Integrative approach to classifying neuronal cell types of the mouse hippocampus

项目摘要/摘要 识别神经系统中细胞类型的多样性将允许对它们进行选择性操作并揭示 他们在健康和疾病方面的功能贡献。然而，这不是一项微不足道的事业，而且受到以下因素的阻碍 在使用哪些属性进行分类方面缺乏共识。特征，比如解剖位置， 连接性、形态、分子轮廓和电生理特性被用作分类 系统，但单独来说，没有一个系统提供所有这些特征的综合视图。为了解决这个问题，我们提出了一项 多学科方法，将为小鼠海马体的每种细胞类型提供所有这些信息 和下丘脑(HPF/SUB)。我们最近确定了所有的HPF/亚分子结构域，并组装了它们的 使用来自鼠标连接项目(www.MouseConnectome.org)的跟踪数据的连接网络。 我们的多次逆行示踪剂注射显示，HPF/SUB含有多个混合种群 具有唯一投影目标的单元格，建议可以根据其 具有解剖学特异性的连接起点和终点。因此，在这里，我们建议使用四元组 逆行追踪法，根据这些联系对这些神经元进行初步分类。随后，一个两个- 依赖于步骤CRE的AAV跟踪方法将确定每种信元类型的所有输出。以确定它们的分子 鉴定，seqFISH，与预先选择的海马体标记基因，将在来自 四倍逆行追踪数据。重要的是，seqFISH保留了空间信息，因此精确的 示踪剂标记的细胞和基因的解剖位置将被保留。接下来，狂犬病疫苗注射放在 每种HPF/亚细胞类型的靶点将揭示其形态。清晰度和双光子显微镜将使 用于进一步分析的3D和神经元重建的形态评估。检视 电生理特性，每种细胞类型都将用逆行示踪剂标记以供识别 并对标记的细胞进行体外细胞膜片钳。最后，依赖cre的病毒追踪(TRIO) 将确定不同HPF/子小区类型的输入。借助膨胀显微镜和双光子 通过成像，顺行/狂犬病联合追踪策略将显示细胞类型的选定输入的准确位置。 如果成功，这个项目可以应用于描述整个大脑的神经细胞类型。所有数据都将 公开分享。来自四重逆行、两步Cre-AAV和三重追踪实验的图像将被 在iConnectome单元类型查看器中可用。这些实验中的标签图形重建将 通过细胞类型连接图在一个通用的神经解剖学框架内汇编和展示。这个 IConnectome细胞类型形态查看器将展示双重狂犬病实验和 提供3D重建及其形态和电生理特性等细节。单元格类型 连接将在交互式Web连接矩阵中可视化。我们的内部信息学管道和 将进一步开发和优化算法，以支持所有观众的拟议功能。

PROJECT SUMMARY/ABSTRACT Identifying the diversity of cell types in the nervous system will allow for their selective manipulation and reveal their functional contributions in health and disease. However, this is not a trivial undertaking and is hindered by the lack of consensus on which properties to use for classification. Characteristics like anatomical location, connectivity, morphology, molecular profile, and electrophysiological properties have been used as classification systems, but singly, none provide a combined view of all these characteristics. To address this, we propose a multidisciplinary approach that will provide all of this information for each cell type of the mouse hippocampus and subiculum (HPF/SUB). We recently identified all HPF/SUB molecular domains and assembled their connectivity networks using tracing data from the Mouse Connectome Project (www.MouseConnectome.org). Our multiple retrograde tracer injections revealed that the HPF/SUB contain multiple intermixed populations of cells with unique projection targets, suggesting different cell types that could be defined based on their connectional start and end points with anatomic specificity. Therefore, here, we propose to use a quadruple retrograde tracing method to initially classify these neurons based on these connections. Subsequently, a two- step cre-dependent AAV tracing method will determine all outputs of each cell type. To determine their molecular identities, seqFISH, with preselected hippocampal marker genes, will be performed on the tissue from the quadruple retrograde tracing data. Importantly, seqFISH preserves spatial information so that the precise anatomic locations of the tracer-labeled cells and the genes will be retained. Next, rabies injections placed in targets of each HPF/SUB cell type will reveal their morphology. CLARITY and two-photon microscopy will enable morphological assessment in 3D and neuronal reconstructions for further analysis. To examine electrophysiologcial properties, each cell type will be labeled with retrograde tracers for identification purposes and ex vivo cell patch clamping will be performed on the labeled cells. Finally, cre-dependent viral tracing (TRIO) will determine inputs to the different HPF/SUB cells types. With the aid of Expansion Microscopy and two-photon imaging, a combined anterograde/rabies tracing strategy will show precise locations of select inputs to cell types. If successful, this project can be applied to characterize neuronal cell types of the entire brain. All data will be publicly shared. Images from the quadruple retrograde, two-step cre-AAV, and TRIO tracing experiments will be available in the iConnectome Cell Type Viewer. Graphic reconstructions of labeling from these experiments will be compiled and presented within a common neuroanatomic frame through a Cell Type Connectivity Map. The iConnectome Cell Type Morphology Viewer will showcase labeling from the double rabies experiments and provide details like 3D reconstructions and their morphological and electrophysiological properties. Cell type connections will be visualized in an interactive Web Connectivity Matrix. Our in-house informatics pipelines and algorithms will be further developed and optimized to support the proposed features of all viewers.