Selective Susceptibility to Inhibitors of GABA Synthesis and Antagonists of GABAA Receptor in Rats with Genetic Absence Epilepsy

Experimental Neurology 161, 714–723 (2000) doi:10.1006/exnr.1999.7302, available online at http://www.idealibrary.com on

Selective Susceptibility to Inhibitors of GABA Synthesis and Antagonists of GABAA Receptor in Rats with Genetic Absence Epilepsy Marguerite Vergnes, Any Boehrer, Sophie Reibel, Simone Simler, and Christian Marescaux Faculte´ de Me´decine, INSERM U 398, 11 rue Humann, 67085 Strasbourg Cedex, France Received May 12, 1999; accepted October 19, 1999

Thalamocortical spike-and-wave discharges characterize the nonconvulsive absence seizures that occur spontaneously in genetic absence epilepsy rats from Strasbourg (GAERS), a selected strain of Wistar rats. GABA is crucial in the generation of absence seizures. The susceptibility to convulsions induced by threshold doses of various GABA receptor antagonists and inhibitors of GABA synthesis, kainic acid and strychnine, was compared in GAERS and in nonepileptic rats from a selected control strain (NE). The brain structures involved in the drug-elicited convulsive seizures were mapped by c-Fos immunohistochemistry. Injection of various antagonists of the GABAA receptor, bicuculline and picrotoxin, and inverse agonists of the benzodiazepine site (FG 7142 and DMCM) induced myoclonic spike-and-wave discharges followed by clonic or tonic– clonic seizures with high paroxysmal activity on the cortical EEG. The incidence of the convulsions was dose-dependent and was higher in GAERS than in NE rats. Mapping of c-Fos expression showed that the frontoparietal cortex was constantly involved in the convulsive seizures elicited by a threshold convulsant dose, whereas limbic participation was variable. In contrast, GAERS were less susceptible than NE rats to the tonic–clonic convulsions induced by the inhibitors of glutamate decarboxylase, isoniazide and 3-mercaptopropionic acid. The GABAB receptor antagonist CGP 56999 and kainic acid induced a similar incidence of seizures in GAERS and NE rats and predominantly activated the hippocampus. No difference in the tonic seizures elicited by strychnine could be evidenced between the strains. These results suggest that an abnormal cortical GABAergic activity may underlie absence seizures in GAERS. r 2000 Academic Press Key Words: absence epilepsy; cortex; GABA receptor antagonists; convulsions; c-Fos immunoreactivity; rat.

INTRODUCTION

Absence epilepsy is characterized by generalized nonconvulsive seizures associated with bilateral spikeand-wave discharges (SWD) and behavioral arrest. 0014-4886/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.

Electroencephalogram (EEG) and behavior return to normal immediately at the end of the seizure (26). Absence seizures, studied in a number of animal models, have a well-defined thalamocortical substrate and do not spread to other brain systems (1, 38, 44). Various rodents spontaneously display cortical SWD characteristic of absence seizures. In mice, independent locus mutations produce generalized spike-wave seizure disorders, sometimes associated with neurological abnormalities (35). Spontaneous SWD have also been recorded in several strains of rats, with the frequency of seizures and their age-dependency varying with the strain (9, 22, 44). Genetic absence epilepsy rats from Strasbourg (GAERS), used in the present experiments, were selected from a Wistar colony for occurrence of spontaneous absence-like seizures (48) and inbred for more than 30 generations in our laboratory. The SWD in GAERS fulfill the requirements for an experimental model of generalized nonconvulsive absence epilepsy (44). Simultaneously, a nonepileptic control strain (NE) free of SWD was inbred from rats selected in the same Wistar colony. In GAERS, thalamocortical SWD occur constantly (about 1/min) and last up to 60 s, when animals are awake and inactive. The specific pharmacological reactivity of absence seizures is identical in GAERS and humans and distinct from that of convulsive seizures (30). All GABA mimetics aggravate absence seizures in GAERS and induce permanent SWD with increasing doses (47). The generation of SWD may be due to dysfunctions either at the level of the thalamus where rhythmic activities are generated or at the level of their cortical projections. The thalamus and in particular the GABAergic projections from the reticular nucleus on relay nuclei are considered to play a crucial role in the generation and synchronization of SWD (for a review see 12). Alternatively, generation of SWD has been attributed to an excessive cortical excitability or synchronization, with SWD representing an abnormal cortical response to afferent thalamocortical volleys (17). Impaired inhibitory activity of local circuits and/or excitatory synaptic mechanisms could account for these cortical alterations.

