Abstracts

Cortical malformations are a common cause of epilepsy, although the mechanism behind this association remains poorly understood. As such, several animal models have been developed to explore this association. A genetic model, the Tish rat, displays bilateral subcortical band heterotopia and recurrent spontaneous seizure activity. Previous work has demonstrated altered GABA-mediated synaptic inhibition, and in the work presented here, we focused on determining potential mechanisms for this alteration. Whole-cell voltage clamp recordings from brain slices obtained from 15 day old heterozygotic and homozygotic rats were performed using standard techniques to study GABA-mediated IPSCs. Layer V pyramidal neurons from heterozygous (control cells) and homozygous cortices (normotopic cells) were selected. The heterotopia in homozygotes is not layered, so large pyramidal cells were selected for recording (heterotopic cells). Excitatory glutamatergic activity was blocked to facilitate recording of multi-synaptic spontaneous IPSCs (sIPSCs) followed by addition of TTX thus permitting recording single vesicle-mediated miniature IPSCs. Immunohistochemistry was done to indicate the relative distribution of inhibitory interneurons by labeling GAD-67. Amplitude: sIPSC amplitude in normotopic cells (52.7[plusmn]1.9pA) was reduced comared to control (58.0[plusmn]2.8pA) and heterotopic (58.4[plusmn]3.4pA) cells. In contrast, mIPSC amplitude of heterotopic cells (40.8[plusmn]1.5 pA) was significantly smaller than control (49.2+/-2.2 pA) and normotopic (48.7[plusmn]2.6 pA) cells (p=0.3, One-way ANOVA).
Frequency: sIPSC frequency recorded from control cells (4.3[plusmn]0.8 Hz) was higher than that recorded from heterotopic (3.4[plusmn]0.5 Hz) and normotopic (2.1[plusmn]0.7[plusmn] Hz) cells. Similarly, mIPSCs frequency recorded from control cells (2.0[plusmn]0.3 Hz) was significatly higher than heterotopic (1.1[plusmn]0.3 Hz) and normotopic (1.2[plusmn]0.2 Hz) cells (p=0.04, One-way ANOVA).
Immunohistochemistry: The heterotopia showed enhanced labeling of GAD-67 compared to control and normotopic cells. Moreover, normotopic cortex showed reduced labeling of GAD-67 compared to control. Heterotopic cells of the Tish brain showed postsynaptic alterations. Smaller mIPSC amplitude but not sIPSC amplitude suggest an alteration of the GABA[sub]A[/sub] receptor, possibly from altered subunit expression. These cells also showed a change in the presynaptic properties, as mIPSC frequency was reduced when compared to control. Futhermore, normotopic cells of the Tish brain show presynaptic alterations: reduced sIPSC amplitude but not mIPSC amplitude, lower mIPSC frequency, and reduced GAD-67 labeling when compared to control. Overall, these results reveal mechanisms of altered synaptic inhibition in the Tish rat cortex, and may have implications for strategies to treat human epilepsy associated with cortical malformation. (Supported by NIH:NS34124, RO1:NS040281, NARSAD Foundation)