Models of Seizures and Epilepsy

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

xiii

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,

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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 (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 (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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Chapter 6/Hippocampal Slices: Designing and Interpreting Studies in Epilepsy Research

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

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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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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

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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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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.

References Anderson, W.W., Lewis, D.V., Swartzwelder, H.S., and Wilson, W.A. 1986. Magnesium-free medium activates seizure-like events in the rat hippocampal slice. Brain Res 398: 215–219. Bartolomei, F., Wendling, F., Vignal, J.P., Kochen, S., Bellanger, J.J., Badier, J.M., Bouquin-Jeannes, R. et al. 1999. Seizures of temporal lobe epilepsy: identification of subtypes by coherence analysis using stereoelectro-encephalography. Clin Neurophysiol 110: 1741–1754. Ben Ari, Y. 2002. Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci 3: 728–739. Ben Ari, Y., Cherubini, E., Corradetti, R., and Gaiarsa, J.L. 1989. Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol 416: 303–325. Ben Ari, Y., and Cossart, R. 2000. Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci 23: 580–587. 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. Bernard, C., Cossart, R., Hirsch, J.C., Esclapez, M., and Ben Ari, Y. 2000. What is GABAergic inhibition? How is it modified in epilepsy? Epilepsia 41(Suppl 6): S90–S95. Bernasconi, N., Bernasconi, A., Andermann, F., Dubeau, F., Feindel, W., and Reutens, D.C. 1999. Entorhinal cortex in temporal lobe epilepsy: a quantitative MRI study. Neurology 52: 1870–1876. Bernasconi, N., Bernasconi, A., Caramanos, Z., Andermann, F., Dubeau, F., and Arnold, D.L. 2000. Morphometric MRI analysis of the parahip-

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A

]~ Controlrat 1

Normal

0 ms Calcium free

3 ms

2

0 ms

3 ms

I

2

6ms

20ms

N

6rns

20 ms

KArat Normal

B 0ms Calcium free

0m~

~

t

~ 3rns

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

C

H

A

P

T

E

R

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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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

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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-

77

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

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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

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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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Chapter 7/Thalamic, Thalamocortical, and Corticocortical Models of Epilepsy

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;

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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.)

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.

Outflow (Ethanolvapor) Ethanol (via pump)

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FIGURE 1 3--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 chambers. 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 animals free access to food and water. The sample port allows air within the experimental chamber to be sampled for determination of ethanol concentrations.

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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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Chapter 8/Studying Epilepsy in the Human Brain

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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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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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 (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 (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

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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 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

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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.

References Atkin, L. 1986. The Auditory Midbrain: Structure and Function in the Central Auditory Pathway. Clifton: Humana Press. Ballenger, J.C., and Post, R.M. 1978. Kindling as a model for alcohol withdrawal syndromes. Br J Psychiatry 133: 1–14.

