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Understanding brain function requires the identification of neuronal connections that mediate cognition and behavior. Conventional labeling methods and reconstruction by serial electron microscopy uncover synaptic partners but do not permit manipulation of identified circuits. trans-Tango, first developed in Drosophila, is a genetic approach for anterograde labeling across the synapse that not only identifies post-synaptic neurons but also permits their transcriptional regulation. A ligand modified from human glucagon is expressed under Gal4 control and tethered to the pre-synaptic membrane of defined neurons where it activates a signaling pathway in the corresponding post-synaptic neurons. Binding of the ligand to the human glucagon receptor coupled to the QF transcription factor through a protease cleavage site recruits an arrestin-protease fusion protein, that cleaves QF. This allows QF to translocate to the nucleus and promote the expression of any gene under the control of the upstream activating sequence (QUAS) where it binds. We adapted trans-Tango for use in the zebrafish nervous system and validated its effectiveness both in transient assays using Tol2 trans-Tango plasmids and in stable transgenic lines. We injected the trans-Tango ligand, receptor, and arrestin constructs along with Tol2 mRNA into embryos from fish-bearing different neuron-specific Gal4 driver lines and UAS:GFP mated to fish containing Tg(QUAS:mApple-CAAX). GFP labeled pre-synaptic neurons were detected in close proximity to mApple labeled putative post-synaptic neurons in Gal4-dependent patterns. Connectivity was validated by examining known synaptic partners in the retina as well as tectal neurons with known morphology. To confirm that gene expression is regulated through signaling across the synapse, we used optogenetics to selectively activate retinal ganglion cells and Tg(QUAS:GCaMP6f) to detect calcium transients in post-synaptic neurons in the optic tectum. These exciting results lay the foundation for genetic approaches to reveal synaptic partners and to access an arsenal of tools to monitor or modulate neural circuits.
