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.

Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent "helper" adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (∆G) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.

Monosynaptic tracing using deletion-mutant rabies virus allows whole-brain mapping of neurons that are directly presynaptic to a targeted population of neurons. The most common and robust way of implementing it is to use Cre mouse lines in combination with Cre-dependent adeno-associated viral vectors for expression of the required genes in the targeted neurons before subsequent injection of the rabies virus. Here we present a step-by-step protocol for performing such experiments using first-generation (∆G) rabies viral vectors. INTRODUCTION"Monosynaptic tracing" refers to the use of a deletion-mutant neurotropic virus that is complemented in vivo selectively within a targeted neuronal population of interest, so that the virus can replicate within them and spread to other neurons that are in direct synaptic contact 1 . In the original monosynaptic tracing system, which remains the primary version in use in neuroscience, the virus in question is a rabies virus from which the "G" gene, encoding its envelope glycoprotein, has been deleted 1,2 . This deletion makes the virus unable to spread beyond infected cells, unless they are engineered to express G in trans. As a second modification to the virus, it is coated with "EnvA", the envelope protein of an avian retrovirus. This makes the rabies virus unable to infect mammalian cells unless they have been engineered to express EnvA's receptor, "TVA" 1,3 , a protein which is not native to mammals.Monosynaptic tracing therefore requires engineered expression of two genes in the targeted population of starting neurons: the gene encoding the receptor TVA and the G gene encoding the rabies virus glycoprotein. The rabies virus can then infect the starting cells via the receptor TVA and, complemented by expression of G in trans, spread beyond them. Because rabies virus naturally spreads in a retrograde direction in the central nervous system, the virus spreads to cells presynaptic to the starting cells. If the engineered G expression has been carefully restricted to the targeted postsynaptic cells and is not expressed in cells of other types that are presynaptic to them, the deletion-mutant virus cannot spread beyond them. The result is labeling of neurons that are monosynaptically connected to the targeted starting cells.The usual implementation of all this is to use a Cre mouse line to provide genetic access to the targeted group of postsynaptic neurons in conjunction with one or two Cre-dependent "helper" adeno-associated viral vectors (AAVs) that deliver the TVA and glycoprotein genes. The AAVs are injected in one surgery, then the rabies virus is injected some time later once the AAVs have had a chance to express the required gene. Some time after injection of the rabies virus, the mouse is perfused, imaged, etc. and the results are obtained.Within this broad description, many variations are possible, and the devil is in the details. Many different helper AAVs have been used by different laboratories, and the degree of success varies widely. Here we present reagents and a ...