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

通过双光子显微镜进行的体内成像彻底改变了我们的 了解神经生物学和免疫学过程，并揭示了意想不到的信息 中枢神经系统结构和免疫过程的动态形态学， 疾病目前，细胞内信号级联只能在孤立的细胞或切片中进行研究。 脑组织尤里卡计划的目标是对活体中的信号转导通路进行成像 小鼠大脑，目的是研究信号事件，因为它们在真实的时间内受到创伤的调节， 炎症和缺氧。在这项提案中，我们将（1）开发转基因生物传感器表达， 小鼠成像五个主要信号转导途径-氧化还原变化，NFB， Rho、PKA和Ras-在体内的小胶质细胞和巨噬细胞中;（2）产生新的遗传编码的 PKA，Rho和Ras途径的生物传感器，优化了双光子体内成像 （3）开发第一个药理学工具（生物传感器缀合的量子点）， 脑中的小胶质细胞和巨噬细胞的生物传感器;以及（4）证明脑中的动态信号传导事件。 缺氧、炎症或创伤性损伤期间活体小鼠大脑中的小胶质细胞的真实的变化。的工具 将从根本上改变我们用来研究动态激活的方法 细胞内信号传导途径。从这个建议的结果将阐明炎症信号 通过关联小胶质细胞的分子机制， 激活与动态形态学改变，以响应细胞外刺激在脑中的一个 活生生的动物这一建议将产生工具，使体内信号转导成像成为可能。 它将为科学界提供新的生物传感器，传递方法和动物 模型研究的时间和空间激活的五个主要信号转导途径， 活生生的动物在小胶质细胞和巨噬细胞中的信号转导途径的体内成像可以是 应用于具有炎症成分的广泛疾病，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.