PROJECT SUMMARY Presynaptic Ca2+ drives neurotransmission at the trillions of chemical synapses that mediate most communication in the nervous system. Ca2+ influx through voltage-gated Ca2+ channels raises localized intracellular Ca2+, which binds to Ca2+ sensors on synaptic vesicle fusion machinery, resulting in vesicle fusion and release of neurotransmitter at specialized areas of synapses called active zones. The abundance and precise location of Ca2+ channels have a profound impact on synapse function, yet the relationship between Ca2+ channel organization and synaptic function has been difficult to study. The Drosophila neuromuscular junction (NMJ) provides an attractive model for studying this question at endogenous synapses by allowing us to compare Ca2+ channel composition, organization and dynamic regulation at two related motor neuron subtypes with very different neurotransmitter release properties. In Aim 1, I will investigate the role of Ca2+ channel auxiliary subunits and nanoscale organization in establishing synapse-specific release properties using CRISPR gene editing, super-resolution imaging, and electron microscopy. Synapses must be reliable, but also malleable to adapt their responses to a dynamic environment. Presynaptic homeostatic potentiation (PHP) is a conserved mechanism for maintaining effective neural communication within a dynamic range through an increase in synaptic probability of release. Previous work from our lab has shown that manipulations to induce PHP result in a rapid recruitment of voltage-gated Ca2+ channels to active zones, but how new channels are trafficked to and organized at active zones remains unknown. In Aim 2, I will use genetics and pharmacology to investigate the cellular mechanisms by which Ca2+ channels are trafficked, inserted in the membrane, and clustered to facilitate changes in release properties during homeostatic plasticity. This research will reveal how Ca2+ channels are differentially and dynamically regulated to achieve and maintain synapse-specific release properties, and advance our understanding of communication in the nervous system. By completing these aims, the applicant will gain scientific and technical expertise in cellular neurobiology, neurogenetics, CRISPR gene editing, super-resolution imaging, and electrophysiology. Through a comprehensive training plan, this fellowship will support the professional development of the applicant in robust experimental design and data analysis; written and oral scientific communication; and effective and inclusive mentoring. Successful completion of the research and training goals are fully supported by the interactive and supportive institutional environment of Brown University and in the Neuroscience Graduate Program, and will prepare the applicant for the next steps towards an independent scientific career.