Project Description Neuropeptides and neurotrophins are packaged in dense-core vesicles (DCVs) in the soma and released at synaptic terminals to control development, mood and numerous behaviors. Despite the importance of neuronal DCVs, little was known about how they are delivered to terminals and release their contents at synapses. Our GFP-based imaging studies combined with Drosophila genetics have revealed that control of synaptic peptide stores and exocytotic release do not operate as advertised in text books. First, instead of a one-way trip mediated by anterograde axonal transport, DCVs circulate in and out of synapses with capture occurring during both anterograde and retrograde transport. Moreover, contrary to long-held assumptions, capture limits the size of presynaptic neuropeptide pool. Furthermore, we have recently found that capture efficiency is directionally controlled by activity, neuron subtype and disease-related genes. Second, preliminary studies suggest that DCV transport appears to be adjusted for extensive innervation with many boutons (e.g. ~1000) and injury in the terminal. The latter effect is associated with intraterminal Ca2+ release, which may potentiate synaptic function to compensate for loss of boutons induced by injury. Third, a monoamine neuromodulator and intracellular cAMP evoke robust synaptic peptide release, with the latter displaying mechanistic differences from release evoked by action potentials. These results suggest that, in addition to conventional transmission evoked by electrical activity, synaptic peptide release is evoked by intracellular Ca2+ release and cAMP signaling. Here fluorescent protein imaging in Drosophila neurons addresses three questions posed by our prior work on this project: 1. What molecular mechanisms selectively control presynaptic DCV capture and neuropeptide release? 2. How is vesicle circulation regulated to support extensive, diverse and injured terminals? 3. What is the release mechanism and physiological impact of intracellular Ca2+ and/or cAMP signaling evoked by injury and neuromodulators? These studies will yield fundamental insights into the maintenance of terminals and the regulation of synaptic release of neuropeptides and neurotrophins. Furthermore, the proposal will show how these processes are affected by acute injury and disease-related genes.