Glutamate is a potent excitatory neurotransmitter. High levels of glutamate released at synapses can caused neuronal hyperexcitability and seizures as well as consequent neuronal death. Extracellular (ECF) glutamate accumulation has been demonstrated by in vivo dialysis in the hippocampi of patients with temporal lobe epilepsy with hippocampal sclerosis. This project examines the cellular mechanisms underlying this glutamate release at high concentration in the temporal lobe by analysis of surgically removed temporal lobe tissue. Two potential mechanisms will be investigated. (1) Increase in ECF glutamate results from defective glutamate (GLU) transporter function, and/or (2) defective mitochondrial function resulting in impaired energy metabolism. The role of GLU transporters will be studied by examining its expression in the epileptogenic temporal lobe by high microscopic immunohistochemistry, in situ hybridization and Northern and Western blot analysis as well as quantitative electron microscopic immunocytochemistry. Transporter function will be assessed by examining GLU uptake and release in primary astrocyte cultures from seizure foci, as will be assessed by examining GLU uptake and release in primary astrocyte cultures from seizure foci, as well as membrane currents due to molecular biological techniques. Defects in mitochondrial function will be studied by evaluating mitochondrial densities and their anatomical pathology, in particular looking for evidence of such pathology in regions of high glutamatergic activity (glutamate synapses). The contribution of defective mitochondrial metabolism to excitatory amino acid metabolism: glutamate dehydrogenase, phosphate activated glutaminase and glutamate synthatase. These tissue studies together with in vivo and ex vivo studies of glutamate/glutamine levels and energy metabolism in the same patients in projects 1 and 2 will provide detailed assessment of glutamatergic function in temporal lobe epilepsy, thereby opening the way to better diagnosis and management of these patients and critical insights to new pharmacotherapeutic interventions.
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