PATHOPHYSIOLOGICAL MECHANISMS OF GENETIC ABSENCE EPILEPSY IN THE RAT

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1 Progress in Neurobiology Vol. 55, pp. 27 to 57, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain /98/$19.00 PII: S (97) PATHOPHYSIOLOGICAL MECHANISMS OF GENETIC ABSENCE EPILEPSY IN THE RAT L. DANOBER*, C. DERANSART, A. DEPAULIS, M. VERGNES and C. MARESCAUX INSERM U. 398, Strasbourg, F France (Received 17 October 1997) AbstractÐGeneralized non-convulsive absence seizures are characterized by the occurrence of synchronous and bilateral spike and wave discharges (SWDs) on the electroencephalogram, that are concomitant with a behavioral arrest. Many similarities between rodent and human absence seizures support the use of genetic rodent models, in which spontaneous SWDs occur. This review summarizes data obtained on the neurophysiological and neurochemical mechanisms of absence seizures with special emphasis on the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). EEG recordings from various brain regions and lesion experiments showed that the cortex, the reticular nucleus and the relay nuclei of the thalamus play a predominant role in the development of SWDs. Neither the cortex, nor the thalamus alone can sustain SWDs, indicating that both structures are intimely involved in the genesis of SWDs. Pharmacological data con rmed that both inhibitory and excitatory neurotransmissions are involved in the genesis and control of absence seizures. Whether the generation of SWDs is the result of an excessive cortical excitability, due to an unbalance between inhibition and excitation, or excessive thalamic oscillations, due to abnormal intrinsic neuronal properties under the control of inhibitory GABAergic mechanisms, remains controversial. The thalamo-cortical activity is regulated by several monoaminergic and cholinergic projections. An alteration of the activity of these di erent ascending inputs may induce a temporary inadequation of the functional state between the cortex and the thalamus and thus promote SWDs. The experimental data are discussed in view of these possible pathophysiological mechanisms. # 1998 Elsevier Science Ltd. All rights reserved CONTENTS 1. Introduction The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) EEG and behavioral characteristics of spike and wave discharges Relationship between spike and wave discharges and wakefulness Pharmacological characteristics of spike and wave discharges Ontogenesis of spike and wave discharges Genetic transmission of spike and wave discharges Conclusion Seizure network: the thalamo-cortical substrate of absence seizures Mapping of spike and wave discharges Mapping of local cerebral metabolism and blood ow Bilateralization and synchronization of spike and wave discharges: role of callosal and thalamic pathways E ects of cortical and thalamic lesions Cortical and thalamic cellular correlates of spike and wave discharges Thalamo-cortical neurotransmissions and absence seizures GABAergic neurotransmission and absence seizures Glutamatergic neurotransmission and absence seizures Conclusion Thalamo-cortical mechanisms in absence epilepsy Anatomy of the thalamo-cortical circuit Physiological functioning of the thalamo-cortical circuit Cortical and/or thalamic mechanisms in absence epilepsy Hypothesis of a thalamic hypersynchronization Role of GABAergic synaptic mechanisms Role of intrinsic mechanisms 39 * Author for correspondence: Dr Laurence Danober, INSERM U. 398, Neurobiologie et Neuropharmacologie des eâ pilepsies geâ neâ raliseâ es, Faculte de Me decine, 11 rue Humann, F Strasbourg Ce dex, France. Tel.: ; Fax: ; U398@neurochem.u-strasbg.fr. 27

2 28 L. Danober et al. CONTENTS (continued) Hypothesis of a cortical hyperexcitability Role of glutamatergic synaptic mechanisms Role of GABAergic synaptic mechanisms Conclusion The thalamo-cortical ascending control of absence seizures Role of the thalamo-cortical inputs in EEG desynchronization Interconnections between structures projecting to the thalamo-cortical circuit Role of the serotonergic inputs Role of the noradrenergic inputs Role of the cholinergic inputs Role of the nucleus basalis projections Role of the pedunculopontine and laterodorsal tegmental nuclei projections Could ascending neuromodulation of the thalamo-cortical circuit underlie absence seizures? The nigro-collicular control of absence seizures Role of the subtantia nigra pars reticulata Role of the GABAergic transmission Role of the glutamatergic transmission Nigral outputs The role of the basal ganglia and dopamine E ects of drugs interacting with dopaminergic neurotransmission Role of the nigro-striatal dopaminergic projection Conclusion General conclusion 49 Acknowledgements 49 References 49 AMPA EEG GABA GAD GAERS i.p. ipsp I T ABBREVIATIONS a-amino-3-hydroxy-5-methyl-4- LCMR isoxazolepropionic acid LDT electroencephalogram NMDA gamma-aminubutyric acid PPTg glutamic acid decarboxylase REM sleep Genetic Absence Epilepsy Rat from Strasbourg SNpr intraperitoneal SWDs inhibitory postsynaptic potential THIP low-threshold Ca 2+ current local cerebral metabolic rates for glucose laterodorsal tegmental nucleus N-methyl-D-aspartate pedonculopontic tegmental nucleus rapid-eye-movement sleep substantia nigra pars reticulata spike and wave discharges 4,5,6,7-tetrahydroxyisoxazolo- 4,5c-pyridine-3-ol 1. INTRODUCTION Absences are generalized non-convulsive seizures that di er in many respects from other forms of epileptic seizures (Berkovic et al., 1987; Porter, 1993; Loiseau et al., 1995; Niedermeyer, 1996). Typical absence seizures are characterized by a brief unresponsiveness to environmental stimuli and cessation of activity, which can be accompanied by automatisms or moderate tonic or clonic components a ecting the limbs, eyeballs or eyelids. Typical absences are associated on the electroencephalogram (EEG) with bilateral, synchronous and regular 3 c/s spike and wave discharges (SWDs) which start and end abruptly. In contrast to generalized convulsive or partial seizures, typical absences leave no postictal depression. They occur as frequently as several hundred times a day, mainly during quiet wakefulness, inattention and in the transition between sleep and awakening. The pharmacological reactivity of absence seizures is also unique: they are suppressed by ethosuximide, which is ine ective in all other forms of seizures, while they are aggravated by carbamazepine, phenytoine and other anticonvulsants e ective in generalized convulsive and partial epilepsies. The heterogeneity of typical absence epilepsies is well accepted (Porter, 1993; Hirsch et al., 1994; Loiseau et al., 1995). Typical absences associated with a regular 3 c/s SWDs are found in ve nonlesional idiopathic