Project Summary Animals have evolved circadian (near-24 h) rhythms to anticipate and adjust their behavior to daily opportunities and challenges such as mating, food availability, and predation. These behavioral rhythms are synchronized to the solar day by the central circadian pacemaker, the suprachiasmatic nucleus (SCN). SCN neurons exhibit daily rhythms in firing rate and clock gene expression that communicate circadian time to the rest of the brain and body. However, critically, we do not know how SCN signals interact with molecular and neuronal clocks in downstream neurons to generate circadian outputs. Our lab’s overarching goal is thus to understand how circadian input from the SCN is encoded by target neurons to ultimately generate diverse behavioral rhythms that peak at different times of day. To address this, over the next five years, our research program will focus on several interrelated but independent themes, including defining the “transfer function” for circadian output circuits, determining how molecular clocks in target neurons contribute to behavioral rhythmicity, and understanding how target neurons integrate diverse inputs to generate behavioral rhythms. We propose that endogenous rhythmicity in downstream neurons and daily input from SCN neurons are each required to drive appropriately timed circadian behavioral outputs. Here, we will use multi-level analysis at the molecular, circuit, and behavioral levels including targeted genomic editing of clock genes, in vivo and ex vivo imaging of rhythmic neurons, and machine learning analysis of behavior to dissect circadian output circuitry in two complementary species, the nocturnal laboratory mouse and the diurnal African striped mouse. Curiously, molecular and neuronal activity rhythms in the SCN peak at similar times in diurnal and nocturnal animals. How does an ostensibly identical SCN rhythm determine these dramatically different temporal niches? Our approach will allow us to address this and other long-standing questions in chronobiology by identifying both the mechanisms that temporally organize behaviors and the differences in molecular and neural function that decide an animal’s temporal niche preference. Identifying the genes, neurons, and circuits that regulate the timing of behavior in both laboratory mice and striped mice will also provide a novel framework for understanding the biological basis of chronotype in humans and the etiology of circadian rhythm sleep disorders. The discoveries we will make through our research program can generalize beyond circadian biology to reveal fundamental mechanisms linking genes and circuits to behavior.