Project Summary Neurons communicate with each other by the quantal release of neurotransmitters stored in synaptic vesicles (SVs), and the strength and efficacy of neurotransmitter release are dynamically altered during physiological activity. This process is essential for learning and memory and is disrupted in many neurological disorders. The neurotransmitter release occurs from a pool of SVs docked at the presynaptic active zone and is tightly controlled by activity-dependent changes in the presynaptic calcium ions concentration ([Ca2+]). At nerve terminals, Ca2+- sensing proteins (Synaptotagmins) couple vesicular release machinery (SNAREs) to Ca2+ signals, thus orchestrating neuronal communication. Despite years of research, there remains a substantial gap between a qualitative description of how the vesicle fusion machinery operates and the millisecond-precision and Ca2+- dependent kinetics of neurotransmitter release observed at the neuronal synapses. In this proposal, we describe the first systematic and comprehensive effort to reconcile the `molecular biochemistry' of vesicle fusion with the `physiology' of Ca2+-evoked neurotransmitter release in the neuronal synapses using a combination of complementary in vitro and in vivo experimental systems. Specifically, we aim to resolve whether Synaptotagmin isoforms with distinct biochemical properties (Syt1 and Syt7), along with the synaptic SNAREs represent the minimal protein machinery for different modes of neurotransmitter release and short-term plasticity. We also propose to quantitatively test the hypothesis that the `dual-binding' of Syt1 and Syt7 to SNAREs allows for synergistic regulation of vesicular release kinetics. For in vitro analysis, we will deploy a biochemically-defined, high-throughput fusion system capable of tracking individual vesicle docking and Ca2+ triggered fusion on a millisecond timescale, which is integrated with fast Ca2+-uncaging protocols to generate [Ca2+] transients mimicking presynaptic calcium dynamics. This will be complemented by physiological analysis in cultured neurons utilizing fast, fluorescent glutamate sensor (iGluSnFR) and Ca2+ dyes to image quantal glutamate release and presynaptic Ca2+ dynamics in individual presynaptic boutons with 2-4 millisecond temporal resolution. We anticipate that this project will provide important insights into the molecular mechanisms underlying neurotransmitter release and build toward a detailed mechanistic model of Ca2+-evoked synaptic vesicle fusion. Overall, this will greatly enhance our understanding of neurotransmission and of how it is tuned across different nerve cells allowing specific yet diverse communication in neuronal networks.