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Idiopathic and acquired forms of epilepsy are commonly associated with alterations in GABAergic inhibition, resulting in increased susceptibility to epileptogenic agents. In genetically epilepsy-prone rats, a model of generalized clonic–tonic convulsions induced by sound, increased seizure susceptibility to a variety of convulsants has been reported (6, 15, 23) and a diminished GABAA receptor-mediated inhibition in the inferior colliculus has been suggested to underlie the audiogenic seizure susceptibility (14). Administration of pentylenetetrazol (PTZ) at low doses in nonepileptic rats induces SWD and is considered a pharmacological model of absence seizures. Repeated injections of subconvulsant doses of PTZ produce chemical kindling, which has been associated with enhanced susceptibility to convulsions induced by GABAA receptor antagonists (11). It has also been demonstrated that a small decrease in the efficacy of the intracortical GABAergic system can lead to propagating synchronized discharges, which might play a role in susceptibility to epileptogenesis (7, 28). The impairment underlying generation of SWD, especially a possible alteration in GABA function and/or cortical excitability, may concomitantly modify seizure susceptibility to a variety of convulsants. Conversely, selective susceptibility to convulsants in GAERS could provide information on the pathogenesis of absence epilepsy. Previous results have shown that GAERS are more susceptible than NE rats to cortical GABA withdrawal-induced focal seizures and to PTZ-induced generalized convulsions (4). Therefore, the seizure susceptibility to various convulsant drugs was compared in GAERS and NE control rats. Small doses of convulsants were administered systemically, with emphasis on inhibitors of GABAergic neurotransmission, such as antagonists of GABA receptors and inhibitors of GABA synthesis. In addition, two non-GABAergic convulsants were used: an agonist of the kainate–AMPA receptor, kainic acid, and the antagonist of the strychninesensitive glycine receptor, strychnine; these drugs induce seizures primarily in limbic and medullary structures, respectively. The seizures were characterized by their electroencephalographic and behavioral correlates. Moreover, local expression of c-Fos protein was examined after administration of threshold convulsant doses, in order to localize the drug-induced seizure activity and to correlate the EEG and clinical data with the involvement of particular anatomical and functional circuits. We focused our interest on the forebrain, including the neural substrate of absence seizures and the highly seizure-prone limbic structures, in order to specify the substrates of the seizures induced by the different drugs. The expression of the nuclear protooncogene c-fos and related immediate-early genes can rapidly and transiently be induced in specific brain areas in response to a wide range of epileptic seizures

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(32). Immunohistochemical labeling of the protein product Fos is commonly used to map activated cells and pathways involved in epileptic seizures. METHODS

Drugs Representative compounds of various types of GABAergic inhibitors were used: GABAA receptor antagonists, (1)-bicuculline and picrotoxin; benzodiazepine receptor inverse agonists (3), DMCM (methyl-6,7dimethoxy-4-ethyl-b-carboline-3-carboxylate) and FG 7142 (N-methyl-b-carboline-3-carboxamide); inhibitors of glutamate decarboxylase activity (GAD) blocking GABA synthesis, isoniazide (isonicotinic acid hydrazide) (19) and 3-mercaptopropionic acid (3-MPA) (34); and a high-affinity GABAB antagonist (2), CGP 56999, [3[[1-R-(3-carboxyphenyl) ethyl] amino]-2-(S)-hydroxypropyl]cyclohexyl-methyl-phosphinic acid), kindly provided by Dr. W. Froestl (Ciba-Geigy, Basel, Switzerland). Two non-GABAergic convulsants, kainic acid and strychnine, were used. The drugs were dissolved in saline, or in molecussol when necessary (bicuculline, FG 7142, and DMCM), and injected intraperitoneally in a volume of 2 ml/kg. At least 3 days of recovery were allowed between injections. In order to prevent a facilitation of convulsions due to repeated seizures, drugs were administered with increasing doses in order to find the threshold dose eliciting convulsions in groups of six or more animals. Repeated seizures and status epilepticus were avoided by limiting the doses injected. When status epilepticus occurred, it was interrupted rapidly by administration of diazepam (1 mg/rat). As elicitation of some seizures can be facilitated by handling, manipulation of the animals was avoided during the experiment. Different series of animals were used for each drug. Animals Adult male Wistar rats (300–450 g) from inbred GAERS and NE strains selected in our laboratory were used. The animals were housed individually, fed ad libitum, submitted to a 12-h light/dark cycle, and gently handled before the experiments were started. In GAERS, SWD occur spontaneously when animals are kept awake. Control NE rats are free of any spontaneous SWD. Experiments were performed in accordance with the guidelines of the European Community Council Directive and the French Ministry of Agriculture. Surgery Surgical procedures were performed under pentobarbitone anesthesia (45 mg/kg ip) in a stereotaxic frame. For EEG recordings, all rats were fitted with four single-contact electrodes connected to a microconnec-

