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Abstract

Techniques for directional tracing of neural circuits will propel our understanding of the organization and function of the brain. We adapted trans-Tango, a powerful genetic approach for anterograde circuit tracing in Drosophila, to a vertebrate nervous system, that of zebrafish. trans-Tango involves a synthetic signaling pathway introduced into all neurons. Specificity comes from genetically defined neurons presenting a tethered ligand at the synapse. Binding of ligand to a G protein-coupled receptor activates the pathway in post-synaptic neurons, resulting in recruitment of an arrestin-protease fusion protein, site-specific proteolysis of the receptor, and release of the QF transcription factor allowing its translocation to the nucleus. There QF binds to upstream regulatory sequences (QUAS) to drive expression of genes such as fluorescent reporters, which label and identify post-synaptic cells. The trans-Tango components were modified for zebrafish by placing receptor and arrestin fusion protein constructs downstream of the elavl3 promoter in Tol2 transposition vectors. Ligand variants differing in their pre-synaptic targeting sequences were also cloned into Tol2 plasmids under control of the Gal4 transcription factor. To test functionality, plasmids encoding ligand, receptor and arrestin, as well as Tol2 transposase RNA, were co-injected into 1-cell stage embryos, the progeny of a Gal4 driver that promotes strong expression of Tg(UAS:GFP) in the hindbrain mated with the Tg(QUAS:mApple-CAAX) reporter. Confocal imaging revealed GFP labeled axon terminals closely apposed to mApple labeled neurons, suggesting that trans-Tango is effective in transient expression assays. With such assays, we determined the optimal ligand for activating the receptor in post-synaptic neurons without off-target labeling. We are currently characterizing trans-synaptic labeling in stable transgenic lines with verified expression of trans-Tango components. We are also using optogenetics and calcium imaging to confirm that identified connections are functional synapses and developing a related configuration for retrograde circuit tracing. This work lays the foundation for mapping neural connectivity in vivo for both invertebrate and vertebrate nervous systems.