Chronic epilepsy; Status epilepticus in immature brain, 341–349 flurothyl model, 341–344, 342f, 4343f kainic acid model, 346–347 model applications, 348–349 model limitations, 348–349 pilocarpine model, 138, 346–347 PTZ model, 345–346 Reference memory, 614f, 615 Reflex testing, 617–618, 617f Repetitive seizures. See Recurrent seizures Resident intruder test, 624, 624t Reticular thalamic neurons, spike-wave discharges in, in absence seizures, 76–77, 76f, 241 Reversal test, 619, 619t, 620f Risk factors, 653–654 RNA-interference (RNAi) techniques, 219 Ro 5-3663 model, 128t, 132–133 Rocker mouse, 226, 226t Rodents. See Mouse; Rat Rotarod test, 617, 617f, 624t
S Sarin, 138–139, 139t Scn2a transgenic mouse model, 207t, 208 Seizure(s). See also Epilepsy; Epileptiform activity and specific types absence, 6, 6t, 8t, 68, 73–86. See also Absence seizures/epilepsy acute, 2
alcohol withdrawal, 161–173 atonic (astatic), 6, 6t, 8t behavioral manifestations, 12–13 chemically induced acute, 127–146 classification, 6–7, 6t, 8t, 653–654 clonic, 6, 6t, 8t complex partial, 6, 6t, 8t definition, 5 electrophysiologic prediction, 3 electroshock, 153–155, 154f. See also Electrical stimulation models recurrent seizures in immature brain, 347–349 emprosthotonic, 136 febrile, 333–339 flexion, 136, 606, 606f focal, 6t, 7, 8t alumina gel injection model, 179–183 in baboons, 262 in brain slices, 43 tetanus toxin model, 145–146, 407–412 forebrain, in baboons, 262 generalized, 6, 6t, 7, 8t long-term consequences of, 11–12 maximal electroshock, 153–155, 154f, 541 myoclonic, 6, 6t, 8t neuronal death from, 50 neuronal injury from. See also Excitotoxicity epileptogenicity of, 51–52, 52f partial, 6–7, 6t, 8t phases of, 10–11 prevention of, 5 progressive nature of, 361. See also Kindling remission, 73, 74t, 233, 274 sensory-evoked in baboons, 261–265 in genetically epilepsy-prone rats, 265–269 simple partial, 6, 6t, 8t sleep-related, in kindled cats, 368 spontaneous. See Spontaneous seizures surrogate markers for, 227–228 temporal lobe, 6, 6t, 8t. See also Temporal lobe epilepsy termination, 11, 661 tonic, 6, 6t, 8t tonic-clonic, 6, 6t, 8t. See also Tonic-clonic seizures Seizure induction. See also Epileptogenesis chemical agents in, 127–143. See also Pharmacologic models for absence seizures, 74, 75, 111–123 Seizure models. See Animal models and specific models Seizure monitoring. See also specific models animal-machine connections, 574–576, 575f, 576f, 578, 579 cables, 575–576, 575f, 576f, 579, 580 cages, 575–576, 575f, 576f, 578 cameras, 570–571 connector board, 575, 576f EEG recording system, 572–574, 580 electrical artifacts, 578 electrode implantation, 578–579 equipment, 570–580
685
Index method selection, 580–581 multiple rodents, 572, 577f, 578 noise control, 572, 578 preamplifiers, 572 purposes, 569–570, 570t, 581 radiotelemetry, 572 recorders, 571 surgical issues, 578–579 swivels, 574–576, 575f, 579–580 video recording system, 570–571, 570–572, 575–576, 575f, 576f, 579 Seizure predisposition in baboons, 262–263, 264 in humans, 262, 263 “Seizures beget seizures” dictum, 51 Seizure threshold, in mutation screening, 228 Selection cassettes, 211, 216 Sensorimotor cortex in absence seizures, 74t, 75–77, 76f, 85, 240–243, 240f alumina gel injection in, 179–183. See also Alumina gel injection models Sensorimotor function testing, 617–618, 617f Sensory-evoked seizures in baboons, 261–265 in genetically epilepsy-prone rats, 265–268 Serotonin, in pilocarpine model, 438–439, 443 Sex differences, 