generalized syndromes: childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, myoclonic absence epilepsy and eyelid myoclonia with absences. Beside absence seizures, patients do not present any other neurological or neuropsychological disorders. Atypical absences occur during the course of symptomatic generalized epilepsies, such as the Lennox-Gastaut syndrome, in patients su ering from severe neurological de cits. Impairment of consciousness is less pronounced and less abrupt for atypical absences than for typical ones, and the ictal EEG discharges are made of irregular slow spikes and waves and/or polyspikes (Porter, 1993). No structural lesion of any kind has ever been identi ed as the substrate of typical absence epilepsies (Berkovic et al., 1987; Niedermeyer, 1996). Their cause is increasingly regarded as genetic (Lennox and Lennox, 1960; Doose et al., 1973; McNamara, 1994). Because typical absence epilepsies mainly a ect children and adolescents and have moderate consequences, studies of their pathophysiological mechanisms cannot be conducted in humans for ethical reasons. Therefore, much of the recent information available about the pathophysiology of absences derived from studies in animal models. To be valid as a model of human disease, animal models should ideally exhibit similar clinical

3 Pathophysiological Mechanisms of Genetic Absence Epilepsy in the Rat 29 (isomorphism) and pharmacological (predictivity) characteristics to those occurring in humans. In addition, they should have a similar etiology (homology) to the human disease (Kornetsky, 1977). Models displaying clinical and pharmacological characteristics of absence seizures are either experimentally induced or genetically determined. SWDs can be pharmacologically induced in rodents, cats or primates by injection of pentylenetetrazol, penicillin, gamma-hydroxybutyrate or GABA agonists (for review see: Snead, 1988, 1994). The thalamic stimulation model of absence epilepsy (Hunter and Jasper, 1949) is now rarely used because it requires continuous stimulation during testing. The spontaneous occurrence of genetically determined high voltage rhythmic activities on cortical EEG of laboratory rodents has been described by many authors. Libouban and Oswaldo-Cruz (1958) rst observed such patterns, which they related to facial twitching. Klingberg and Pickenhain (1968) found that twenty per cent of their rats presented large ``spindle-like'' discharges predominantly in the frontal cortex and occurring in awake, but quiet, animals. Since these rst descriptions, many authors have reported similar paroxystic EEG patterns in di erent strains of rats and mice (Noebels and Sidman, 1979; Vergnes et al., 1982; Chocholova, 1983; Noebels, 1984; Semba and Komisaruk, 1984; van Luijtelaar and Coenen, 1986; BuzsaÁ ki et al., 1990; Coenen et al., 1992; Hosford et al., 1992; Marescaux et al., 1992a; Snead, 1994; Jando et al., 1995). Although they were rst considered to correspond either to an artefact or to a physiological state typical of the rodent's brain, they were later validated as models of spontaneous absences seizures (Marescaux et al., 1992a). Such genetic models include the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) selected in our laboratory (Vergnes et al., 1982; Marescaux et al., 1992a), the WAG/Rij rats (van Luijtelaar and Coenen, 1986; Coenen et al., 1992) and numerous strains of mice (Noebels, 1984). Unlike genetic rat models, spontaneous absence seizures in mice are usually associated with other neurological disorders. Lethargic mice (lh/lh) exhibit spontaneous absence seizures concomitantly with ataxia and lethargic behavior (Hosford et al., 1992). The tottering mice also display spontaneous absence seizures but exhibit paroxysmal abnormal movements and postures (Noebels and Sidman, 1979). The main advantage of using strains of rats with spontaneous SWDs instead of pharmacological models is to avoid methodological bias that may overemphasize the role of a cerebral structure electrically stimulated or neurotransmission systems pharmacologically manipulated. Furthermore, seizures induced by injection of a drug cannot mimic the chronicity that characterizes absence epilepsy. Genetic models with spontaneous SWDs more closely reproduce the state of chronically recurrent spontaneous seizures observed in human. Some data have demonstrated that absence seizures are generated in a speci c neuronal network involving cortical and thalamic areas from both hemispheres (Pen eld and Jasper, 1947; Avoli and Gloor, 1982; Vergnes et al., 1987; Gloor and Fariello, 1988; Gloor et al., 1990; Vergnes et al., 1990; Marescaux et al., 1992a; Vergnes and Marescaux, 1992; Inoue et al., 1993; Niedermeyer, 1996). This thalamo-cortical circuitry is under the control of several speci c inhibitory and excitatory systems arising from the forebrain and brainstem. The purpose of this article is to review recent ndings on the pathophysiological mechanisms of absence seizures collected in di erent rodent models and especially in genetic models. Most of this review will be devoted to a description of how thalamo-cortical mechanisms are involved in the genesis of spontaneous SWDs. Finally, the control of these mechanisms by other circuits involving several brain structures, such as the cholinergic and noradrenergic projections and the basal ganglia, will be discussed. 2. THE GENETIC ABSENCE EPILEPSY RATS FROM STRASBOURG (GAERS) Thirty per cent of the Wistar rats from the initial breeding colony in our laboratory in Strasbourg presented spontaneous SWDs which were bilateral and synchronous over the cerebral cortex. A strain in which all animals displayed SWDs was selected after several generations by selecting breeders with SWDs. This strain was named ``Genetic Absence Epilepsy Rats from Strasbourg'' (GAERS) and has now been bred through 37 generations. Similarly, a control strain, free of any spontaneous SWD, has been outbred over 30 generations EEG and Behavioral Characteristics of Spike and Wave Discharges In GAERS, SWDs start and end abruptly on a normal EEG background (Fig. 1). In 4-month-old GAERS from the 30th generation, the mean frequency of spike and wave complexes within a discharge is c/s. Their voltage varies from 300 to 1,000 mv. When the animals are maintained in a state of quiet wakefulness, SWDs last for s, occur 1.3 times/min on average and their mean cumulated duration per minute is 2528 s (Vergnes et al., 1982; Marescaux et al., 1992a). SWDs are concomitant with behavioral immobility and rhythmic twitching of the vibrissae and facial muscles. Muscle tone in the neck is sometimes diminished, inducing a gradual and slight lowering of the head. In some instances, SWDs appear while the rat is moving: the movement is then suddenly interrupted and resumes as soon as the discharge stops (Vergnes et al., 1982; Marescaux et al., 1992a). During SWDs, the responsiveness to mild sensory stimuli is abolished. However, SWDs are immediately interrupted by strong and unexpected sensory stimulations or during performance of various motivated tasks (Vergnes et al., 1991; Getova et al., 1997). Comparisons with non-epileptic rats have shown that spontaneous activity, exploration, feeding, social interactions or learning of positively or negatively reinforced tasks are not impaired in GAERS. Sexual and reproductive behaviors also appear normal (Vergnes et al., 1991). Similar results

