PROJECT SUMMARY Tools for exclusively targeting neuronal and glial subtypes are needed to advance our understanding of the brain. “Intersectional” systems improve targeting by restricting “reporter/effector” transgenes to a subdomain defined by the expression overlap between two activating factors. “Split-driver” systems have enhanced targeting precision in flies and are operable in fish, but have yet to be systematically deployed in vertebrate systems. Our goal is to improve transgene expression precision in the vertebrate nervous system by creating a series of intersectional vectors designed to enable exclusive targeting of discrete neuronal and glial cell subtypes. Binary systems, such as Gal4/UAS, separate transgene expression into “drivers” and “reporter/effectors”. The combinatorial nature ensures versatility; however, most driver lines fail to target specific cell types. In turn, this can compromise the integrity of reporter/effector-based manipulations. To enhance expression specificity, drivers have been split into two “hemidriver” components: a DNA-binding domain (DBD) and transactivation domain (AD). Hemidrivers can only be assembled where DBD and AD expression overlaps, thus restricting driver-dependent reporter/effectors to the “intersect”. DBD-AD and reporter/effector activity can be further refined by expressing repressor proteins in non-targeted domains. Moreover, efficient knock-in methods and single-cell transcriptomics now afford an unprecedented level of transgene expression fidelity and targeting precision. We propose to leverage these advances to create a series of DBD hemidriver, AD hemidriver, and repressor toolsets for targeting discrete neuronal and glial subtypes. To further enhance targeting precision, repressor resources will developed to inhibit effector activity in non-targeted cells. Given its utility in dissecting cell function, we propose to identify genetic repressors of the nitroreductase (NTR) system of inducible targeted cell ablation. While the utility of these resources will be validated in the zebrafish, universal vectors will be created to facilitate adaptation to any other vertebrate model amenable to transgenesis. Three aims are proposed: Aim 1: Create and validate tools for labeling and functionally dissecting discrete neuronal cell subtypes. Aim 2: Create and validate tools for labeling and functionally dissecting discrete glial cell subtypes. Aim 3: Create and validate tools for repressing effector activity non-targeted brain regions/cells. Funding will allow us to create toolsets facilitating transgenic targeting at unparalleled levels of cellular and circuit precision in the brain. We anticipate creating ~25 DBD, ~50 AD, and ~25 repressor vectors/lines, allowing targeting of thousands of unique neuronal and glial subtypes. In combination with existing reporter/effectors expressing optogenetic, cell ablation, transsynaptic and other tools for monitoring and manipulating cells and circuits, the propo...