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tor. The electrodes, made from stainless-steel screws, were implanted bilaterally over the frontal and parietal cortex. The electrodes were embedded in dental acrylic cement. The animals were then allowed 1 week of recovery before drug injections. EEG Recording The left and right frontoparietal EEG was recorded in freely moving animals placed in a Plexiglas cage and connected to the EEG apparatus with flexible wires, for 20 min before drug injection, and for 1 to 3 h after drug injection, according to the kinetics of the compound. The animals were continuously observed and the behavioral seizure pattern was noted on the EEG. The latency, duration, and number of seizures were evaluated on the EEG. Some experiments were replicated before and after surgery but revealed no difference in susceptibility to convulsions.

noted for each experimental group in the considered structures. The semiquantitative rating of c-Fos labeling, based on the intensity of immunostaining and the density of stained cells in forebrain structures, provides information on the origin and/or circuitry of the seizure, but statistical analysis of these results would be inappropriate. Analysis of Results The proportion of animals convulsing in each group was compared using the x2 test. The latency and duration of the seizures were measured on the EEG and groups were compared with the nonparametric Mann–Whitney test for independent samples. A correlation between duration of seizures and intensity of cortical c-Fos labeling was calculated using a nonparametric regression analysis. RESULTS

C-Fos Protein Immunohistochemistry Two hours after occurrence of a seizure or after injection of the solvent for controls, the animals were anesthetized and decapitated, and the brains were removed and rapidly frozen in isopentane at 235°C and stored at 280°C. Serial 25-µm-thick coronal sections were collected, mounted on chromalun gelatin-coated slides, and left to dry at room temperature for 1 h. Sections were postfixed in freshly prepared 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.3) for 7 min and sequentially rinsed in phosphate-buffered saline (PBS), PBS containing 0.2% H2O2 to quench endogenous peroxidase activity, and PBS with 2% bovine serum albumin, 10% normal goat serum, and 0.25% Triton X-100 (PBS1) to reduce nonspecific binding. The immunohistochemical procedure consisted of sequential incubations at room temperature. Sections were incubated with the primary rabbit polyclonal antibody against c-Fos (Santa Cruz Biotechnology SC052), 1/300, for 2 h. Slides were then rinsed three times in PBS1 and incubated with secondary biotinylated goat anti-rabbit antibody, 1/200, for 1 h, followed by avidin–biotin peroxidase complex using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Fos immunostaining was revealed by nickel (0.01% nickel chloride) enhancement of the chromogen diaminobenzidine (0.025%, Sigma). Slides were rinsed, dehydrated, and coverslipped. C-Fos immunoreactivity, appearing as a dark brown staining in the cell nucleus, was rated in forebrain structures. The distribution and density of Fos-stained cells were rated by direct visual examination using a four-point grading scale from 0 to 3, with 0 corresponding to the absence of immunoreactive cells and 1, 2, and 3 indicating low, moderate, and intense labeling, respectively. The median and extreme values (range) were