4 30 L. Danober et al. Fig. 1. Mapping of spike and wave discharges (SWDs) in GAERS. Left panel:, schematic mapping of SWDs on a coronal section of rat brain. Left hatched areas: high amplitude SWDs; right hatched areas: small amplitude SWDs; dots: no SWDs recorded; white areas: no recording performed. Right panel:, simultaneous EEG recordings from the cortex, dorsal hippocampus, ventrobasal thalamus and amygdala. On the top of the gure, SWDs were shown at a faster scale time. Am: amygdala; Cx: cortex; ic: internal capsule; MD mediodorsal thalamic nucleus, Rt: reticular thalamic nucleus; VB: ventrobasal thalamic nucleus. have been obtained in WAG/Rij rats (Coenen et al., 1991; van Luijtelaar et al., 1991). The behavioral and EEG patterns of the rat during SWDs appear similar to those observed in humans during absence seizures (Jung, 1962; Loiseau and Cohadon, 1970; Loiseau et al., 1995). However, the frequency of spike-and-wave complexes within a discharge di ers. In humans, it is classically around 3 c/s, whereas in GAERS it varies from 7 to 11 c/s. However, it seems impossible to elicit absences with 3 c/s SWDs in rodents (McQueen and Woodbury, 1975; Avoli, 1980). In penicillininduced absences in cats, the mean frequency is 4.5 c/s (Gloor and Fariello, 1988; Gloor et al., 1990). Only in primates can 3 c/s SWDs be observed during pharmacologically induced absence seizures (Marcus and Watson, 1968; Snead, 1978). The frequency of SWDs during generalized non-convulsive seizures therefore seems to be species-dependent Relationship Between Spike and Wave Discharges and Wakefulness The level of vigilance plays a critical role in the occurrence of absence seizures, both in humans and in animal models (Mirsky et al., 1986; Lannes et al., 1988; Coenen et al., 1991; Drinkenburg et al., 1991; Niedermeyer, 1996). In humans, SWDs occur within restricted limits of vigilance state, corresponding to quiet wakefulness or somnolence (Mirsky et al., 1986; Niedermeyer, 1996). To determine the conditions in which the brain can generate SWDs, the relationship between the occurrence of SWDs and the level of vigilance was examined in GAERS equipped with cortical, hippocampal and myographic electrodes, and recorded during 12-hr periods (Lannes et al., 1988). SWDs appear commonly during quiet wakefulness and disappear during active arousal, slow-wave sleep and rapideye-movement (REM) sleep. Twenty per cent of SWDs occur during transition from wakefulness to slow-wave sleep, and 7% during transition from slow-wave sleep to arousal. Less than 7% start and end in slow-wave sleep, and then usually during the rst minute of a sleep episode. SWDs are sporadic during REM-sleep (Lannes et al., 1988). Similar relationships between spontaneous SWDs and vigilance have been described for di erent strains of rodents (Vanderwolf, 1975; Radil et al., 1982; Kaplan, 1985; Coenen et al., 1991; Drinkenburg et al., 1991). In WAG/Rij rats, the occurrence of SWDs was studied in relation to the daily uctuation of vigilance level. Thirty three per cent of SWDs are preceded by passive wakefulness and 48% by light slow-wave sleep. SWDs rarely occur during active wakefulness, deep slow-wave sleep and REM sleep (Coenen et al., 1991; Drinkenburg et al., 1991, 1993). All these studies of absence epilepsy on genetic rat models con rm that the occurrence of SWDs is limited to a speci c vigilance states. A high degree of neuronal desynchronization, observed during both arousal and REM sleep, precludes the occurrence of SWDs. Reciprocally, SWDs do not commonly occur during slow-wave sleep, which is associated with a high degree of neuronal synchronization. The strong

5 Pathophysiological Mechanisms of Genetic Absence Epilepsy in the Rat 31 Table 1. E ects of antiepileptic drugs on SWD in GAERS suppressed by increased by una ected by Ethosuximide Carbamazepine Progabide Valproate Phenytoin Lamotrige Trimethadione Vigabatrin Benzodiazepines (Diazepam, Tiagabine Clonazepam, Nitrazepam) Gabapentin link between vigilance and SWDs has therefore suggested that regulatory mechanisms of arousal may be involved in the occurrence of SWDs (e.g., Section 5) Pharmacological Characteristics of Spike and Wave Discharges The therapeutic pro le of GAERS and human absences is identical (Micheletti et al., 1985; Marescaux et al., 1992a). In GAERS, SWDs are suppressed by the four main antiepileptic drugs which are e ective against human absences (ethosuximide, trimethadione, valproate and benzodiazepines) and worsened by the two drugs which are either ine ective or aggravating in humans (carbamazepine, phenytoin) (Micheletti et al., 1985; Loiseau et al., 1995; Mattson, 1995) (Table 1). Phenobarbital evokes biphasic e ects: it is suppressive at 2.5 to 10 mg/kg, but not at 20 mg/kg (Micheletti et al., 1985). The results obtained in GAERS with new potential antiepileptic drugs are, up to now, predictive of their e ects in human absences: vigabatrin, tiagabine and gabapentine aggravate SWDs, whereas progabide is ine ective (Marescaux et al., 1992a; personal observations). Lamotrigine, which is e ective in humans against simple absences but ine ective against absences with myoclonic components (Schlumberger et al., 1994), is ine ective in GAERS. Finally, SWDs in GAERS are increased by many drugs (pentylenetetrazol, gamma-hydroxybutyrate, THIP and penicillin) that are commonly used to induce bilateral synchronous SWDs in normal rats (Marescaux et al., 1984, 1992a; Snead, 1988, 1994). The pharmacological reactivity of SWDs in GAERS is similar to what has been reported in other genetic or pharmacological models of absence seizures (Peeters et al., 1988; Hosford et al., 1992; Snead, 1994; Van Rijn et al., 1994; Coenen et al., 1995; Hosford and Wang, 1997) Ontogenesis of Spike and Wave Discharges No SWDs are observed in rats younger than 30 days of age in GAERS (Vergnes et al., 1986). At 40 days, only 30% of rats are a ected. The number of GAERS with SWDs then increases gradually with age and reaches 100% at the age of 3 months. The rst SWDs are rare (1 or 2 per hr) and shortlasting (1±3 s), with a low frequency