At the presynaptic terminal, the central nervous system can change the efficacy of synaptic transmission over a large temporal range. G protein coupled receptors (GPCRs) commonly mediate this plasticity. We have shown, in a central presynaptic terminal, that Gbetagamma microinjection mimics 5-HT's inhibition of neurotransmission. Gbetagamma is critical for this effect since a Gbetagamma scavenger blocks inhibition by 5-HT. Gbetagamma has no effect on fast, action potential evoked intracellular Ca2+ transients that trigger neurotransmission. Thus, Gbetagamma blocks the exocytosis downstream of Ca2+ entry to inhibit neurotransmitter release. 5-HT-mediated presynaptic inhibition is still seen after blockade of classical Gbetagamma effector pathways. We have also shown that Gbetagamma interacts with SNARE proteins in vitro and inhibits release in a second (PC 12 cell) assay of secretion. Our preliminary data lead us to hypothesize that Gbetagamma directly targets the fusion machinery at the presynaptic terminal to inhibit release. We will investigate this hypothesis. Is Gbetagamma signalling cytosolic and relatively slow, involving intermediate phosphorylation or dephosphorylation events, or is this rapid involving direct binding of Gbetagamma to proteins within the membrane, for example SNARE? We will probe the timing of GPCR-mediated presynaptic inhibition and will also investigate the possible role of cytosolic messengers. Gbetagamma binds both to core complex protein components and to the assembled t-SNARE. We will probe at what state of core complex formation Gbetagamma acts to prevent synaptic vesicular fusion. We will inject proteins, peptides and toxins into a functioning giant synapse or apply these to cracked PC12 cells. We will use botulinum toxins to determine if activation of GPCRs inhibits neurotransmitter release prior to or after docking or priming of the fusion machinery. We will use Gbetagamma mutants which result in both gain and loss of functions against classical Gbetagamma effectors and identify Gbeta mutations whose binding properties to components of the core complex have changed. These Gbetagamma mutants will then be used to screen for gain or loss of inhibition of exocytosis. We will identify Gbetagamma binding sites on the SNARE proteins responsible for Gbetagamma interactions and then transfect PC 12 cell lines with proteins in which these sites have been altered to identify whether alterations in these sites can lead to a loss of function of Gbetagamma-mediated inhibition of neurotransmitter release. These studies will serve to elucidate the molecular mechanisms of synaptic modulation and thus contribute to our understanding of the regulation of information encoding in the brain thought to underlie learning and memory.