Abstracts

Rationale: Glutamate is the most abundant neurotransmitter in the brain mediating excitatory neurotransmission. Together with the inhibitory neurotransmitter GABA, it plays a crucial role in the pathophysiology of epilepsy. Psychomotor seizures are a type of focal seizures, which involves parts of the limbic portion such as hippocampus, resulting from a lesion producing localized reverberating circuits which gradually recruit adjacent cortical regions. Antiepileptic drugs (AEDs) act on various molecular targets such as voltage gated Na+, Ca2+ and K+ channels to reduce the seizure activity. In this study, we measured the effect of N-1'- N-3'- disubstituted trione derivatives of spirohydantoin on the basal, K+- and veratridine - evoked endogenous glutamate release from hippocampal synaptosomes. Methods: Glutamate release was measured using a glutamate dehydrogenase dependant NADP+ reduction assay using a highthroughput fluorescence 96-well plate method. As stoichiometry exists between the glutamate and the NADPH produced in the reaction, the amount of glutamate can be estimated. Hippocampi were dissected from 14-21day old Sprague-Dawley rat pups and synaptosomes were isolated using percoll density gradients. Results: Phenytoin at 500 μM, carbamazepine at 166 μM, gabapentin at 100 μM and the N-1'-p-nitrophenyl, N-3'-ethyl-2'H, 3H, 5'H-spiro-(2-benzofuran-1,4'- imidazolidine)-2',3,5'- trione at 133 μM significantly reduced the basal and veratridine-evoked glutamate release from the presynaptic nerve terminals of CA1 and CA3 regions of hippocampus. Neither phenytoin at 500 μM, nor carbamazepine at 166 μM, was able to significantly reduce the potassium-evoked glutamate release, whereas they significantly reduced veratridine-evoked glutamate release. On the other hand, gabapentin at 100 μM, and levetiracetam at 50 nM, were able to significantly reduce the potassium-evoked glutamate release. Conclusions: This approach should enable us to evaluate the relative efficacy and difference in the potential mechanism of action of the N-1'- N-3'- disubstituted trione derivatives of spirohydantoin compared with the common AEDs.