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Sleep is a complex and plastic behavior regulated by multiple brain regions and influenced by numerous internal and external factors. Thus, to fully uncover the function(s) of sleep, cellular resolution of sleep-regulating neurons needs to be achieved. 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. 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), and show that two 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 two different types of sleep-regulating neurons controlling distinct aspects of sleep. Our work highlights the importance of using tools (GAL4 drivers in this case) that are as specific as possible when trying to link a behavior with a neuron or group of neurons.