13 alcohol withdrawal seizures, 162 morphologic changes, 645–646 spatial learning, 622 Sexual dysfunction, in kindled cats, 367, 367t Shaffer collateral transfection, in organotypic slice cultures, 49 Shaker flies, 189 Short-term memory, 614f, 615 Signal transduction, in pilocarpine model, 439–440 Silver-staining, 642 Simple partial seizures, 6, 6t, 8t Single-cell labeling, 642–643 Single-cell molecular profiling, 28–29 Single nerve cells, acutely dissociated, 15–21. See also Acutely dissociated cell preparations Site-specific recombinase (SSR) systems, 212, 213f 6 Hz psychomotor seizure model, 153–155, 154f drug-refractory epilepsy, 557–559 drug screening, 542t, 543, 543t Sleep-related seizures, in kindled cats, 368 Slow-wave discharges, in absence epilepsy, 73, 74t, 75–76, 76f Smith-Magenis syndrome, mouse model of, 214 Socialization tests, 623f, 624 Social recognition task, 623f, 624, 624t Sodium channel blockers, mechanism of action, in cell cultures, 29 Sodium channels/currents. See also ion channels/currents in acutely dissociated cell preparations, 17–19, 19f
anticonvulsant activity, 556 in drug-refractory epilepsy, 556 Sodium-potassium ATPase, in pilocarpine model, 442 Soman, 138–139, 139t Sound-induced seizures, 266, 267 cerebral blood flow mapping, 587 Spasms epileptic, 10 infantile, 7 Spatial bias test, 619, 619t, 620f Spatial memory tests, 618–622, 619f, 620f Species, morphologic changes, 645–646 Species differences, 13 SPECT, 589–590, 596–597 Spike-wave discharges in absence epilepsy, 74t, 75–85, 122, 233 in corticocortical models, 80–85 in ferret dorsal lateral geniculate nucleus model, 77–79, 78f, 79f in mice models, 234 in rat models, 73–77, 234–243 in thalamic models, 74t, 75–79, 76f, 78f, 79f in thalamocortical models, 79–80, 80f, 239–243, 240f in drug screening, 542t in protothrombosis model, 512, 513f–515 Spontaneous mutations. See also Mutations cortical malformation and, 249–258. See also Cortical malformation models in mice, 223–230. See also Mouse epilepsy models, spontaneous mutation Spontaneous recurrent epileptiform discharges, 30 Spontaneous seizures, 544t. See also specific models age-related issues, 579–580 in drug screening, 544–545, 544t monitoring, 569–581. See also Seizure monitoring species-related issues, 579–580 SSR (site-specific recombinase) systems, in gene replacement, 212, 213f Staining, 39–40 fluorescent, 39–40, 47, 48f, 642 immunocytochemistry, 643–644 silver, 642 techniques, 39–40, 642–643 Timm’s, 424, 425f, 426–428, 470, 643 with voltage-sensitive dyes, 68 Standby neurotransmitter hypothesis, 28 Status epilepticus models. See also Recurrent seizures acetylcholine-related substances, 138 acute period, 433–434 age factors, 585, 586–587 autoradiography, 585–588 behavioral manifestations, 606–607. See also Behavioral manifestations blood-brain barrier permeability, 588–589 chronic period, 434 development, 433, 434f domoic acid, 135, 416
drug-refractory epilepsy, 559–564 electrical stimulation, 347–349, 449–462, 559, 562–564, 563f. See also Electrical stimulation models, status epilepticus flurothyl, 341–344, 342f, 344f hippocampal sclerosis, 416, 423–424, 636 hypoxia-induced seizures/hypoxic encephalopathy, 329–330 imaging, 585–588 insights into human disorders, 348–349 kainic acid, 133–135, 143–145, 346–347, 415–430. See also kainic acid/kainate latent period, 434 limitations, 348–349 neuropathology, 359, 360 pilocarpine, 138, 346–347, 433–444, 559–562, 561f. See also Pilocarpine model PTZ, 129–130, 345–346. See