Rabies Virus CVS-N2c(ΔG) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability

Abstract

Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19(ΔG) transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2c(ΔG), and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19(ΔG), the CVS-N2c(ΔG) strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2c(ΔG) strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS.

FIGURE 1. Construction and Packaging of CVS-N2c^ΔG for monosynaptic tracing

The CVS-N2c deletion mutant was created in the manner pioneered by Wickersham et al (2007a,. A key difference is the use of a neural cell line for viral packaging. (A) Schematic illustrating monosynaptic restriction of viral spread from starter to secondary neurons. (B) CVS-N2c genome and recombinant CVS-N2c^ΔG vector with XmaI/NheI insert restriction sites flanking dsRed insert as well as rescued virus expressing dsRed in Neuro2A cells (top) and complementation vector expressing CVS-N2c glycoprotein inside murine leukosis virus (MLV) (middle) stably transfected into Neuro2A cells for amplification of virus CVS-N2c^ΔG to create the line Neuro2A-N2c(G). Levels of GFP expression correspond to expression of N2c(G) (bottom). (C) Complementation vector expressing a chimeric EnvA glycoprotein with short CVS glycoprotein tail (top) transfected into Neuro2A cells for packaging of pseudotyped virus. 293-TVA cells show infection by EnvA-pseudotyped CVS-N2c^ΔG and Neuro2A and Hek293 cells lacking the TVA receptor show no infection by the same virus (bottom).

FIGURE 2. Greater transsynaptic transfer for CVS-N2c than SAD-B19 in the forebrain

(A) Schematic for corticostriatal retrograde transsynaptic infection. Primary neurons in striatum infected by conditional AAV expressing RABV(G) and nuclear GFP (histone2B-GFP fusion: nGFP) and AAV expressing TVA and mCherry after recombination via germline Adora2a-Cre, and re-infected two weeks later by pseudotyped RABV^ΔG–dsRed[EnvA]. (B–C) Confocal images of primary infection for SAD-B19 and CVS-N2c. Primary cells display both nGFP as well as dsRed. (D–E) Representative image of monosynaptic viral spread with 20× confocal image inset from anatomical plot showing all RABV+ neurons. (F) Plot of infection spread from primary striatal to secondary cortical neurons in 6 mice, 3 for each viral strain. Primary infection was marked by expression of both dsRed from RABV and nGFP from the AAV complementation vector. Primary infection was constrained to small populations in the same region of anterior dorsal striatum. Secondary infection was calculated by sampling the same 5 coronal sections from each animal as identified by position relative to Bregma. Pan-cortical secondary infection is greater, but scales with numbers shown here.

FIGURE 3. CVS-N2c^ΔG from lumbar motor neurons shows enhanced transsynaptic spread compared to SAD-B19^ΔG

(A) Schematic of RABV-monosynaptic tracing from lumbar motor neurons. (B–D) Representative images for each viral strain showing transsynaptically transferred infection at lumbar (B), thoracic (C) and cervical (D) levels. (E) Total primary to secondary ratios for SAD-B19^ΔG (grey) and CVS-N2c^ΔG (red). (F,G) Plot of infection spread from primary to secondary neurons in 6 mice, 3 for each viral strain, at local lumbar (F), and (G) distant thoracic, cervical and hindbrain levels.

FIGURE 4. Restricted transsynaptic transfer of CVS-N2c^ΔG

(A) Experimental design to examine overlap of GS and TA motor neuron dendrites. (B) Fluorescent labeled motor neurons showing overlap of GS and TA dendrites. (C) Assay to test for selective trans-synaptic transfer of RABV. (D) Example images of GS and TA muscle spindles showing dsRed positive sensory endings in GS (top) but not TA (bottom). (E) Quantitation of labeled sensory endings in GS or TA muscles.

FIGURE 5. Reduced expression level and cytotoxicity of CVS-N2c compared to SAD-B19

(A) Neuro2A cells infected by GFP-expressing SAD-B19^ΔG or (B) CVS-N2c^ΔG. (C) Intensometric plot of GFP expressed by each RABV strain at 2 and 4 days post-infection, normalized to single infected cells. (D) Neuro2A cells infected with SAD-B19^ΔG or (E) CVS-N2c^ΔG after 4 days and treated with propidium iodide, a proxy marker for cell death at 4 days post-infection. (F) Relative cell death after infection by each RABV strain. Error bars in C and F represent ± SEM.