of spike and wave complexes during a discharge (4±5 c/s). With age, the number, duration and frequency of SWDs increases, suggesting that they require complete cortical maturation. The number of SWDs reaches a maximum around 4 to 6 months. SWDs can be recorded until the death of the animals (Vergnes et al., 1986). A similar ontogenesis of spontaneous SWDs has been described in other strains of rats (Chocholova, 1983; Coenen and van Luijtelaar, 1987). Likewise, pentylenetetrazol-induced SWDs appear only at 3 weeks of age (Schickerova et al., 1984) and gamma-hydroxybutyrate-induced SWDs at 4 weeks of age (Snead, 1988). In humans, typical childhood absence epilepsy occurs between the ages of 2 and 8 years and its evolution is most often benign with remission before puberty (Loiseau and Cohadon, 1970; Loiseau et al., 1995). Juvenile absence epilepsy and eyelid myoclonia with absences occur after the ages of 10±12 years and persist in adulthood (Porter, 1993; Hirsch et al., 1994). Absence epilepsies appear, therefore, to be associated with brain development and maturation Genetic Transmission of Spike and Wave Discharges In the initial colony of Wistar rats in our laboratory, 30% of the animals showed spontaneous SWDs. Breeding of selected pairs over a few generations produced a strain in which 100% of the rats were a ected. Similarly, a control strain, free of SWDs, was selected. The data demonstrate that transmission of SWDs are inherited (Marescaux et al., 1992a). A classical Mendelian cross-breeding study with rats from the 13 to 15th generation showed that all o spring from the control nonabsence strain had a normal EEG up to 12 months. All o spring from the GAERS strain presented SWDs. More than 95% of the F1 (control GAERS) showed SWDs at 12 months, demonstrating that there is a dominant transmission. Similar SWDs in males and females F1 indicate that the transmission is autosomal. Interindividual variability for age of appearance and duration of SWDs is extremely high, suggesting that the inheritance of SWDs is probably not due to a single gene locus. This data are con rmed in F2 (F1 F1) and backcross (F1 control) generations (Marescaux et al., 1992a). The selection of homozygous inbred strains of GAERS and control non-epileptic rats upon 37 generations will favor investigations of the genetic background and mode of transmission of absence seizures. Genetic linkage analysis may provide valuable clues to identify genetic loci and genes associated with absence epilepsy. Similar data from the Mendelian cross-breeding study were reported in WAG/Rij rats, where SWDs are apparently controlled by several genes: one dominant gene may determine whether a rat is epileptic or not, while other genes could regulate the number and duration of seizures (Peeters et al., 1990a). However, the inheritance of SWDs in rodents may vary from one strain to another.

6 32 L. Danober et al. In mice, SWDs are transmitted in a recessive way, and may result from several di erent mutations responsible for the same EEG phenotype, which is associated with di erent neurological disorders (Noebels, 1984; Qiao and Noebels, 1991; Hosford et al., 1992). Recently, a mutation of the a 1A voltagesensitive calcium channel gene was identi ed in tottering mice (Fletcher et al., 1996). A mutation of the b subunit of the calcium channel was reported in lethargic mouse (Burgess et al., 1997). Although the tottering and lethargic phenotypes include not only absence seizures but also ataxia, lethargic behavior and motor abnormalities, these are the rst genes that have been proposed to be involved in absence seizures. Human absence epilepsies are also believed to be genetically determined (Lennox and Lennox, 1960; Doose et al., 1973; McNamara, 1994). In homozygous twins, concordance rates of 84% for EEG discharges and of 75% for absence seizures were found, while heterozygous twins showed no concordance (Lennox and Lennox, 1960). A high concordance rate in homozygous twins in comparison to heterozygous twins was also reported by Berkovic et al. (1993). In some families, the high incidence of the presence of 3 c/s SWDs in rst-degree relatives suggests a monogenic autosomal dominant mode of inheritance, the gene having its highest penetrance in childhood and early adolescence (Gloor et al., 1982). In other families multigenic transmission has been suggested (Doose et al., 1973) Conclusion According to their EEG, behavioral and pharmacological similarities with human absence seizures, SWDs in GAERS are now considered as a valid isomorphic and predictive model of absence epilepsy (LoÈ scher and Schmidt, 1988; Jobe et al., 1991; Marescaux et al., 1992a; Buchhalter, 1993). Furthermore, similarities in the genetic inheritance and in the cerebral structures involved in the genesis of SWDs (e.g., Section 3) to the human disorders suggest that GAERS could be a homologous model of absence epilepsy. Since absence seizures in GAERS are spontaneous, numerous and persist for months, this genetic model is particularly useful for pharmacological drug evaluation. Although no animal model completely mimics the human SWDs (Niedermeyer, 1996), the genetic models (i.e. GAERS and WAG/Rij) are helpful in further understanding the pathogenesis of some forms of human absence epilepsies (Jobe et al., 1991; Buchhalter, 1993). 3. SEIZURE NETWORK: THE THALAMO- CORTICAL SUBSTRATE OF ABSENCE SEIZURES Since the 1940s, two main hypotheses concerning the origin of SWDs have been proposed. Gibbs and Gibbs (1952) considered SWDs to be dependent only on a di use cortical process. Bancaud and coworkers suggested that SWDs are always secondary to a focal discharge in the frontal cortex which is rapidly propagated to the whole cortex through various cortico-cortical pathways (Bancaud, 1969). In contrast to this cortical theory, Pen eld and Jasper (1947); Pen eld and Jasper, 1954) assigned to a hypothetic neuronal systemðthe ``centrencephalon''ða cardinal role in initiating SWDs. The generalized nature of SWDs and their simultaneous onset in all areas was attributed to the fact that the centrencephalic system was thought to project di usely to the cerebral cortex. Both the cortical and centrencephalic hypotheses were supported by studies in experimental models and in humans (Hunter and Jasper, 1949; Williams, 1953; Weir, 1964; Marcus and Watson, 1968; Pollen, 1968; Bancaud, 1969). In the animal experiments cited in support of either hypothesis, cortical or subcortical structures had selectively been manipulated and thus the methodology itself overemphasized the role of cortical or centrencephalic mechanisms. Using a model in which SWDs were produced by a systemic acting drug, the feline generalized penicillin epilepsy, Gloor and coworkers attempted to