The mechanisms of excitatory synaptic transmission at single synaptic contracts in the CNS are not well understood. Questions of postsynaptic receptor occupancy, receptor number, mechanisms of clearance, synapse specificity and crosstalk, although beginning to be addressed, have not been answered even in simplified preparations including culture and slice. The proposed work will examine these questions by determining the time course of clearance of transmitter at hippocampal synapses in cultures and at Schaffer collateral/commissural inputs to CA1 pyramidal cells in slices from rat. The first hypothesis to be tested is that glutamate transporters, in addition to maintaining extracellular levels of glutamate below those that can induce excitotoxicity, also buffer glutamate by rapid and capacious binding thereby diminishing the lifetime of elevated transmitter in the synaptic cleft. In particular, the differential effects of glial and neuronal glutamate transporters on the kinetics of postsynaptic responses will be assayed using newly developed transporter blockers and selective antisense oligonucleotides to test the effects of synaptic and extrasynaptic transporters on clearance rates and functional separation of synaptic sites. The second hypothesis to be tested is that, despite powerful uptake mechanisms, transmitter can diffuse to neighboring inactive synaptic contacts in quantities high enough to activate significant numbers of NMDA receptors and desensitize AMPA receptors. If crosstalk is sufficient to activate NMDA receptors at neighboring sites, recent proposals that the expression of LTP involves rapid increases in the number of functional AMPA receptors may not be correct or only partially correct. This issue will be directly addressed with electrophysiological techniques in hippocampal slices using single and dual pathway stimulation. Reuptake and crosstalk are inter-related processes. Regulation of the former will clearly affect the latter. Determination of the capacity and kinetics of uptake mechanisms and the degree of crosstalk at distinct synapses will help define the meaning of synapse specificity, a central conception in theories of neuronal integration and information processing.
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