Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. Both in mammalian and invertebrate models, there are known requirements for glial cells, in addition to neuronal circuits, in the regulation of circadian behavior and sleep 1-4. Using the Drosophila model, our lab was the first to demonstrate that astrocytes were required, in vivo, for normal circadian behavior 5, 6, and we show in this proposal that astrocytes also modulate sleep. In preliminary studies for this application, we have demonstrated that activation of fly astrocytes, by increasing intracellular Ca2+, promotes sleep whereas inhibition of astrocyte vesicle trafficking and release leads to decreased sleep. In related studies (now published), we have shown that an astrocyte-enriched, secreted protein known as NKT promotes sleep 7. Based on these findings, we hypothesize that neuronal sleep-regulating circuits are subject to astrocyte modulation. Aim 1a will test this hypothesis by simultaneously activating or inhibiting astrocytes and recording the excitability of known sleep- regulating neuronal populations using genetically encoded voltage sensors. This will identify neuronal groups that show altered excitability with activation or inhibition of astrocytes. In complementary studies of Aim 1b, we will express membrane-tethered versions of NKT in all known sleep-regulating neuronal groups to identify those subject to modulation by the glial protein. NKT is a secreted astrocyte protein, but this subaim will utilize neuronal expression of membrane-tethered NKTs to identify its cellular targets. Together, Aims 1a and 1b will identify sleep-regulating neurons that are subject to astrocyte modulation. Aim 2 will use Translating Ribosome Affinity Purification (TRAP) methods combined with genome-wide RNA-seq to test the hypothesis that the gene expression profile of sleep-regulating neurons is altered by astrocyte modulation. The studies of this aim will focus on identification of neuronal factors (receptors, intracellular signaling components) with vertebrate orthologs that may mediate the astrocyte modulation of sleep. Preliminary results documenting feasibility for the aims are included in the proposal. Together, the two aims will provide valuable information about neuronal targets of astrocyte modulation and identify neuronal molecular pathways that respond to astrocyte signals. Such pathways may be important for glia-to-neuron communication or more directly the regulation of sleep.