Abstracts

Domestic Mongolian gerbils begin having spontaneous seizures at [sim]1.5 months of age, making it possible to evaluate them at a pre-epileptic stage before the confounding effects of seizures. In epileptic and pre-epileptic gerbils the substantia nigra [italic]pars reticulata[/italic] (SNr) displays reduced GABA-binding compared with non-epileptic controls (Olsen et al, 1985, PNAS 82:6701), suggesting possible differences in GABA[sub]A[/sub] receptors. We tested this hypothesis by assaying expression levels of the [alpha]1 subunit (the dominant [alpha] subunit expressed in SNr) and by evaluating GABA[sub]A[/sub] receptor-mediated postsynaptic currents. Real-time RT-PCR and Western blot analysis were done using substantia nigra-rich tissue harvested from 1-month-old pre-epileptic seizure-susceptible gerbils and non-domestic non-epileptic age-matched controls. Quantitative PCR was performed with 0.8 ng of RNA (n=6 animals/group) for both the target gene and an internal control (GAPDH), and cycle threshold values were analyzed using the comparative C[sub]T[/sub] method (Applied Biosystems). [alpha]1-protein levels were measured by densitometry of Western blots using [beta]-actin as an internal control (n=3 groups with [ge]8 animals/group). Anti-GABA[sub]A[/sub] receptor [alpha]1 polyclonal serum (Upstate Biotechnology) was used for both Westerns and for immunocytochemistry on brainstem sections. Whole-cell voltage-clamp recordings from visualized SNr neurons were obtained in 350[micro]m-thick acute coronal slices (32[deg]C) from 1-month-old pre-epileptic and control gerbils. mIPSCs were isolated in TTX (1[micro]M). GABA[sub]A[/sub] [alpha]1 subunit mRNA levels in pre-epileptic gerbils were [sim]96% of controls (p[gt]0.9, t-test, range for pre-epileptics: 0.6-1.8, controls: 0.6-1.7). [alpha]1 protein levels in pre-epileptics were similar to controls (0.70[plusmn]0.01 vs 0.72[plusmn]0.03, p[gt]0.7). [alpha]1 subunit immunocytochemistry revealed similarly strong expression in SNr in both groups. The frequency and amplitude of sIPSCs in SNr neurons from pre-epileptic gerbils were comparable to controls (23 vs 18 Hz, p[gt]0.1; 36 vs 34 pA, p[gt]0.7; n=13, 15 cells respectively) and mIPSC frequency was similar between groups (17 vs 13 Hz, p[gt]0.2; n=11, 12). However, averaged mIPSC amplitude in pre-epileptics was significantly larger than in controls (26 vs 18 pA, p[lt]0.007). Zolpidem (200nM), an [alpha]1 subunit-specific modulator of the GABA[sub]A[/sub] receptor, was equally efficacious in broadening mIPSCs in both groups (% change in weighted decay time for pre-epileptics: 32% vs controls: 35%, p[gt]0.8, n=6). Contrary to our expectations neurons of the SNr in pre-epileptic gerbils display normal or enhanced IPSC frequencies and amplitudes. And, mRNA and protein expression levels of the major GABA[sub]A[/sub] receptor [alpha] subunit are normal. These findings suggest that defects in GABA[sub]A[/sub]-mediated inhibition in the SNr are an unlikely cause of seizures in this model of inherited epilepsy. (Supported by NIH / NINDS.)