Mammalian circadian rhythms, which orchestrate the daily temporal structure of biological processes, including the sleep-wake cycle, are primarily regulated by the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). The SCN clock is also implicated in providing an arousal 'signal,' particularly during the wake-maintenance zone (WMZ) of our biological day, essential for sustaining normal levels of wakefulness in the presence of mounting sleep pressure. Here we identify a role for SCN Neuromedin-S (SCN) neurons in regulating the level of arousal, especially during the WMZ. We used chemogenetic and optogenetic methods to activate SCNneurons in vivo, which potently drove wakefulness. Fiber photometry confirmed the wake-active profile of SCNneurons. Genetically ablating SCNneurons disrupted the sleep-wake cycle, reducing wakefulness during the dark period and abolished the circadian rhythm of body temperature. SCNneurons target the dorsomedial hypothalamic nucleus (DMH), and photostimulation of their terminals within the DMH rapidly produces arousal from sleep. Presynaptic inputs to SCNneurons were also identified, including regions known to influence SCN clock regulation. Unexpectedly, we discovered strong input from the preoptic area (POA), which itself receives substantial inhibitory input from the DMH, forming a possible arousal-promoting circuit (SCN->DMH->POA->SCN). Finally, we analyzed the transcriptional profile of SCNneurons via single-nuclei RNA-Seq, revealing three distinct subtypes. Our findings link molecularly-defined SCN neurons to sleep-wake patterns, body temperature rhythms, and arousal control. NMS NMS NM NMS NMS NMS NMS
