PROJECT SUMMARY While sleep is evolutionarily conserved across all animals studied, the precise function of sleep remains unknown. It is vital that organisms receive an adequate amount of sleep, as sleep deprivation has profound widespread physiological effects including cognitive impairment, compromised immune system function, increased risk of cardiovascular disease, and in extreme cases can be fatal. In mammals, sleep is characterized by transitions between rapid eye movement (REM) and non-REM (NREM) sleep states. Sleep states and transitions between them are regulated by diverse neuronal populations found throughout the brain and are under the influence of homeostatic pressure. While numerous sleep-active and wake-active brain regions have been identified, it remains unknown how diverse neuronal populations coordinate their actions to regulate sleep and wakefulness. The preoptic area (POA) of the hypothalamus contains sleep-active GABAergic neurons and activation of their axons innervating the tuberomammillary nucleus (TMN) are critical for sleep regulation. However, it is yet understood exactly how the activity of POA GABAergic axonal projections to the TMN changes in response to increased sleep need and whether they are necessary to integrate homeostatic pressure. Conversely, the TMN histaminergic neurons are wake-active and innervate the POA. It is unknown how the activation of axonal projections of TMN histaminergic neurons or other neuronal subtypes to the POA promotes wakefulness. Understanding how these cell-type specific nuclei coordinate as a circuit via axonal projections will help elucidate the mechanism of sleep and wake regulation. The central hypothesis of this proposal is that the POA and TMN contain cell-type specific reciprocal projections that encode homeostatic sleep need and mediate sleep and wakefulness. To address this hypothesis, this proposal will integrate genetic mouse models for specifically labeling specific cell types, in vivo and ex vivo measurements of activity using fiber photometry, optrode and whole-cell patch-clamp recordings, and optogenetic and chemogenetic techniques to modulate neuronal activity. Aim 1 will investigate the role of POA GABAergic axonal projections to the TMN in sleep homeostasis. Aim 2 will define cell-type specific roles of TMN axons innervating the POA in wake regulation. Together, these studies will reveal novel circuit mechanisms by which the POA and TMN coordinate their activity during sleep/wake and periods of homeostatic sleep pressure.