Context-dependent arousal via wake-promoting pontine neurons in the Drosophila melanogaster fan-shaped body.

Neurons projecting into the Drosophila dorsal fan-shaped body (dFB) respond electrically during rising sleep pressure to implement sleep. While the molecular and circuit mechanisms that track sleep pressure have been an area of intense focus, less attention has been placed on the downstream neurons targeted by dFB cells to change behavioral state. To identify relevant circuitry, we first used an anterograde transsynaptic labeling tool, trans-Tango, to identify postsynaptic partners of dFB. We found that neurons downstream of dFB resemble hΔF cells and through thermogenetic stimulation identify their activity to be wake promoting. We validated these results using independent genetic lines, including highly specific split-Gal4 drivers. Next, we found that hΔF neurons express the glutamate transporter VGLUT and the acetylcholine biosynthetic enzyme, Choline acetyltransferase (ChAT). Consistent with their wake promoting role, RNAi-mediated knock down of VGlut and ChAT in hΔF increases baseline sleep by sustaining sleep episodes. Furthermore, RNAi for VGlut but not ChAT in h∆F cells reduced night-time sleep loss when flies were exposed to night-time light, indicating that these cells promote arousal in response to specific sensory cues. In contrast, VGlut and ChAT knock-down in h∆F neurons resulted in enhanced sleep loss while flies were deprived of food overnight. These results suggest that hΔF cells may selectively release glutamate and acetylcholine to fine tune arousal responses to changing environmental inputs.