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 left transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolished cytotoxicity and reduced transgene expression to trace levels but left 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 appeared unperturbed at 1 year postinjection. Whole-cell patch-clamp recordings showed no physiological abnormalities at 8 weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to 4 months, showed that transduced neurons did not die but retained stable visual response properties even at the longest time points imaged.
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S.C. thanks D. Williams and S. de Vries for software, A. Cheng and N. Orlova for hardware construction, and R. D’Aleo and N. Ouellette for experimental assistance. J.A.H. thanks K.E. Hirokawa and P. Bohn for technical assistance. I.R.W. thanks W. Salmon for assistance with confocal imaging. The authors thank the founders of the Allen Institute for Brain Science, Paul G. Allen and Jody Allen, for their vision, encouragement and support. Research reported in this publication was supported primarily by the National Institute of Mental Health under award number U01MH106018 (BRAIN Initiative) to I.R.W. and in part by the National Institute on Aging under award number R01AG047589 to J.A.H. and the National Eye Institute under award numbers R01EY010115 and R01EY018742 to R.C.R. K.M.T. was supported in part by a New York Stem Cell Foundation Robertson Investigator award and McKnight Scholar Award, and G.G.C. was supported by a JFDP fellowship and a NARSAD Young Investigator Award. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.