Scalable Approaches for Bidirectional Brain-wide Trans-Neuronal Connectivity Mapping of Defined Cell Types

A major goal in modern neuroscience is to comprehensively map circuits of synaptically connected cell types throughout the mammalian brain. The key technological gap this proposal will address is the need for systematic, high-throughput methods to define neuronal wiring diagrams at the level of defined cell types. The overall objectives of this application are to establish a suite of tools that combine spatial transcriptomics and connectomics into scalable, high-throughput methods and analytical tools for linking the molecular identities of neural cell types to their synaptic connectivity. The rationale for the proposed work is that scalable, rapid means of unraveling circuit connectivity with cell type-specificity will accelerate efforts to unravel circuit structure and function throughout the brain. 

These goals will be pursued in three specific aims: 1) Establish and validate a spatial transcriptomic approach, TransA-MERFISH, for multiplexed, brain-wide mapping of the postsynaptic neurons of genetically defined starter cells; 2) Establish and validate a spatial transcriptomic approach, TransR- MERFISH, for multiplexed, brain-wide mapping of the presynaptic neurons of genetically defined starter cells; and 3) Establish computational platforms to decode the information gathered from TransA- and TransR- MERFISH. These collaborative experiments will draw on diverse expertise to merge connectomic methods developed by the applicants with spatial transcriptomic methods and analytical tools. These methods will be validated in the mouse brain, including multiple cortical and midbrain areas. 

The research proposed in this application is innovative because it establishes new tools to comprehensively map in situ synaptic inputs and outputs at the level of molecularly defined cell types. The proposed research will use commercial equipment to facilitate the easy adoption of these techniques by other labs. However, the approaches are also easily extensible to other spatial transcriptomics methods. The proposed research is significant because it is expected to yield a scalable, user-friendly, rapid means of unraveling circuit connectivity with cell type-specificity and defining circuit connectivity. Ultimately, the scalable tools developed here have the potential to accelerate investigations of neural circuit assembly and function in a variety of model organisms.