Transcriptomic characterization of preoptic area in homeostatic sleep controls using single-nucleus RNA-sequencing

点击翻译按钮获取中文摘要

ABSTRACT Sleep homeostasis maintains the balance between sleep and wakefulness. Homeostatic sleep regulation is essential for cellular health and sleep disorders are implicated in many neurological disorders and age-related diseases. Understanding sleep homeostatic mechanisms is necessary for developing new therapies for sleep disorders. The preoptic area (POA) of the hypothalamus is essential for sleep homeostasis. Multiple nuclei of POA, including the ventrolateral preoptic area (VLPO) and the median preoptic nucleus (MnPO), contain sleep- active neurons that display increased activity during sleep compared with wake. The numbers of c-Fos positive neurons in VLPO and MnPO increase under high sleep pressure, e.g. after sleep deprivation and during recovery sleep following sleep deprivation. The complete makeup of the sleep-active neurons in POA is unknown. The galanin-expressing GABAergic neurons in VLPO are the most widely studied sleep-active neurons. However, not all c-Fos positive sleep-active neurons express galanin and not all galanin neurons are c-Fos positive during sleep at any given time in POA. Given the heterogeneous molecular and functional makeup of POA, it is important to comprehensively characterize the sleep-active neurons in POA at the individual cell level in an unbiased way. Towards this end, we will apply the recently advanced single-nucleus RNA sequencing (snRNA- seq) technique to POA and compare gene expression changes in individual cells between mice during recovery sleep following sleep deprivation (high sleep pressure) and mice after long periods of spontaneous sleep (low sleep pressure). Aim 1 will comprehensively map all neuronal groups that are activated under high sleep pressure based on a panel of activity-regulated genes. We expect to find that specific subtypes of galanin- expressing inhibitory neurons, as well as non-galanin expressing inhibitory neuronal groups that express other neuronal markers, are activated with high sleep pressure. Aim 2 will reveal the transcriptional changes regulated by homeostatic sleep pressure in all cell groups, including neurons and non-neuronal cells. For example, astrocytes play key roles in maintenance of sleep homeostasis. However, little is known about transcriptional regulation of astrocytes involved in sleep homeostasis in POA. Given the recent discovery of the molecular and regional specificity of astrocytes, we hypothesize that we will reveal region-specific and cell-specific changes in astrocytes. Aim 3 will use multiplex fluorescent in situ hybridization (RNAscope) to characterize the anatomical localization of the identified sleep-active neurons based on the molecular markers identified by snRNA-seq. This combination of molecular and spatial characterization of the sleep-active neurons in POA will enable future dissection and manipulation of the sleep circuit.