Abstracts

Rationale: Neurosteroids exert anticonvulsant action in several animal models of epilepsy. Neurosteroids modulate GABAA receptors (GABARs). The δ subunit-containing GABARs are highly sensitive to neurosteroids and they also mediate tonic inhibition in hippocampal dentate granule cells (DGCs). We reported diminished neurosteroid modulation of whole cell GABAR mediated currents in DGCs of animals with temporal lobe epilepsy (epileptic DGCs). The present study investigated the mechanisms underlying diminished neurosteroid sensitivity in the epileptic DGCs. Methods: Adult male Sprague Dawley rats were made epileptic using the continuous hippocampal stimulation model. GABAR mediated tonic currents were recorded from control and epileptic DGCs using whole cell slice patch electrophysiology. Standard immunohistochemical techniques were used. Surface expression was studied by biotinylating hippocampal slices, purifying the biotin tagged surface proteins using neutravidin beads, and determining protein expression by Western blotting. Expression of δ subunit was studied in the microsomal fraction isolated by subcellular fractionation. Results: Electrophysiological studies revealed that the robust effect of allopregnanolone (60nM) on tonic currents in control DGCs (25.6 ± 8.3 pA, n=6) was lost in epileptic DGCs (3.18 ± 1.6 pA, n=6, p<0.05). Similarly, a lower concentration of allopregnanolone (30nM) also enhanced tonic currents in control (6.61 ± 1.9 pA, n=8) but not in epileptic DGCs (0.10 ± 0.9 pA, n=7, p<0.05). The δ subunit immunoreactivity (IR) was reduced in the epileptic DGCs (OD=85.6 ± 1.64, n=64 section, 6 animals) compared to that from controls DGCs (95.1 ± 2.0, n=64 sections, 6 animals, p<0.05). The cell surface expression of the δ subunit was reduced by 66% (p<0.01). Furthermore, there was an increased retention (by 32%) of the δ subunit within the microsomal fraction containing the ER in the epileptic hippocampi. Despite reduced δ subunit surface expression, tonic currents were similar in both control (46.59 ± 5.1 pA, n=9) and epileptic (50.86 ± 7.8 pA, n=6) DGCs. GABARs containing α5γ2 subunits may also contribute to tonic currents in DGCs, however, the α5 specific drug, L655708 (1μM) inhibited tonic currents in all control DGCs (n=7) but not in 5/6 epileptic DGCs. Further, there was reduced α5-IR in the DGCs of epileptic animals. The presence of a novel α4γ2 subunit containing GABARs was then tested. LaCl (100μM) inhibited tonic currents in control (12.60 ± 2.9 pA, n=10) but not in epileptic DGCs (0.95 ± 6.3 pA, n=7, p<0.05). Likewise, Ro-154513 (300nM) did not alter tonic currents in control DGCs (0.40 ± 1.5 pA, n=6), but robustly inhibited tonic currents in epileptic DGCs (20.26 ± 5.9 pA, n=10, p<0.05).