achieve a synthesis of these two opposing views (Gloor, 1968; for review see Gloor and Fariello, 1988). This cortico-reticular hypothesis postulated that SWDs represent the manifestation of an abnormal interaction between the cortex and di usely projecting subcortical neuronal systems centered upon several thalamic nuclei. Genetic animal models of absence seizures have provided observations validating this hypothesis Mapping of Spike and Wave Discharges Brain structures involved in the elaboration of SWDs have been determined in GAERS by depth EEG recordings, performed with bipolar electrodes. SWDs have never been recorded in the hippocampus or in any limbic structure (septum, amygdala, cingular and piriform cortex) in GAERS (Vergnes et al., 1987, 1990; Marescaux et al., 1992a) (Fig. 1). Similar observations have been reported in other genetic and pharmacological models (Inoue et al., 1993; Hosford et al., 1995; Snead, 1994). In contrast, SWDs could be recorded from the dorsal hippocampus in some mice models (Noebels, 1984; Fariello and Golden, 1987). This discrepancy may be related to di erences between species or to technical bias resulting from recording procedures. In our study on GAERS, SWDs were consistently recorded from the lateral frontoparietal cortex and the posterolateral thalamus (Fig. 1), suggesting that these structures play a critical role in the initiation of SWDs (Vergnes et al., 1987). SWDs predominated in lateral and fronto-parietal cortical sites. They were recorded simultaneously over the cortex, starting usually in the frontoparietal regions and more rarely in the occipital cortex (Marescaux et al., 1992a). SWDs were absent or strongly reduced in the anterior and midline nuclei of the thalamus. Small-amplitude or delayed SWDs were recorded in the striatum, lateral hypothalamus and ventral tegmentum (Vergnes et al., 1990; Marescaux et al., 1992a). Discharges involving the cortex and the thalamus were also observed in other strains of rodents with spontaneous or pharmacologicallyinduced SWDs (Klingberg and Pickenhain, 1968; Chocholova, 1983; Semba and Komisaruk, 1984;

7 Pathophysiological Mechanisms of Genetic Absence Epilepsy in the Rat 33 BuzsaÁ ki et al., 1990; Inoue et al., 1993; Snead, 1994; Hosford et al., 1995) and in generalized absences induced by penicillin in cats (Avoli and Gloor, 1982; Gloor and Fariello, 1988; Gloor et al., 1990). All experimental studies on animal models agree that absence seizures are generated in a circuitry that involves the cerebral cortex and the thalamus, although the site of primary dysfunctioning is uncertain Mapping of Local Cerebral Metabolism and Blood Flow Local cerebral energy metabolic examination is often used to determine the neuronal network involved in the generation of convulsive seizures (for review see Handforth and Ackermann, 1995). In order to further characterize the neuronal network involved in the generation of SWDs, local cerebral metabolic rates for glucose (LCMR) have been measured by using the [ 14 C]-2-deoxyglucose method in adult GAERS (Nehlig et al., 1991). An overall increase in LCMR was recorded in GAERS compared to controls. This 16 to 50% increase concerned all structures whether they exhibited SWDs (i.e. neocortex and thalamus) or not (e.g. limbic and brainstem structures) (Nehlig et al., 1991). Similarly, using PET measurements in humans with typical childhood absence epilepsy Engel et al. (1985), showed a general increase of cerebral metabolism during the 45-min scan, without any focal hyperactivity. These data suggest that SWDs are not by themselves responsible for the increase in cerebral energy metabolism (Nehlig et al., 1991). However, because of the long duration of a 2- deoxyglucose experiment, i.e. 45 min, the cerebral metabolic level recorded is always a combination of ictal and interictal phases. Therefore, in order to record continuous changes in cerebral blood ow during SWDs in GAERS, laser-doppler owmetry was applied on the cortex (Nehlig et al., 1996). SWDs were accompanied by a decrease in the level of cerebral blood ow which started 2±7 s after the beginning of the cortical SWDs. At the end of SWDs, cerebral blood ow returned to baseline levels (Nehlig et al., 1996). Cerebral blood ow was signi cantly decreased under basal levels during absences in humans (Sperling and Skolnick, 1995; Nehlig et al., 1996). The metabolic studies suggest that the underlying alterations of absence epilepsy may not be speci cally expressed within the cerebral circuitry involved in the genesis of SWDs, but may be di used throughout the central nervous system Bilateralization and Synchronization of Spike and Wave Discharges: Role of Callosal and Thalamic Pathways On both EEG and oscilloscopic recordings, SWDs are always bilateral and synchronous on both cerebral hemispheres (Vergnes et al., 1989). Cortical neurons from one hemisphere project massively to the contralateral cortex through the corpus callosum (Innocenti, 1986; Kolb and Tees, 1990). Some intrathalamic connections have also been observed (Rinvik, 1984; Jones, 1985; Chen et al., 1992). These two pathways may thus be involved in the bilateralization and synchronization of SWDs. To examine this hypothesis, transection of the corpus callosum and/or midline cut of the thalamus were performed in GAERS. After transection of the corpus callosum, the bilateral synchronism was partially abolished and two patterns emerged: (i) SWDs occurred unilaterally and independently on each hemisphere and sometimes alternated from one side to the other; (ii) SWDs started on one side and then continued bilaterally after delays varying from 0.5 to several seconds (Vergnes et al., 1989). However, some bilateral synchronous SWDs still occurred in most animals. When SWDs were bilaterally present, each single spike-and-wave complex was synchronous on both hemispheres. The total amount of seizures, measured on both hemispheres, was comparable to the values found in intact GAERS (Vergnes et al., 1989). Similarly, in other animal models with bilateral and synchronous SWDs, the corpus callosum appears to be the main structure ensuring bilateral synchronization (Marcus et al., 1969; Naquet et al., 1975; Musgrave and Gloor, 1980). On the contrary, the midline thalamus appears to play a minor role in bilateral transfer of SWDs. In GAERS, speci c transection of this area separating the thalamus in two lateral parts did not a ect SWDs, which occurred in the same amounts as in intact animals and synchronously on both hemispheres. However, when the transection of the corpus callosum was associated with a midline cut through the thalamus, the bilateral desynchronization was more complete: during a bilateral SWDs, the single spike-and-wave complexes occurred independently