Abstract
Defining the direct postsynaptic targets of selected neuronal populations remains a 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 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, we found that efficient one-step VSVdG spread required disruption of cytokine-mediated antiviral responses triggered by AAV transduction and VSV infection. This was achieved by loss of type I interferon signaling in IFNAR1 knock-out mice, together with suppression of additional antiviral responses either through AAV-mediated expression of CVS-N2c rabies virus phosphoprotein or administration of cytokine-blocking antibody cocktail. These adaptations enabled VSVdG spread from the dorsal striatum to expected downstream targets in mice of both sexes. However, 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, rather than providing a ready-to-use tracing tool, this study presents a 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 an efficient and specific monosynaptic anterograde viral tracer.