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

摘要 睡眠动态平衡维持睡眠和清醒之间的平衡。动态平衡睡眠调节 对细胞健康和睡眠障碍是必不可少的，与许多神经疾病和年龄相关 疾病。了解睡眠稳态机制对于开发新的睡眠疗法是必要的。 精神错乱。下丘脑的视前区(POA)是维持睡眠稳态所必需的。多个原子核 视前外侧区(VLPO)和视前正中核(MnPO)等脑区含有睡眠。 活跃的神经元在睡眠中表现出比清醒时更活跃的活动。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 睡眠不足后的睡眠。POA中睡眠活动神经元的完整组成尚不清楚。这个 VLPO内表达Galanin的GABA能神经元是目前研究最广泛的睡眠活动神经元。然而， 并不是所有c-Fos阳性的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 在小一年级的任何指定时间睡觉。考虑到POA的分子和功能组成的异质性，它是 重要的是要在单个细胞水平上全面表征POA中的睡眠活跃神经元 不偏不倚。为此，我们将应用最近先进的单核RNA测序(SnRNA- SEQ)技术检测POA并比较恢复过程中小鼠个体细胞基因表达的变化 睡眠剥夺后的睡眠(高睡眠压力)和长时间自然睡眠后的小鼠(低睡眠 睡眠压力)。目标1将全面映射在高睡眠状态下激活的所有神经元组 基于一组活动调节基因的压力。我们希望发现甘丙素的特定亚型- 表达抑制性神经元，以及表达非甘丙素的表达其他 神经元标记物在高睡眠压力下被激活。目标2将揭示转录调控的变化 包括神经元和非神经元细胞在内的所有细胞组中的动态平衡睡眠压力。例如, 星形胶质细胞在维持睡眠动态平衡方面起着关键作用。然而，人们对转录因子知之甚少。 星形胶质细胞参与POA睡眠动态平衡的调节。鉴于最近发现的分子和 星形胶质细胞的区域特异性，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。目标3将使用多重荧光原位杂交(RNAScope)来表征解剖结构 基于SNRNA-seq鉴定的分子标记对已鉴定的睡眠活跃神经元进行定位。这 POA中睡眠活动神经元的分子和空间特征的结合将使未来 对睡眠回路的解剖和操纵。

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.