Long-term labeling and imaging of synaptically-connected neuronal networksin vivousing double-deletion-mutant rabies viruses

Abstract

SummaryThe highly specific and complex connectivity between neurons is the hallmark of nervous systems, but techniques for identifying, imaging, and manipulating synaptically-connected networks of neurons are limited. Monosynaptic tracing, or the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons1, is the most widely-used technique for mapping neural circuitry, but the rapid cytotoxicity of first-generation rabies viral vectors has restricted its use almost entirely to anatomical applications. We recently introduced double-deletion-mutant second-generation rabies viral vectors, showing that they have little or no detectable toxicity and are efficient means of retrogradely targeting neurons projecting to an injection site2, but they have not previously been shown to be capable of gated replication in vivo, the basis of monosynaptic tracing. Here we present a complete second-generation system for labeling direct inputs to genetically-targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires complementation of both of the deleted viral genes in trans in the starting postsynaptic cells; suppressing the expression of these viral genes following an initial period of viral replication, using the Tet-Off system, reduces toxicity to the starting cells without decreasing the efficiency of viral spread. Using longitudinal two- photon imaging of live monosynaptic tracing in visual cortex, we found that 94.4% of all labeled cells, and an estimated 92.3% of starting cells, survived for the full twelve-week course of imaging. Two-photon imaging of calcium responses in labeled networks of neurons in vivo over ten weeks showed that labeled neurons’ visual response properties remained stable for as long as we followed them. This nontoxic labeling of inputs to genetically-targeted neurons in vivo is a long-held goal in neuroscience, with transformative applications including nonperturbative transcriptomic and epigenomic profiling, long-term functional imaging and behavioral studies, and optogenetic and chemogenetic manipulation of synaptically-connected neuronal networks over the lifetimes of experimental animals.

Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency

“…Beginning one week later, we began imaged the calcium signals in the labeled neurons in a series of imaging sessions that continued until 16 weeks postinjection, in the awake mice viewed visual stimuli consisting of drifting gratings of different orientations and frequencies. Just as we found previously for ∆GL viruses (Chatterjee et al, 2018;Jin et al, 2021b), we found no signs of dysfunction in cells labeled by the thirdgeneration vector for as long as we followed them (Figure 4; see also Figures S4 and S5. See File S4 for all counts and statistical comparisons and Video S2 for an example of calcium responses in a group of cortical neurons 16 weeks after injection of RV∆L-Cre).…”

“…Beginning one week later, we began imaged the calcium signals in the labeled neurons in a series of imaging sessions that continued until 16 weeks postinjection, in the awake mice viewed visual stimuli consisting of drifting gratings of different orientations and frequencies. Just as we found previously for ∆GL viruses 4,5 , cells labeled by the third-generation vector showed no signs of dysfunction for as long as we followed them (Fig. 4; see also Extended Data Figs.…”

“…The BHK-B19L cell line, expressing the SAD B19 strain rabies virus polymerase gene, was made by transfecting BHK-21 cells (ATCC CCL-10) with pCAG-hypBase (Jin et al, 2021a) and pB-CAG-B19L-IRES-mCherry-WPRE-BGHpA (Jin et al, 2021b) using Lipofectamine 2000 (Thermo Fisher 11668019), then expanding the cells and sorting on a FACS Aria sorter (BD) to collect the brightest 5%, as well as the next brightest 5%, of mCherry-expressing cells. The two collected populations were expanded and refrozen, then thawed and tested for their efficacy at supporting replication of ∆L virus; the second-brightest population ("BHK-B19L_2") was found to result in higher titers and is referred to here as BHK-B19L.…”

“…Since their introduction to neuroscience in 2007 1,2 , recombinant rabies viral vectors have become widely-adopted tools in neuroscience, allowing "monosynaptic tracing" of direct inputs to genetically-targeted starting postsynaptic neuronal populations [2][3][4] as well as simple retrograde targeting of projection neurons when injected at the sites of these projection neurons' axonal arborizations 1,5 . These vectors are now used in a large number of laboratories worldwide and have contributed to many high-impact studies of a wide variety of neural systems [6][7][8][9][10][11][12][13][14] .…”

“…Because first generation ("∆G") rabies viral vectors (which have only the glycoprotein gene G deleted from their genomes) are cytotoxic (Chatterjee et al, 2018;Jin et al, 2021a;Jin et al, 2021b;Wickersham et al, 2007a), we recently introduced second-generation, "∆GL" rabies viral vectors, which have both the glycoprotein gene G and the viral polymerase gene L (for "large" protein) deleted from their genomes (Chatterjee et al, 2018). Because the viral polymerase is absolutely required for transcription of all genesviruses, we washed the cells twice with DPBS and applied fresh medium, then collected supernatant samples every 24 hours for five days after infection, then titered the samples on reporter cells.…”