Dissecting the role of neuronal-astroglial interactions in sleep homeostasis

剖析神经元-星形胶质细胞相互作用在睡眠稳态中的作用

• 批准号: 10299917
• 负责人: Ashley Miranda Ingiosi
• 金额: $ 12.25万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10299917
• 关键词: Address; Architecture; Arousal; Astrocytes; BRAIN initiative; Behavior; Biological; Brain; Calcium; Cells; Cognition; Connexins; Electric Stimulation; Electroencephalography; Electromyography; Equilibrium; Event; Feedback; Frequencies; Future; Goals; Health; Homeostasis; Image; Impairment; Investigation; Link; Liquid Chromatography; Measures; Mediating; Mental disorders; Metabolic; Metabolic Diseases; Methods; Microscope; Molecular; Monitor; Mus; Mutant Strains Mice; Neurodegenerative Disorders; Neuroglia; Neurons; Neurotransmitters; Norepinephrine; Pathway interactions; Performance; Phosphoproteins; Phosphorylation; Physiological; Play; Post-Translational Protein Processing; Process; Proteins; Proteomics; Recovery; Regulation; Rest; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Sleep; Sleep Deprivation; Sleep Disorders; Synapses; System; Testing; Time; United States; Wakefulness; Wild Type Mouse; arm; awake; base; brain cell; cell type; frontal lobe; in vivo; innovation; link protein; locus ceruleus structure; multimodality; neurotransmission; non rapid eye movement; optogenetics; response; sleep abnormalities; sleep physiology; sleep regulation; tandem mass spectrometry

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.

项目摘要 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异常的睡眠稳态有关： 平衡睡眠需求、睡眠强度和睡眠量的调节过程，作为先前时间的函数 醒着度过睡眠稳态需要一个反馈回路来维持系统在限定的范围内。 然而，这种反馈回路的细胞组分和蛋白质信号通路仍然不完全 定义了到目前为止，我们对睡眠稳态的理解主要基于对神经元的研究，但我 表明非神经元细胞（即星形胶质细胞）也起作用。我认为自我平衡反馈回路 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细胞整合器 信号了我建议，唤醒促进神经递质去甲肾上腺素（NA）是一个候选的神经元 与星形胶质细胞相互作用的唤醒信号。我进一步提出，钙（Ca 2+）是星形胶质细胞的整合剂， 睡眠需要，因为1）NA增加星形胶质细胞Ca 2+活性，2）我表明星形胶质细胞Ca 2+在 睡眠稳态我的总体假设是，唤醒促进神经元增加星形胶质细胞的钙信号 在睡眠需求增加的情况下。我将在两个目标中测试这个假设：1）确定NA如何影响星形胶质细胞 睡眠剥夺（SD）之前、期间和之后的Ca 2+动态; 2）确定睡眠丧失如何影响星形胶质细胞 蛋白质信号对于AIM 1，我将使用多方面的方法来光遗传学抑制或刺激NA 神经元，同时成像邻近星形胶质细胞中的Ca 2+动力学并记录脑电图脑 在自由行为的小鼠中的状态活动。光遗传学、钙离子成像和脑电图记录将 在基线条件下和SD和恢复期间同时发生。使用这种多模式方法，我 可以在不同的唤醒中暂时记录细胞类型特异性神经元活动和星形胶质细胞Ca 2+动力学 在自由活动的老鼠身上。对于AIM 2，我将确定哪些星形胶质细胞蛋白对睡眠变化做出反应 需要的我会用超高效液相色谱-串联质谱法来定量星形胶质细胞 使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 NA-和Ca 2+相关的信号蛋白以及突触，代谢和间隙连接蛋白，因为这些 蛋白质与睡眠稳态有关。我也会确定这些蛋白质的磷酸化状态 因为磷酸化状态随着睡眠需要而改变，并且是睡眠中重要的翻译后修饰， 星形胶质细胞星形胶质细胞将从星形胶质细胞减少的野生型小鼠和突变小鼠的脑中分离。 Ca 2+信号。通过这种方式，我可以确定哪些星形胶质细胞蛋白1）对睡眠的变化做出反应 2）Ca ~（2+）依赖。拟议的研究使用创新的方法来定义生物基质， 睡眠稳态这些发现，反过来，将进一步表征非神经元细胞在神经细胞中的作用。 调节睡眠-觉醒行为，并将扩大我们对生理和睡眠障碍的理解。