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.
We propose a novel multidisciplinary approach that combines classic neuroanatomy, with the most advanced techniques currently available in viral circuit tracing, and molecular and electrophysiological profiling to reveal different cell types in the mouse hippocampus and subiculum. Combined with CLARITY, Expansion Microscopy, and multiphoton imaging, our approach will reveal the anatomic location, the specific connectional inputs/outputs, the morphology, the molecular expression, and the electrophysiological characteristics of each cell type. Our in-house informatics pipelines and algorithms will be optimized to analyze and visualize all the data, which will be publicly shared through the design and development of novel web viewers (www.MouseConnectome.org).
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