Neuropeptides are crucial in development and for a wide variety of behaviors including circadian rhythms and sleep. This project has been using imaging to study synaptic neuropeptide release. For example, a new optical method based on a fluorogen activating protein (FAP) has shown that activity-evoked synaptic neuropeptide release occurs solely via fusion pores formed by kiss and run exocytosis. These experiments also established that synaptic fusion pores open spontaneously at a low rate, thereby providing a basis for resting peptidergic transmission. Furthermore, FAP imaging in the brain showed that Drosophila clock neurons release neuropeptides daily with different rhythms from terminals and the soma. This differential timing is based on the use of independent triggers: synaptic release requires Ca2+ influx as expected for release induced by electrical activity, but somatic release is mediated by the biochemical signal IP3. Remarkably, perturbing the somatic release mechanism alters specific features of circadian behavior and sleep. Thus, spatially and temporally partitioned neuropeptide release controls rhythmic behaviors. In parallel, GFP-tagged neuropeptide imaging led to the discovery of regulation of neuropeptide packaging by tyrosine phosphatases and new insights into neuropeptide delivery, capture and release at synapses. Finally, G-protein coupled receptor GPCR activation- based (GRAB) fluorescence sensors have been incorporated into our research to provide a new approach for resolving release and the subsequent spread of neuropeptides in the brain. Here the above optical tools will be used with Drosophila genetics to answer three questions posed by prior work on this project: (a) How is daily IP3 signaling induced in clock neurons and coupled to somatic neuropeptide release and regulation of rhythmic behaviors? (b) How does multi-compartmental fusion pore opening and dilation orchestrate neuropeptide release at native release sites? (c) How are neuropeptide packaging, axonal transport and synaptic capture controlled to support neuropeptide stores in release sites undergoing morphological plasticity (i.e., as occurs daily in clock neurons and in motor neuron axonal arbors in response to retrograde signaling)? These studies will reveal how surprising signaling-induced neuropeptide release from the soma participates the peptidergic connectome. Furthermore, in vivo imaging will elucidate the process of neuropeptide release by fusion pores. Finally, the cell biology underlying the support of peptidergic transmission during plasticity will be determined for the first time. Thus, this project will yield fundamental insights into neuropeptide release and the control of behavior, which are critical to the operation of the nervous system.