Multimodal imaging of neuronal and glial contributions to sleep homeostasis in vivo

项目摘要 疾病控制和预防中心表示，睡眠不足是一种流行病，它会导致 对临床和经济产生重大影响。睡眠不佳可由睡眠动态平衡受损引起 根据事先的清醒程度来调节睡眠需求。因此，确定睡眠的细胞基础 动态平衡是理解睡眠异常的根本原因所必需的。细菌的生物底物 睡眠稳态尚不完全清楚，但神经元-神经胶质反馈机制可能在其中起重要作用。 角色。因为神经元和星形胶质细胞的化学信号在一定程度上是由细胞内钙波介导的，所以我们 假设神经元和星形胶质细胞内钙动力学参与了细胞内钙的积聚和 解除睡眠需要。这一假说将使用多模式成像方法在体内进行测试 神经元和星形胶质细胞胞体和突起的细胞内钙活性。基因编码的钙 指示物将选择性地在神经元或星形胶质细胞中表达，以评估细胞内钙动力学。 同一只未麻醉小鼠的两种显微镜方法：1)轻巧的头盔 允许自由运动和行为的荧光显微镜和2)双光子显微镜相结合 有一个平台，允许头戴着头巾的笼子航行。细胞内的钙动力学将同时 记录有睡眠-觉醒行为，如脑电图仪和肌电图仪所确定的 生理条件和对睡眠剥夺的反应。因此，本项目将开发一个可扩展的平台 不同脑细胞类型在自由活动和非麻醉状态下的功能评估方法 动物。我们的方法将互补的显微技术与标准的行为分析相结合 在复杂的过程和行为中对神经元和神经胶质活动的评估。因此，建议的 设计可以扩展到成瘾、衰老、学习和记忆的研究，以及更多地进一步阐明 中枢调节过程和行为的基础神经生物学。

Project Summary The Centers for Disease Control and Prevention have stated that insufficient sleep is an epidemic that poses significant clinical and economic impacts. Poor sleep can be caused by impaired sleep homeostasis which regulates sleep need as a function of prior wakefulness. Therefore, determining the cellular basis of sleep homeostasis is necessary to understand the underlying causes of abnormal sleep. The biological substrates of sleep homeostasis are incompletely understood, but neuronal-glial feedback mechanisms may play a central role. Because neuronal and astroglial chemical signaling is mediated, in part, by intracellular calcium waves, we hypothesize that neuronal and astroglial intracellular calcium dynamics contribute to the accumulation and discharge of sleep need. This hypothesis will be tested using a multimodal imaging approach to measure in vivo intracellular calcium activity in neuronal and astroglial somata and processes. Genetically encoded calcium indicators will be selectively expressed in neurons or in astrocytes to assess intracellular calcium dynamics using two microscopy methodologies in the same, unanesthetized mouse: 1) a lightweight, head-mounted epifluorescent microscope that permits free movement and behavior and 2) two-photon microscopy combined with a platform that allows for head-restrained cage navigation. Cellular calcium dynamics will be simultaneously recorded with sleep-wake behavior, as determined by electroencephalography and electromyography, under physiological conditions and in response to sleep deprivation. Thus, this project will develop a scalable platform methodology for the functional assessment of different brain cell types in freely behaving and unanesthetized animals. Our approach integrates complementary microscopy techniques with standard behavioral analyses for the assessment of neuronal and glial activity during complex processes and behavior. As such, the proposed design can be extended to studies of addiction, aging, learning and memory, and more to further elucidate the underlying neurobiology of centrally mediated processes and behaviors.