Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons

Recombinant rabies viral vectors have proven useful for applications including retrograde targeting of projection neurons and monosynaptic tracing, but their cytotoxicity has limited their use to short-term experiments. Here we introduce a new class of double-deletion-mutant rabies viral vectors that leave transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolishes cytotoxicity and reduces transgene expression to trace levels but leaves vectors still able to retrogradely infect projection neurons and express recombinases, allowing downstream expression of other transgene products such as fluorophores and calcium indicators. The morphology of retrogradely targeted cells appears unperturbed at one year postinjection. Whole-cell patch-clamp recordings show no physiological abnormalities at eight weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to four months, shows that transduced neurons do not die and retain stable visual response properties even at the longest time points imaged.