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

Abstract

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.

Keywords: channelrhodopsin(H134R); equine infectious anemia virus; rabies glycoprotein; tTA.

Fig. 1.

Rabies glycoprotein pseudotyping of recombinant equine infectious anemia virus (rEIAV) improved synapsin promoter (iSynP) GFP does not allow robust expression but allows immunodetection of neurons projecting to the injection site. Three weeks after virus injection into dorsolateral geniculate nucleus (DLG), the expression of GFP is barely visible under native fluorescence at the site of injection (C) and visual cortex (A), which projects to DLG. The expression can be readily detected in vast numbers of neurons after immunoenhancement with a GFP antibody at the injection site (D) and V1 (B). Other neurons labeled that are known to project to DLG include superior colliculus neurons (E) and retinal ganglion cells (F). The schematic of the viral construct is depicted in the inset in A.

Fig. 2.

TLoop rEIAV vector is able to express robust channelrhodopsin-2-YFP (ChR2YFP) after retrograde uptake as visualized with native fluorescence. Injection site at the S1 barrel field (BF) is conveniently marked with the glial expression since the virus is taken up also by glia only locally (A). Cell bodies and dendrites of neurons projecting to the site of injection are labeled within the S1 barrel field (B), granular and agranular retrosplenial cortices (RSG, RSA) (C), posterior medial complex (PoM) and ventral posteromedial nucleus (VPM) (D), Meynert nucleus of the basal forebrain (MFB) (E), and secondary somatosensory cortex (S2) (F). The schematic of the viral construct is depicted in the inset in A.

Fig. 3.

Retrograde infection using TLoop rEIAV ChR2YFP viruses allows efficient optogenetic control. A recorded biocytin-filled neuron (A) is among many other neighboring ChR2YFP-expressing neurons (B) located in RSG (green filled circle in C) that are retrogradely infected from the injection site (red filled circle in C). Action potentials are reliably evoked by stimulating this recorded neuron with 2-ms pulses of blue light (D).

Fig. 4.

Neurons that are infected retrogradely by the TLoop rEIAV ChR2YFP virus can continue expressing the gene products as long as 6 mo. A layer V pyramidal neuron cell body is shown at high magnification with a nucleus that is indistinguishable from neighboring cells [YFP-DAPI (blue) overlay, A; cell is marked with an arrow]. Neuronal processes from many different areas from retrogradely infected neurons look normal in morphology (dendrites in C and axons in D). In rare instances, neurons that are infected by the TLoop rEIAV ChR2YFP virus that continue expressing the gene products for 6 mo show signs of fluorescent aggregations (B). Dendritic processes from neurons located in the ventral medial nucleus of the thalamus that were retrogradely infected from the barrel cortex injection and have been expressing the gene products for 6 mo show rare signs of protein Chr2YFP aggregation that may be due to uncontrolled expression (indicated with arrows).

Fig. 5.

Gene expression from the TLoop rEIAV construct in infected neurons can be tightly controlled by doxycycline administration in drinking water. Four littermates were injected with the same batch of rabies glycoprotein-pseudotyped TLoop rEIAV ChR2YFP virus in barrel cortices. For a duration of 2 wk, 2 of the animals were given doxycycline whereas the other 2 were not. Mostly faint glia expression is visible in doxycycline-treated littermates (A and B; red blank channel was used to see the anatomical landmarks), whereas strong ChR2YFP signal can be observed in the remaining untreated mice (C and D).

Fig. 6.

Early-onset and Cre-conditional expression with TLoop vectors. Native fluorescence can be detected as early as 48 h after injection in mouse barrel cortex with TLoop EIAVs (A). A DsRedX-expressing TLoop rEIAV virus was pseudotyped with vesicular stomatitis virus glycoprotein (VSVgp) and injected into the mouse barrel cortex. The expression can be detected after 2 days without any need of staining. Expression is mainly nuclear, indicating that the 2A skipping peptide has not been efficient enough and the majority of the DsRedX (arrows in A) was targeted to nucleus alongside with tTA, which contains a nuclear targeting signal (nuclei stained with DAPI in blue). Native ChR2YFP expression using Cre-dependent TLoop ChR2 vectors: VSVgp-pseudotyped Cre-dependent TLoop ChR2YFP vector was injected into layer V barrel cortex of a Nex-Cre mouse (B). Five days after injection, the brain was removed and sectioned. The native fluorescence from ChR2YFP is readily visible at the soma and processes of Cre-expressing neurons without any immunoenhancement. The schematics of the viral constructs are depicted in insets in A and B.