Uncovering the Molecular Determinants of Cell Type-Specific Connectivity

The numerous functions of the brain are carried out by circuits that are very precisely wired. Within a brain region, there can be hundreds of distinct cell types, but a specific pathway may only innervate some cell types and not others. These synaptic connectivity patterns are largely established during development by the interactions of varied pre- and postsynaptic cell adhesion molecules (CAMs) expressed by each cell type. Large-scale transcriptomic atlasing methods have now described thousands of cell types, with hundreds of CAM genes expressed throughout the brain. Consequently, uncovering the CAMs essential for establishing cell type-specific connections onto neuronal populations has proved incredibly challenging. Tackling this research area requires two key methodological advancements. First, it necessitates the development of a high-throughput method for mapping connectivity across transcriptomically defined cell types. I recently developed such an approach by leveraging all-optical tools for scalable synaptic mapping (SynMap) across a neuronal population. With SynMap, I can measure postsynaptic responses of < 0.25 mV across hundreds of cells per experiment, and determine the identity of each cell using spatial transcriptomic methods. But understanding how these circuits are constructed now requires the development of a second technology for screening the functional roles of many molecules across an entire population of cells in intact tissue. To this end, in the K99 phase of this proposal I will demonstrate in vivo Perturb-FISH, a method for performing and characterizing stochastic CRISPR-based genetic perturbations across distinct cell types in the brain using spatial transcriptomics. In the independent R00 phase of this work, I will combine Perturb-FISH and SynMap to determine which CAMs are essential for establishing cell type-specific synaptic connections between the motor thalamus and the motor cortex– a pathway in which cell-type specific wiring is thought to be crucial for circuit function. The general connectivity principles that I uncover as part of this study will greatly advance our understanding of the molecular basis of cell type-specific wiring throughout the brain.