Astroglial mechanisms in sleep homeostasis

摘要 白天过度嗜睡和失眠等睡眠问题在美国很常见。他们 在许多精神和神经疾病中被发现，并导致注意力、学习和记忆障碍。 有些睡眠问题可能是由昼夜节律紊乱引起的，但另一些可能反映了睡眠的变化 动态平衡；一种神秘的调节机制，可以增加睡眠动力、睡眠时间和睡眠 强度是之前醒着的时间的函数。睡眠稳态的细胞机制是不完全的。 被描述但传统上被认为是神经元的。然而，我们已经证明，神经胶质星形胶质细胞是 这种机制的一部分。更具体地说，我们认为睡眠动态平衡产生于 星形胶质细胞和神经元。因此我们假设对失眠的正常代偿性反应 涉及星形胶质细胞的细胞内和分子变化。此A1提交文件已在 与初步审查一致。包括了新的实验和初步数据(用红色字体表示)。 我们将在体内用三种创新的方法来检验这一总体假设。在目标1中，我们通过基因结合 编码钙指示剂(GECI)星形胶质细胞显像与多导睡眠图同步记录 体内未麻醉的小鼠。这使我们能够测量星形胶质细胞钙动态在自然状态下的快速- 使用双光子和荧光的眼动睡眠、非(N)REM睡眠和觉醒 显微镜。我们还通过诱导的方法更直接地测试了细胞内钙在睡眠稳态中的必要性 在体内减少这种信号，并测量睡眠表达和动态平衡的变化。在目标2中，我们使用 诱导分子技术改变已知存在于星形胶质细胞中的主要信号通路(即GQ，GI 和Gs蛋白)，并检查由此导致的睡眠表达和内稳态的变化。在目标3中，我们使用 下一代测序技术(单细胞RNA测序(scRNA-seq))，以分离额外的(但 目前尚不清楚)参与星形胶质细胞介导的睡眠稳态的信号通路。 哺乳动物星形胶质细胞在形态、细胞特异性标记物(如GFAP)、离子 通道、谷氨酸转运体和代谢底物。这些不同因素的相对贡献 星形胶质细胞能否入睡尚不清楚。ScRNA-seq为解决这一问题提供了一种新的强有力的方法。 影响：我们对一种新的神经胶质睡眠机制的刻画将为研究高血压的病因提供新的见解。 不正常的睡眠和觉醒。我们的实验还将为非神经元的功能提供新的见解 脑细胞。这反过来可以导致针对胶质细胞而不是神经元的新疗法的开发。

Summary Sleep problems such as excessive daytime sleepiness and insomnia are common in the United States. They are found in many psychiatric and neurological disorders and cause deficits in attention, learning and memory. Some sleep problems may be caused by disrupted circadian rhythms, but others may reflect changes in sleep homeostasis; an enigmatic regulatory mechanism that increases sleep drive, sleep amounts and sleep intensity as a function of prior time awake. The cellular mechanisms of sleep homeostasis are incompletely described but have traditionally thought to be neuronal. We, however, have shown that glial astrocytes are part of this mechanism. More specifically, we propose that sleep homeostasis arises from interactions between astrocytes and neurons. We therefore hypothesize that the normal compensatory response to sleep loss involves intracellular and molecular changes in astrocytes. This A1 submission has been extensively revised in accordance with initial review. New experiments and preliminary data are included (indicated by red font). We will test this overall hypothesis with three innovative approaches in vivo. In Aim 1, we combine genetically encoded Ca2+ indicator (GECI) astrocyte imaging with simultaneous polysomnographic recording in unanesthetized mice in vivo. This allows us to measure astrocyte Ca2+ dynamics in natural states of rapid- eye-movement (REM) sleep, non(N)REM sleep and wakefulness using both 2-photon and epiflorescent microscopy. We also more directly test the necessity of intracellular Ca2+ in sleep homeostasis by inducibly reducing this signal in vivo and measuring changes in sleep expression and homeostasis. In Aim 2, we use inducible molecular techniques to alter the major signaling pathways known to exist in astrocytes (i.e. Gq, Gi and Gs proteins) and examine the resulting changes in sleep expression and homeostasis. In Aim 3, we use next generation sequencing technology (single-cell RNA sequencing (scRNA-seq)) to isolate additional (but currently unknown) signaling pathways that are involved in astrocyte-mediated sleep homeostasis. Mammalian astrocytes are highly diverse based on morphology, cell-specific markers (e.g. GFAP+), ion channels, glutamate transporters and metabolic substrates. The relative contribution of these different astrocytes to sleep is unknown. scRNA-seq provides a new and powerful method to address this problem. Impact: Our characterization of a novel glial sleep mechanism will provide new insights into the etiology of abnormal sleep and arousal. Our experiments will also provide new insights into the function of non-neuronal brain cells. This in turn can lead to the development of new therapeutics that target glia, rather than neurons.