Regulation of pre-dawn arousal in Drosophila by a pair of trissinergic descending neurons of the visual and circadian networks.

Circadian neurons form a complex neural network that generates circadian oscillations. How the circadian neural network transmits circadian signals to other brain regions, thereby regulating the activity patterns in fruit flies, is not well known. Using the FlyWire database, we identified a cluster of descending neurons, DNp27, which is densely connected with key circadian neurons and the visual circuit, projecting extensively across the brain. DNp27 receives excitatory inputs from the circadian neurons DN3s at night and photo-inhibitory signals predominantly during the day, resulting in calcium oscillations that peak in the early morning and dip at dusk. Experimental manipulation of DNp27 revealed its role in activity regulation: artificial activation of DNp27 decreased flies' activity, while ablation or silencing led to an advance in the morning anticipatory peak. Similar alterations in the morning peak were observed following pan-neuronal knockdown of either Trissin or TrissinR, suggesting the involvement of this neuropeptide signaling pathway in DNp27 function. Moreover, neural circuitry and connectivity analyses indicate that DNp27 may regulate circadian neurons via extra-clock electrical oscillators (xCEOs). Lastly, we found that DNp27 modulates arousal thresholds by inhibiting light-responsive activity in the central brain, thereby promoting sleep stability, particularly in the pre-dawn period. Together, these findings suggest that DNp27 plays a crucial role in maintaining stable sleep patterns.