Comparative distribution of the hypothalamic neurons activated during Wakefulness and Paradoxical (REM) sleep using TRAP2-red mice: contribution of Orexin, MCH, Lhx6 and a new marker Meis2

Study objectivesParadoxical 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. MethodIn the same TRAP2-red mouse, neurons activated during Wakefulness (4h) and PSR (2h) 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. ResultsTwelve 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) and supraoptic (SO) nuclei were predominantly activated during Wakefulness. MCH and Lhx6 neurons were mainly recruited during PSR, whereas Orx neurons were activated during both. A ventral subpopulation of MCH neurons showed higher activation during PSR than the dorsal subpopulation. Additionally, [~]30% of the c-Fos+ neurons in ZI and AHA express Meis2. A similar proportion of TdTomato+ neurons positive for Meis2 were encountered in PVN and SO. Overall, [~]20% of all hypothalamic neurons activated during PSR are now neurochemically identified. ConclusionOur study identifies new neuronal populations activated during PSR in AHA and tuberal nucleus. We further get evidence that Meis2 delineates novel neuronal populations activated during PSR. In summary, our results using TRAP2-red mice characterize new cell populations activated during Wakefulness or PSR, opening experimental paths for determining their function regarding vigilance states. Statement of significanceWakefulness 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 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 as Narcolepsy (Orx neurons), Amyotrophic Lateral Sclerosis (MCH and Orx signaling) or neurodegenerative diseases (Parkinsons and Alzheimer diseases).