Combined Disruption of Multiple Cytokine Signaling Pathways Enables One-Step Anterograde Tracing with Vesicular Stomatitis Virus

Defining the direct postsynaptic targets of selected neuronal populations remains a major challenge for neural circuit mapping. Vesicular stomatitis virus (VSV) spreads efficiently in the anterograde direction, but replication-competent VSV undergoes multistep spread and therefore cannot distinguish direct from indirect downstream targets. Here, we developed a glycoprotein-deleted VSV (VSVdG)-based strategy for one-step anterograde tracing using AAV-mediated trans-complementation with several adaptations. In this system, VSVdG was engineered to encode Cre, allowing a Cre-dependent AAV to express VSV-G only after VSVdG infected the same cells, thereby limiting VSV-G expression to a short time window. To reduce VSV-M-mediated cytotoxicity, we introduced the M33A/M51R double-mutant VSV-Md variant. Using the basal ganglia circuit as a model, these adaptations enabled VSVdG spread from the striatum to expected downstream targets in mice of both sexes. Efficient VSVdG-based one-step spread required loss of type I interferon signaling in IFNAR1-knockout mice and additional suppression of cytokine-mediated antiviral responses that were independent of type I and type II interferon signaling. This was achieved either by AAV-mediated delivery of rabies virus phosphoprotein from the CVS-N2c strain or by a cytokine-blocking antibody cocktail. Although cells labeled by VSV transmission were confined to expected brain regions, the downstream labeled cells included both neurons and glia, revealing an important limitation for interpreting this approach as strictly neuron-to-neuron monosynaptic anterograde spread. Overall, this study provides a proof-of-concept VSVdG strategy for one-step anterograde circuit tracing and defines viral toxicity, innate immunity, and cell-type specificity constraints that must be addressed to develop a monosynaptic anterograde viral tracer.