Sleep disturbances are increasingly common and are associated with a variety of comorbidities and other public health consequences. It is therefore critical to improve our understanding of the neural mechanisms that control the timing and quality of sleep. Key signaling molecules that regulate sleep in animals ranging from flies to humans come from the family of neuropeptide transmitters. These molecules have sparse expression levels and selective effects on behavior, including sleep, making them prime candidates for the development of focused drug treatments with minimal side effects. However, the mechanisms by which these molecules act individually and in concert to regulate target cells in the brain and thus behavior are poorly understood. This renewal application will take advantage of the powerful genetics and relatively simple sleep network organization of the fruit fly, Drosophila melanogaster, to address how neuropeptides function at the molecular, cellular, and behavioral levels to regulate sleep. In Aim 1, we will utilize optogenetic activation of specific subsets of neurons expressing the sleep-promoting transmitter short neuropeptide F (sNPF) in order to determine their particular roles in sleep regulation. In Aim 2, we will coactivate sleep-promoting sNPF neurons and wake-promoting neurons to determine their hierarchical organization in sleep regulation. In Aim 3, we will utilize live fluorescent imaging to test the hypothesis that sNPF acts as an inhibitory output signal onto leucokinin neurons that regulate sleep. In combination, the proposed work will allow us to establish how sNPF neurons function within sleep regulatory networks in the brain, expanding our understanding of the mechanisms that control sleep and wakefulness at the molecular and circuit levels. In turn, this knowledge will provide a basis for the design of more effective treatments of human sleep abnormalities. This R15 AREA renewal proposal will directly involve undergraduate students in all aspects of the research, including designing and carrying out studies involving techniques of genetic manipulation, molecular and cellular neurobiology, and behavioral analysis. Students will also take an active role in disseminating the scientific knowledge acquired through their research, in the form of conference presentations and published manuscripts. Their experiences will provide formative training for future careers in biomedical fields.