Project Summary/Abstract Circadian rhythms are important for human health. At a molecular level, they are controlled by a cell-autonomous molecular clock which is generally synchronized across different tissues. In mammals, these local clocks are coordinated by the suprachiasmatic nucleus (SCN), the circadian pacemaker. Most previous work in this field has focused on how the SCN regulates neuronal physiology and behavior, and the function of local clocks in these processes is relatively understudied. In particular, the functions of extra-SCN oscillators in the brain are poorly understood. Our lab previously identified a molecule Wide Awake (WAKE) in Drosophila, which acts downstream of the clock to reduce arousal at night by inhibiting neuronal activity in a cell-autonomous manner. WAKE is conserved from flies to mammals, including mice and humans. In mice, our lab has recently found that WAKE in mice is enriched in the subfornical organ (SFO). This brain region is known to regulate behaviors controlling fluid balance and water consumption. Moreover, the SFO has been shown to exhibit robust cycling of local clock oscillators, and furthermore water consumption is under circadian control. My preliminary data suggest that loss of mWAKE results in an increase in drinking, specifically during the night. Taken together, these findings lead me to hypothesize that mWAKE normally inhibits thirst-promoting SFO neurons at night. Because SCN-specific mechanisms have also been shown to regulate circadian aspects of drinking, this system may allow for characterization of the interplay between local and central clock mechanisms in regulating a motivated behavior. In Aim 1, I propose to examine whether mWAKE expression in the SFO is rhythmic and under clock control and to characterize the nature of mWAKE+ cells in the SFO. In Aim 2, I will investigate the effects of mWAKE on SFO neuronal activity and the function of mWAKE and mWAKE+ cells in regulating rhythmic water consumption. These studies should provide new insights into how local clocks help tune neuronal activity to modulate rhythmic behaviors. Because desynchrony between local body clocks and the SCN can be produced by common activities such as shift work and is likely associated with poor health outcomes, these studies may one day inform future therapies related to dysregulation of circadian clocks. Finally, the proposed experiments will provide a solid platform for me to strengthen my technical skills and conceptual background and, combined with my proposed training in career and professional development, will position me for future success as an independent investigator and a mentor for diverse students in biomedical research.