High Throughput Approaches for Cell-Specific Synapse Characterization

项目说明 突触在发育过程中动态形成、断裂和重塑，正常 功能和对活动的响应。尽管这一总原则已经确立，但在 这体现在复杂网络中特定的神经元亚型中，以及如何 突触输入模式的改变将决定网络功能，但尚未量化 调查过了。在这里，我们建议开发分子遗传学工具来定义突触 利用荧光素激活蛋白(FAPs)在小鼠大脑中的组织和连接 强大的模块化系统，能够对突触和 特定于细胞的连接。我们的初步数据表明，我们可以将FAP定位到突触 定量分析，以及为自动突触导入3D荧光图像数据 使用图像处理平台Imaris进行检测。在这里，我们将创建和验证Pre-and 分别以荧光蛋白和FAP蛋白为突触后靶点，促进跨突触 具有高信号背景敏化发射的FRET信号，允许选择性地检测 在两个遗传选择的细胞群体之间形成突触连接。这些 构造，以及相关的成像和分析方法，建立了一条管道，用于 用于单元类型特定触点分配的吞吐量数据采集和分析。作为一项测试- 对于这项技术，我们将利用它来确定突触的一个重要的输入映射 小鼠皮质中间神经元亚群--表达生长抑素的GABA能细胞 大脑皮层。

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.