Anatomical characterization of neuronal cell types of the mouse brain

项目总结和摘要 全面了解神经元细胞类型的多样性是选择性操作的重要指导， 阐明细胞类型对健康和疾病的特定功能贡献。因此，大脑 倡议细胞普查网络（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，并创建一个通用的3D图谱， 类型数据。为此，我们提议的合作实验室旨在从解剖学上表征大脑皮层的神经细胞类型。 小鼠边缘系统使用中尺度四重逆行追踪，我们将首先表征细胞类型 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）]。使用高级技术的两步依赖于cre的腺相关病毒追踪策略 病毒工具随后将验证和改进特定的轴突投射、侧枝和投射野 [e.g., ACB/X/Y←BLAa→ACB/X/Y]。在经免疫处理的组织中注射G-缺失狂犬病疫苗将标记 细胞类型的形态特征。依赖于Cre的TRIO病毒追踪将确定每个病毒的离散输入。 细胞类型，提供更深入的连通性表征。在cre中使用flp重组酶的新型TRIO- 依赖性小鼠将确定遗传上确定的细胞类型的投射模式。新构建的AAV和 狂犬病病毒标记到意大利面条怪物荧光蛋白，与扩展组合应用 显微镜和多光子成像将确定不同突触输入的空间组织。 细胞类型。总的来说，实验将揭示细胞类型解剖位置，形态，和全面的 连通性。最初的努力将集中在边缘系统，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， 分析二维和三维的细胞类型解剖数据。一个在线可视化工具，功能类似于我们的 iConnectome查看器将显示四重逆行和TRIO追踪图像。数字化，重建 四重逆行、cre-AAV和TRIO标签将放置在艾伦参考图谱（ARA）上， 创建一个在线2D连接图，允许轻松比较细胞类型特定的输入和输出。 Cre-AV标记实验的通用坐标框架（CCF）配准和重建将 提供投影的细胞类型特异性3D背景，其中输入和形态信息被集成到 观众。一个交互式的、加权的和有向的矩阵将呈现所有连通性的直观可视化 数据3D重建的神经元也将在Neuromorpho.org上进行物种间比较。我们 将扩展和优化现有的信息管道，以支持拟议的查看器功能。我们 我希望我们的技术能够阐明不同细胞类型的特异性网络，并为研究细胞间的相互作用提供基础。 BICCN的首要目标是创建一个全面的3D细胞类型图谱。

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.