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Bayard, M., McIntyre, J., Hill, K.R., and Woodside, J. Jr. 2004. Alcohol withdrawal syndrome. Am Fam Physician 69: 1443–1450. Becker, H.C., and Hale, R.L. 1993. Repeated episodes of ethanol withdrawal potentiate the severity of subsequent withdrawal seizures—an animal model of alcohol withdrawal kindling. Alcohol Clin Exp Res 17: 94–98. Becker, H.C., Diaz-Granados, J.L., and Hale, R.L. 1997. Exacerbation of ethanol withdrawal seizures in mice with a history of multiple withdrawal experience. Pharmacol Biochem Behav 57: 179–183. Becker, H.C., Veatch, L.M., and Diaz-Granados, J.L. 1998. Repeated ethanol withdrawal experience selectively alters sensitivity to different chemoconvulsant drugs in mice. Psychopharmacology (Berl) 139: 145–153. Belknap, J.K., Metten, P., Helms, M.L., O’Toole, L.A., Angeli-Gade, S., Crabbe, J.C., and Phillips, T.J. 1993. Quantitative trait loci (QTL) applications to substances of abuse: physical dependence studies with nitrous oxide and ethanol in BXD mice. Behav Genet 23: 213–222. Bonnet, U., Banger, M., Leweke, F.M., Maschke, M., Kowalski, T., and Gastpar, M. 1999. Treatment of alcohol withdrawal syndrome with gabapentin. Pharmacopsychiatry 32: 107–109. Bozikas, V., Petrikis, P., Gamvrula, K., Savvidou, I., and Karavatos, A. 2002. Treatment of alcohol withdrawal with gabapentin. Prog Neuropsychopharmacol Biol Psychiatry 26: 197–199. Brailowsky, S., Kunimoto, M., Menini, C., Silva-Barrat, C., Riche, and Naquet, R. 1988. The GABA-withdrawal syndrome: a new model of focal epileptogenesis. Brain Res 442: 175–179. Brown, D.J., and Long, W.C. 1988. Quality control in blood alcohol analysis: simultaneous quantitation and confirmation. J Anal Toxicol 12: 279–283. Buck, K.J., Rademacher, B.S., Metten, P., and Crabbe, J.C. 2002. Mapping murine loci for physical dependence on ethanol. Psychopharmacology (Berl) 160: 398–407. Busto, U., Sellers, E.M., Naranjo, C.A., Cappell H., Sanchez-Craig, M., and Sykora, K. 1986. Withdrawal reaction after long-term therapeutic use of benzodiazepines. N Engl J Med 315: 854–859. Cadete-Leite, A., Tavares, M.A., Pacheco, M.M., Volk, B., and PaulaBarbosa, M.M. 1989. Hippocampal mossy fiber-CA3 synapses after chronic alcohol consumption and withdrawal. Alcohol 6: 303–310. Cagetti, E., Liang, J., Spigelman, I., and Olsen, R.W. 2003. Withdrawal from chronic intermittent ethanol treatment changes subunit composition, reduces synaptic function, and decreases behavioral responses to positive allosteric modulators of GABAA receptors. Mol Pharmacol 63: 53–64. Cagetti, E., Baicy, K.J., and Olsen, R.W. 2004. Topiramate attenuates withdrawal signs after chronic intermittent ethanol in rats. Neuroreport 15: 207–210. Carpenter-Hyland, E.P., Woodward, J.J., and Chandler L.J. 2004. Chronic ethanol induces synaptic but not extrasynaptic targeting of NMDA receptors. J Neurosci 24: 7859–7868. Carta, M., Ariwodola, O.J., Weiner, J.L., and Valenzuela, C.F. 2003. Alcohol potently inhibits the kainate receptor-dependent excitatory drive of hippocampal interneurons. Proc Natl Acad Sci USA 100: 6813–6818. Carta, M., Mameli, M., and Valenzuela, C.F. 2004. Alcohol enhances GABAergic transmission to cerebellar granule cells via an increase in Golgi cell excitability. J Neurosci 24: 3746–3751. Carta, M., Olivera, D.S., Dettmer, T.S., and Valenzuela, C.F. 2002. Ethanol withdrawal upregulates kainate receptors in cultured rat hippocampal neurons. Neurosci Lett 327: 128–132. Chakravarty, D.N., and Faingold, C.L. 1998. Comparison of neuronal response patterns in the external and central nuclei of inferior colliculus during ethanol administration and ethanol withdrawal. Brain Res 783: 102–108. Chapman, A.G., Croucher, M.J., and Meldrum, B.S. 1984. Evaluation of anticonvulsant drugs in DBA/2 mice with sound-induced seizures. Arzneimittelforschung 34: 1261–1264.

