PROJECT SUMMARY/ABSTRACT It is estimated that the human brain contains an overwhelming 1015 synapses, structures essential for the normal functioning of neural circuits. Our knowledge of the connections that form these critical signaling sites, in even the simplest vertebrate nervous systems, is sorely lacking. Thus, a stated goal of this BRAIN initiative is to “develop and validate novel tools to facilitate the detailed analysis of complex circuits and provide insights into cellular interactions that underlie brain function”. This multi-PI collaborative project precisely addresses this goal. It takes advantage of a powerful genetic technique, trans-Tango, that directs signaling across synapses to identify both pre-synaptic neurons and their specific post-synaptic targets. The overall objectives of the proposed experiments are three-fold: First, we will adapt the trans-Tango anterograde trans-synaptic signaling platform, which was initially established and successfully implemented in the Drosophila model, to a vertebrate brain - that of the zebrafish. The zebrafish is the organism of choice because of the ability to assay trans-Tango components efficiently from injections of plasmid constructs into 1-cell embryos, and the ease and rapidity of generating transgenic animals to activate trans-Tango in defined neuronal populations. Second, we will independently and rigorously validate the neural connections revealed by trans-Tango as functional synaptic connections, capitalizing on optogenetics, imaging techniques, and advanced microscopy methods. Owing to its transparency, the larval zebrafish is ideally suited to verify synaptic connectivity in vivo using optical approaches. Third, we will develop a new retrograde version of trans-Tango, which will allow identification of the pre-synaptic input of given post-synaptic neurons. The modularity of trans-Tango permits efficient reconfiguration and optimization of the system for accurate circuit map ping. The “retro-Tango” version will first be applied to Drosophila, building upon lessons learned from the establishment of trans-Tango and, once optimal, introduced to the zebrafish nervous system. By assembling the proposed genetic toolkit for anterograde and retrograde trans-synaptic tracing in both invertebrate and vertebrate nervous systems, we expect these techniques to become widely used by the neuroscience community and applied to additional experimental models. The strengths of this proposal are the innovative strategies used to map neural connectivity, the compelling preliminary data, and the unique and complementary expertise in molecular genetics, circuit neuroscience and microscopy design that the collaborating researchers bring to the project.