The goal of this project is to understand the molecular mechanisms of extracellular vesicle (EV) trafficking in the nervous system in vivo. EVs are small vesicles secreted from donor cells that can carry a diverse array of cargoes, and have recently come to the forefront as a novel mode of intercellular traffic and communication in the brain. EVs are thought to contribute to many human health conditions, including the spread of pathological proteins in neurodegenerative disease. However, because most studies of EVs are conducted with cells in culture, little is known about the release, uptake and fate of EVs in the diverse interacting cell types of the intact nervous system. We have developed a system in which to study traffic of endogenous neuronal EV cargoes in a living animal, using cutting edge genetic and cell biological tools available in Drosophila. Using this system, we discovered an unexpected role for synaptic periactive zone (PAZ) membrane remodeling machinery in EV cargo sorting, stability and release from axon terminals at the Drosophila larval neuromuscular junction. We also found that endogenous EV cargo release is dynamically regulated by neuronal activity. Finally, we made the surprising discovery that released EVs are partially protected from the target cell cytoplasm, suggesting the possibility that additional regulatory steps may govern their exposure and/or degradation upon internalization to recipient cells. Our proposed research will elucidate how cellular membrane traffic machinery controls the release, uptake, and fate of EV cargoes. To achieve these goals we will use advanced Drosophila genetics, live cell imaging techniques, structured illumination microscopy, electron microscopy, and tissue-specific detection and manipulation of EV cargoes in donor and recipient cells. Specifically, we propose: 1) To elucidate in vivo mechanisms of EV traffic and release by PAZ proteins. 2) to determine how activity regulates neuronal traffic of endogenous EV cargoes and 3) to determine the fate of NMJ EV cargoes, at rest and in response to neuronal activity. Given the conserved nature of synaptic membrane trafficking machinery, our findings and tools will lay the foundation for new insights into endogenous EV traffic in many aspects of nervous system function, including in human neurological disease.