also PTZ model self-sustaining, 449 spontaneous seizures, 544t vs. kindling, 360 Stereology, 642 Stereotypies, 603–604. See also Behavioral manifestations Strain, morphologic changes, 645–646 Strain specificity, 13 Stress testing, 622–623, 623f Stroke models, 501–518 middle cerebral artery occlusion, 501–509 protothrombosis, 509–518 Strychnine model, 139 Subcortical band heterotopia, 257 Subpial undercut model, 467, 477. See also Chronic partial cortical isolation model Superior colliculus, in alcohol withdrawal seizures, 166–167 Superoxide dismutase, in pilocarpine model, 441, 444 Susceptibility genes, 13 Swivels, 574–576, 575f, 579–580 Synapses excitatory, potentiation of, epileptiform activity and, 52, 52f inhibitory, in cell cultures, 26–27 Synaptic interactions assessment of, 644–645 in cell cultures, 30 Synaptic plasticity. See also Neuroplasticity in cell cultures, 27–28 in human brain slices, 98 Synaptic reorganization, 638–640 in kainic acid model, 426–428, 427f, 429 Syndromes, seizures in, 653–654, 654t Syphilis, 521–522
T T1/2-weighted magnetic resonance imaging, 593. See also Magnetic resonance imaging Tabun, 138–139, 139t Tail twisting, 606, 606f
686 Tapeworm infections, 521–524 Temporal lobe epilepsy, 6, 6t, 8t, 410. See also Mesial temporal lobe epilepsy alumina gel injection model, 179–183 drug-refractory, 551–565. See also Drug-refractory epilepsy febrile seizures, 333 functional consequences, 388–389 hippocampal slices, 60–61, 63–68 kindling model, 360, 368, 396, 402–403 Mongolian gerbil model, 285 morphologic changes in, 629–647. See also under Cortical dysplasia; Morphologic post-status epileptic models, 559–564 seizure duration, 421–422 sleep disturbances in, 368 vs. absence seizures, 421–422 Teratogens, for lesion models, 306–307 Tertiary syphilis, 521–522 Tetanic stimulation, of isolated guinea pig brain, 106 Tetanus toxin model, 145–146, 407–412, 411 animal issues, 407–408 behavioral features, 409–410 clinical features, 409–410 dose, 408 drug screening, 411, 412 ease of development, 408–409 ease of use, 411 EEG features, 408 electromyography, 408 epilepsia partialis continua, 411, 412 experimental procedures, 408 genetics, 411 hippocampal sclerosis, 410 hypometabolism, 411 imaging, 411 injection sites, 408, 409, 412 insights into human disorders, 412 limitations, 411 mechanisms, 412 methodology, 407–409 molecular changes, 411 monitoring, 408 neuropathology, 410 neuroplasticity, 411 neurotoxicity, 407, 408 overview, 407 pathology modeled by, 407 reliability, 408–409 remission, 409 reproducibility, 411 response to anticonvulsants, 411, 412 safety, 408 seizure rate, 410 spontaneous seizures, 409 usefulness, 407, 412 Tetanus toxin vaccination, 408 Tetrodotoxin in chronic partial cortical isolation model, 487, 490 dendritic hyperexcitability and, 54–55, 55f
Index Thalamic models, of absence seizures, 74t, 75, 77–79, 78f, 79f, 85, 239–242, 240f Thalamic slices, horizontal, preparation of, 79 Thalamic spike-wave discharges, in absence seizures, 77–79, 78f, 79f Thalamocortical models, of absence seizures, 74t, 75–77, 76f, 79–80, 80f, 85, 240–243, 240f Thalamocortical slices, preparation of, 79–80, 80f Theophylline model, 139 Therapeutic assays. See Drug screening Therapeutic index, 389 Therapy, disease-modifying, 546–547 Therapy-resistant epilepsy. See Drug-refractory epilepsy THIP model, of absence seizures, 112–113, 113t, 115 Timm’s stain, 643 mossy fiber sprouting and, 424, 425f, 426–428, 470 Tish mutant rat, cortical malformations