Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses

The highly specific and complex connectivity between neurons is the hallmark of nervous systems, but techniques for identifying, imaging, and manipulating synaptically-connected networks of neurons are limited. Monosynaptic tracing, or the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons1, is the most widely-used technique for mapping neural circuitry, but the rapid cytotoxicity of first-generation rabies viral vectors has restricted its use almost entirely to anatomical applications. We recently introduced double-deletion-mutant second-generation rabies viral vectors, showing that they have little or no detectable toxicity and are efficient means of retrogradely targeting neurons projecting to an injection site2, but they have not previously been shown to be capable of gated replication in vivo, the basis of monosynaptic tracing. Here we present a complete second-generation system for labeling direct inputs to genetically-targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires complementation of both of the deleted viral genes in trans in the starting postsynaptic cells; suppressing the expression of these viral genes following an initial period of viral replication, using the Tet-Off system, reduces toxicity to the starting cells without decreasing the efficiency of viral spread. Using longitudinal two-photon imaging of live monosynaptic tracing in visual cortex, we found that 94.4% of all labeled cells, and an estimated 92.3% of starting cells, survived for the full twelve-week course of imaging. Two-photon imaging of calcium responses in labeled networks of neurons in vivo over ten weeks showed that labeled neurons' visual response properties remained stable for as long as we followed them. This nontoxic labeling of inputs to genetically-targeted neurons in vivo is a long-held goal in neuroscience, with transformative applications including nonperturbative transcriptomic and epigenomic profiling, long-term functional imaging and behavioral studies, and optogenetic and chemogenetic manipulation of synaptically-connected neuronal networks over the lifetimes of experimental animals. ### Competing Interest Statement I.R.W. is a consultant for Monosynaptix, LLC, advising on design of neuroscientific experiments.