Abstract Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed in Drosophila melanogaster , there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here we describe retro -Tango, a method for transsynaptic, retrograde circuit tracing and manipulation in Drosophila . In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validate retro -Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of the Drosophila hemibrain. Our experiments establish retro -Tango as a key method for circuit tracing in neuroscience research.

retro-Tango enables versatile retrograde circuit tracing inDrosophila