High Throughput Approaches for Cell-Specific Synapse Characterization

用于细胞特异性突触表征的高通量方法

• 批准号: 9380589
• 负责人: ALISON L BARTH
• 金额: $ 219.44万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9380589
• 关键词: Anatomy; Axon; Beds; Behavioral; Brain; Cells; Cerebral cortex; Chimeric Proteins; Cognitive; Complement; Complex; Data; Data Analyses; Data Set; Dendritic Spines; Detection; Development; Disease; Dyes; Electrophysiology (science); Energy Transfer; Esthesia; Fluorescence; Genetic; Goals; Human; Image; Image Analysis; Interneurons; Knowledge; Label; Lesion; Link; Maps; Mediating; Memory; Methodology; Methods; Modeling; Molecular Genetics; Mus; Neocortex; Neural Pathways; Neurons; Noise; Output; Pattern; Plasticizers; Population; Positioning Attribute; Property; Proteins; Rabies virus; Reagent; Science; Side; Signal Transduction; Site; Somatostatin; Stereotyping; Structure; Synapses; System; Technology; Testing; Thalamic Nuclei; Three-Dimensional Image; Validation; Viral; Vitronectin; aerobic respiration control protein; base; cell type; data acquisition; experience; fluorescence imaging; high throughput analysis; hippocampal pyramidal neuron; image processing; in vivo; in vivo imaging; information processing; neocortical; neuroligin 1; neuronal cell body; postsynaptic; presynaptic; public health relevance; reconstitution; relating to nervous system; response; time interval; tool

PROJECT DESCRIPTION Synapses are formed, broken and reformed dynamically both during development, normal function and in response to activity. Although this general principle is well-established, the way in which this is manifested in specific subtypes of neurons across a complex network, and how altered patterns of synaptic input will determine network function, have not been quantitatively investigated. Here we propose to develop molecular genetic tools for defining synaptic organization and connectivity in the mouse brain using fluorogen activating proteins (FAPs), a robust and modular system that enables multiplexed fluorescence identification of synapses and cell-specific connectivity. Our preliminary data indicate that we can target FAPs to synapses for quantitative analysis, as well as import 3D fluorescence image data for automated synapse detection using the image processing platform Imaris. Here we will create and validate pre- and postsynaptic targeting of fluorescent and FAP proteins respectively, acheiveing trans-synaptic FRET signal with high signal-to-background sensitized emission, allowing selective detection of synaptic connections formed between two genetically selected cell populations. These constructs, and the associated imaging and analysis approach, establish a pipeline for high- throughput data acquisition and analysis for assignment of cell-type specific contacts. As a test- bed for this technology, we will employ it to determine the synaptic input map for an important subset of cortical interneurons, somatostatin-expressing GABAergic cells, in the mouse neocortex.

项目描述 在开发过程中，突触形成，破碎和改革既是动态的，正常 功能和对活动的响应。尽管这一一般原则是完善的，但 这表现在复杂网络的神经元的特定亚型中，以及如何 突触输入的改变模式将确定网络功能，尚未定量 调查。在这里，我们建议开发用于定义突触的分子遗传工具 使用荧光激活蛋白（FAP）的组织和连通性，A 强大而模块化的系统，可实现突触的多重荧光鉴定和 细胞特异性连通性。我们的初步数据表明，我们可以针对FAP到突触 定量分析以及自动突触的进口3D荧光图像数据 使用图像处理平台Imaris检测。在这里，我们将创建并验证 荧光和FAP蛋白的突触后靶向靶向，可实现反式突触 具有高信噪比敏化发射的FRET信号，可以选择性检测 突触连接在两个基因选择的细胞群之间形成。这些 构造以及相关的成像和分析方法，建立了高级的管道 吞吐量数据采集和分析用于分配细胞类型的特定接触。作为测试 - 这项技术的床，我们将采用它来确定重要的突触输入图 皮质中间神经元的子集，表达生长抑素的GABA能细胞的子集，在小鼠中 新皮层。