Loss of GABAA receptors during partial status epilepticus

There is a critical role for loss of GABA-mediated inhibition in the CA1 region of the hippocampus in the emergence of partial status epilepticus (SE) in experimental animals. ' We demonstrated loss of GABA-mediated inhibition in the CA1 region of the hippocampus by the paired-pulse method in several different experimental models of partial SE. ' The cellular mechanism underlying loss of GABA-mediated inhibition during SE remains unclear. This study investigates the effect of experimental SE on rat forebrain GABA receptor. We studied the effect of a combination of lithium and pilocarpine and lithium alone on GABA-mediated inhibition in the CA1 region of the hippocampus by paired-pulse inhibition technique.2 Methods. SE was induced by a method described by Honchar et aL3 Briefly, adult male Sprague-Dawley rats weighing 175 to 250 grams were each given 3 mEqlkg lithium chloride as a single intraperitoneal injection followed 20 hours later by 50 mglkg pilocarpine. Animals started having seizures 13 to 46 minutes after pilocarpine injection, and these seizures continued for 30 to 47 minutes before termination. The seizures were terminated by ether anesthesia and the forebrains were rapidly removed from the skulls, placed in a chilled sucrose buffer, and homogenized. The sucrose buffer contained 0.32 M sucrose, 100 mM PIPES, 10 mM EGTA, and 20 mM EDTA. The homogenate was spun at 5,000 g for 10 minutes in a Beckman 52-21 centrifuge. The supernatant was then spun at 18,000 g for 20 minutes. The resultant pellet (P2 fraction) was osmotically shocked in a lox volume of distilled water. The synaptic plasma membrane fractions derived by this procedure were suspended in 50 mM Tris citrate buffer (pH 7.4) and washed three times in Tris buffer. After three washes, the membranes were stored at -70 "C. Fifty p1 of 3H-muscimol was added to 100 pl membranes and 850 p1 50 mM Tris citrate to reach final muscimol concentrations varying from 3 nM to 100 nM. Based on preliminary experiments, nonspecific binding was obtained in the presence of 100 mM GABA. The membranes were incubated with radioligand for 20 minutes and then reaction was terminated by rapid filtration under vacuum on Whatman GF/B filters. Filters were washed three times, then digested in 3 ml of Insta-Gel Packard scintillation fluid. Radioactivity was measured by conventional scintillation counting. Binding data is presented as specific binding, calculated by subtracting binding in presence of GABA from total binding. Paired-pulse inhibition technique was utilized to measure GABA-mediated inhibition in the CA1 region of the hippocampus of urethane (1.3 to 1.5 gkgbanesthetized animals. We have previously described* this procedure for studying paired-pulse inhibition in detail. In one set of experiments, naive animals were studied; in the second set of experiments, rats were given 3 mEqlkg lithium chloride intraperitoneally 20 hours prior to urethane anesthesia. In the lithium-pretreated animals, inhibition was measured, 50 m g k g pilocarpine was administered, and 15 minutes later paired-pulse inhibition was measured again. Results. All animals injected with lithium and pilocarpine experienced seizures for at least 30 minutes. No seizures were observed in animals treated with lithium alone or in naive animals. A specific high-affinity saturable binding site for 3H-muscimol was defined in rat forebrain synaptic plasma membranes. In three separate experiments, a saturation curve for specific 3H-muscimol binding was generated by measuring total and nonspecific binding for free 3H-muscimol concentration ranging from 6 nM to 100 nM. Specific binding was approximately 50% of total binding. For each experiment, synaptic plasma membranes from five naive rats and from five animals undergoing SE were pooled; for each 3H-muscimol concentration, three separate measurements of total and nonspecific binding were made. Each measurement was repeated once. Synaptic plasma membranes from naive rats and from those undergoing SE were 0 8 -