FIGURE 6. CVS-N2c^ΔG is an effective transsynaptic vector for optogenetic manipulation

(A) Confocal image of neurons after 7 days of infection by SAD-B19^ΔG virus expressing hChR2-YFP (cortex). (B) Same proteins via virus CVS-N2c^ΔG. (C) Assay of cell health by morphological irregularity, blebs along proximal dendrites per RABV-infected neuron. (D) Schematic of corticostriatal retrograde infection using CVS-N2c^ΔG-hChR2-YFP in the dorsal striatum in wild type mice and retrograde spread into cortex (top) and patch-clamp recording of infected cortical neurons (bottom). (E) Schematic of whole-cell current-clamp recordings obtained from neurons in acute cortical slices (top). In vitro two-photon images of a recorded cortical neuron retrogradely infected by CVS-N2c^ΔG-hChR2-YFP (green) and filled with fluorescent dye from patch pipette (red). (F) Example hChR2-photostimulation-evoked voltage responses recorded from CVS-N2c^ΔG-hChR2-YFP-infected neurons at 6, 10, 14, and 28 days post infection as seen in (B). (G) Spike probability at each 6/10/14/21/28 DPI showing increasing effectiveness of hChR2 expressed by the CVS-N2c^ΔG vector. (H) Schematic and in vitro two-photon image showing CVS-N2c^ΔG-infected (green) neuron in the vicinity of a non-infected cortical neuron filled with red fluorescent dye via patch pipette (top). Example averaged postsynaptic responses recorded from the non-infected neuron confirming effective synaptic release at 8 days post infection (bottom).

FIGURE 7. CVS-N2c^ΔG is an effective transsynaptic vector for optogenetic monitoring

(A) Confocal image of neurons after 10 days of infection by SAD-B19^ΔG virus expressing GCaMP6f (medial septum). (B) Same protein via virus CVS-N2c^ΔG. (C) Assay of cell health by morphological irregularity, blebs along proximal dendrites per RABV-infection neuron. (D) Schematic of infection of CVS-N2c^ΔG-GCaMP6f in medial septum and retrograde spread to the dorsal hippocampus. (E) Schematic of head-fixed two-photon imaging of dorsal hippocampus of mouse running on an environmentally enriched treadmill (top), in vivo two-photon image of an example septal projecting neuron imaged for activity in the area CA1 of the dorsal hippocampus (middle), and time-series of neural activity, expressed as relative changes in GCaMP6f fluorescence (ΔF/F, black traces) of neuron during running bouts (grey traces) on belt, imaged via in vivo two-photon microscopy (bottom). (F) Population summary of the magnitude of running-evoked of Ca²⁺ signals indicating comparable responses over extended periods after infection (4–6 DPI: n = 13 cells in n = 2 animal; 10–17 DPI: n = 31 cells in n = 3 animals, Wilcoxon-Mann-Whitney two sample rank test, p = 0.592). (G) In vivo two-photon image of the same septal-projecting neuron in area CA1 of the dorsal hippocampus (left), and GCaMP6f fluorescence Ca²⁺ signals (ΔF/F, black traces) recorded from the same neuron during running (grey traces) at 11, 14, and 17 days post-injection (right). Error bars in F represent ± SEM.

FIGURE 8. CVS-N2c^ΔG drives Cre- or Flp-dependent recombination

(A) Schematic depicting injection of CVS-N2c^ΔG-mCherry-Flpo and recombination in Frt-STOP-Frt-GFP mouse line. (B) Confocal images of thoracic spinal cord showing recombination in retrogradely infected neurons. (C) Schematic depicting injection and recombination by CVS-N2c^ΔG-mCherry-Cre. (D) Images in parabrachial nucleus (PBN) showing neurons after recombination and expression from a local AAV-FLEX-GFP injection and a corresponding infection in VTA and retrograde uptake of Cre-expressing virus by PBN neurons.