G gene-deficient single-round rabies viruses for neuronal circuit analysis

•Rabies virus is a natural poly-transsynaptic tracer.•Single-round ΔG rabies can be pseudotyped for retrograde or anterograde labeling.•Single-round ΔG rabies virus can be complemented in situ to perform a single transsynaptic jump.•Mono transsynaptic tracers for identification and analysis of directly connected neurons.

Rhabdoviruses like the neurotropic rabies virus are fully amenable to pseudotyping with homologous and heterologous membrane proteins, which is being harnessed for the study of viral envelope proteins, viral retargeting, or immunization purposes. Particularly, pseudotyped delta G rabies viruses are emerging as safe and superb tools for mapping direct synaptic connections and analyzing neuronal circuits in the central and peripheral nervous system, which is a fundamental pillar of modern neuroscience. Such retrograde rabies mono-transsynaptic tracers in combination with optogenetics and modern in vivo imaging methods are opening entirely new avenues of investigation in neuroscience and help in answering major outstanding questions of connectivity and function of the nervous system. Here, we provide a brief overview on the biology and life cycle of rabies virus with emphasis on neuronal infection via axon ends, transport, and transsynaptic transmission of the virus. Pseudotyping of single-round, G-deleted virus with foreign glycoproteins allows to determine tropism and entry route, resulting in either retro- or anterograde labeling of neurons. Pseudotyping in vitro also allows specific targeting of cells that serve as starter cells for transsynaptic tracing, and pseudotyping in situ for a single (mono-transsynaptic) step of transmission to presynaptic neurons. We describe principle and experimental variations for defining “starter” cells for mono-transsynaptic tracing with ΔG rabies virus and outline open questions and limitations of the approach.