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: 8605557
• 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/8605557
• 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; non-invasive imaging; 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 Our recent unpublished findings show that Lck-GCaMP3 allows for non-invasive imaging with spectacular clarity.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(申请人提供)：本申请的重点是开发迫切需要的、易于使用的光学和遗传工具，以允许在遗传指定和易处理的细胞群体的生理隔间中研究星形胶质细胞的功能。星形胶质细胞通过称为外周星形胶质细胞突起(PAPs)的精细专门化远端延伸与神经元相互作用。然而，进展的一个瓶颈是缺乏监测PAP中钙信号的方法，这是了解大脑不同部分星形胶质细胞不同功能所需克服的关键障碍。这一应用是基于我们实验室取得的进展，使我们能够直接测量PAPs中的钙信号。为了开发这样一种方法，我们修改了一种名为GCaMP2的基因编码的钙指示剂(GECI)，使其携带膜系留结构域(Lck)，从而产生了Lck-GCaMP2。然后我们将其改进了3倍，产生了LCK-GCaMP3，并用腺相关病毒(AAV)在体内表达了它。我们最近未发表的发现表明，LCK-GCaMP3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，将首次允许研究人员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，我们制备了LCK-GCaMP5G，其性能是LCK-GCaMP3的~3倍，是LCK-GCaMP2的~10倍。我们现在准备开发新的体内工具，以便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。在目标1中，我们将产生在rosa26基因座表达Lck-GCaMP5G的敲入小鼠。在目标2中，我们将建立新的BAC转基因小鼠，在基因指定的星形胶质细胞中表达CRE/ERT。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。