PROJECT SUMMARY/ ABSTRACT The purpose of an internal circadian clock is to generate a series of phases - time markers across the day and night by which different aspects of physiology and behavior (e.g., sleep, hormone release, temperature elevations) may be aligned to local time for optimal efficiency. We know a great deal about the molecular mechanisms of the clock (the timekeeping system) and how it is sensitive to local time. We know much less about circadian output, and specifically how the clock generates multiple phases across the entire solar day. Such phases are used by other clock cells, and by non-clock bearing downstream cells and circuits. Our laboratory studies circadian neurophysiology and the overall goal of this project is to understand the generation and usage of different circadian phasic outputs. The work is performed in the model system Drosophila. It builds on observations and a model we created from our past studies of the neural circuit in the Drosophila brain that controls daily locomotor behavior. In the fly brain, ~150 dedicated circadian pacemaker neurons direct daily rhythmic physiology and behavior. These 150 pacemakers are highly synchronized: they all tell the same time. Our model features the cell-intrinsic molecular clock in all pacemakers directing a morning phase of heightened neuronal activity. Yet, different subsets of pacemakers are not all active in the morning, but at different and stereotyped times of the day and night. The diversity of active periods (circadian phases) is generated primarily by cell interactions (especially neuropeptide modulation) and together these activity periods represent the multi-phasic outputs of the pacemaker system. Overall, this research program aims to extend and test this model by providing a cell- and molecular-level understanding for how circadian phase information is transmitted beyond the pacemaker system and received by downstream target circuits. Our work in Drosophila will likely inform our understanding of circadian output in the mammalian brain, and will also be relevant more generally to the mechanisms of neural circuit modulation by neuropeptides.