Clock-dependent regulation of a homeostatic sleep center maintains daytime sleep and evening activity

Homeostatic sleep centers promote sleep in response to prolonged wakefulness, but their contribution to circadian-regulated daily sleep is still unclear. Do neuronal circuits driving rebound sleep after extended wakefulness also drive circadian-gated sleep, or does rebound sleep differ on a neurophysiological level from daily baseline sleep? We observed in Drosophila that 23E10+ neurons, which include a homeostatic sleep center, the dorsal fan-shaped body (dFSB),¹^,²^,³^,⁴^,⁵ promote sleep in a time-of-day-dependent manner-the neurons play the strongest role in the maintenance of daytime sleep, and this effect on the siesta maps to cholinergic neurons within the dFSB. We asked whether 23E10+ neurons interact with the circadian clock to regulate daily sleep and find their role in maintaining the daytime siesta is at least partially dependent on the period gene. Through in vivo imaging, we show that calcium levels in the dFSB display a circadian rhythm, with a peak coinciding with the daytime siesta. In the absence of a period, the 24-h rhythm is lost, but a daytime increase in calcium activity is maintained. Loss of pigment dispersing factor (PDF) signaling causes premature downregulation of calcium activity in the dFSB, coinciding with the earlier truncation of the siesta in pdfr mutants and resulting in an earlier onset of night sleep. Silencing the dFSB is sufficient to rescue the timing of night sleep onset in pdfr mutants. These results indicate that the dFSB, a homeostatic sleep center, relies on the circadian clock to restrict sleep drive to specific times of day.