in each hemisphere (Vergnes and Marescaux, 1995). These data show that although the corpus callosum is the major pathway involved in bilateralization of spontaneous SWDs, synchronization may also develop through the midline thalamus. However, this pathway is not critical for synchronization E ects of Cortical and Thalamic Lesions To examine cortico-thalamic relationships during the generation of SWDs, various cortical and thalamic areas were lesioned in GAERS. The corpus callosum was previously transected to prevent the interhemispheric propagation of SWDs (e.g., Section 3.3). This preparation, therefore, allows the study of the e ects on the lesion in one hemisphere and control on the contralateral non-lesioned hemisphere in the same rat. When a functional cortical lesion was produced by a unilateral injection of KCl into the super cial layers of the cortex, SWDs were immediately suppressed, not only in the injected cortex, but also in the ipsilateral thalamus (Fig. 2A) (Vergnes and Marescaux, 1992). Only after full recovery of the cortical activity (30 to 50 min after the KCl injection), did the rst SWDs reappear simultaneously in the cortex and the thalamus on the injected side. Controlateral SWDs were unchanged in both cerebral structures throughout the experiment (Vergnes

8 34 L. Danober et al. Fig. 2. E ects of cortical functional inhibition and thalamic lesion on the occurrence of absence seizures in GAERS. A, Left panel: coronal section showing an unilateral cortical spreading depression consecutive to cortical application of 1 ml of KCl solution from a brain with transected corpus callosum. Right panel: EEG recordings in the cortex and thalamus from both hemispheres. SWDs are suppressed in both the thalamus and cortex ipsilateral to the KCl application. SWDs are unchanged in the thalamus and cortex of the contralateral non-injected side. B, Left panel: coronal section showing an unilateral lesion of the lateral thalamus, associated with a transection of the corpus callosum. Right panel: EEG recordings in the cortex from both hemispheres. SWDs are abolished on the cortex ipsilateral to the thalamic lesion. Cx: cortex; Hi: hippocampus; ic: internal capsule; Rt: reticular thalamic nucleus; VB: ventrobasal thalamic nucleus. and Marescaux, 1992). This data demonstrates that SWDs cannot occur in a thalamus that is disconnected from the cortex. This is in agreement with penicillin-induced SWDs in cats which are also suppressed by cortical spreading depression (Avoli and Gloor, 1982; Gloor and Fariello, 1988; Gloor et al., 1990). Bilateral electrolytic lesions of the anterior, the ventromedial or the medial thalamus did not a ect the number and duration of SWDs in GAERS. On the contrary, large bilateral lesions of the lateral thalamus completely suppressed SWDs (Vergnes and Marescaux, 1992). After unilateral electrolytic lesions of the lateral thalamus, including the ventrobasal and reticular nuclei, cortical SWDs were de nitively suppressed on the ipsilateral side, whereas SWDs occurred normally on the unlesioned side (Fig. 2B). Moreover, in these animals, THIP, gamma-hydroxybutyrate and pentylenetetrazol, which usually induce SWDs in non-epileptic rats, never evoked SWDs on the lesioned side, whereas they markedly increased spontaneous SWDs on the unlesioned side (Vergnes and Marescaux, 1992). These results clearly show that SWDs cannot occur in a cortex deprived of its thalamic inputs. Furthermore, a unilateral excitotoxic lesion limited to the reticular nucleus completely suppressed SWDs on the ipsilateral hemisphere during the rst 3 days, indicating a predominant role of this nucleus in the generation of SWDs in GAERS. On the fourth day, low frequency (3±5 c/s) sharp waves appeared on the lesioned side and persisted for the duration of the experiments but classical SWDs never reappeared (Avanzini et al., 1992). Finally, local injections in the reticular thalamic nucleus of the calcium channels blocker, cadmium, reversibly suppressed SWDs (Avanzini et al., 1993). Similarly to GAERS, cortical SWDs were suppressed by thalamic lesions in several other experimental models. BuzsaÁ ki et al. (1988) showed that

9 Pathophysiological Mechanisms of Genetic Absence Epilepsy in the Rat 35 lesions of the reticular thalamic nucleus suppressed cortical high-voltage SWDs in old rats. Pentylenetetrazol-induced cortical SWDs in rats were abolished when thalamic activity was blocked by spreading depression (Pohl and Mares, 1983). In the model of penicillin-induced generalized epilepsy in the cat, large lesions of the lateral thalamus abolished cortical SWDs, whereas lesions of the anterior nuclei or the ventromedial thalamus were ine ective (Gloor and Fariello, 1988; Gloor et al., 1990) Cortical and Thalamic Cellular Correlates of Spike and Wave Discharges Several electrophysiological studies have con- rmed that cortical and thalamic events underlie SWDs. In penicillin-induced SWDs in the cat, the spike component of SWDs was associated with a large barrage of excitatory postsynaptic potentials and repetitive synchronous ring in cortical and thalamic neurons, whereas the wave component was associated with neuronal silence due to long-lasting hyperpolarizing potentials (Avoli et al., 1983; Gloor and Fariello, 1988). Similar observations were made in primate (Steriade, 1974), in WAG/Rij rats (Inoue et al., 1993), in old Fischer rats (BuzsaÁ ki et al., 1988) and in GAERS (personal observations). Recent in vivo experiments in anesthetized cat showed that during SWDs, cortical neurons display a progressive synchronization along with a tonic depolarization, sculptured by rhythmic hyperpolarizations, whereas relay thalamocortical neurons were inhibited, probably by GABAergic reticular thalamic neurons (Steriade and Amzica, 1994; Steriade and Contreras, 1995) Thalamo-Cortical Neurotransmissions and Absence Seizures As the thalamus and cortex were shown to be the structures of the central nervous system that underlie absence seizures, several studies have examined the role of neurotransmitters in this circuitry in the occurrence of absence seizures. Thalamocortical and corticothalamic projections are mainly glutamatergic. Several intracortical and intrathalamic inhibitory GABAergic projections have also been described (e.g., Section 4.1). Therefore, pharmacological studies have focused primarily upon GABAergic and excitatory glutamatergic systems GABAergic Neurotransmission and Absence Seizures In GAERS, intraperitoneal (i.p.) administration of GABA A -mimetics (GABA A agonists: muscimol and THIP; GABA transaminase inhibitors: g-vinyl GABA and L-cycloserine; or GABA reuptake inhibitors: SKF and tiagabine) induced a dosedependent increase in the duration of SWDs (Table 2) (Vergnes et al., 