It is now generally accepted that excitatory transmission in many areas of the mammalian CNS depends on presynaptic release of L-glutamate and subsequent activation of at least two classes of glutamate-gated ion channels in postsynaptic neurons. AMPA (or non-NMDA) receptor channels mediate a very quickly activating and deactivating conductance that is responsible for most information transfer on the millisecond time scale. NMDA receptors have much slower kinetics and are required during synaptogenesis for the formation of appropriate connections and, in many excitatory pathways, for altering the strength of synaptic transmission. Although some of the underlying properties that govern the behavior of AMPA and NMDA receptors are known, very little is known about the basic mechanisms of excitatory synaptic activation. The objective of the proposed work is to characterize these basic phenomena: 1) how long free glutamate remains elevated in the synaptic cleft after presynaptic release, 2) how the time course of unbound transmitter concentration can be altered by processes that change the probability of release, 3) how the time course of unbound transmitter is affected by changing the efficacy of the mechanisms responsible for transmitter clearance, and finally, 4) how such alterations affect receptor occupancy and the frequency of channel opening. Knowledge of these basic phenomena in normal conditions is essential to understand 1) how synaptic release at one site may affect responses to subsequent or adjacent events, 2) what mechanisms are available for changing synaptic efficacy in both the short and long term, and 3) how pathological states such as stroke and epilepsy might affect synaptic processing at sites peripheral to those directly affected. We will use patch clamp and rapid solution exchange techniques to study NMDA and AMPA channel kinetics in isolated patches of membrane and single and dual whole cell recording to study autaptic and synaptic mechanisms. In addition, several techniques to isolate responses from individual synaptic contacts will be used to simplify the interpretation of results from whole cell synaptic events.
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