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发挥着良好的支持作用。此外，星形胶质细胞的新作用还包括向神经元传递信号和调节局部血流。星形胶质细胞的某些功能与胞质钙瞬变相关或受其调节，而胞质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，但目前不可能在体外、组织切片制剂或体内无创地测量星形胶质细胞突起中的钙信号。在切片和体内测量星形胶质细胞过程中的钙的方法将能够严格测试关于星形胶质细胞在健康中枢神经系统中脑功能中的作用的机制假设，并将开辟新的方法来研究反应性星形胶质细胞形成的影响，它发生在对所有形式的损伤和疾病的反应中，对这些相同的中枢神经系统功能。为了开发这种方法，我们修改了一种名为GCaMP2的基因编码钙传感器，使其在其N端携带膜系结结构域（称为Lck），从而产生Lck-GCaMP2。到目前为止，我们的研究结果表明，Lck-GCaMP2可以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无创成像。这种分辨率水平是可能的，因为Lck-GCaMP2在质膜中选择性地高度表达，提供微米尺度的空间信息。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。在这一建议中，我们寻求利用我们的新方法，并通过产生和表征新的转基因小鼠来为星形胶质细胞信号社区提供重要的新资源，这些小鼠将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，我们将制造针对星形胶质细胞的Lck-GCaMP2基因构建体，并使用这些构建体建立转基因小鼠的创始系。在Aim 2中，我们将描述并使用这些Lck- GCaMP2转基因小鼠来研究海马切片星形胶质细胞过程中钙信号传导的机制和功能。我们将验证两个假设：(i)星形胶质细胞显示空间区域化的钙信号传导；（ii） TGF2在反应性星形胶质细胞过程中对钙信号传导具有直接的受体介导作用，从而提供了一种反应性星形胶质形成影响神经元的机制。这里提出的工作将提供新颖的，具有良好特征的光学报告小鼠，这将使我们和其他人能够在完整的组织结构（如脑切片）和体内精确测量星形细胞体细胞和过程中的局部钙信号。这些新的报告小鼠将成为星形胶质细胞群落有价值的通用工具，使研究人员能够测量目前传统成像方法无法达到的星形胶质细胞局部钙信号。

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.