Abstracts

Cortical malformations are commonly associated with intractable epilepsy, however the functional relationship between malformations and seizures remains unclear. The tish rat is a seizure-prone genetic mutant that displays bilateral subcortical heterotopia reminiscent of those observed in some patients with intractable epilepsy. The mechanisms underlying seizures in the tish brain are unknown, but recent studies indicate that GABA-mediated synaptic inhibition is disturbed in the tish cortex. The present study investigated possible alterations in subunit composition of the GABA[sub]A[/sub] receptor (GABA[sub]A[/sub]R) in the tish cortex. Postnatal day 15 male tish rats and wildtype rats were chosen because seizures have not been observed in animals younger than 30 days. They were perfused intracardially with 0.1M PB followed by 4% paraformaldehyde in 0.1M PB pH 7.4. Brains were removed and post-fixed overnight. Samples were cryoprotected, frozen by CO2, and coronally sectioned at 30um on a cryostat. Slices were washed and incubated at room temperature for 1 hour in a blocking solution containing 5% serum. Slices were incubated at 4[deg]C for 72 hours in subunit antibody with 1% BSA. Slices were washed and incubated in NeuN antibody at 4[deg]C for 48 hours. Slices were washed and incubated in secondary antibodies conjugated with Alexa fluor (4ug/ml) at room temperature for 1 hour in darkness. Slices were mounted, air dried, and cover slipped. Wildtype layer V pyramidal neuron somata were strongly immunoreactive (IR) to [alpha]2 and [alpha]5 subunits, with a paucity of [alpha]1-IR. Layer II/III cells displayed strong [alpha]2-IR on the somata with low-level IR for [alpha]1 or [alpha]5 subunits, however the neuropil in this region showed strong [alpha]1-IR. Tish normotopic layer V pyramidal neuron somata, in contrast to wildtype, exhibited IR for all three subunits at almost equal levels, with a slight predominance for [alpha]2 and [alpha]5. Layer II/III somata showed greater expression of all subunits as compared to wildtype, but the neuropil in this region was less intensely labeled as compared to [alpha]1-IR in wildtype. Heterotopic cells were not organized in layers, however [alpha]1 and [alpha]2 expression predominated in a patchy, rather than laminar, pattern such that regions of intense somata and neuropil labeling were adjacent to regions of weak immunoreactivity. [alpha]5-IR was nearly absent from somata, and the neuropil displayed weak, patchy labeling. The current study demonstrated altered IR patterns of GABA[sub]A[/sub]R subunits in the immature tish rat brain. These changes may provide a molecular mechanism underlying the altered GABAergic synaptic transmission in the tish brain observed in previous electrophysiological experiments. Future studies will focus on examination of developmental changes in subunit expression, as well as the distribution of interneuronal subtypes and their association with specific GABA[sub]A[/sub]R subunit IR. (Supported by NIH:NS34124;RO1:NS040281;Individual NRSA.)