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Chapman, A.G., and Meldrum, B.S. 1989. Non-competitive N-methyl-daspartate antagonists protect against sound-induced seizures in DBA/2 mice. Eur J Pharmacol 166: 201–211. Chu, N.S. 1979. Carbamazepine: prevention of alcohol withdrawal seizures. Neurology 29: 1397–1401. Chu, N.S. 1981. Prevention of alcohol withdrawal seizures with phenytoin in rats. Epilepsia 22: 179–184. Clemmesen, L., Ingvar, M., Hemmingsen, R., and Bolwig, T.G. 1988. Local cerebral glucose consumption during ethanol withdrawal in the rat: effects of single and multiple episodes and previous convulsive seizures. Brain Res 453: 204–214. Costa, E.T., Soto, E.E., Cardoso, R.A., Olivera, D.S., and Valenzuela, C.F. 2000. Acute effects of ethanol on kainate receptors in cultured hippocampal neurons. Alcohol Clin Exp Res 24: 220–225. Crabbe, J.C. 1992. Antagonism of ethanol withdrawal convulsions in withdrawal seizure prone mice by diazepam and abecarnil. Eur J Pharmacol 221: 85–90. Crabbe, JC. 2002. Alcohol and genetics: new models. Am J Med Genet 114: 969–974. Crabbe, J., and Kosobud, A. 1990 Alcohol withdrawal seizures: genetic animal models. In Alcohol and Seizures: Basic Mechanisms and Clinical Concepts. Eds. R.J. Porter, R.H. Mattson. pp. 126–139. Philadelphia: F.A. Davis. Crews, F.T., Morrow, A.L., Criswell, H., and Breese, G. 1996. Effects of ethanol on ion channels. Int Rev Neurobiol 39: 283–367. 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. Davies, M. 2003. The role of GABAA receptors in mediating the effects of alcohol in the central nervous system. J Psychiatry Neurosci 28: 263–274. Deitrich, R.A., and Erwin, V.G. 1996. Pharmacological Effects of Ethanol on the Nervous System. Boca Raton: CRC Press. Devaud, L.L., Matthews, D.B., and Morrow AL. 1999. Gender impacts behavioral and neurochemical adaptations in ethanol-dependent rats. Pharmacol Biochem Behav 64: 841–849. Devaud, L.L., Fritschy, J.M., Sieghart, W., and Morrow, A.L. 1997. Bidirectional alterations of GABAA receptor subunit peptide levels in rat cortex during chronic ethanol consumption and withdrawal. J Neurochem 69: 126–130. Dodd, P.R., Beckmann, A.M., Davidson, M.S., and Wilce, P.A. 2000. Glutamate-mediated transmission, alcohol, and alcoholism. Neurochem Int 37: 509–533. D’Onofrio, G., Rathlev, N.K., Ulrich, A.S., Fish, S.S., and Freedland, E.S. 1999. Lorazepam for the prevention of recurrent seizures related to alcohol. N Engl J Med 340: 915–919. Duka, T., Gentry, J., Malcolm, R., Ripley, T.L., Borlikova, G., Stephens, D.N., Veatch, L.M. et al. 2004. Consequences of multiple withdrawals from alcohol. Alcohol Clin Exp Res 28: 233–246. Eckardt, M.J., Campbell, G.A., Marietta, C.A., Majchrowicz, E., Rawlings, R.R., and Weight, F.F. 1992. Ethanol dependence and withdrawal selectively alter localized cerebral glucose utilization. Brain Res 584: 244–250. Ellis, F.W., and Pick, J.R. 1970. Experimentally induced ethanol dependence in rhesus monkeys. J Pharmacol Exp Ther 175: 88–93. 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. Essardas-Daryanani, H., Santolaria, F.J., Gonzalex Reimers, E., Jorge, J.A., Batista Lopez, N., Martin Hernandez, F., Martinez Riera, A., et al. 1994. Alcoholic withdrawal syndrome and seizures. Alcohol Alcohol 29: 323–328. Essig, C.F., Jones, B.E., and Lam, R.C. 1969. The effects of pentobarbital on alcohol withdrawal in dogs. Arch Neurol 20: 554–558.

Evans, M.S., Li, Y., and Faingold, C.L. 2000. Inferior colliculus intracellular response abnormalities in vitro associated with susceptibility to ethanol withdrawal seizures. Alcoholism Clin Exp Res 24: 1180–1186. Faingold, C.L. 1999. Neuronal networks in the genetically epilepsy-prone rat. Adv Neurol 79: 311–321. 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., and Riaz, A. 1995. Ethanol withdrawal induces increased firing in inferior colliculus neurons associated with audiogenic seizure susceptibility. Exp Neurol 132: 91–98. Faingold, C.L., and Riaz, A. 1994. Increased responsiveness of pontine reticular formation neurons associated with audiogenic seizures susceptibility during ethanol. Brain Res 663: 69–76. Faingold, C.L., and Randall, M.E. 1995. Pontine reticular formation neurons exhibit a premature and precipitous increase in acoustic responses prior to audiogenic seizures in genetically epilepsy-prone rats. Brain Res 704: 218–226. Faingold, C.L., Li, Y., and Evans, M.S. 2000. Decreased GABA and increased glutamate receptor-mediated activity on inferior colliculus neurons in vitro are associated with susceptibility to ethanol withdrawal seizures. Brain Res 868: 287–295. Faingold, C.L., N’Gouemo, P., and Riaz, A. 1998. Ethanol and neurotransmitter interaction. From molecular to integrative effects. Prog Neurobiol 55: 509–535. Fehr, C., Shirley, R.L., Metten, P., Kosobud, A.E., Belknap, J.K., Crabbe, J.C., and Buck, K.J. 2004. Potential pleiotropic effects of Mpdz on vulnerability to seizures. Genes Brain Behav 3: 8–19. Fehr, C., Shirley, R.L., Belknap, J.K., Crabbe, J.C., and Buck, K.J. 2002. Congenic mapping of alcohol and pentobarbital withdrawal liability loci to a 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 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

471

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

Models of Seizures and Epilepsy

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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 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

Chapter 38/Chronic Partial Cortical 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

.. ..

-

; D

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 (~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

A

C

D

PIA

PIA

somadendrites

somadendrites axonal arbor IV V

IV V

soma

200 mm

soma

200 mm

axonal arbor

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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Chapter 39/Head Trauma: Hemorrhage-Iron Deposition

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