Axonal and subcellular labelling using modified rabies viral vectors

Abstract

An important aspect of any neural circuit is the placement of its output synapses, at levels ranging from macroscopic to subcellular. The many new molecular tools for locating and manipulating synapses are limited by the viral vectors available for delivering them. Adeno-associated viruses are the best current means of labelling and manipulating axons and synapses, but they have never expressed more than one transgene highly enough to label fine axonal structure while also labelling or perturbing synapses. Their slow expression also makes them incompatible with retrograde and transsynaptic vectors, preventing powerful combinatorial experiments. Here we show that deletion-mutant rabies virus can be specifically targeted to cells local to an injection site, brightly labelling axons even when coexpressing two other transgenes. We demonstrate several novel capabilities: simultaneously labelling axons and presynaptic terminals, labelling both dendrites and postsynaptic densities, and simultaneously labelling a region's inputs and outputs using co-injected vectors.

Fig. 1

Packaging deletion-mutant RV with the VSV envelope protein abolishes retrograde infectivity. A) Schematic of packaging G-deleted RV with its own envelope protein for efficient infection of distant neurons projecting to an injection site, as described in Wickersham et al. ’07a. B) Injection site in mouse barrel cortex of RV-4GFP packaged with RV G. Many cortical neurons with cell bodies distant from the injection site are labeled. C) Numerous retrogradely infected cells are also present in the somatosensory thalamus (VPM and Po nuclei) of the same mouse. D) Schematic of packaging G-deleted RV with the VSV envelope protein for local instead of retrograde infection. E) Injection site in mouse barrel cortex of RV-4GFP packaged with VSV G, with injection volume and viral titer equal to those of the injection depicted in panels B and C. Infected neurons are restricted to a tight cluster at the injection site, with bright GFP labeling of their axons. F) The somatosensory thalamus of the same animal contains dense arborizations of cortical axons in VPM and Po brightly labeled with GFP, but no retrogradely labeled cell bodies. Overlays indicating approximate structural boundaries are adapted from Franklin and Paxinos. Abbreviations: S1BF = primary somatosensory cortex, barrel field; ec = external capsule; CPu = caudate putamen; Po = posterior thalamic nuclear group; VPM = ventral posteromedial thalamic nucleus. Scale bars: B & E 500 um, C & F 200 um.

Fig. 2

Expression of multiple transgenes from single vectors allows simultaneous labeling of neurites and synapses. A) Transgene location within the viral genome determines expression level, as determined by flow cytometric analysis of EGFP brightness. Two transgenes inserted in the G locus have only slightly lower expression than a single one. Addition of a transgene in the promoter-proximal locus prior to the first viral gene, with a second transgene in the original G locus, results in expression 2.4-fold higher than that of a single gene in the G locus. Error bars denote standard error of the mean (n=?). B–C) Two-color brightly fluorescent labeling of the apical dendrites and postsynaptic densities of a layer 5 cortical pyramidal cell. Cytoplasmic mOrange2 is expressed at a high level from the promoter-proximal locus; PSD-95-EGFP fusion is expressed at a lower level from the G locus. Solid line indicates approximate location of pia; dashed square indicates region enlarged in panels D–E; dashed rectangle indicates region enlarged in Supplementary Figure S4. D–E) Higher-resolution image of region enclosed by dashed square in panel B. EGFP puncta are located on dendritic spines. Scale bars: B–D 50 um, E–G 10 um.

Fig. 3

Simultaneous multicolor brightly fluorescent labeling of three subcellular compartments. A) Injection site in mouse somatosensory cortex of anterograde (locally-infecting) rabies virus encoding cytoplasmic EGFP, nuclearly-localized mTagBFP, and synaptophysin-TagRFP-T fusion protein. A tight cluster of cells primarily in layer 6 resulted from this 5 nl injection, with little labeling in more superficial layers. Descending and locally arborizing axons are brightly labeled. Overlay indicating approximate structural boundaries is adapted from Franklin and Paxinos. B–D) Higher-power image of the layer 6 cells, with separate green and blue channels showing cytoplasmic EGFP and nuclear mTagBFP. E–G) High-power image of thalamocortical axonal processes (green) and synaptic terminals (red) in primary somatosensory thalamus (ventral posteriomedial nucleus). Abbreviations: S1BF = primary somatosensory cortex, barrel field; ec = external capsule; CPu = caudate putamen. Scale bars: A 250 um, B–D 100 um, E–G 5 um.

Fig. 4

Joint retrograde and anterograde tracing of inputs and outputs of a brain region. A–C) Injection site in mouse somatosensory thalamus of an equal mixture of two deletion-mutant rabies viral vectors: an anterograde (i.e. locally-infecting) vector encoding mOrange2 (yellow), and a retrograde (i.e. infecting remote neurons projecting to the injection site) vector encoding mCherry (red). The anterograde virus prolifically infects thalamic neurons at the injection site, while the retrograde virus infects far fewer cells locally. D–F) In somatosensory cortex of the same mouse, mOrange2-filled thalamocortical axons ascend to layer 4 and densely ramify among the apical dendrites of mCherry-filled layer 6 corticothalamic cells retrogradely infected by the coinjected retrograde virus. Scale bar 200 um, applies to all panels.

Fig. 5

Strategies now available for targeting multiple neuronal populations with different RV vector classes. A) Anterograde targeting with VSVG-enveloped RV, introduced in this paper. B) Retrograde targeting with RVG-enveloped RV. C) Genetic targeting with EnvA-enveloped RV. Packaging RV with an avian retroviral envelope protein renders it unable to infect mammalian cells in the absence of engineered expression of an exogenous receptor. D) Monosynaptic targeting with RV. When complemented by engineered expression of RVG in trans, G-deleted RV spreads to neurons directly presynaptic to the G-expressing cells.