Antagonists of the GABA Receptor All antagonists of the GABAA receptor, bicuculline, picrotoxin, DMCM, and FG7142, induced short (1–7 s) SWD in NE rats and modified clinical expression of spontaneous SWD in GAERS. These SWD were rapidly accompanied by myoclonus, expressed as isolated or irregular jerks of the head and the body. Myoclonus already appeared at doses below the convulsant doses and always preceded the occurrence of convulsive seizures. These preconvulsive phenomena were not considered convulsive seizures. The clonic or tonic–clonic convulsion usually appeared following a myoclonic discharge, simultaneously with high-amplitude spikes and waves on the EEG (Fig. 1). With all antagonists of the GABAA receptor the convulsant doses were significantly lower in GAERS than in NE rats (Table 1). The evaluated CD50, in GAERS and NE, respectively, was 1.7 and 3.7 mg/kg for picrotoxin, 0.6 and 1.3 mg/kg for DMCM, and 0.4 and 23 mg/kg for FG 7142. Picrotoxin induced convulsions in a dose-dependent manner, with higher incidence of seizures and shorter latencies in GAERS than in NE rats. Similar results were obtained in three age groups: 2-, 7-, and 12-monthold animals. Bicuculline, at 4 mg/kg, induced significantly more convulsions in GAERS than in NE rats. The difference in sensitivity to convulsions in GAERS and NE rats was particularly remarkable with the inverse agonists of the benzodiazepine receptor. In GAERS, the threshold convulsant dose was about two times lower with DMCM, a full inverse agonist, and four times lower with FG 7142, a partial inverse agonist (Tables 1 and 2, Fig. 2). The GABAB receptor antagonist CGP 56999 produced similar seizures in GAERS and in NE rats, with CD50 5 2.6 and 2.4 mg/kg, respectively (Tables 1 and 2,

FIG. 1. EEG recording of seizures. (A) Picrotoxin: 1, Spontaneous spike-wave discharge (absence seizure) before drug injection in a GAERS. 2, In the same animal, a clonic seizure induced 18 min after picrotoxin injection (2 mg/kg). (B) CGP 56999: In a NE rat, seizure without motor expression 93 min after CGP 56999 injection (2.5 mg/kg). (C) Kainate: 1, Spontaneous spike-wave discharge (absence seizure) before drug injection. 2, In the same GAERS, seizure with immobility induced by kainic acid (5 mg/kg) 40 min after injection. 3, Clonic generalized seizure in a NE, 47 min after kainic acid injection. Calibration: 1 s, 200 µV.

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Fig. 2). At the threshold dose the seizures were recorded on the EEG as rhythmic spike discharges, without any motor behavior (Fig. 1). In most animals, several seizures occurred. Their duration increased and they finally generalized as clonic or tonic–clonic motor seizures with large spike waves on the EEG.

Isoniazide, 200 mg/kg, provoked one or two generalized tonic–clonic seizures in all NE rats, whereas no GAERS displayed convulsions. The incidence of tonic– clonic convulsions secondary to the injection of 3-MPA was higher in NE rats than in GAERS at the dose of 22.5 mg/kg (Tables 1 and 2). Non-GABAergic Convulsants: Kainic Acid and Strychnine At a threshold dose (5 mg/kg), kainate first produced seizures with no motor expression and recorded as regular EEG spiking, varying from 3 to 7/s (Fig. 1). TABLE 1 Incidence of Drug-Induced Convulsions in GAERS and NE Rats

7 months

12 months Bicuculline DMCM

FG 7142

CGP 56999

Isoniazide 3-MPA

Kainic acid Strychnine

Latency and Duration of Seizures in GAERS and NE Rats Latency (min) Drug Picrotoxin

Inhibitors of GAD: Isoniazide and 3-Mercaptopropionic Acid

Picrotoxin 2 months

TABLE 2

Dose (mg/kg)

GAERS

NE rats

2.5 4 5 2 2.5 4 6 2 3 4 0.25 0.5 1 2 2.5 5 10 20 2 2.5 3 120 200 20 22.5 25 5 7 2.5 3

3/12 6/12* 7/8* 3/7* 6/8** 7/8* — 6/6*** 0/6 6/7** 0/6 5/12* 7/8* — 2/8 7/11** 15/15*** — 0/6 5/11 8/8 0/8 0/8*** 1/8 1/8* 6/6 3/8 10/12 0/6 3/6

0/12 1/12 3/8 0/8 0/8 4/11 7/8 0/6 0/6 1/8 — 0/10 4/12 6/7 — 0/8 0/8 5/13 0/6 9/13 7/8 0/8 8/8 2/8 6/8 6/6 6/8 10/12 0/6 3/6

Note. x2, * P , 0.05, ** P , 0.01, *** P , 0.001.