and spontaneous seizures in, 250, 250t, 251, 253–255, 256. See also Cortical malformation models, genetic Tissue inhibitors of metalloproteinases, in pilocarpine model, 442 Tonic-clonic seizures, 6, 6t, 8t absence seizures and, 73. See also Absence seizures/epilepsy alcohol withdrawal, 161. See also Alcohol withdrawal seizures in baboons, 262–263 behavioral manifestations, 603, 603f, 606. See also Behavioral manifestations in genetically epilepsy-prone rats, 265, 266 triggers, 266 Tonic seizures, 6, 6t, 8t Topiramate for alcohol withdrawal, 172–173 in hypoxia-induced seizures/hypoxic encephalopathy model, 328 in kainate model, 428–429, 428f response to, in kindled rats, 553, 555t Tottering mouse, 225, 226t spike-wave discharges in, 234 Transfer test, 619, 619t, 620f Transgenic animals, organotypic slice cultures from, 48–49 Transgenic mouse models, 202–209. See also Mouse epilepsy models, transgenic Traumatic brain injury. See Posttraumatic epilepsy Treatment. See Therapy Trimethadione in pilocarpine model, 437 therapeutic assay for, 541 TSC2 rat, 632 T-type calcium current, 19 Tuberous sclerosis, 632, 633f Tumor-associated epilepsy model, 527–532 imaging, 528, 529–532, 530f–532f metabolic changes, 528
methodology, 529–532, 530f–532f response to anticonvulsants, 528 seizure frequency/incidence, 527–528, 528t treatment issues, 528–529 tumor types/sites, 527–528 TUNEL labeling, 642 2DG method, 584–587. See also Autoradiography
U Undercut model, 467, 477. See also Chronic partial cortical isolation model University of Utah Anticonvulsant Drug Development Program, 540–549
V Vaccination, tetanus toxin, 408 Vagus nerve stimulation, 5 Valproic acid for alcohol withdrawal, 169–170, 170t, 172–173 response to in alumina gel injection models, 182 in kindled rats, 553, 555t in pilocarpine model, 437 6 Hz test, 545, 545t Vasogenic brain injury, autoradiography and, 587–589 Ventral hippocampus. See also Hippocampus electrical stimulation of, for status epilepticus induction, 460–462 kindling in, 356t, 358 in rats, 373, 373t Video-EEG monitoring. See also Seizure monitoring electrical artifacts, 578 equipment, 576–578, 577f, 580 purposes, 570t Vigabatrin, response to, in kindled rats, 553, 555t Viral encephalitis, 521–522, 524–525 Visual agnosia, in kindled cats, 367, 367t Visual-spatial memory tests, 618–622, 619f, 620f Voltage-sensitive dyes, for hippocampal slices, 68 VR (isobutyl S-2-diethylamino ethyl methylphosphonothioate) (nerve agent), 138–139, 139t VX (ethyl S-2–diisopropylamino ethyl ethylphosphonothioate) (nerve agent), 138–139, 139t
W Water maze, 618–621, 619t, 620f, 624t Weapons-grade organophosphorus compounds, 138–139, 139t
Index West syndrome, 7, 369 Wet dog shakes, 606, 606f in kainic acid model, 134, 420f kindling and, 358 Willis circle, in isolated guinea pig brain, 104, 105f Wistar Albino Glaxo Rats, absence seizures and, 74t, 75, 233–243 Withdrawal seizures alcoholic, 161–170, 171–173. See also Alcohol withdrawal seizures nonalcoholic, 170–171
Woodbury criteria, 195 Working memory, 614f, 615
Z Zebrafish model, 189–196 advantages, 195–196 applications, 189–190 behavioral manifestations, 191, 192f correlation with infant development, 190 drug screening, 190–191, 195
687 electrophysiologic manifestations, 191–193, 193f, 194f future directions, 196 genetics, 193–195 larval form, 190 limitations, 196 methodology, 190–191 molecular alterations, 194–195 response to anticonvulsants, 195, 195f utility, 189–190 Zinc model, 143 Zolpidem withdrawal, 171
