Function of Astrocytic GPCR Signaling Cascades in Physiology and Mental Illness

描述（由申请人提供）：该资助提案的目标是开发可用于激活或激活严格鉴定的星形胶质细胞群体中的GPCR信号级联的遗传工具，使用研究界易于使用的方法;我们相信，这些工具的可用性将在理解星形胶质细胞在生理学，行为学，和精神疾病虽然星形胶质细胞占CNS的很大一部分，并且表达在神经元活动期间被激活的多种GPCR，但对星形胶质细胞GPCR的作用知之甚少。在这方面的信息缺乏源于我们无法选择性地激活或星形胶质细胞信号级联，同时测量生理和行为参数。重要的是，临床上用于治疗精神疾病的大多数治疗剂影响GPCR信号级联。最近，已经开发了遗传工具，可用于选择性地激活或阻断特定细胞类型中的GPCR信号级联。一种这样的药物遗传学方法利用被统称为DREADD受体的受体家族（用于由设计者药物专门激活的设计者受体）来激活特定细胞类型中的GPCR信号传导级联。DREADD受体通过M3-ACh受体DREADD受体的定向分子进化制备，不能响应任何已知的内源性GPCR配体，并且被氯氮平-N-氧化物（CNO）激活。重要的是，CNO穿过血脑屏障，首次通过腹膜内（ip）注射CNO选择性激活特定星形胶质细胞群体中的GPRC信号传导。我们计划使用交叉基因激活，它需要两个细胞类型特异性启动子来激活基因表达，并大大增加基因表达的细胞特异性。交叉基因激活将用于在严格定义的星形胶质细胞群体中表达Gq-、Gs-和Gi-DREADD受体。任何药理学方法的关注点是所产生的表型（例如，LTP、行为等）反映了所研究系统的过度刺激。我们计划开发可用于在严格定义的星形胶质细胞群体中检测GPCR信号级联的遗传工具;这些遗传工具还将利用交叉基因激活。能够在严格定义的星形胶质细胞群体中激活或抑制GPCR信号级联的组合将提供一种强有力的方法来整理星形胶质细胞在生理学，行为和精神疾病中的作用;这些小鼠模型在研究界的分布可能是转型的。本提案中开发的遗传工具将用于测试星形胶质细胞GPCR信号级联激活调节突触传递的假设;这一假设对于我们理解星形胶质细胞在脑功能中的作用至关重要，目前在研究界存在极大争议。

DESCRIPTION (provided by applicant): The goal of this grant proposal is to develop genetic tools that can be used to activate or inactivate G-protein coupled receptor (GPCR) signaling cascades in rigorously identified populations of astrocytes using approaches that can be readily used by the research community; we believe the availability of such tools will be transformational with respect to understanding the role of astrocytes in physiology, behavior, and mental illness. While astrocytes make up a large portion of the CNS and express a wide variety of GPCRs that are activated during neuronal activity, the role of astrocytic GPCRs is poorly understood. The lack of information in this area stems from our inability to selectively activate or inactivate astrocytic signaling cascades while measuring physiological and behavioral parameters. Importantly, the majority of therapeutic agents used clinically to treat mental illness effect GPCR signaling cascades. Recently, genetic tools have been developed that can be used to selectively activate or inactivate GPCR signaling cascades in specific cell types. One such pharmacogenetic approach utilizes a receptor family referred to collectively as DREADD receptors (for Designer Receptor Exclusively Activated by Designer Drug) to activate GPCR signaling cascades in specific cell types. DREADD receptors were prepared by directed molecular evolution of M3-ACh receptor DREADD receptors, fail to respond to any known endogenous GPCR ligand, and are activated by clozepine-N-oxide (CNO). Importantly, CNO crosses the blood-brain-barrier enabling, for the first time, the selective activation of GPRC signaling in specific populations of astrocytes via an intraperitoneal (ip) injection of CNO. We plan to use intersectional gene activation which requires two cell- type specific promoters to activate gene expression and greatly increases the cell specificity of gene expression. Intersectional gene activation will be used to express Gq-, Gs-, and Gi-DREADD receptors in rigorously defined populations of astrocytes. A concern with any pharmacological approach is whether the resulting phenotype (e.g., LTP, behavior, etc.) reflects the over stimulation of the system under study. We plan to develop genetic tools that can be used to inactivate GPCR signaling cascades in rigorously defined populations of astrocytes; these genetic tools will also take advantage of intersectional gene activation. The combination of being able to activate or inactivate GPCR signaling cascades in rigorously defined populations of astrocytes will provide a powerful approach to sorting out the role of astrocytes in physiology, behavior, and mental illness; distribution of these mouse models to the research community is likely to be transformational. The genetic tools developed in this proposal will be used to test the hypothesis that the activation of astrocytic GPCR signaling cascades modulates synaptic transmission; this hypothesis is fundamental to our understanding of the role of astrocytes in brain function and is currently extremely controversial within the research community.