Bicuculline DMCM

FG 7142

CGP 56999 Isoniazide 3-MPA Kainic acid

Dose (mg/kg) 2.5 4 4 0.5 1 2 5 10 20 2.5 200 22.5 5 7

GAERS 20 6 2 12 6 9* 25 6 5 12 6 1 7 6 1* 32 6 9 30 6 4 109 6 20 969 75 6 22* 36 6 4

NE rats 21 6 3 21 6 21 15 6 1 961 25 6 6 118 6 10 51 6 1 861 38 6 4 29 6 3

Duration (s) GAERS

NE rats

34 6 3 36 6 6 24 6 6 20 6 1 32 6 8

0 71 6 19 60 6 60 0 45 6 11 57 6 17 0 0 78 6 11 41 6 10 36 6 3 42 6 13 27 6 2 28 6 4

33 6 3 38 6 8 33 6 1 0 46 6 46 17 6 3* 24 6 3

* P , 0.05.

Occasionally, these seizures ended with rapid head shakes and could be followed by a recurrent seizure with low-frequency spikes and no behavioral concomitants. When the dose was increased to 7 mg/kg, repeated seizures occurred, their duration increased, and they generalized to full clonic seizures with an EEG pattern of large spike waves, ending sometimes in status epilepticus. GAERS and NE rats did not significantly differ in the incidence of kainate-induced seizures (Table 1, Fig. 2). However, at the lowest dose, the first seizure was more severe in NE rats, with shorter latency, longer duration (Table 2), and more seizures per rat: 1.3 6 0.3 in GAERS versus 4.5 6 0.9 in NE rats, P , 0.05. At the highest dose, many repeated seizures occurred in both strains, ending in status epilepticus more frequently in NE rats than in GAERS: 7/10 and 1/10, respectively. Strychnine induced full tonic seizures with desynchronized low-voltage EEG activity similarly in GAERS and in NE rats (Table 1). C-Fos Immunohistochemistry The distribution and intensity of c-Fos labeling varied with the drug and the characteristics of the seizures induced, independent of the strain of rats, GAERS or NE. The seizures were induced with a minimal dose of convulsant, in order to localize the structure(s) most susceptible to seizure initiation and to avoid generalized activation due to spread of the seizure. Results are summarized in Table 3. In control rats, injected with saline, either no c-Fos or low and diffuse c-Fos labeling occurred in the cortex. In all animals, isolated cells were labeled in the medial thalamus and the central mesencephalic gray. This labeling appeared to be in-

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FIG. 2. Percentage of convulsing animals per dose of picrotoxin, FG 7142, CGP 56999, and kainic acid in GAERS (s) and in NE rats (d). The dose/effect curve of GAERS is significantly shifted to the left for picrotoxin and FG 7142.

duced by exposure to the experimental procedure and was not rated. When only myoclonic spike waves were elicited by a subconvulsant dose of a GABAA receptor antagonist, low-level expression of c-Fos was found in cortical and limbic areas (results not shown). Picrotoxin-elicited convulsions induced clear c-Fos expression in the frontoparietal, piriform, and perirhinal cortex and low-level

expression in the amygdala. Only after the most severe tonic–clonic seizures, lasting more than 60 s, did the hippocampus also become labeled in two animals. When all picrotoxin-injected animals were pooled, the duration of convulsive seizures (myoclonus being scored as zero) was significantly correlated with the intensity of c-Fos labeling in the cortex (regression analysis, P , 0.01). Similar results were obtained after FG 7142-

TABLE 3 C-Fos Rating: Median (Range) Drug

n

CxFP

Piriform

Perirhinal

AmBasLat

AmCoMe

Hipp DG

CA1–3

Saline Picrotoxin FG 7142 DMCM Isoniazide CGP 56999 Kainic acid Strychnine

6 9 6 2 5 3 2 3

0.5 (0–1) 2 (1–2) 2 (2–2) 2 (2–2) 3 (2–3) 2 (1–2) 1 (1–1) 1 (1–2)

0 2 (1–2) 2 (2–2) 3 (3–3) 3 (2–3) 2 (2–2) 1 (1–1) 0 (0–0)

0 2 (0–2) 2.5 (1–2) 2 (2–2) 3 (2–3) 1 (1–2) 0 (0–0) 0 (0–0)

