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

研究星形胶质细胞中多种钙信号的新光学和遗传工具

• 批准号: 8442163
• 负责人: Baljit Khakh
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8442163
• 关键词: Astrocytes; Brain; Brain Part; Brain region; Breeding; Calcium; Calcium Signaling; Cell Nucleus; Cells; Communities; Data; Dependovirus; Development; Distal; Dynamite; Fluorescence-Activated Cell Sorting; GLAST Protein; Gene Deletion; Gene Expression Profile; Genes; Genetic; Glial Fibrillary Acidic Protein; Goals; Heterogeneity; Image; Knock-in Mouse; Laboratories; Lead; Measurement; Measures; Membrane; Mental disorders; Methods; Molecular; Monitor; Morphology; Mus; Neurons; Optical reporter; Optics; Peripheral; Physiological; Population; Process; Property; Recording of previous events; Regulation; Reporter; Research Personnel; Role; Schizophrenia; Signal Transduction; Sleep; Sleeplessness; Slice; Specific qualifier value; Structure; Tamoxifen; Testing; Thalamic structure; Time; Transgenic Mice; Transgenic Organisms; base; calcium indicator; improved; in vivo; innovation; interest; nervous system disorder; neuronal cell body; novel; promoter; public health relevance; recombinase; research study; response; tool

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 Our recent unpublished findings show that Lck-GCaMP3 allows for non-invasive imaging with spectacular clarity.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.

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，以允许研究基因特定且易于处理的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）的精细特化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，这是了解大脑不同部位的不同星形胶质细胞功能需要克服的关键障碍。该应用程序基于我们实验室取得的进展，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 GCaMP2 的基因编码钙指示剂 (GECI)，以携带膜束缚结构域 (Lck)，从而生成 Lck-GCaMP2。然后我们将其改进了约 3 倍以生成 Lck-GCaMP3 并用腺相关病毒 (AAV) 在体内表达它。我们最近未发表的研究结果表明，Lck-GCaMP3 可以实现具有惊人清晰度的非侵入性成像。这是一个非常令人兴奋的创新突破，它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，我们制造了 Lck-GCaMP5G，它比 Lck-GCaMP3 好约 3 倍，比 Lck-GCaMP2 好约 10 倍。我们现在准备开发新颖的体内工具，以便任何人都可以使用 Lck-GCaMP5G，从而推广研究星形胶质细胞功能和多样性的精确方法。在目标 1 中，我们将生成在 Rosa26 基因座表达 Lck-GCaMP5G 的敲入小鼠。在目标 2 中，我们将生成在基因指定的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对丘脑皮质切片中的星形胶质细胞钙信号进行成像。