Glial Modulation of Autonomic Nervous System Activity

描述（由申请人提供）：星形细胞信号系统在行为和疾病中的作用基本上尚未被探索，尽管这些细胞表现出多种g蛋白偶联受体（gpcr），这些受体在神经元活动期间被激活。在很大程度上，这一领域信息的缺乏源于我们在测量行为参数时无法选择性地激活这些细胞。为了解决这个问题，我们开发了一种转基因小鼠系，在星形胶质细胞中表达一种新的gq偶联GPCR (Gq-DREADD)，这种gq偶联GPCR对穿过血脑屏障（bbb）的配体氯氮平- n -氧化物（CNO）有反应。所有研究表明Gq- DREADD的功能与天然Gq- gpcr相似。我们在整个中枢神经系统进行了广泛的免疫组织化学和钙成像研究，发现Gq-DREADD的表达和活性仅限于星形胶质细胞。在GFAP-Gq- DREADD小鼠中，ip注射CNO后观察到的行为/生理表型范围令人震惊。单次ip注射CNO导致血压、心率、唾液形成和阴茎勃起明显增加，体温下降；这些参数都是由自主神经系统调节的。重要的是，CNO对同窝wt小鼠没有影响，星形细胞Gq-DREADD小鼠在注射载药后与同窝对照小鼠没有区别。尽管这些发现令人兴奋，但在进行机制研究之前，有几个重要的问题需要解决。首先，有必要确定调节ANS活性的GFAP+细胞的解剖位置。虽然我们不排除GFAP+雪旺细胞介导Gq- DREADD激活对ANS活性的影响的可能性，但鉴于大量涉及星形细胞调节神经元活性的文献，这种影响似乎更有可能是由于星形细胞信号级联的激活。其次，虽然我们所有的免疫细胞化学和钙成像实验都表明Gq-DREADD信号传导仅限于GFAP+胶质细胞，但使用这些方法几乎不可能排除一小部分神经元表达Gq-DREADD并负责观察到的表型的可能性。Specific Aim 1中描述的实验旨在确定Gq-DREADD细胞调节ANS功能的解剖位置，并进一步研究ANS活动的调节是一小部分神经元激活的结果的可能性。第三，当使用药物遗传学方法在体内激活信号级联时，出现的一个问题是，所产生的表型是否反映了生理过程或我们过度刺激信号级联的能力。在Specific Aim 2中，我们建议使用最近发现的磷脂酶¿1 （PLC¿1）的显性/阴性（d/n）突变来开发一种在体内选择性和可逆地阻断胶质Gq-GPCR信号级联的方法。

DESCRIPTION (provided by applicant): The role of astrocytic signaling systems in behavior and disease remains essentially unexplored in spite of the fact that these cells exhibit a wide variety of G-protein coupled receptors (GPCRs) that are activated during neuronal activity. The lack of information in this area stems, in large part, from our inability to selectively activate tese cells while measuring behavioral parameters. To circumvent this problem, we developed a transgenic line of mice that expresses a novel Gq-coupled GPCR (Gq-DREADD) in astrocytes that responds to a ligand, clozepine-N-oxide (CNO), which crosses the blood-brain barrier (bbb). All studies indicate that Gq- DREADD functions similarly to native Gq-GPCRs. We have performed extensive immunohistochemical and calcium imaging studies throughout the CNS and find that the expression and activity of Gq-DREADD are restricted to astrocytes. The scope of the behavioral/physiological phenotype observed following an ip injection of CNO in GFAP-Gq- DREADD mice is stunning. A single ip injection of CNO leads to a marked increase in blood pressure, heart rate, saliva formation, and piloerection, and a decrease in body temperature; each of these parameters are regulated by the autonomic nervous system. Importantly, CNO has no effect in littermate wt mice, and astrocytic Gq-DREADD mice are not different from littermate control mice when injected with vehicle. As exciting as these findings are, several important questions need to be addressed before proceeding to mechanistic studies. First, it is essential to define the anatomical location of the GFAP+ cells modulating ANS activity. While we have not ruled out the possibility that GFAP+ Schwann cells mediate the effect of Gq- DREADD activation on ANS activity, it seems more likely that the effect is due to the activation of astrocytic signaling cascades given the extensive literature involving astrocytic modulation of neuronal activity. Second, while all of our immunocytochemical and calcium imaging experiments suggest that Gq-DREADD signaling is restricted to GFAP+ glia, using these methods it is nearly impossible to rule out the possibility that a small population of neurons express Gq-DREADD and are responsible for the observed phenotype. Experiments described in Specific Aim 1 are designed to define the anatomical location of Gq-DREADD cells modulating ANS function and further examine the possibility that the modulation of ANS activity is the result of the activation of a small population of neurons. Third, a question that arises whe using a pharmacogenetic approach to activate signaling cascades in vivo is whether the resulting phenotype reflects a physiological process or our ability to over-stimulate a signaling cascade. In Specific Aim 2 we propose to use a recently identified dominant/negative (d/n) mutation in phospholipase ¿1 (PLC ¿1) to develop an approach for selectively and reversibly blocking glial Gq-GPCR signaling cascades in vivo.