Optical control of retrogradely infected neurons using drug-regulated "TLoop" lentiviral vectors

Many approaches that use viral vectors to deliver transgenes have limited transduction efficiency yet require high levels of transgene expression. In particular, infection via axon terminals is relatively inefficient but is a powerful means of achieving infection of specific neuron types. Combining this with optogenetic approaches requires high gene expression levels that are not typically achieved with nontoxic retrogradely infecting vectors. We generated rabies glycoprotein-pseudotyped lentiviral vectors that use a positive feedback loop composed of a Tet promoter driving both its own tetracycline-dependent transcription activator (tTA) ("TLoop") and channelrhodopsin-2-YFP (ChR2YFP). We show that TLoop vectors strongly express proteins in a drug-controllable manner in neurons that project to injection sites within the mouse brain. After initial infection, the virus travels retrogradely, stably integrates into the host genome, and expresses gene products. The expression is robust and allows optogenetic studies of neurons projecting to the location of virus injection, as demonstrated by fluorescence-targeted intracellular recordings. ChR2YFP expression did not cause observable signs of toxicity and continued for up to 6 mo after infection. Expression can be reversibly blocked by administration of doxycycline, if necessary, for expression of gene products that might be more toxic. Overall, we present a system that will allow researchers to achieve high levels of gene expression even in the face of inefficient viral transduction. The particular vectors that we demonstrate may enhance efforts to gain a precise understanding of the contributions of specific types of projection neurons to brain function.