Regulation of sleep by cholinergic neurons located outside the central brain in Drosophila

Sleep is a complex and plastic behavior regulated by multiple brain regions and influenced by numerous internal and external stimuli. Thus, to fully uncover the function(s) of sleep, cellular resolution of sleep-regulating neurons needs to be achieved. Doing so will help to unequivocally assign a role or function to a given neuron or group of neurons in sleep behavior. In the Drosophila brain, neurons projecting to the dorsal fan-shaped body (dFB) have emerged as a key sleep-regulating area. To dissect the contribution of individual dFB neurons to sleep, we undertook an intersectional Split-GAL4 genetic screen focusing on cells contained within the 23E10-GAL4 driver, the most widely used tool to manipulate dFB neurons. In this study, we demonstrate that 23E10-GAL4 expresses in neurons outside the dFB and in the fly equivalent of the spinal cord, the ventral nerve cord (VNC). Furthermore, we show that 2 VNC cholinergic neurons strongly contribute to the sleep-promoting capacity of the 23E10-GAL4 driver under baseline conditions. However, in contrast to other 23E10-GAL4 neurons, silencing these VNC cells does not block sleep homeostasis. Thus, our data demonstrate that the 23E10-GAL4 driver contains at least 2 different types of sleep-regulating neurons controlling distinct aspects of sleep behavior.