Anatomical characterization of neuronal cell types of the mouse brain

项目摘要和摘要 全面了解神经细胞类型的多样性是选择性操作和 阐明细胞类型的特定功能对健康和疾病的贡献。因此，大脑 首创细胞普查网络(BICCN)正在统一具有独特解剖学专业知识的实验室的努力， 遗传学、电生理学和功能对神经元进行分类，并创建具有集成细胞的通用3D图谱 键入数据。为此，我们提议的合作旨在从解剖学的角度描述大鼠脑内神经细胞的类型。 小鼠边缘系统。使用中尺度四重逆行追踪，我们将初步确定细胞类型 基于它们的连接起点和终点的解剖位置[例如， Acb(对侧)←blaa→Acb(同侧)]。一种两步依赖cre的AAV跟踪策略 病毒工具随后将验证和改进特定的轴突投射、侧支和投射区域 [例如，acb/X/Y←blaa→acb/X/Y]。在透明处理的组织中注射G缺失狂犬病将标记 细胞类型的形态特征。依赖Cre的三重病毒跟踪将确定每个病毒的离散输入 单元格类型，提供更深层次的连接性特征。利用Flp重组酶在cre-Re中构建新的三联体 依赖的小鼠将定义基因定义的细胞类型的投影模式。新建成的AAV和 标记在意大利面条怪物荧光蛋白上的狂犬病病毒，与扩增结合应用 显微镜和多光子成像将确定不同突触输入的空间组织 单元类型。总的来说，实验将揭示细胞类型的解剖位置、形态和综合 连通性。最初的努力将集中在边缘系统上，设计可扩展到神经元 整个大脑的特征。将开发一个基于网络的可视化平台，以便能够查看和 细胞类型解剖数据的二维和三维分析。一个在线可视化工具，功能类似于我们的 IConnectome查看器将呈现四重逆行和三重跟踪图像。数字化，重建 将在艾伦参考地图集(ARA)上放置四重逆行、cre-AAV和三重标记，以 创建在线2D连接地图，可轻松比较特定单元类型的输入和输出。 共用坐标框架(CCF)注册和重建cre-AAV标记实验将 提供投影的单元格类型特定3D上下文，并将输入和形态信息集成到 观众。交互式、加权和定向矩阵将呈现所有连接的直观可视化 数据。3D重建的神经元也将被托管在NeuromorPho.org上，用于物种间的比较。我们的 目前的信息学管道将得到扩展和优化，以支持拟议的查看器功能。我们 希望我们的技术能够阐明不同的细胞类型特定网络，并为 BICCN的首要目标是创建一份全面的3D单元类型地图集。

PROJECT SUMMARY AND ABSTRACT A comprehensive understanding neuronal cell type diversity is an essential guide to selective manipulation and illuminating cell type specific functional contributions toward health and disease. Accordingly, the Brain Initiative Cell Census Network (BICCN) is unifying the efforts of laboratories with unique expertise in anatomy, genetics, electrophysiology, and function to classify neurons and create a common 3D atlas with integrated cell type data. To this end, our proposed collaboratory aims to anatomically characterize neuronal cell types of the mouse limbic system. Using mesoscale quadruple retrograde tracing, we will initially characterize cell types based on the anatomical location of their connectional start and end points [e.g., ACB(contralateral)←BLAa→ACB(ipsilateral)]. A two-step cre-dependent AAV tracing strategy using advanced viral tools will subsequently validate and refine specific axonal projections, collaterals, and projection fields [e.g., ACB/X/Y←BLAa→ACB/X/Y]. Injections of G-deleted rabies in CLARITY-processed tissue will label morphological features of cell types. Cre-dependent TRIO viral tracing will determine discrete inputs to each cell type, providing deeper characterization of connectivity. Novel TRIO using flp recombinase in cre- dependent mice will define projection patterns of genetically-defined cell types. Newly constructed AAV and rabies viruses tagged to spaghetti monster fluorescent proteins, applied in combination with Expansion Microscopy and multiphoton imaging, will determine the spatial organization of different synaptic inputs to the cell types. Collectively, experiments will reveal cell type anatomic location, morphology, and comprehensive connectivity. Initial efforts will focus on the limbic system, with the design extensible to neuronal characterization of the entire brain. A web-based visualization platform will be developed to enable viewing and analysis of cell type anatomy data in 2D and 3D. An online visualization tool similar in function to our iConnectome viewer will present quadruple retrograde and TRIO tracing images. Digitized, reconstructed quadruple retrograde, cre-AAV, and TRIO labeling will be placed atop the Allen Reference Atlas (ARA) to create an online 2D connectivity map, allowing easy comparison of cell type specific inputs and outputs. Common Coordinate Framework (CCF) registration and reconstruction of cre-AAV labeling experiments will provide the cell type specific 3D context of projections, with input and morphological information integrated into the viewer. An interactive, weighted and directed matrix will present an intuitive visualization of all connectivity data. 3D reconstructed neurons will also be hosted on Neuromorpho.org for interspecies comparison. Our current informatics pipelines will be extended and optimized to support the proposed viewer features. We expect our technologies to elucidate diverse cell type specific networks and provide foundations for the overarching goal of the BICCN of creating a comprehensive 3D cell type atlas.