Summary Sleep problems such as excessive daytime sleepiness and insomnia are common in the United States. They are found in many psychiatric and neurological disorders and cause deficits in attention, learning and memory. Some sleep problems may be caused by disrupted circadian rhythms, but others may reflect changes in sleep homeostasis; an enigmatic regulatory mechanism that increases sleep drive, sleep amounts and sleep intensity as a function of prior time awake. The cellular mechanisms of sleep homeostasis are incompletely described but have traditionally thought to be neuronal. We, however, have shown that glial astrocytes are part of this mechanism. More specifically, we propose that sleep homeostasis arises from interactions between astrocytes and neurons. We therefore hypothesize that the normal compensatory response to sleep loss involves intracellular and molecular changes in astrocytes. This A1 submission has been extensively revised in accordance with initial review. New experiments and preliminary data are included (indicated by red font). We will test this overall hypothesis with three innovative approaches in vivo. In Aim 1, we combine genetically encoded Ca2+ indicator (GECI) astrocyte imaging with simultaneous polysomnographic recording in unanesthetized mice in vivo. This allows us to measure astrocyte Ca2+ dynamics in natural states of rapid- eye-movement (REM) sleep, non(N)REM sleep and wakefulness using both 2-photon and epiflorescent microscopy. We also more directly test the necessity of intracellular Ca2+ in sleep homeostasis by inducibly reducing this signal in vivo and measuring changes in sleep expression and homeostasis. In Aim 2, we use inducible molecular techniques to alter the major signaling pathways known to exist in astrocytes (i.e. Gq, Gi and Gs proteins) and examine the resulting changes in sleep expression and homeostasis. In Aim 3, we use next generation sequencing technology (single-cell RNA sequencing (scRNA-seq)) to isolate additional (but currently unknown) signaling pathways that are involved in astrocyte-mediated sleep homeostasis. Mammalian astrocytes are highly diverse based on morphology, cell-specific markers (e.g. GFAP+), ion channels, glutamate transporters and metabolic substrates. The relative contribution of these different astrocytes to sleep is unknown. scRNA-seq provides a new and powerful method to address this problem. Impact: Our characterization of a novel glial sleep mechanism will provide new insights into the etiology of abnormal sleep and arousal. Our experiments will also provide new insights into the function of non-neuronal brain cells. This in turn can lead to the development of new therapeutics that target glia, rather than neurons.