Glial Modulation of Autonomic Nervous System Activity

描述（由申请人提供）：星形胶质细胞信号系统在行为和疾病中的作用基本上仍未被探索，尽管这些细胞表现出在神经元活动期间被激活的多种G蛋白偶联受体（GPCR）。这方面信息的缺乏在很大程度上是因为我们在测量行为参数时无法选择性地激活tese细胞。为了解决这个问题，我们开发了一种转基因小鼠系，其在星形胶质细胞中表达新型Gq偶联GPCR（Gq-DREADD），该星形胶质细胞对穿过血脑屏障（bbb）的配体氯氮平-N-氧化物（CNO）作出反应。所有研究表明，Gq-DREADD功能类似于天然Gq-GPCR。我们已经进行了广泛的免疫组织化学和钙成像研究，整个中枢神经系统，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/生理表型的范围是惊人的。单次ip注射CNO导致血压、心率、唾液形成和立毛显著增加，以及体温降低;这些参数中的每一个都由自主神经系统调节。重要的是，CNO在同窝野生型小鼠中没有影响，并且当注射媒介物时，星形胶质细胞Gq-DREADD小鼠与同窝对照小鼠没有差异。尽管这些发现令人兴奋，但在进行机理研究之前，需要解决几个重要问题。首先，必须确定调节ANS活性的GFAP+细胞的解剖位置。虽然我们没有排除GFAP+ Schwann细胞介导Gq-DREADD激活对ANS活性的影响的可能性，但考虑到涉及神经元活性的星形胶质细胞调节的大量文献，该影响似乎更可能是由于星形胶质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表明，Gq-DREADD信号仅限于GFAP+神经胶质细胞，使用这些方法，几乎不可能排除一小群神经元表达Gq-DREADD并负责观察到的表型的可能性。具体目标1中描述的实验旨在确定调节ANS功能的Gq-DREADD细胞的解剖学位置，并进一步检查ANS活性的调节是一小群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问题是所得到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。在具体目标2中，我们建议使用最近鉴定的磷脂酶<$1（PLC <$1）中的显性/阴性（d/n）突变来开发一种选择性和可逆性阻断体内胶质细胞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.