Project Summary/Abstract 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 diverse 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 diverse 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 diverse populations 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. To achieve these goals, I will utilize the many resources available to me at Boston University, the guidance of my mentor and co-mentor, Drs. Michael Economo and Brian Cleary, and the expert Advisory Committee that I have assembled to provide career and scientific support. Along with the resources and network provided by the BRAIN Initiative, this training plan will enable me to enhance diversity in the neuroscience community, and will position me for a successful career as a role model, a mentor, and an independent scientist.