Neural control of NREM sleep in the medulla

项目总结/摘要 在这个建议中，我们利用光遗传学和化学遗传学工具，体内钙离子， 成像，RNA-seq，基于病毒的电路跟踪和遗传靶向技术，以解剖 哺乳动物大脑中控制睡眠行为的神经回路。在基本层面上， 本研究建议中提出的工作可能为开发 治疗各种人类睡眠障碍的新方法，如失眠症。 几十年的研究表明，神经调节回路是睡眠的关键调节器， 清醒然而，这些研究大多集中在唤醒系统。促睡眠 神经元理论上是睡眠状态的启动和维持所必需的， 唤醒-睡眠转换的触发器模型，其中促进睡眠的神经元相互 具有促进唤醒的神经元的抑制性。以前的研究已经确定了几个大脑 促进非快速眼动（或NREM）睡眠的结构，但这些结构是否 结构参与启动或维持仍然在很大程度上未知。在这 研究项目，我们将主要集中在睡眠开始的神经控制，即过渡 从清醒到睡眠-在生理条件下，总是到NREM睡眠。我们 旨在破译唤醒-睡眠转换背后的电路机制。 在初步研究中，我们已经确定了一个新的群体的多巴胺能神经元， 投射到视前区（POA）的腹外侧延髓（VLM）， 中心我们目前的初步数据表明，这些VLM神经元被激活， 这些神经元的觉醒-睡眠转换和光遗传学激活诱导持久的 清醒小鼠的NREM睡眠。这些结果导致我们的工作假设：VLM 多巴胺能神经元诱导从觉醒到NREM睡眠的过渡， 随后激活其下游靶点以维持NREM睡眠。在目标1中，我们 激活和激活VLM能神经元，并检查其充分性和必要性 它们在清醒-睡眠过渡期的活动。在目标2中，我们将进行显微内镜钙 VLM神经元成像并检查其在睡眠期间的活动。那就 使用单细胞RNA-seq来鉴定睡眠活跃的VLM神经递质的遗传标记 用于进一步的体内实验。在目标3中，我们将使用基于病毒的电路跟踪， 描述VLM神经元的输入和输出，并检查它们在睡眠中的功能 调控总之，从这一建议中获得的结果将扩大我们对以下问题的理解： 睡眠行为的神经基础

Project Summary/Abstract In this proposal, we make use of optogenetic and chemogenetic tools, in vivo calcium imaging, RNA-seq, viral-based circuit tracing, and genetic targeting techniques to dissect the neural circuits that control sleep behavior in the mammalian brain. At a fundamental level, the work presented in this research proposal may provide valuable information for developing new treatments for various human sleep disorders, such as insomnia. Decades of studies have revealed neuromodulatory circuits as key regulators of sleep and wakefulness. However, most of these studies focus on the arousal system. Sleep-promoting neurons are theoretically required for the initiation and maintenance of sleep states based on the flip-flop model of wake-sleep switching, in which sleep-promoting neurons are mutually inhibitory with wake-promoting neurons. Previous studies have identified several brain structures that promote non-rapid eye movement (or NREM) sleep, but whether these structures are involved in the initiation or the maintenance remains largely unknown. In this research project, we will primarily focus on neural control of sleep initiation, i.e. the transition from wakefulness to sleep – under physiological conditions, always to NREM sleep. We aim to decipher the circuit mechanisms underlying the wake-sleep transition. In pilot studies, we have identified a novel population of glutamatergic neurons in the ventrolateral medulla (VLM) that project to the preoptic area (POA), a prominent sleep center. We present preliminary data showing that these VLM neurons are activated during wake-sleep transitions and optogenetic activation of these neurons induces long-lasting NREM sleep in awake mice. These results lead to our working hypothesis: VLM glutamatergic neurons induce the transition from wakefulness to NREM sleep, which subsequently activate their downstream targets to maintain NREM sleep. In Aim 1, we will activate and inactivate VLM glutamatergic neurons and examine the sufficiency and necessity of their activity in the wake-sleep transition. In Aim2, we will perform microendoscopic calcium imaging in VLM glutamatergic neurons and examine their activity during sleep. Then, we will use single-cell RNA-seq to identify genetic markers of sleep-active VLM glutamatergic neurons for further in vivo experiments. In Aim 3, we will use viral-based circuit tracing to characterize the inputs and outputs of VLM neurons and examine their function in sleep regulation. Together, the results obtained from this proposal will expand our understanding of the neural basis of sleep behavior.