Imaging signal transduction in the living mouse brain using two-photon microscopy

双光子显微镜的活体成像已经彻底改变了我们的 了解神经生物学和免疫学过程，并透露意外信息 健康人群中枢神经系统结构和免疫过程的动态形态研究 疾病。目前，细胞内信号级联只能在分离的细胞或脑片中进行研究。 脑组织。这个尤里卡计划的目标是成像活体中的信号转导通路 小鼠的大脑，目的是研究信号事件，因为它们受到创伤的实时调节， 炎症和缺氧。在这项建议中，我们将(1)开发转基因生物传感器 成像小鼠五个主要信号转导通路的激活-氧化还原变化，NFB， Rho、PKA和Ras-1在体内的小胶质细胞和巨噬细胞中的表达；(2)产生新的基因编码 用于PKA、Rho和RAS通路的生物传感器，通过双光子优化用于活体成像 显微镜；(3)开发第一个药理工具(生物传感器结合量子点)以提供 对大脑中的小胶质细胞和巨噬细胞的生物传感器；以及(4)显示动态信号事件 在缺氧、炎症或创伤期间，实时观察活小鼠脑内的小胶质细胞。这些工具 我们提出的开发将从根本上改变我们用来研究动态激活的方法 细胞内信号通路。这项提议的结果将阐明炎症信号 一种前所未有的转导途径--通过关联小胶质细胞的分子机制 脑组织对细胞外刺激反应的动态形态变化激活 活着的动物。这一提议将产生使体内信号转导成像成为可能的工具。 它将向科学界提供新的生物传感器、输送方法和动物 大鼠脑内五条主要信号转导通路的时空激活模型 活着的动物。小胶质细胞和巨噬细胞信号转导通路的体内成像 适用于多种含有炎症成分的疾病，包括癌症、糖尿病、 哮喘、感染和自身免疫性疾病，以及以小胶质细胞为特征的神经系统疾病 激活，如多发性硬化症、阿尔茨海默病、肌萎缩侧索硬化症、疼痛和 脊髓损伤。

In vivo imaging by two-photon microscopy has revolutionized our understanding of neurobiological and immunological processes and revealed unexpected information on the dynamic morphology of central nervous system structures and immune processes in health and disease. Currently, intracellular signaling cascades can only be studied in isolated cells or slices of brain tissue. The goal of this EUREKA proposal is to image signal transduction pathways in the living brain in mice, with the aim to study signaling events as they are regulated in real time by trauma, inflammation, and hypoxia. In this proposal we will (1) develop genetically modified biosensorexpressing mice to image the activation of five major signal transduction pathways-redox changes, NFB, Rho, PKA, and Ras- in microglia and macrophages in vivo; (2) generate novel genetically encoded biosensors for the PKA, Rho, and Ras pathways, optimized for in vivo imaging by two-photon microscopy; (3) develop the first pharmacologic tools (biosensor-conjugated Quantum Dots) to deliver biosensors to microglia and macrophages in the brain; and (4) demonstrate dynamic signaling events in microglia in living mouse brain in real time during hypoxia, inflammation, or traumatic injury. The tools we propose to develop will fundamentally change the methods we use to study the dynamic activation of intracellular signaling pathways. Results from this proposal will elucidate inflammatory signal transduction pathways in an unprecedented way-by correlating molecular mechanisms of microglial activation with dynamic morphologic alterations in response to extracellular stimuli in the brain of a living animal. This proposal will generate tools to make possible imaging of signal transduction in vivo. It will make available to the scientific community novel biosensors, delivery methods, and animal models to study the temporal and spatial activation of five major signal transduction pathways in the living animal. In vivo imaging of signal transduction pathways in microglia and macrophages can be applied to a wide range of diseases with an inflammatory component, including cancer, diabetes, asthma, infection, and autoimmune disorders, and nervous system disorders characterized by microglia activation, such as multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, pain, and spinal cord injury.