Glial Modulation of Autonomic Nervous System Activity

神经胶质对自主神经系统活动的调节

• 批准号: 8535858
• 负责人: Ken Douglas McCarthy
• 金额: $ 17.85万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535858
• 关键词: Address; Agonist; Applications Grants; Area; Astrocytes; Autonomic nervous system; Behavior; Behavioral; Blood - brain barrier anatomy; Blood Pressure; Body Temperature; Brain; Calcium; Cells; Cholinergic Receptors; Coupled; Data; Directed Molecular Evolution; Disease; Dominant-Negative Mutation; Dyes; Exhibits; G Protein-Coupled Receptor Signaling; G alpha q Protein; G-Protein-Coupled Receptors; Glial Fibrillary Acidic Protein; Heart Rate; Hippocampus (Brain); Image; Intraperitoneal Injections; Label; Laboratories; Lead; Ligands; Literature; Location; Measures; Mediating; Methods; Mus; Muscarinics; Nervous System Physiology; Neuroglia; Neurons; Optics; Oxides; Pain; Perfusion; Pharmacogenetics; Phenotype; Phospholipase; Phospholipase A1; Photons; Physiological; Physiological Processes; Physiology; Piloerection; Play; Population; Research Proposals; Role; Saliva; Schwann Cells; Signal Transduction; Slice; Staining method; Stains; System; Therapeutic Agents; Transgenic Organisms; Visual Cortex; behavior measurement; calcium indicator; cell type; cholinergic; design; in vivo; lead oxide; novel; novel therapeutics; premature; promoter; receptor; research study; response; stem

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.

描述（由适用提供）：尽管这些细胞表现出在神经元活性期间被激活的多种G蛋白偶联受体（GPCR），但星形胶质细胞信号系统在行为和疾病中的作用基本上仍然是意外的。在这一领域缺乏信息，很大程度上是我们无法选择性地激活TESE细胞的同时测量行为参数的。为了解决这个问题，我们开发了一小鼠的转基因系，该系在星形胶质细胞中表达新型的GQ耦合GPCR（GQ-DREADD），该星形胶质细胞对配体氯糖蛋白-N-氧化物（CNO）的反应，该配体穿过血脑屏障（BBB）。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中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/生理表型的范围令人惊叹。 CNO的一次IP注入导致血压，心率，唾液形成和毛毛的显着升高以及体温降低。这些参数中的每一个都由自主神经系统调节。重要的是，CNO对同窝型WT小鼠没有影响，当注射媒介物时，星形胶质细胞GQ-DreadD小鼠与同窝控制小鼠没有差异。尽管这些发现令人兴奋，但在进行机械研究之前，需要解决一些重要的问题。首先，定义调节ANS活性的GFAP+细胞的解剖位置至关重要。尽管我们没有排除GFAP+ Schwann细胞介导GQ-Dreadd激活对ANS活性的影响的可能性，但由于涉及涉及神经元活性星形胶质细胞模型的大量文献，似乎更有可能是由于星形胶质细胞信号级联的激活所致。其次，尽管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+ Glia，使用这些方法几乎不可能排除少数神经元表达GQ-Dreadd的可能性，并且在特定目标1中所描述的实验构成了模态的模态及其模态的可能性。 ANS活性是少量神经元激活的结果。第三，使用药物遗传学方法在体内激活信号级联的问题是，所得的表型是否反映了物理过程还是我们过度刺激信号级联的能力。在特定目标2中，我们建议在磷脂酶»1（PLC€1）中使用最近确定的显性/阴性（D/N）突变，以开发一种方法，以选择性和可逆地阻断Vivo的Glial GQ-GPCR信号级联。