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 他们了解神经生物学和免疫过程，揭示了有关健康和疾病中中枢神经系统结构和免疫过程的动态形态的意想不到的信息。目前，细胞内信号级联只能在分离的细胞或脑组织切片中进行研究。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、炎症和缺氧的实时调节。在这项建议中，我们将(1)开发表达小鼠的转基因生物传感器，以成像体内小胶质细胞和巨噬细胞中氧化还原变化、NFB、Rho、PKA和Ras这五条主要信号转导途径的激活情况；(2)为PKA、Rho和Ras途径产生新的遗传编码生物传感器，并优化双光子显微镜在体内的成像；(3)开发第一个药物工具(生物传感器连接的量子点)来提供 (4)在缺氧、炎症或创伤性损伤期间，实时显示活小鼠脑内小胶质细胞的动态信号事件。我们计划开发的工具将从根本上改变我们用来研究细胞内信号通路动态激活的方法。这一提议的结果将以一种前所未有的方式阐明炎症信号转导途径-通过关联小胶质细胞的分子机制 在活体动物的大脑中，对细胞外刺激作出反应的动态形态变化的激活。这一提议将产生使体内信号转导成像成为可能的工具。 它将为科学界提供新的生物传感器、递送方法和动物模型，以研究活体动物中五条主要信号转导通路的时空激活。小胶质细胞和巨噬细胞中信号转导通路的活体成像可以应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 biosensor expressing 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.