Dissecting the role of neuronal-astroglial interactions in sleep homeostasis

项目总结 睡眠不足、睡眠障碍以及由此导致的健康和认知问题越来越普遍。 在美国。许多睡眠障碍可能与睡眠动态平衡异常有关：先天的 作为先前时间的函数来平衡睡眠需求、睡眠强度和睡眠数量的调节过程 一直醒着。睡眠动态平衡需要一个反馈电路来将系统维持在规定的范围内。 然而，这种反馈回路的细胞成分和蛋白质信号通路仍然不完整。 已定义。到目前为止，我们对睡眠稳态的理解主要是基于对神经元的研究，但我 表明非神经细胞(即星形胶质细胞)也起到了作用。我假设体内平衡反馈电路 包括反映睡眠需要的神经元唤醒信号和神经元唤醒的星形胶质整合因子 信号。我认为促进觉醒的神经递质去甲肾上腺素(NA)是神经元的候选物质。 与星形胶质细胞相互作用的唤醒信号。我进一步提出，钙是星形胶质细胞的整合因子。 睡眠需要是因为1)NA增加了星形胶质细胞的钙活性，2)我证明了星形胶质细胞的钙在 睡眠动态平衡。我的总体假设是，促进觉醒的神经元增加了星形胶质细胞的钙信号 在睡眠需求升高的时候。我将从两个方面检验这一假说：1)确定NA如何影响星形胶质细胞 睡眠剥夺(SD)前、中、后的CA2动态变化；2)决定睡眠不足对星形胶质细胞的影响 蛋白质信号。对于AIM 1，我将使用多方面的方法来光基因抑制或刺激NA 相邻星形胶质细胞钙动态成像和脑电记录时的神经元 在行为自由的小鼠中的状态活动。光遗传学、钙成像和脑电记录将 在基线条件下和在SD和恢复期间同时发生。使用这种多模式方法，我 在不同的觉醒过程中，可以在时间上记录细胞类型的特定神经元活动和星形胶质细胞的钙动力学 在行为自由的老鼠中处于这种状态。对于AIM 2，我将确定哪些星形胶质蛋白对睡眠变化做出反应 需要。我将使用超高效液相色谱-串联质谱仪对星形胶质细胞进行定量 利用靶向和非靶向蛋白质组学从休息和SD小鼠中提取蛋白质。靶向蛋白质组学将包括 钠和钙相关的信号蛋白以及突触、代谢和缝隙连接蛋白，因为这些 蛋白质与睡眠动态平衡有关。我还将确定这些蛋白质的磷酸化状态。 因为磷酸化状态会随着睡眠需要而改变，并且是一种重要的翻译后修饰 星形胶质细胞。将从星形胶质细胞减少的野生型和突变小鼠的脑组织中分离星形胶质细胞 CA2信令。通过这种方式，我可以确定哪些星形胶质蛋白对睡眠变化有反应 需要和2)钙依赖。拟议的研究使用创新的方法来定义生物底物 睡眠动态平衡。这些发现反过来将进一步表征非神经细胞在脑内的作用。 调节睡眠-觉醒行为，并将扩大我们对生理睡眠和睡眠紊乱的理解。

PROJECT SUMMARY Insufficient sleep, sleep disorders, and resulting problems with health and cognition are increasingly common in the United States. Many sleep disorders may be associated with abnormal sleep homeostasis: an innate regulatory process that balances sleep need, sleep intensity, and sleep amount as a function of prior time spent awake. Sleep homeostasis requires a feedback circuit to maintain the system within defined limits. However, the cellular components and protein signaling pathways of this feedback circuit remain incompletely defined. Our understanding of sleep homeostasis thus far is primarily based on the study of neurons, but I showed that non-neuronal cells (i.e. astrocytes) also play a role. I posit that the homeostatic feedback circuit includes a neuronal waking signal that reflects sleep need and an astroglial integrator of the neuronal waking signal. I propose that the wake-promoting neurotransmitter noradrenaline (NA) is a candidate for the neuronal waking signal that interacts with astrocytes. I further propose that calcium (Ca2+) is the astroglial integrator of sleep need because 1) NA increases astroglial Ca2+ activity and 2) I showed that astroglial Ca2+ plays a role in sleep homeostasis. My overall hypothesis is that wake-promoting neurons increase astroglial Ca2+ signaling during elevated sleep need. I will test this hypothesis in two AIMS: 1) Determine how NA impacts astroglial Ca2+ dynamics before, during, and after sleep deprivation (SD); 2) Determine how sleep loss impacts astroglial protein signaling. For AIM 1, I will use a multifaceted approach to optogenetically inhibit or stimulate NA neurons while imaging Ca2+ dynamics in adjacent astrocytes and recording electroencephalographic brain state activity in freely behaving mice. Optogenetics, Ca2+ imaging, and electroencephalographic recordings will occur simultaneously under baseline conditions and during SD & recovery. Using this multimodal approach, I can temporally register cell-type specific neuronal activity and astroglial Ca2+ dynamics within distinct arousal states in freely behaving mice. For AIM 2, I will determine which astroglial proteins respond to changes in sleep need. I will use ultra-performance liquid chromatography-tandem mass spectrometry to quantify astroglial proteins from rested and SD mice using targeted and untargeted proteomics. Targeted proteomics will include NA- & Ca2+-related signaling proteins as well as synaptic, metabolic, and gap junction proteins because these proteins are implicated in sleep homeostasis. I will also determine the phosphorylation status of these proteins because phosphorylation status changes with sleep need and is an important post-translational modification in astrocytes. Astrocytes will be isolated from brains of wild type mice and mutant mice with reduced astroglial Ca2+ signaling. In this way, I can determine which astroglial proteins are 1) responsive to changes in sleep need and 2) Ca2+-dependent. The proposed studies use innovative methods to define biological substrates of sleep homeostasis. These findings, in turn, will further characterize the contribution of non-neuronal cells in the regulation of sleep-wake behavior and will expand our understanding of physiological and disordered sleep.