Recurrent circadian circuitry regulates central brain activity to maintain sleep

Animal brains have discrete circadian neurons, but little is known about how they are coordinated to influence and maintain sleep. Here, through a systematic optogenetic screening, we identified a subtype of uncharacterized circadian DN3 neurons that is strongly sleep promoting in Drosophila. These anterior-projecting DN3s (APDN3s) receive signals from DN1 circadian neurons and then output to newly identified noncircadian "claw" neurons (CLs). CLs have a daily Ca²⁺ cycle, which peaks at night and correlates with DN1 and DN3 Ca²⁺ cycles. The CLs feedback onto a subset of DN1s to form a positive recurrent loop that maintains sleep. Using trans-synaptic photoactivatable green fluorescent protein (PA-GFP) tracing and functional in vivo imaging, we demonstrated that the CLs drive sleep by interacting with and releasing acetylcholine onto the mushroom body γ lobe. Taken together, the data identify a novel self-reinforcing loop within the circadian network and a new sleep-promoting neuropile that are both essential for maintaining normal sleep.