A Hyperkinetic-dependent redox-sensing mechanism operates specifically in dorsal fan-shaped body neurons to promote sleep.

Sleep is a universal and tightly regulated process that is controlled by both circadian and homeostatic mechanisms1. Work in Drosophila melanogaster has shown that sleep homeostasis is largely governed by the dorsal fan-shaped body (dFB). Within this region, some dFB neurons monitor the need to sleep through changes in intrinsic excitability. As sleep pressure builds, their input-output function becomes biased toward spike generation, whereas excitability returns toward baseline after rebound sleep2 in a process linked to mitochondrial reactive oxygen species (ROS)3. Prolonged periods of wakefulness elevate ROS-derived carbonyls, which are reduced by Hyperkinetic, an aldoketoreductase enzyme binding the cofactor NADP(H)3,4. The resulting change in cofactor redox state decelerates potassium channel inactivation, increases excitability and promotes sleep3,4. In line with this mechanism, dampening ROS levels and disrupting the excitability shift, or the Hyperkinetic-dependent redox-sensing mechanism, results in insomnia2,3. Conversely, production of non-radical ROS at the plasma membrane, i.e., where the functional Shaker-Hyperkinetic ion channel complex is localized, increases the excitability of dFB neurons and promotes sleep3,4. Together, these observations suggest that changes in mitochondrial oxidation in dFB neurons convey sleep need by coupling metabolic state to neuronal excitability3,4,5. However, the original R23E10-GAL4 driver line used to identify this mechanism has been recently shown to also label sleep-promoting ventral nerve cord (VNC-SP) cells in addition to dFB neurons6. Although prior electrophysiological and imaging experiments only targeted dFB neurons3,4,5, the interpretation of the sleep phenotypes may be confounded by contributions from both dFB and VNC-SP neurons. Indeed, a recent study suggested that dFB neurons may in fact not have a sleep-promoting role and that the redox-dependent mechanism may act in the ventral nerve cord instead7. To resolve this uncertainty, we directly compared the functional roles of dFB and VNC-SP neurons in redox-dependent sleep control using behavioral sleep assays, redox-state manipulations, and split-GAL4 (Sp-GAL4) lines that segregate these neuronal populations. As a result, we demonstrate that the redox-sensing mechanism operates specifically in dFB neurons to promote sleep.