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

Abstract 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-labeling 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, supraoptic, 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 per cent of the c-Fos+ neurons in ZI and ~20 per cent in LHA expressed Meis2. Overall, ~20 per cent 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. Statement of Significance Wakefulness and paradoxical sleep are very similar at the electroencephalographic level. It remains relevant to determine the potential overlap of the neurons active during each vigilance state. We here took advantage of the powerful transgenic male TRAP2-red mice to directly compare in the same animal the brain cell activation during both states, with a focus on the hypothalamus. A deeper knowledge of each individual subpopulation of hypothalamic neurons within complex brain circuits underlying the sleep-waking cycle will help the understanding and validation of treatments of sleep disorders, at least those directly linked to demonstrated hypothalamic dysfunction such as narcolepsy (Orx neurons), amyotrophic lateral sclerosis (MCH and Orx signaling) or neurodegenerative diseases (Parkinson’s and Alzheimer diseases).