Asytrocytes, sleep and neuroinflammation

 描述(申请人提供)：睡眠研究历史上一直是以神经元为中心的；很多工作都集中在确定神经元电路和/或神经元产生的递质物质的作用上。在历史上，神经胶质细胞被认为只是被动的大脑居民，然而新的研究表明，神经胶质细胞，特别是星形胶质细胞和小胶质细胞，是睡眠调节和睡眠免疫相互作用的积极参与者。例如，最近的研究表明，抑制星形胶质细胞释放囊泡可以减少脑电慢波活动，并减轻睡眠剥夺导致的非快速眼动睡眠(NREM)睡眠的增加。然而，关于星形胶质细胞在免疫挑战期间睡眠改变的潜在机制中的相对贡献，仍有许多有待了解。细胞因子白介素1β和肿瘤坏死因子α参与了睡眠的调节和免疫攻击时睡眠的改变。重要的是，IL-1和TNF由神经元和神经胶质细胞产生，它们的受体存在于神经元和神经胶质细胞上，它们构成了神经元-神经胶质细胞通讯的重要机制纽带。如果星形胶质细胞的活动能够在体内得到特异、独立和可逆的调节，我们对星形胶质细胞在睡眠免疫相互作用中的贡献的知识将会进步。因此，R21探索/发展研究资助奖的总体目标是选择性地刺激星形胶质细胞中的G蛋白偶联信号级联，以阐明它们对调节的贡献 在免疫挑战期间的睡眠。为了完成这项任务，我们将使用专门由设计师药物(DREADD)激活的设计师受体。DREADD是一种经过改造的人类毒扁豆碱G蛋白偶联受体，无法结合其内源性配体乙酰胆碱。相反，它们与化学惰性的合成配体氯氮平-N-氧化物(CNO)结合，然后刺激细胞内信号传递。到目前为止，DREADDS几乎只用于调节神经元的活动。本申请中提出的实验将检验的中心假设是，刺激星形胶质细胞中的G蛋白偶联信号级联将减弱免疫挑战引起的睡眠变化。我们将使用Cre-loxP系统、商业上可用的GFAP：Cre小鼠和腺相关病毒载体选择性地在星形胶质细胞上表达DREADD。然后，我们将在两个目标的背景下进行实验，以检验我们的总体假设。在目标1中，我们将在没有免疫挑战的情况下刺激GQ或GIG蛋白偶联信号，并量化小鼠脑内细胞因子蛋白的区域性变化。在目标2中，我们将激活这些相同的信号级联，并确定这种操作对内毒素(LPS)诱导的小鼠睡眠模式和脑细胞因子蛋白变化的影响。在完成这些研究后，我们不仅将拥有一个新的工具包，而且我们将拥有有助于我们理解睡眠免疫相互作用的机制和功能的新数据。这些成就将填补我们在免疫挑战期间睡眠和神经胶质传递方面的知识空白，从而对这一领域产生持久和持久的影响。

 DESCRIPTION (provided by applicant): Sleep research has historically been neuron-centric; much effort has focused on determining neuronal circuits and/or actions of transmitter substances produced by neurons. Historically, glia were thought to be merely passive brain residents, yet new research demonstrates that glial cells, particularly astrocytes and microglia, are active participants in sleep regulation and in sleep-immune interactions. For example, recent studies demonstrate that inhibiting vesicular release from astrocytes reduces EEG slow wave activity and attenuates sleep deprivation-induced increases in non-rapid eye movement (NREM) sleep. However, much remains to be learned about the relative contribution of astrocytes to mechanisms underlying alterations in sleep during immune challenge. The cytokines interleukin-1β (IL-1) and tumor necrosis factor-α (TNF) are involved in the regulation of sleep and in the alterations in sleep that occur during immune challenge. Importantly, IL-1 and TNF are produced by neurons and glia, their receptors are present on neurons and glia, and they constitute an important mechanistic link in neuronal-glial communication. Our knowledge about astrocyte contributions to sleep-immune interactions will be advanced if actions of this cell type can be specifically, independently, and reversibly modulated in vivo. Thus, the overall objective of this R21 Exploratory / Developmental Research Grant Award is to selectively stimulate G protein-coupled signaling cascades in astrocytes to elucidate their contributions to the regulation of sleep during immune challenge. To accomplish this task we will use designer receptors exclusively activated by designer drugs (DREADDs). DREADDs are engineered human muscarinic G protein-coupled receptors that are unable to bind their endogenous ligand, acetylcholine. Instead, they bind a chemically inert synthetic ligand, clozapine-N-oxide (CNO), which then stimulates intracellular signaling. To date, DREADDs have almost exclusively been used to modulate activity of neurons. The central hypothesis to be tested by experiments proposed in this application is that stimulating G protein-coupled signaling cascades in astrocytes will attenuate changes in sleep that are induced by immune challenge. We will use the Cre-loxP system, commercially-available GFAP:Cre mice, and adeno-associated virus vectors to selectively express DREADDs on astrocytes. We will then test our overall hypothesis by conducting experiments within the context of two Aims. In Aim 1 we will stimulate Gq or Gi G protein-coupled signaling in the absence of immune challenge and quantify regional changes in mouse brain cytokine protein. In Aim 2 we will activate these same signaling cascades and determine the impact of this manipulation on lipopolysaccharide (LPS)-induced alterations in mouse sleep patterns and brain cytokine protein. Upon completing these studies we will not only possess a new toolkit, but we will have novel data that will contribute to our understanding of mechanisms and functions for sleep-immune interactions. These achievements will have a prolonged and sustained impact on the field by filling a gap in our knowledge of sleep and gliotransmission during immune challenge.