The objective of the proposed work is the understanding of the mechanisms that control the kinetics and plasticity of glutamate mediated excitatory synaptic neurotransmission in the mammalian CNS. The glutamate receptors directly gating ion channels and those coupled to G-proteins will be studied using whole cell and single channel patch clamp techniques in primary cultures of hippocampal neurons. Glutamate agonists and antagonists will be applied externally to cells by fast perfusion techniques. Controlled internal dialysis involving patch pipette perfusion and dual whole cell recording will be utilized to pharmacologically manipulate intracellular second messenger systems. The fast non-NMDA and slow NMDA receptor components of the excitatory postsynaptic current will be examined and compared to responses induced by glutamate analogues to determine if channel kinetics, receptor desensitization, transmitter diffusion and uptake or some combination of these factors can account for the differences in their time courses. The cellular mechanisms that regulate the efficacy of excitatory synaptic transmission will be studied by investigating the influence of activation of the G-protein coupled glutamate receptor and other second messenger systems on both currents induced synaptically and by exogenous glutamate ligands. Synaptic transmission through non-NMDA receptor gated ion channels is likely to represent the majority of fast cell to cell communication in the CNS, and both the NMDA component and the G-protein coupled receptor are involved in intracellular signalling and plasticity. The glutamate receptor system is capable of rapid up-and down-regulation and has been implicated in both developmental and adult plasticity. Over activation can result in epileptiform behavior and excitotoxicity. A detailed knowledge of the way in which the activation of kinetics of glutamate receptor subtypes and their biochemical regulation govern the behavior of glutamate synapses is therefore fundamental to the understanding of the CNS in both normal and abnormal states.
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