Molecularly Distinct Subtypes of Lhx6-positive Neurons of the Zona Incerta Differentially Regulate Sleep Pressure and Recovery Sleep

Sleep pressure is regulated not only by circadian rhythms, but also by sleep homeostasis, an activity-dependent process that dissipates during sleep. Recent work implicates Lhx6-positive GABAergic neurons of the zona incerta (ZI) in regulating sleep pressure, but their precise role remains unclear. Using sleep deprivation and HiPlex single-molecule fISH, we show that Lhx6-positive ZI neurons are broadly activated by both natural and induced increases in sleep pressure and remain active for more than 3 h into recovery sleep. Anterior Lhx6-positive neurons showed stronger activation. Fos induction differed across molecularly distinct subpopulations, with Nkx2-2-positive cells showing robust responses and Calb2-positive cells showing reduced activation. We also identified distinct sleep pressure-responsive Lhx6-negative Slc32a1-positive GABAergic ZI subpopulations. Finally, intersectional genetic loss of Nkx2-2 reduced and redistributed Lhx6-positive neurons, blunted their activation, and increased total sleep time. These findings reveal a central, heterogeneous role for Lhx6-positive ZI neurons in sleep homeostasis.