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，以研究遗传指定和可处理细胞群体的生理区室中的星形胶质细胞功能。星形胶质细胞通过被称为外周星形胶质突（PAPs）的精细特殊的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星形胶质细胞的不同功能而需要克服的关键障碍。这个应用是基于我们实验室取得的进步，使我们能够直接测量pap中的钙信号。为了开发这种方法，我们修改了一种叫做GCaMP2的基因编码钙指示剂（GECI），使其携带一个膜系缚结构域（Lck），从而产生了Lck-GCaMP2。然后我们将其改进3倍，生成Lck-GCaMP3，并在腺相关病毒（adeno associated virus， AAV）体内表达。我们最近未发表的研究结果表明，Lck-GCaMP3允许无创成像，具有惊人的清晰度。这是一个非常令人兴奋的创新突破，这将使研究人员第一次能够直接测量与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，我们制作了Lck-GCaMP5G，其性能比Lck-GCaMP3好~3倍，比Lck-GCaMP2好~10倍。我们现在准备开发新的体内工具，使Lck-GCaMP5G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Aim 1中，我们将产生在Rosa26位点表达Lck-GCaMP5G的敲入小鼠。在目标2中，我们将在基因指定的星形胶质细胞中产生表达Cre/ERT的新型BAC转基因小鼠。在Aim 3中，我们将利用我们的新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 Our recent unpublished findings show that Lck-GCaMP3 allows for non-invasive imaging with spectacular clarity.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.