The broad goals of this project are to understand the cellular and molecular mechanisms underlying vesicle exocytosis at synapses that tonically release transmitter. The proposal is divided into three specific aims in which optical, electrophysiological, and molecular biological techniques will be utilized to examine vesicle exocytosis at the first two synapses in the visual system;i.e., the output synapses of photoreceptors and retinal bipolar cells. These synapses release transmitter continuously at high rates, but the underlying mechanisms are poorly understood. The presynaptic terminals of photoreceptors and bipolar cells contain varying numbers of an organelle, the synaptic ribbon, which tethers vesicles close to active zones. It has been postulated that vesicles may fuse together along the ribbon in response to a stimulus, but direct evidence for compound fusion at synapses has been lacking. To examine mechanisms of exocytosis at tonic synapses, I will generate transgenic zebrafish that express a novel fluorescent reporter of exocytosis, sypHy. Bipolar cells and cone photoreceptors from these transgenic zebrafish will be isolated, stimulated, and monitored for changes in fluorescence along ribbons. If multivesicular release due to compound fusion occurs in response to a stimulus, an increase in fluorescence should be detected along the ribbon. Homotypic and heterotypic membrane fusion is driven by a core SNARE complex that is thought to be stabilized, by a protein called complexin, in a fusion-ready state before calcium enters the presynaptic terminal and binds to synaptotagmin. A recent study has identified two mammalian complexins at retinal ribbon-containing synapses, but their functions at these synapses are unknown. To understand the roles of these complexins in regulating vesicle fusion, I will knockdown their expression with morpholino antisense oligos in transgenic zebrafish that express sypHy. Furthermore, I will exogenously add complexins to sypHy-expressing retinal neurons to determine if complexin overexpression perturbs exocytosis. To identify and localize complexins in the zebrafish retina, in situ hybridization and immunocytochemistry will be performed. Taken together, these studies will clarify how visual signals are processed at retinal synapses and may shed light on mechanisms of fast, sustained transmitter release at other synapses. Understanding the general mechanisms of synaptic vesicle exocytosis will provide insights into neuropsychiatric and neurodegenerative diseases where synaptic dysfunction plays a crucial role. The importance of complexins in normal synaptic function is underscored by several findings showing decreased levels of complexins in patients with schizophrenia, Huntington's Disease, and Alzheimer's Disease.