Abstracts

Rationale: Neurosteroids have anticonvulsant action and regulate seizure frequency in epileptic animals. Physiological concentrations of neurosteroids allosterically potentiate GABA-A receptors (GABARs). Extrasynaptic ? subunit-containing GABARs (?GABARs), which mediate tonic inhibition, are particularly sensitive to neurosteroids. In the hippocampus of animals with temporal lobe epilepsy surface expression of ?GABARs is reduced. Neurosteroid modulation of tonic inhibition is also diminished in the dentate granule neurons (DGCs) of these animals. In the experimental animals status epilepticus (SE) often results in the development of spontaneous seizures following a period of active plasticity changes in GABARs. We tested whether NMDA receptor activation triggers down regulation of the ?GABARs and plays a role in epileptogenesis.Methods: SE was induced by lithium-pilocarpine method. Progression of SE and development of spontaneous seizures was monitored by 24 hours a day, 7 days a week video-EEG recordings through intra hippocampal electrodes. In the cultured hippocampal pyramidal neurons (HN), or organotypic hippocampal slice cultures (SC), in vitro epileptiform activity was induced by application of high extracellular potassium (HK, 10 mM KCl) and NMDA (10 ?M). Surface expression of ?GABARs was studied by biotinylation and Western blotting. Neurosteroid modulation of tonic inhibition was determined by whole cell patch clamp technique. Results: In the cultured HN, prolonged incubation (48 hr) in HKNMDA-containing medium attenuated allopregnanolone (THP, 10 nM) modulation of tonic inhibition, and reduced surface expression of ?GABARs in SC. Similar changes also occurred following NMDA treatment but not HK treatment for 48 hrs. Furthermore, inhibiting TrkB receptor activation neither reversed NMDA induced reduction in surface expression of ?GABARs nor blocked decrease in THP potentiation of tonic inhibition. In contrast, exogenously applied BDNF (100 ng/ml) increased surface expression of ?GABARs and augmented THP effect on tonic inhibition. These results revealed that NMDA receptor activation decreased surface ?GABARs and reduced neurosteroid modulation of tonic inhibition. In the experimental animals, surface expression of ?GABARs decreased at 7 days post-SE and THP modulation of tonic current was diminished. We studied whether blocking NMDA receptors during SE could prevent these changes and alter development of spontaneous seizures. Injection of ketamine (10 mg/kg/hr) during SE prevented reduction in surface expression of ?GABARs at 7 days post-SE. Furthermore latency to the development of spontaneous seizures was also substantially prolonged. Conclusions: These studies suggest that NMDA receptor activation triggers down regulation of ?GABARs. Impact of these changes on epileptogenesis is under investigation.