0 1 (0–2) 1 (1–1) 2 (2–2) 3 (3–3) 2 (1–2) 0 (0–0) 0 (0–0)

0 1 (0–2) 1 (1–1) 2 (2–2) 2 (1–3) 0 (0–1) 1 (1–1) 0 (0–0)

0 0 (0–2) 0 (0–0) 3 (3–3) 3 (2–3) 3 (2–3) 3 (3–3) 0 (0–0)

0 0 (0–2) 0 (0–1) 2 (2–2) 2 (2–2) 2 (1–2) 2.5 (2–3) 0 (0–0)

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elicited seizures: correlation between duration of FG 7142-elicited seizure and cortical c-Fos was highly significant (P , 0.001), but no hippocampal labeling was seen (Fig. 3). After DMCM- and isoniazide-induced tonic–clonic convulsions, a strong immunoreactivity was observed in the cortex and also in the amygdala, dentate gyrus, and CA fields of the hippocampus. After CGP 56999 injection, when a single nonconvulsive seizure was recorded on the EEG, c-Fos immunoreactivity was detected in the hippocampus only. When clonic–tonic generalized seizures were also elicited, additional c-Fos immunostaining was observed in the hippocampus, cortex, and amygdala. Kainate-elicited nonconvulsive seizures induced high-level and selective expression of c-Fos in the dentate gyrus and CA fields, while cortical c-Fos labeling remained low (Fig. 3). Except for a weak c-Fos staining in the cortex, tonic seizures provoked by strychnine did not induce c-Fos expression in the forebrain. DISCUSSION

Absence epilepsy has been related to an excess in cortical excitability (17). Nevertheless, thalamocortical absence seizures are aggravated by increased GABAergic transmission (44) and they have been repeatedly considered seizures of the inhibitory GABA system (16, 17, 33). An increase in susceptibility to convulsions initiated in the cortex and/or aberrant sensitivity to drugs interfering with GABAergic transmission could indicate possible abnormalities involved in the generation of spontaneous SWD in GAERS. In this study, GAERS appeared more susceptible to all GABAA receptor antagonists than NE, as revealed by the lower doses necessary to induce convulsions. This difference in susceptibility was not age-related in adult GAERS and NE, as shown for picrotoxin-induced seizures. The occurrence of myoclonic seizures and the predominance of clonic manifestations after administration of threshold doses of antagonists of the GABAA receptor point to the forebrain, and more specifically the cortex, as a likely site where seizures are initiated (5, 37). The primary involvement of the cortex is confirmed by our c-Fos data. The convulsive seizures induced by picrotoxin and the inverse agonists of the benzodiazepine site, FG 7142 and DMCM, constantly involved the neocortex and the piriform cortex. The clear correlation between the duration of picrotoxin- or FG 7142-induced seizures and the intensity of cortical c-Fos expression suggests that the cortex is primarily involved in these seizures. These results are in agreement with data indicating that picrotoxin-induced convulsive seizures involve cortical neurons (18) and are led by the frontal cortex (31). Moreover, bicuculline-induced seizures have been shown to be generated in the cortex and occur also in the neocortex of thalamectomized animals (42).

