Single-nucleus gene expression and chromatin accessibility profiling of sleep and wake driven changes in murine hypothalamus - ABSTRACT There are developing data that in the hypothalamus neurons that promote sleep are intermingled with neurons that promote wakefulness. Moreover, there is a considerable molecular heterogeneity even within neuronal groups that express the same major neurotransmitter. Thus, new techniques capable of simultaneously detecting both molecular and functional heterogeneity are needed to achieve a new understanding of the neurons involved in sleep and wake regulation. We have proven that single-nucleus RNA sequencing is suitable for this task by clustering cell-based gene expression patterns to determine the molecular identity of cell types, combined with examination of changes in activity regulated genes (ARGs) to determine functional involvement. When we applied this approach to the preoptic area of the hypothalamus, we successfully identified a subset of galanin neurons that were activated during sleep with high sleep drive (high delta power). We now plan to expand this approach to the whole hypothalamus. In our previous study we found that there was increased expression of a particular pyrimidine and of a serotonin receptor during recovery sleep following six hours of sleep deprivation only in the subset of galanin cells activated during high sleep drive. This finding could reveal novel signaling mechanisms for sleep homeostasis. We hypothesize that if these receptor genes are involved in sleep homeostasis, their expression should increase during the period of sleep deprivation, even prior to sleep onset. Thus, in addition to comparing gene expression during sleep with high sleep drive (recovery sleep following sleep deprivation) to low sleep drive, we will compare gene expression between animals kept awake and animals sleeping. All groups will be sacrificed at same diurnal time to remove circadian effects. In addition to detecting changes in gene expression, we propose to measure changes in chromatin accessibility in the same nuclei using the new Multiome assay from 10xGenomics. This will for the first time allow detection of cell-type specific regulatory elements in the hypothalamus involved in sleep, wake, and sleep homeostatic regulation. While this study will address specific and important questions, it will also allow us to provide a single-cell gene expression and ATAC-seq atlas related to sleep regulation that will facilitate the work of other investigators in the field of sleep research.
