High Throughput Approaches for Cell-Specific Synapse Characterization

项目描述 突触在发育过程中、正常过程中动态地形成、断裂和重组 功能和响应活动。虽然这一一般原则是既定的， 这在复杂网络中的特定神经元亚型中表现出来， 突触输入的改变模式将决定网络功能，尚未定量 研究了在这里，我们建议开发分子遗传学工具来定义突触 组织和连接在小鼠大脑中使用荧光激活蛋白（FAP）， 一个强大的模块化系统，能够对突触进行多路荧光识别， 细胞特异性连接。我们的初步数据表明，我们可以将FAP靶向突触， 定量分析，以及导入3D荧光图像数据进行自动化突触 使用图像处理平台Imaris进行检测。在这里，我们将创建并验证pre-and 荧光蛋白和FAP蛋白的突触后靶向，实现跨突触 FRET信号具有高信号背景敏化发射，允许选择性检测 两个基因选择的细胞群之间形成的突触连接。这些 结构，以及相关的成像和分析方法，建立了一个管道， 用于分配小区类型特定联系人的吞吐量数据采集和分析。作为一个测试- 床这项技术，我们将采用它来确定突触输入地图的一个重要的 小鼠皮质中间神经元的一个亚群，生长抑素表达GABA能细胞 新皮层

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.