New Optical and Genetic Tools to Study Diverse Calcium Signals in Astrocytes

描述（由申请人提供）：本申请的重点是开发急需且易于使用的光学和遗传工具，以允许研究遗传特异性和易处理细胞群的生理区室中的星形胶质细胞功能。星形胶质细胞通过称为外周星形胶质细胞突起（PAP）的精细专门远端延伸与神经元相互作用。然而，进展的瓶颈是缺乏监测PAP中钙信号的方法，这是为了了解大脑不同部位的星形胶质细胞功能而必须克服的关键障碍。该应用基于我们实验室的进步，使我们能够直接测量PAP中的钙信号。为了开发这种方法，我们修改了一种称为GCaMP 2的遗传编码钙指示剂（GECI），使其携带一个膜束缚结构域（Lck），从而产生Lck-GCaMP 2。然后，我们将其改进~3倍以产生Lck-GCaMP 3，并用腺相关病毒（AAV）在体内表达。我们最近未发表的研究结果表明，Lck-GCaMP 3允许以惊人的清晰度进行非侵入性成像。这是一项非常令人兴奋的创新突破，将首次使研究人员能够直接测量生理相关的星形胶质细胞信号和功能。此外，通过最近基于结构的改进，我们制备了Lck-GCaMP 5G，其比Lck-GCaMP 3好约3倍，比Lck-GCaMP 2好约10倍。我们现在准备开发新的体内工具，使Lck-GCaMP 5G可以被任何人使用，从而推广一种精确的方法来研究星形胶质细胞的功能和多样性。在目标1中，我们将产生在Rosa 26基因座表达Lck-GCaMP 5G的敲入小鼠。在目标2中，我们将产生在遗传特异性星形胶质细胞中表达Cre/ERT的新型BAC转基因小鼠。在目标3中，我们将利用我们的新型小鼠在丘脑皮质切片中成像星形胶质细胞钙信号。

DESCRIPTION (provided by applicant): The focus of this application is to develop much needed and easy to use optical and genetic tools to permit the study of astrocyte function in physiological compartments for genetically specified and tractable cell populations. Astrocytes interact with neurons via fine specialised distal extensions called peripheral astrocyte processes (PAPs). However, a bottleneck to progress has been lack of methods to monitor calcium signals in PAPs, which is a crucial hurdle to overcome in order to understand diverse astrocyte functions in different parts of the brain. This application is based on advances made in our laboratory that allow us to directly measure calcium signals in PAPs. To develop such a method we modified a genetically encoded calcium indicator (GECI) called GCaMP2 to carry a membrane tethering domain (Lck), thus generating Lck-GCaMP2. Then we improved this ~3-fold to generate Lck-GCaMP3 and expressed this in vivo with adeno associated viruses (AAV). Our recent unpublished findings show that Lck-GCaMP3 allows for non-invasive imaging with spectacular clarity. This is a very exciting innovative breakthrough that for the first time will alow researchers to directly measure physiologically relevant astrocyte signals and functions. Moreover, with recent structure-based refinements we made Lck-GCaMP5G, which is ~3-fold better than Lck-GCaMP3 and ~10-fold better than Lck-GCaMP2. We are now ready to develop novel in vivo tools so that Lck-GCaMP5G can be used by anyone and thus generalise a precise way to study astrocyte function and diversity. In Aim 1 we will generate knock-in mice expressing Lck-GCaMP5G at the Rosa26 locus. In Aim 2 we will generate novel BAC transgenic mice expressing Cre/ERT in genetically specified astrocytes. In Aim 3 we will exploit our novel mice to image astrocyte calcium signals in thalamocortical slices.