Astroglial mechanisms in sleep homeostasis

总结 睡眠问题，如白天过度嗜睡和失眠在美国很常见。他们 在许多精神和神经系统疾病中发现，并导致注意力、学习和记忆缺陷。 有些睡眠问题可能是由昼夜节律紊乱引起的，但其他问题可能反映了睡眠的变化 稳态;一种神秘的调节机制，增加睡眠驱动力，睡眠量和睡眠 强度作为先前清醒时间的函数。睡眠稳态的细胞机制是不完全的 但传统上认为是神经元的。然而，我们已经表明，胶质星形胶质细胞是部分 这个机制。更具体地说，我们认为睡眠稳态来自于 星形胶质细胞和神经元。因此，我们假设，正常的补偿反应，睡眠不足， 涉及星形胶质细胞的细胞内和分子变化。本A1提交文件已在 根据初步审查。包括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，我们将联合收割机 编码Ca 2+指示剂（GECI）星形胶质细胞成像与同步多导睡眠图记录 未麻醉的小鼠体内。这使我们能够测量星形胶质细胞在快速- 使用双光子和表荧光素的眼动（REM）睡眠、非（N）REM睡眠和觉醒 显微镜我们还更直接地测试了细胞内Ca 2+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体内平衡的变化。在目标2中，我们使用 可诱导的分子技术来改变已知存在于星形胶质细胞中的主要信号传导途径（即Gq、Gi 和Gs蛋白），并检查由此产生的睡眠表达和体内平衡的变化。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 目前未知）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基于形态学、细胞特异性标志物（例如GFAP+）、离子通道（例如NH 4+）、细胞内基质（例如NH 4+）和细胞外基质（例如NH 4+）而高度多样化。 通道，谷氨酸转运蛋白和代谢底物。这些不同的相对贡献 星形胶质细胞睡眠是未知的。scRNA-seq为解决这一问题提供了一种新的强大方法。 影响：我们对一种新的胶质细胞睡眠机制的表征将为神经胶质细胞睡眠的病因学提供新的见解。 异常的睡眠和觉醒我们的实验也将为非神经元的功能提供新的见解。 脑细胞这反过来又可以导致针对神经胶质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.