PROJECT SUMMARY Identifying how neurons are connected to each other in the brain is an important and necessary step towards understanding how brain activity gives rise to behavior, and how it is perturbed by disease. Currently available methods have limitations that make it challenging to visualize these brain wiring diagrams. In addition, there is an urgent need for a method that will make it possible not only to unveil brain connectivity, but also to genetically modify the functional properties of neurons connected in a circuit. We recently developed a genetic system named TRACT and showed using Drosophila that it possesses both of these features. However, many complex brain functions cannot be examined in Drosophila, and understanding them will require the use of vertebrate animals. The zebrafish has emerged as a useful vertebrate animal model to study complex brain processes due to its relatively simple yet conserved vertebrate brain, optical transparency, amenability to large-scale behavioral assays, the emergence of complex behaviors after only 5 days of development, and a growing suite of genetic tools that allow observation and manipulation of neuronal circuits in behaving animals. However, the usefulness of zebrafish is constrained by a lack of methods to identify and perturb synaptically connected neurons. In preliminary studies, we developed a TRACT system that can identify anterograde monosynaptic connections between neurons in the zebrafish brain. In the parent grant, we propose to further develop TRACT as a tool for transneuronal tracing in zebrafish by developing additional anatomical tracing modalities, and by establishing the use of TRACT to genetically manipulate synaptically connected neurons. This diversity supplement application describes an experimental and conceptual career development plan for a graduate student whose experimental goals are to (1) determine whether TRACT can function in all neurons in the brain, (2) determine whether transient expression or electroporation can be used to avoid the need to generate transgenic fish and thus expedite the use of TRACT for diverse neuronal populations, and (3) use flow cytometry and single cell RNA sequencing to identify synaptically connected neurons in a comprehensive and high-throughput manner. This experimental plan directly relates to the parent grant by further developing the TRACT system in zebrafish in experiments that are separate from, yet synergize with, the experiments described in the parent grant. Together, the parent grant and diversity supplement have the potential to establish a powerful new technology for mapping brain circuits that will increase the usefulness of zebrafish as a model system to study vertebrate neuronal circuit function, to reveal general principles of neuronal circuits that underlie specific behaviors, and to model complex brain disorders such as autism, Alzheimer’s disease and schizophrenia.