An approach to image calcium in small volumes of astrocytes in vivo

描述(由申请人提供)：星形胶质细胞在大脑中扮演着良好的支持性角色。此外，星形胶质细胞的新兴作用包括向神经元发送信号和从神经元发出信号，以及调节局部血流量。某些星形胶质细胞的功能与胞浆钙瞬变相关或受其调节，钙瞬变被认为是反映星形胶质细胞兴奋性的生理信号。星形胶质细胞主要通过其远端突起与神经元和血管相互作用，但目前还不可能在体外、组织切片制备或体内非侵入性地测量星形胶质细胞突起中的钙信号。一种在切片和体内测量星形胶质细胞突起中钙离子的方法将使关于星形胶质细胞在健康中枢神经系统中的作用的机制假说得到严格的检验，并将开辟新的途径来研究反应性星形胶质细胞增生症对这些相同的中枢神经系统功能的影响。为了开发这样一种方法，我们修改了一种名为GCaMP2的基因编码的钙传感器，使其N端带有一个膜系留结构域(称为Lck)，从而产生Lck-GCaMP2。到目前为止，我们的发现表明，LCK-GCaMP2允许对细胞膜附近的星形胶质细胞和细胞培养过程中的钙水平进行非侵入性成像。这种水平的分辨率是可能的，因为LCK-GCaMP2选择性地在质膜上高度表达，提供微米级的空间信息。这种方法将有助于揭示星形胶质细胞在生理和病理生理过程中何时、何地以及如何被激活。在这项提议中，我们试图开发我们的新方法，并通过产生和鉴定新型转基因小鼠来为星形胶质细胞信号转导社区提供重要的新资源，这些转基因小鼠将允许在组织切片和体内的星形胶质细胞过程中进行钙成像。我们有两个具体目标。在目标1中，我们将制造针对LCK-GCaMP2的星形胶质细胞的基因构建物，并利用这些构建物建立转基因小鼠的建立系。在目标2中，我们将表征并使用这些Lck-GCaMP2转基因小鼠来研究钙信号在海马片星形胶质细胞突起中的机制和功能。我们将检验两个假设：(I)星形胶质细胞表现出空间分区的钙信号；(Ii)TGF2在反应性星形胶质细胞的过程中对钙信号具有直接、受体介导的作用，从而提供了一种反应性星形胶质细胞增生症影响神经元的机制。这里提出的工作将提供新颖的、具有良好特性的光学报告小鼠，使我们和其他人能够精确测量星形胶质细胞胞体和完整组织结构(如脑片和活体)内的突起中的局部钙信号。这些新的报告小鼠将成为星形胶质细胞社区有价值的通用工具，使研究人员能够在传统成像方法无法获得的过程中测量局部星形胶质细胞钙信号。 与公众健康相关：我们将开发小鼠模型，使我们和其他研究人员能够在体外和体内监测和跟踪星形胶质细胞中的钙信号。这些小鼠的出现将成为研究星形胶质细胞在正常健康大脑和神经系统疾病(包括癫痫和神经退化)以及脑损伤和修复过程中作用的特殊工具。

DESCRIPTION (provided by applicant): Astrocytes play well documented supportive roles in the brain. In addition, emerging roles for astrocytes include signaling to and from neurons, and regulation of local blood flow. Certain astrocyte functions are correlated with, or regulated by, cytosolic calcium transients, which are considered a physiological signal reflecting astrocyte excitability. Astrocytes interact with neurons and blood vessels primarily with their distal processes, but it is currently not possible to measure calcium signals non-invasively in astrocyte processes either in vitro in tissue slice preparations or in vivo. A method for measuring calcium in astrocyte processes in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding astrocyte roles in brain function in the healthy CNS, and would open up new ways to study the impact of reactive astrogliosis, which occurs in response to all forms of injury and disease, on these same CNS functions. To develop such a method, we have modified a genetically encoded calcium sensor called GCaMP2 to carry a membrane tethering domain (called Lck) on its N terminus, thus generating Lck-GCaMP2. Our findings thus far show that Lck-GCaMP2 allows the non-invasive imaging of calcium levels in astrocytes near the membrane and in processes in cell cultures. This level of resolution is possible because the Lck-GCaMP2 is selectively and highly expressed in the plasma membrane, providing micrometer scale spatial information. This approach will help reveal when, where and how astrocytes are activated during physiological and pathophysiological processes. In this proposal we seek to exploit our new approach and provide important new resources for the astrocyte signaling community by generating and characterizing novel transgenic mice that will allow calcium imaging in astrocyte processes in tissue slices and in vivo. We have two specific aims. In Aim 1 we will manufacture gene constructs that target Lck-GCaMP2 to astrocytes and use these constructs to establish founder lines of transgenic mice. In Aim 2 we will characterize and use these Lck- GCaMP2 transgenic mice to study mechanisms and functions of calcium signaling in astrocyte processes in hippocampal slices. We will test two hypotheses: (i) that astrocytes display spatially compartmentalized calcium signaling and (ii) that TGF2 has direct, receptor mediated effects on calcium signaling in the processes of reactive astrocytes, thereby providing a mechanism through which reactive astrogliosis could influence neurons. The work proposed here will provide novel, well characterized optical reporter mice that will allow us and others to measure precisely localized calcium signals in astrocyte somata and processes within intact tissue structures such as brain slices and in vivo. These new reporter mice will be valuable general tools for the astrocyte community by allowing researchers to measure local astrocyte calcium signals in processes that are currently inaccessible to conventional imaging methods. PUBLIC HEALTH RELEVANCE: We will develop mouse models that will allow us and other researchers to monitor and track calcium signaling in astrocytes in vitro and in vivo. The availability of these mice would constitute exceptional tools with which to study the role of astrocytes in the normal healthy brain and in diseases of the nervous system, including epilepsy and neurodegeneration as well as during brain injury and repair.