Abstracts

RATIONALE: At the end of this activity, the participants should be able to discuss potential novel mechanisms meditating disinhibition in the epileptic hippocampus.
Inhibition is compromised in area CA1 of rats with temporal lobe epilepsy (TLE). Similar decreased inhibition occurs in animals in which expression of the neuronal glutamate transporter, EAAC-1, is reduced by antisense oligonucleotide administration (Cohen et al. SFN Abstracts 25, 1224, 1999). These animals also exhibit seizures. EAAC-1 is enriched in inhibitory synaptic terminals, potentially providing glutamate as a substrate for local GABA synthesis. We therefore hypothesized that epilepsy-associated decreases in inhibition in area CA1 may be secondary to reductions in levels of GABA in the presynaptic terminal.
METHODS: To test this hypothesis, expression levels of EAAC-1, the GABA transporter, GAT-1, and the vesicular GABA transporter, VGAT, were examined using Western blotting and immunocytochemistry in adult rats with TLE induced by pilocarpine, compared to age matched controls. TLE animals were injected with scopolamine, followed by pilocarpine (380 mg/kg, I.P.), which triggered status epilepticus. This was terminated within 3 hrs with diazepam (4-10 mg/kg). Animals were used for experimentation 3-9 months following pilocarpine, and all exhibited spontaneous seizures. Western blots were run using 50 ug per lane of isolated fresh frozen CA1 protein. The same primary antibody was used for both techniques.
RESULTS: Western blot analysis of CA1 protein isolated from TLE animals (n=3) demonstrated a significantly decreased expression of both EAAC-1 and GAT-1, but not VGAT relative to controls (n=3). In immunocytochemical studies, distinct VGAT, GAT-1, and EAAC-1 immunoreactive punctate staining within stratum pyramidale of area CA1 was observed. A significant decrease in both the numbers of immunoreactive puncta and intensity of staining within puncta was observed for both EAAC-1 and GAT-1 in TLE animals. VGAT immunoreactivity did not change in number of puncta or intensity of puncta stained between the control and TLE animals (n=3 each).
CONCLUSIONS: Since VGAT levels were not altered in Western blots, or in numbers or intensity of VGAT immunoreactive puncta, we conclude that the numbers of inhibitory terminals within stratum pyramidale of CA1 were not altered in epileptic animals. However, both EAAC-1 and GAT-1 levels were significantly decreased, suggesting decreased levels of glutamate as a local precursor for GABA synthesis and decreased recycling of GABA in inhibitory terminals, respectively. This could combine to reduce the amount of GABA available for release in inhibitory terminals, and disrupt inhibition in area CA1 of epileptic animals via a presynaptic mechanism.
[Supported by: NIH-NINDS grants NS-32403 and NS-38572.]