NMDA receptors (NMDARs) are one of two major types of ion channels located in the postsynaptic membrane at the vast majority of excitatory synapses in the CNS. They not only mediate excitation but, because of their permeability to calcium, raise intracellular calcium concentrations and thus trigger a variety of intracellular pathways leading to short and long term changes in synaptic strength. They also contribute to neuronal death in pathological conditions. Recent studies suggest that NMDARs are located not only at postsynaptic sites but also at presynaptic release sites and can modify the likelihood of vesicular release. As these receptors are important in both physiological and pathological conditions, determining where they are located will help define their effects. We will test whether NMDARs are expressed presynaptically with a combination of calcium imaging using two photon microscopy and electrophysiology to measure pre- and postsynaptic responses to global and local applications of NMDAR agonists and antagonists. We have chosen two types of neurons for this study: cerebellar stellate cells and neocortical layer 5 pyramidal cells. It is clear that NMDAR activation alters transmitter release from these two cell types but it is unclear if the same mechanisms are involved. Whether these receptors alter release by directly increasing intracellular calcium at release sites or rather by depolarizing release sites is not clear. If the former is true, NMDARs would have to be located very close to sites of release because calcium does not diffuse far intracellularly. If the regulation of release by NMDARs is secondary to their depolarizing influence, however, they need not be located near release sites because depolarizations can spread electrotonically for hundreds of micrometers. These two mechanisms are not mutually exclusive and could be mutually reinforcing. NMDAR-mediated depolarization by itself may have multiple effects on vesicular release by 1) directly activating voltage-sensitive calcium channels (VSCCs) and thereby increasing release by elevating baseline calcium concentrations, 2) inactivating axonal Kv1-type potassium channels leading to a broadening of action potentials, enhanced activation of VSCCs and thus greater release, 3) a calcium-independent, though uncharacterized, regulation of the release process and 4) inactivation of voltage-dependent sodium channels and/or VSCCs and therefore a blockade of action potential propagation to release zones and decreased calcium influx leading to decreased neurotransmitter release. We will determine whether NMDARs are located at presynaptic release zones and whether somatic depolarizations alter release by one or a combination of these mechanisms.

It is accepted that NMDA receptors residing on the postsynaptic side of synapses are key elements for signaling in the central nervous system. They mediate and regulate information flow between neurons on the millisecond time scale, they are required for both short and long term forms of synaptic plasticity that underlie learning and memory and, in pathological conditions including ischemia and neurological diseases, they contribute to neuronal death. The work proposed here will determine whether NMDA receptors are present on the presynaptic side of the synapse and, if so, what role they play in mediating and modulating information transfer and processing in the central nervous system and whether they contribute to pathological events.