1984; Marescaux et al., 1992b). Similar results were obtained when the same drugs were injected bilaterally into relay thalamic nuclei (Table 2) (Liu et al., 1991; Marescaux et al., 1992b). At high doses, i.p. or intrathalamic injections of GABA A -mimetics induced permanent SWDs with a reduced frequency (5±6 c/s) or isolated spikes on a at EEG background. On the contrary, when GABA A mimetics were applied bilaterally within the reticular thalamic nucleus, they suppressed SWDs, probably by preventing oscillatory activities in this nucleus (Table 2) (Liu et al., 1991). Similar results were obtained after local injections of GABA A -mimetics in reticular and ventrobasal thalamic nuclei in lethargic mice (Hosford et al., 1997). Intraperitoneal administration of GABA A -mimetics aggravated seizures in all models of generalized nonconvulsive epilepsy in rodents, as well as in cats (Fariello et al., 1980; Marescaux et al., 1992b; Snead, 1994). They also aggravated the bilateral SWDs induced by light ashes in the baboon Papio papio (Meldrum and Horton, 1980). Moreover, systemic and thalamic administration of GABA A - mimetics in non-epileptic animals induced synchronous oscillations that resemble SWDs (Fariello and Golden, 1987; Liu et al., 1991; Marescaux et al., 1992b). Upon systemic or local intrathalamic injections, GABA A antagonists (picrotoxin or bicuculline) reduced spontaneous SWDs in GAERS, but also induced myoclonic SWDs, followed, at the highest doses, by convulsive seizures (personal observations). R-baclofen, a GABA B agonist increased SWDs in GAERS when injected i.p. or bilaterally in the reticular or relay nuclei. The same injections induced paroxysmal rhythmic oscillations which resemble SWDs in control rats (Marescaux et al., 1992c). On the contrary, systemic administration of CGP 35348, and other GABA B antagonists, suppressed SWDs in GAERS (Marescaux et al., 1992c) (Table 2). Similar results were obtained after intrathalamic microinjections of GABA B antagonists in either ventrobasal or reticular thalamic nuclei (Table 2). Furthermore, intrathalamic injections of Table 2. E ects of intraperitoneal (i.p.) and local intrathalamic injections of GABAergic compounds on SWD in GAERS. Rt: reticular thalamic nucleus; VB: ventrobasal thalamic nucleus. i.p. injections local VB injections local Rt injections GABA transaminase inhibitor (g-vinyl-gaba) increase increase biphasic GABA reuptake inhibitor (SKF 89976) increase not done not done GABA A agonists (Muscimol, THIP) increase increase decrease antagonists (Picrotoxin, Bicuculline, SR 95531) no e ect no e ect no e ect GABA B agonist (Baclofen) increase increase increase antagonists (CGP 35348, Phaclofen) decrease decrease decrease

10 36 L. Danober et al. CGP suppressed both spontaneous SWDs in GAERs and SWDs induced in non epileptic rats by prior injection of GABA A -mimetics (THIP, muscimol, g-vinyl GABA), pentylenetetrazol or g-hydroxybutyrate (Liu et al., 1992; Marescaux et al., 1992c) (Table 2). Similar data were obtained in other models of absence seizures (Hosford et al., 1992; Snead, 1992). These data indicate that both GABA A and GABA B neurotransmissions play a critical role in the generation and control of SWDs in absence epilepsy. It is known that systemic injection of muscimol reduces convulsive seizures, whereas GABA antagonists bicuculline and picrotoxin evoke these seizures (for review, see Bradford, 1995). The di erential e ects of GABAergic compounds on convulsive and non-convulsive epilepsy mean that the GABAergic system is di erently involved in these two types of epilepsy. Absences constitute a particular form of epilepsy which may be related to an excess of GABAergic inhibition within the thalamus (Fromm and Kohli, 1972; Fariello et al., 1980; Gloor and Fariello, 1988; Liu et al., 1991; Hosford et al., 1992; Liu et al., 1992). Conversely, during focal or generalized convulsive seizures, an excess of excitation is prevalent (Meldrum, 1994; Bradford, 1995) Glutamatergic Neurotransmission and Absence Seizures Intra-peritoneal or intra-cerebroventricular injections of both N-methyl-D-aspartate (NMDA) and NMDA antagonists (the competitive NMDA-antagonist CGP 40116, the non-competitive NMDA-antagonist (+)-MK 801 and the antagonist of the glycine modulatory site 5,7-dichlorokynurenic acid) dose-dependently suppressed SWDs in GAERS. Bilateral infusions of the same drugs in the ventrobasal nucleus of the thalamus had similar suppressive e ects (Marescaux et al., 1992b, Koerner et al., 1996). Similar results were obtained with NMDAantagonists in rats, after SWDs were pharmacologically induced by i.p. injection of gamma-hydroxybutyrate (Banerjee and Snead, 1992, 1995), and in WAG/Rij rats (Peeters et al., 1989, 1990b). This data con rms that NMDA receptor-mediated neurotransmission plays a major role in the thalamo-cortical circuit underlying absence seizures. Dysregulation of thalamic NMDA-mediated transmission by NMDA itself or by NMDA antagonists interacting with various sites of the receptor complex, may suppress the thalamo-cortical oscillatory activity which underlies SWDs. Intracerebroventricular injections of a-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) induced a dose-dependent increase of SWDs in WAG/Rij rats, whereas similar injections of AMPA antagonists (glutamic acid diethylester and CNQX) decreased them (Ramakers et al., 1991; Peeters et al., 1994). Kainate had no e ects on the occurrence of SWDs but induced convulsive seizures at high doses (Peeters et al., 1994). These results suggest that AMPA receptors are involved in the occurrence of absence seizures Conclusion In GAERS, as in other models, EEG recordings from various brain regions and lesion experiments suggest that the frontoparietal cortex, reticular nucleus and relay nuclei of the thalamus play a predominant role in the development of SWDs. Neither the cortex, nor the thalamus alone can sustain SWDs, indicating that both structures are intimately involved in the genesis of SWDs. Pharmacological data con rms that both inhibitory and excitatory neurotransmissions are involved in the genesis and control of absence seizures. Whether the generation of SWDs is the result of an excessive cortical hyperexcitability, as was proposed in feline generalized epilepsy (Gloor and Fariello, 1988; Gloor et al., 1990), or an excessive thalamic synchronization, possibly under the control of inhibitory GABAergic mechanisms (Crunelli and Leresche, 1991; Liu et al., 1991, 1992), remains controversial. Possible pathogenic impairment of the thalamo-cortical circuit will be discussed in the following section. 