Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

Abstract

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation ({Delta}G) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant ({Delta}GL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that {Delta}GL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here we introduce third-generation ({Delta}L) rabies viral vectors, which, like first-generation vectors, have only a single gene deleted from their genomes (in this case the viral polymerase gene L) but which appear to be as nontoxic as second-generation ones, based on in vivo longitudinal structural and functional two-photon imaging and ex vivo electrophysiology. Although third-generation vectors are therefore phenotypically very similar to second-generation ones, we show that they have the major advantage of growing to much higher titers, and this key difference results in significantly increased numbers of retrogradely labeled neurons in vivo. These {Delta}L rabies viral vectors therefore constitute a new state of the art for minimally perturbative, pathway-specific expression of recombinases and transactivators in mammalian neurons selected on the basis of their axonal projections. Because replication of deletion-mutant rabies viruses within complementing cells is precisely the process that underlies monosynaptic tracing, the higher replication efficiency of this new class of rabies viral vectors furthermore suggests the potential to provide the foundation of an improved nontoxic monosynaptic tracing system.