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

摘要 睡眠稳态维持睡眠和清醒之间的平衡。稳态睡眠调节是 对细胞健康和睡眠障碍至关重要，与许多神经系统疾病和年龄相关的疾病有关。 疾病了解睡眠稳态机制对于开发新的睡眠疗法是必要的 紊乱下丘脑视前区（POA）对睡眠稳态至关重要。多核 POA，包括腹外侧视前区（VLPO）和正中视前核（MnPO），包含睡眠- 与清醒时相比，睡眠时活跃神经元的活动增加。c-Fos阳性细胞数 VLPO和MnPO中的神经元在高睡眠压力下增加，例如在睡眠剥夺后和恢复期间 睡眠剥夺后的睡眠POA中睡眠活跃神经元的完整组成尚不清楚。的 VLPO中表达甘丙肽的GABA能神经元是研究最广泛的睡眠活性神经元。然而，在这方面， 不是所有的c-Fos阳性睡眠活跃神经元都表达甘丙肽，也不是所有的甘丙肽神经元在睡眠过程中都呈c-Fos阳性。 在POA中的任何给定时间睡觉。考虑到POA的分子和功能组成的异质性， 重要的是在单个细胞水平上全面表征POA中的睡眠活跃神经元， 公正的方式。为此，我们将应用最近先进的单核RNA测序（snRNA- seq）技术与POA进行比较，并比较恢复期间小鼠之间单个细胞中的基因表达变化 睡眠剥夺（高睡眠压力）后的睡眠和长时间自发睡眠（低睡眠压力）后的小鼠 睡眠压力）。Aim 1将全面绘制在高睡眠下激活的所有神经元群 基于一组活动调节基因的压力。我们希望能发现甘丙肽的特殊亚型- 表达抑制性神经元，以及表达其他抑制性神经元的不表达甘丙肽的抑制性神经元组。 神经元标记物在高睡眠压力下被激活。目的2将揭示转录变化的调节 通过在所有细胞群中的稳态睡眠压力，包括神经元和非神经元细胞。比如说， 星形胶质细胞在维持睡眠稳态中起关键作用。然而，人们对转录水平知之甚少。 POA中参与睡眠稳态的星形胶质细胞的调节。鉴于最近发现的分子和 区域特异性的星形胶质细胞，我们假设，我们将揭示区域特异性和细胞特异性的变化， 星形胶质细胞目的3：利用多重荧光原位杂交技术（RNAscope）对肿瘤的解剖结构进行表征， 基于snRNA-seq鉴定的分子标记物对鉴定的睡眠活性神经元进行定位。这 POA中睡眠活跃神经元的分子和空间特征的结合将使未来的研究成为可能。 睡眠回路的解剖和操作

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.