The present results are in accordance with previously demonstrated increases in susceptibility to PTZinduced generalized seizures and to focal seizures induced by withdrawal of cortical infusion of GABA in GAERS (4). PTZ reduces GABAergic inhibition by interaction with the chloride ionophore of the GABAA receptor (11, 41) and affects various voltage-operated potassium channels involved in seizure susceptibility (29). Convulsive seizures evoked by a low dose of PTZ induced c-Fos predominantly in the cortex and also in the amygdala and the hippocampus. When only myoclonic SWD were evoked, c-Fos immunoreactivity was low in the cortex, the amygdala, and the dentate gyrus (personal observations). These data demonstrate a cortical activation in the PTZ-evoked seizures, but this activation appears to be less selective than in seizures evoked by specific antagonists of the GABAA receptor. The distribution and number of neurons immunoreactive for GABA and GAD and the binding of ligands of the GABAA receptor subtypes have been shown to be similar in the cerebral cortex of GAERS and NE rats (12, 24, 39, 40). Only a selective reduction in the intensity of the immunostaining for the b-2,3 subunits of the GABAA receptor and decreased GABA-enhanced [ 3H]flunitrazepam binding have been found in the cortex of adult GAERS (40). These data suggest that the overall increase in the susceptibility of GAERS to convulsions induced by antagonists binding to various sites of the GABAA receptor complex is not simply related to a reduction in binding. In contrast, GAERS were less sensitive to blockers of GABA synthesis than NE, suggesting enhanced availability of synaptic GABA in GAERS. This result is in accordance with the finding of raised extracellular GABA in the thalamus of these animals (36). Expression of mRNA encoding GAD65 and GAD67 isoforms is similar in the cortex of GAERS and NE rats (12). However, differences in GAD activity in vivo cannot be excluded. The amount of synaptic GABA is also dependent on its metabolic inactivation, presynaptic release, and uptake by GABA transporters. No abnormality in these parameters has been described to date in GAERS. The presumed disorder of the inhibitory GABA system involved in the pathogenesis of absence seizures requires further investigation. The incidence of convulsions induced by other convulsants did not differ between GAERS and NE rats. The nonconvulsive seizures elicited by a low dose of kainate or by a GABAB antagonist are initiated in limbic structures that are not involved in absence seizures in GAERS (45). These results confirm the high seizure susceptibility of the hippocampus to GABAB receptor antagonists (46) and to kainate (8, 49). However, kainate-induced seizures were more severe in NE, indicating that elicitation and control of ongoing seizures depend on different mechanisms. It has recently been

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FIG. 3. C-Fos immunolabeling in frontoparietal cortex (A–D) and hippocampus (E–H) in a saline-injected control (A,E), following a picrotoxin-induced tonic–clonic seizure (B,F), a FG 7142-induced clonic seizure (C,G), and a kainic acid-induced EEG seizure without motor expression (D,H).

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shown that activation of kainate receptors downregulates GABAergic inhibition in hippocampal pyramidal neurons (25) and provokes loss of GABAergic inhibition in the dentate gyrus (21). The decreased severity of kainate-induced seizures in GAERS may thus reflect an increased GABAergic inhibition, as suggested above by the data obtained with inhibitors of GAD activity. Strychnine-induced tonic seizures were similar in GAERS and NE rats and did not induce any specific c-Fos expression in the forebrain, confirming, together with the absence of paroxysmal activity on the EEG, that these seizures do not spread to the forebrain. Altered susceptibility to chemoconvulsants could be due to blood–brain barrier differences between GAERS and NE rats. However, this possibility is unlikely, as the drug-specific susceptibility in GAERS versus NE is increased, decreased, or similar, depending on the drug’s central activity. Data obtained in other models of absence epilepsy also suggest that alterations in cortical excitability or cortical GABAergic mechanisms may be associated with absence seizures. In a strain of rats with genetic absence-like epilepsy, the WAG/Rij rats, in vitro experiments on the frontoparietal cortex have demonstrated a reduction in the efficiency of GABAergic inhibition (27). An increase in cortical network excitability has been described in stargazer mice, a genetic model of spike-wave epilepsy (13). An impairment in GABAinduced chloride uptake and aberrant properties of cortical GABAA receptors have been found in the singlelocus mutant tottering mice, a genetic model of absence seizures (43). In the lethargic mouse model of absence epilepsy, a selective increase in cortical GABAB receptors and increased GABAB receptor-mediated synaptic responses have been described (20). Finally, in a study characterizing the intracortical inhibitory control of synchronized thalamic inputs, the authors suggest that a decrease in the strength of fast GABAA-mediated inhibition may contribute to generation of spike-wavetype discharges (10). Altogether, these data show that different types of alterations at the level of the cortex have been associated with absence seizures in various experimental models. In conclusion, the differential susceptibility of adult GAERS and NE rats appears to be specific to convulsions induced by drugs affecting GABA transmission and involving primarily the cortex. The resistance to GABA-depleting drugs in GAERS suggests an increased availability of GABA, while their susceptibility to antagonists of the GABAA receptor is evidence of a decreased efficacy of these receptors. The present results suggest that possible dysfunctions of GABAergic activity in the cortex underlie absence seizures in GAERS. This agrees with previous observations sug-

gesting that absence seizures result from disorders of the inhibitory GABA system. ACKNOWLEDGMENTS We acknowledge the technical assistance of Arielle Ferrandon for immunohistochemical preparations. This study was supported by INSERM.

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