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

使用单核 RNA 测序对稳态睡眠控制中视前区的转录组学特征

• 批准号: 10373184
• 负责人: Xiaofeng Guo
• 金额: $ 44.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-29 至 2024-03-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373184
• 关键词: Address; Adenosine; Affect; Anatomy; Arousal; Astrocytes; Cell Nucleus; Cells; Coupled; Data; Disease; Dissection; Equilibrium; FOS gene; Fluorescent in Situ Hybridization; Freezing; Future; Galanin; Gene Expression; Gene set enrichment analysis; Genes; Genetic Transcription; Health; Homeostasis; Hypothalamic structure; In Situ Hybridization; Individual; Knock-out; Maintenance; Mammals; Maps; Molecular; Mus; Neuroglia; Neurons; Neuropeptides; Pattern; Play; Preoptic Areas; Property; Recovery; Role; Signal Transduction; Sleep; Sleep Deprivation; Sleep Disorders; Sleep Disorders Therapy; Specificity; System; Techniques; Time; Tissues; Transcriptional Regulation; Wakefulness; Zebrafish; age related; base; brain cell; brain tissue; cell type; differential expression; high reward; high risk; inhibitory neuron; molecular marker; nervous system disorder; non rapid eye movement; novel therapeutics; preoptic nucleus; pressure; response; single-cell RNA sequencing; sleep regulation; transcriptome sequencing; transcriptomics

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.

抽象的 睡眠稳态保持睡眠和清醒之间的平衡。稳态睡眠调节是 对于细胞健康和睡眠障碍至关重要 疾病。了解睡眠稳态机制对于开发新的睡眠疗法是必要的 疾病。下丘脑的原始区域（POA）对于睡眠稳态至关重要。多核的 POA，包括腹外侧前区域（VLPO）和中值前核（MNPO），含有睡眠 与唤醒相比，在睡眠过程中显示出增加活性的活性神经元。 c-fos阳性的数量 VLPO和MNPO中的神经元在高睡眠压力下增加，例如睡眠剥夺后和康复期间 睡眠剥夺后睡眠。 POA中睡眠活性神经元的完整构成尚不清楚。这 VLPO中表达GALANIN的GABA能神经元是研究最广泛的睡眠神经元。然而， 并非所有C-FOS阳性睡眠神经元表达甘丙蛋白，而并非所有甘丙蛋白神经元在 在POA中的任何给定时间睡觉。鉴于POA的异质分子和功能构成，它是 重要的是全面表征在单个细胞水平的POA中的睡眠活性神经元 公正的方式。为此，我们将应用最近晚期的单核RNA测序（snRNA- SEQ）技术到POA并比较恢复过程中小鼠之间单个细胞中单个细胞中基因表达的变化 长时间自发睡眠后睡眠剥夺（高睡眠压力）和小鼠的睡眠（低睡眠压力）（低 睡眠压力）。 AIM 1将全面绘制在高睡眠下激活的所有神经元组 基于活性调节基因的压力。我们希望发现galanin-的特定亚型 表达抑制性神经元，以及表达表达其他人的抑制性神经元基团 神经元标记物以高睡眠压力激活。 AIM 2将揭示受调节的转录变化 通过所有细胞组的稳态睡眠压力，包括神经元和非神经元细胞。例如， 星形胶质细胞在维持睡眠体内平衡方面起关键作用。但是，关于转录的知之甚少 对POA睡眠体内稳态涉及的星形胶质细胞的调节。鉴于最近发现了分子和 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变化 星形胶质细胞。 AIM 3将使用多重荧光原位杂交（RNASCOPE）来表征解剖学 基于SnRNA-Seq鉴定的分子标记物的鉴定睡眠神经元的定位。这 POA中睡眠活动神经元的分子和空间表征的结合将使未来 睡眠电路的解剖和操纵。