The synapse is the point of functional contact between neurons, where information is communicated between cells. Each electrical impulse in the presynaptic neuron leads to the secretion of chemical neurotransmitter, which is packaged at the presynaptic active zone in small membrane-bound vesicles. Fusion of the vesicles with the plasma membrane results in liberation of transmitter molecules, which bind postsynaptically to receptor channels and signal the postsynaptic cell of the presence of presynaptic activity. Vesicle fusion is accomplished by a molecular machine consisting of several components. Vesicles are docked at the membrane by the SNARE complex of vesicular membrane protein VAMP (vesicle associated membrane protein) and the plasma membrane proteins syntaxin and SNAP-25 (soluble NSF-attachment protein of 25 kD). Presynaptic impulses open calcium channels to admit calcium to the cytoplasm, where it binds to the vesicular protein synaptotagmin, which interacts with SNAREs and the plasma membrane to initiate fusion. SNAREs must assemble before arrival of action potentials in order for vesicles to be primed for release. After fusion, SNAREs must disassemble before the vesicle membrane is recovered by endocytosis and recycled for reuse. Some models of vesicle fusion involve the tightening of the SNARE complex as an early step in fusion. The timing of these processes is unknown, and they have never been measured directly. This project will use fluorescence resonance energy transfer (FRET) interactions between fluorescent tags on vesicle and plasma membrane components of SNAREs to characterize their assembly, disassembly, and conformational changes on fusion. Specifically, cerulean-SNAP-25B and citrine-VAMP-2 N-terminal interactions, and VAMP-2-cerulean and syntaxin-1-citrine C-terminal interactions, will be studied in cultured rat hippocampal neurons under field stimulation. FRET will be measured by enhanced exciter emission or by reduced donor emission on donor excitation, and by two-photon fluorescence lifetime imaging microscopy (FLIM). The dispersion and re-aggregation of SNAP-25 following secretion will also be analyzed. This project will advance the specific goal of the NIH Roadmap of developing innovative tools to study interactions between individual proteins within single cells. Understanding the molecular mechanisms of synaptic function provides an essential foundation for the development of rational and effective therapeutic interventions to treat synaptic dysfunctions underlying numerous neurological disorders. Understanding the molecular machinery of synaptic transmission is a specific priority of the NINDS Strategic Plan in furtherance of the NINDS primary mission of reducing the burden of neurological disease.