4. THALAMO-CORTICAL MECHANISMS IN ABSENCE EPILEPSY Altogether, the data presented in Section 3 provides evidence for a thalamo-cortical substrate of SWDs. Before discussing the main hypotheses proposing that thalamo-cortical alterations underlie absence seizures, the anatomy and functioning of the thalamo-cortical circuit will be brie y reviewed Anatomy of the Thalamo-Cortical Circuit The thalamo-cortical circuitry includes connections between the cerebral cortex, relay nuclei of the dorsal thalamus and the reticular nucleus of the ventral thalamus (Fig. 3) (For review, see Sherman and Guillery, 1996). The thalamus is the major source of subcortical inputs to the cerebral cortex. Most of the thalamocortical axons terminate in the cortical layers IV and III (Fig. 3) (Jones, 1985; Alloway et al., 1993; Lu and Lin, 1993). Given the abundance of cortical interconnections, information originating in the thalamus is transmitted to other cortical areas. Reciprocally, pyramidal cells from the cortical layers V and VI provide an important innervation to the thalamus (Fig. 3) (Jones, 1985; Chmielowska et al., 1989; Burkhalter and Charles, 1990; Boussara et al., 1995). Thalamocortical and corticothalamic connections appear to be mainly glutamatergic (Fonnum et al., 1981; Ottersen et al., 1983; Kanedo and Mizumo, 1988; Descheà nes and Hu, 1990; Zilles et al., 1990; Kharazia and Weinberg, 1994). NMDA, AMPA and metabotropic receptors are involved in the excitatory synaptic responses recorded in thalamocortical neurons, evoked by stimulation of cortical a erents (McCormick, 1992; McCormick and Von Krosigk, 1992; Gil and Amitai, 1996; Salt and Eaton, 1996). No connections exist between the di erent relay thalamic nuclei. In the rat, GABAergic interneurons are con ned within the lateral geniculate nucleus,

11 Pathophysiological Mechanisms of Genetic Absence Epilepsy in the Rat 37 In the cerebral cortex, the main source of GABAergic inhibition comes from local non-pyramidal interneurons (basket cells, chandelier cells, double bouquet cells and bipolar cells) (Fig. 3) which strongly control the activity of cortical pyramidal neurons (Somogyi and Cowey, 1981; Meinecke and Peters, 1987; Beaulieu, 1993; DeFelipe, 1993). Cortical interneurons receive thalamocortical and extrathalamic input and therefore are involved in the transfer and integration of sensory information coming from the thalamus to the cortex (McCormick, 1992). Fig. 3. Schematic representation of the thalamo-cortical circuit. Thalamocortical and corticothalamic projections are mainly glutamatergic. Each of these pathways sends a collateral projection to the reticular thalamic nucleus. The reticular thalamic nucleus provides, in turn, large GABAergic inhibitory projections to thalamocortical relay neurons. In the cortex, several intracortical GABAergic interneurons control the activity of cortical neurons. contrary to the cat, monkey and guinea pig, in which GABAergic interneurons have been described in all relay nuclei (Ohara et al., 1983; Jones, 1985; Rinvik et al., 1987; Sprea co et al., 1994). All thalamic relay nuclei receive a massive GABAergic inhibitory projection from the reticular thalamic nucleus, which is the primary source of GABA in the rat thalamus (Jones, 1985; De Biasi et al., 1988; Pinault et al., 1995; Cox et al., 1996) (Fig. 3). Therefore, the reticular thalamic nucleus assures the synchronization of thalamic oscillations and thus plays a critical role in the gating of sensory information, passing through the dorsal thalamus (Steriade and Descheà nes, 1984; Steriade et al., 1987; Contreras et al., 1993). The reticular thalamic nucleus is exclusively composed of GABAergic inhibitory neurons (Houser et al., 1980; De Biasi et al., 1986; Sprea co et al., 1991). Interaction between reticular neurons depends on dendro-dendritic synapses and on short axonal collaterals that project laterally to other GABAergic reticular cells (Fig. 3) (Descheà nes et al., 1985; Mulle et al., 1986; Sprea co et al., 1988; Luebke, 1993; Pinault et al., 1997). Reticular thalamic neurons do not project directly to the cerebral cortex but receive excitatory collaterals from thalamocortical and corticothalamic neurons (Fig. 3) (Frassoni et al., 1984; Jones, 1985; Harris, 1987; de Curtis et al., 1989; Sprea co et al., 1991; Contreras et al., 1993; Luebke, 1993; Warren et al., 1994; Boussara et al., 1995) Physiological Functioning of the Thalamo-Cortical Circuit The thalamus is a key structure in the central nervous system for gating the ow of sensory information from the periphery to the cortex. Electrophysiological in vitro and in vivo studies have shown that thalamocortical neurons spontaneously exhibit two di erent modes of intrinsic activity during various stages of the wakefulness/sleep cycle (Steriade and Descheà nes, 1984; Llinas, 1988; McCormick, 1992; Steriade, 1993; Steriade et al., 1993; McCormick and Bal, 1997). Periods of arousal and attentiveness are associated with a tonic mode of discharge of fast, sodium/potassium-mediated action potentials, allowing transmission of information to the cortex. During stages of drowsiness and slow-wave sleep, thalamocortical neurons generate bursts of spikes triggered by rhythmic lowthreshold calcium spikes (Steriade and Descheà nes, 1984; Llinas, 1988; McCormick and Pape, 1990; Leresche et al., 1991; McCormick, 1992; Steriade, 1993; Steriade et al., 1993). This rhythmic oscillatory bursting mode of discharge is involved in the generation of synchronized oscillatory activity in the forebrain, which prevails during spindles and slow/delta waves, that occur respectively, during early and late deep stages of slow-wave sleep. Delta activities, occurring at low frequency (0.5± 4 c/s), are generated by intrinsic mechanisms of thalamocortical neurons. Delta oscillations result from the interaction between two neuronal intrinsic currents: a low-threshold Ca 2+ current (I T ) and a cationic inward rectifying current (I h ) that are, respectively, deinactivated and activated by membrane hyperpolarization (McCormick and Pape, 1990; Leresche et al., 1991; Soltesz et al., 1991; McCormick, 1992; Steriade, 1993; Steriade et al., 1993; McCormick and Bal, 1997). Spindles consist of rhythmic oscillations of 7± 14 c/s that occur every 3±10 s during the initial phase of slow-wave sleep (Steriade and Descheà nes, 1984; Steriade, 1993). Spindles result from interaction between the reticular and relay thalamic nuclei, where both intrinsic and synaptic properties play a critical role (Steriade and Descheà nes, 1984; Bal and McCormick, 1993; Steriade et al., 1993; Von Krosigk et al., 1993; Destexhe et al., 1994; Huguenard and Prince, 1994; Bal et al., 1995; Wang et al., 1995; Destexhe et al., 1996; McCormick and Bal, 1997). In vivo and in vitro recordings have demonstrated that spindles are generated by GABAergic inhibitory neurons of the reticular thal-