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.

Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the dorsal fan-shaped body (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation) in Drosophila. We report that dFB neurons change their physiological response-properties to dopamine (DA) and allatostatin-A (AstA) in response to different types of waking. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons intrinsically regulate their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together, these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism.

As in the mammalian system, sleep in Drosophila is regulated by multiple brain regions. Among them, neurons projecting to the dorsal Fan-Shaped Body (dFB) have been intensively studied and the data suggest they play a critical role for in sleep regulation. The 23E10-GAL4 driver is the most widely used tool to label and manipulate dFB neurons. Multiple studies have reported that activation of 23E10-GAL4 neurons promotes sleep. However, anatomical analysis revealed that 23E10-GAL4 labels 23-30 dFB neurons in the Drosophila brain and many non-dFB neurons in the brain and in the Ventral Nerve Cord (VNC), the fly equivalent of the spinal cord. To better understand the role of individual dFB neurons in sleep regulation, we undertook a Split-GAL4 screen to gain access to subsets of 23E10-GAL expressing cells. In this study, we report the discovery of two VNC cholinergic sleep-promoting neurons labeled by the 23E10-GAL4 driver.