Our long term goal is to elucidate the physiological role of presynaptic glutamate (Glu) receptors. These receptors, of both NMDA and non-NMDA subtypes, have only recently been identified on catecholaminergic nerve terminals, in which their activation directly lead to Ca2+-dependent transmitter release. Recent evidence from the principal investigator indicates that activation of presynaptic Glu receptors increased met- enkephalin release from striatal synaptosomes, to an extent comparable to that of K+-depolarization. Such a robust effect, as compared to the much smaller effect on catecholamine release, would suggest that regulation of peptide release is an important function of these receptors. If proven to be widespread, this effect of presynaptic Glu receptor activation suggests that the receptors may mediate a novel action of Glu as an initiator of local peptidergic modulatory actions, in addition to its role as a fast excitatory classical transmitter. These dual actions of the major excitatory transmitter in the CNS may be involved in a variety of important transmitter interactions that are implicated in physiological and pathological processes, such as neuronal plasticity, aging, schizophrenia, and ischemic neurotoxicity. Yet, little is known about the basic properties of presynaptic Glu receptors. This proposal focuses on three such properties: functionally, on characterizing the regulation of peptide release in different brain regions; biochemically, on demonstrating receptor phosphorylation and studying its modulation of receptor functions; and molecularly, on cloning and characterizing novel and potentially presynaptic Glu receptor isoforms from olfactory sensory neurons, a neuronal model system for studying these receptors in the absence of abundant postsynaptic Glu receptors. The first two properties are examined in synaptosomes, which, because of the absence of intact cells and postsynaptic structures, are most suitable for such studies. Synaptosomes from appropriate regions will be used to characterize the release of several neuropeptides and their regulation by presynaptic Glu receptors will be studied. Receptor- mediated peptide release will also be used to characterize modulation by receptor phosphorylation. Finally, PCR amplification demonstrated the presence of a previously cloned non-NMDA receptor isoform (GluR5), and of a novel isoform related to KA1, in the olfactory epithelium (OE). The full length cDNA of the novel isoform will be cloned from OE libraries, and its localization and functional properties will be characterized. The proposed studies, when completed, will fill large gaps in our knowledge of presynaptic Glu receptors, and will facilitate the study of the significance of these receptors physiological and pathological neuronal processes.