Comparative distribution of the hypothalamic neurons activated during wakefulness and paradoxical (REM) sleep using male TRAP2-red mice: contribution of orexin, MCH, Lhx6 and a new marker Meis2.

Study Objectives Paradoxical sleep (PS) is a state involving numerous hypothalamic neuronal subpopulations, many remaining neurochemically uncharacterized. Our goal was to compare hypothalamic neurons active during Wakefulness or PS rebound (PSR) and explore their potential overlap, with a focus on melanin-concentrating-hormone (MCH), Orexin (Orx), Lhx6 and a new contingent of Meis2-expressing neurons.Methods In the same male TRAP2-red mouse, neurons activated during Wakefulness (4 h) and PSR (2 h) express TdTomato and c-Fos, respectively. Double-labelling and triple immunofluorescence with neurochemical markers were performed to characterize and quantify cell populations in hypothalamic structures.Results Twelve hypothalamic structures showed distinct activation patterns. The anterior hypothalamic area (AHA), zona incerta (ZI) and tuberal nucleus contained more activated neurons during PSR than Wakefulness, whereas the paraventricular hypothalamic (PVN), supraoptic (SO), parasubthalamic nuclei and retrochiasmatic area were predominantly activated during Wakefulness. MCH and Lhx6 neurons were mainly recruited during PSR, whereas Orx neurons were more activated during wakefulness. The ventral subpopulation of MCH neurons showed higher activation during PSR than the dorsal subpopulation. Additionally, ~30% of the c-Fos + neurons in ZI and ~ 20% in LHA expressed Meis2. Overall, ~20% of all hypothalamic neurons activated during PSR are now neurochemically identified.Conclusions Our study identifies new neuronal populations activated during PSR in AHA, ZI and tuberal nucleus. We further provide evidence that Meis2 is expressed in novel populations of neurons activated during PSR. In summary, our results using male TRAP2-red mice characterize the cell populations activated during Wakefulness and PSR, opening experimental paths for determining their function regarding vigilance states.