Abstracts

Studies have shown that GABA[sub]A[/sub] receptor (GABA[sub]A[/sub]R) function is decreased immediately following SE and in association with epilepsy. One mechanism whereby neurons regulate receptor function and overall synaptic efficacy is by internalization and recycling of neurotransmitter receptors at the plasma membrane. This study was initiated to investigate the contribution of altered GABA[sub]A[/sub]R endocytosis towards both the decrease in receptor function and induction and maintenance of seizure discharges associated with SE and epilepsy. SE was induced in hippocampal cultures by exposure to low Mg⁺⁺ media, and spontaneous recurrent epileptiform discharges (SREDs) were produced for the life of the neurons in culture by 3 hours of SE. Electrophysiological analysis of SE and SRED activity was obtained with whole-cell current-clamp recordings. GABA concentration/response curves were generated using the whole-cell voltage-clamp technique. Immunohistochemical detection of membrane GABA[sub]A[/sub]R was carried out using a monoclonal antibody to the [beta]2/3 receptor subunits and analyzed by laser scanning confocal microscopy. GABA[sub]A[/sub]R endocytosis was inhibited in hippocampal cultures with either GDP-[beta]S (600 uM) or a peptide that specifically blocks the dynamin-amphiphysin association step in clathrin-mediated receptor endocytosis. An additional study involved confocal microscopic analysis of membrane GABA[sub]A[/sub]R [beta]2/3 staining on hippocampal sections from the rat pilocarpine model of acquired epilepsy. Low Mg⁺⁺ treatment of hippocampal cultures resulted in SE and the development of SREDs following 3 hr of SE. A 50% reduction in GABA response was observed with the induction of the [ldquo]epileptic[rdquo] condition in this model. Confocal analysis of GABA[sub]A[/sub]R [beta]2/3 immunostaining revealed a significant reduction in GABA[sub]A[/sub]R membrane levels in both acute (SE) and [ldquo]epileptic[rdquo] hippocampal cultures when compared to control. Inhibition of GABA[sub]A[/sub]R endocytosis in [ldquo]epileptic[rdquo] cultures resulted in both the total blockade of SREDs and a recovery of GABA[sub]A[/sub]R [beta]2/3 membrane staining back to control levels. Further studies in the pilocarpine model of acquired epilepsy demonstrated decreased hippocampal GABA[sub]A[/sub]R membrane staining in epileptic compared to control animals one year after the onset of epilepsy. No changes in hippocampal synaptophysin membrane staining were observed between epileptic and control animals. Increased GABA[sub]A[/sub]R endocytosis may contribute to decreased receptor function and the induction and maintenance of seizure discharges observed in both SE and [ldquo]epileptic[rdquo] neuronal cultures and in the intact pilocarpine model of acquired epilepsy. The findings of this study suggest a role for altered GABA[sub]A[/sub]R membrane recycling in the pathophysiology of SE and epilepsy. (Supported by NINDS RO1-NS23350 and P50-NS25630 to RJD.)