Abstract
An exciting frontier in circuit neuroscience lies at the intersection between neural network mapping and single-cell genomics. Monosynaptic rabies viruses provide a promising platform for the merger of circuit mapping methods with -omics approaches. However, three key limitations have hindered the extraction of physiologically meaningful gene expression profiles from rabies-mapped circuits: inherent viral cytotoxicity, high viral immunogenicity and virus-induced alteration of cellular transcriptional regulation. These factors alter the transcriptional and translational profiles of infected neurons and their neighboring cells. To overcome these limitations we applied a self-inactivating genomic modification to the less immunogenic rabies strain, CVS-N2c, to generate a self-inactivating CVS-N2c rabies virus (SiR-N2c). SiR-N2c not only eliminates undesired cytotoxic effects but also substantially reduces gene expression alterations in infected neurons and dampens the recruitment of innate and acquired immune responses, thus enabling open-ended interventions on neural networks and their genetic characterization using single-cell genomic approaches.
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Data availability
All materials described in this paper can be obtained for non-commercial purposes after signing a material transfer agreement (MTA) with the UKRI. Data generated during this study are included in the manuscript and supporting files. The DNA constructs to generate the viral vectors used in this study are available from Addgene (99610, 194352–194356). The raw NGS dataset has been deposited into NCBI’s Sequence Read Archive (SRA) and is accessible through accession number PRJNA901288. Further requests for information, resources and reagents should be directed to the Lead Contact, Marco Tripodi.
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Acknowledgements
The authors thank the creators of the SeqMonk program (Babraham Institute) and the SeqSeek atlas41, which were used in the analysis pipelines. The authors also thank the members of the LMB Biological Service Group for their support with the in vivo work. This study was supported by the Medical Research Council (MC_UP_1201/2) to M.T., the European Research Council (STG 677029) to M.T., the Rosetrees Trust with an MBPhD fellowship to H.L. (M598) and by the Cambridge Philosophical Society and St Edmund’s College (University of Cambridge) with the Henslow Research Fellowship to A.G.-R.
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M.T., H.L. and E.C. conceived the project and analyzed the data; H.L. and E.C. conducted the experiments and wrote the manuscript with input from M.T.; E.C. and H.L. co-developed the SiR-N2c technology; E.C. produced SiR viruses with the help of H.L. and F.M.; H.L. led the in vivo characterization of the virus and performed the immunology and bulk RNA-seq experiments; H.L. performed the long-term survival experiments and peripheral neurotropism experiments with the help of E.C.; E.C. performed the pseudotyped rabies tracing experiment; E.C., E.W. and F.N. performed the single-cell genomic experiments; H.L. and S.M. analyzed the bulk and single-cell genomic data; A.G.-R. performed the electrophysiological recordings.
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E.C. and M.T. are inventors of a patent related to the SiR technology (WO2018203049A1). All other authors have no competing interests.
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Nature Methods thanks Naoshige Uchida and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Sequencing of SiR viral productions confirms viral productions maintain the self- inactivating TEVs-PEST sequence.
a) Scheme of the procedure to sequence individual viral particles from independent SiR preparations: ssRNA genomes from a viral production are extracted and treated with DNAse I. The RNA is retrotranscribed and the entire N-TEVs-PEST sequence and portion of the P gene is PCR amplified. The amplicon is then inserted into a pBluescript KS(+) sequencing vector to create genomic libraries. (b) List of detected mutations divided by batch (47 individual clones per batch). The position of the mutations is calculated referring to +1 as the first base of the nucleoprotein N coding sequence. (c) The CA1 of mice were injected with SiR-N2c virus or PBS control. 1 week p.i. the hippocampi were dissected for total RNA-seq sample preparation. Sequenced reads were aligned to the SiR-N2c viral genome using Burrows-Wheeler Aligner (BWA)54. Aligned reads were then screened for mutations using freebayes variant detector55. (d) Visualization of reads from SiR- N2c-injected samples aligned to the SiR-N2c genome using Seqmonk software. Samples from PBS-injected samples show only few chance alignments in comparison. (e) Zoom in to TEVs-PEST region of the genome (f) Visualization of mutagenic events identified across SiR-N2c genome flanking the TEVs-PEST coding region (yellow) from a total of 3 injected samples.
Extended Data Fig. 2 Rabies viral infection induces innate immune responses in vitro.
a) Immunostaining of neuro2A cells infected with ΔG−Rabies-B19-GFP shows induction of NF-kB (left) and p38 MAPK (right) activation 24 h post-infection. Treatment with PBS or 100 ng TNFα were used as negative and positive controls, respectively, for induction of innate interferon responses. (b) RT–qPCR fold changes of immune response genes IL6 and RIG-I in neuro2A following ΔG-Rabies-B19, ΔG-Rabies-N2c, SiR-B19, SiR-N2c infection. Fold changes are normalized to SiR-N2c induced transcript levels. (mean ± SEM, n = 3, one-way ANOVA with Tukey correction for multiple comparisons; top-down, left to right: P = ****5.47e-5; **1.2e-3; *1.3e-2; *2.7e-2. Scale bars 100 μm for NF-kB images, 50 μm for MAPK images.
Extended Data Fig. 3 ΔG-Rabies-N2c infection induces an extensive immune response and perturbs synaptic function in the mouse hippocampus.
(a) Scatter plot depicting differentially regulated transcripts in ΔG-Rabies-N2c-injected vs. PBS-injected control mice (blue). Gene ontology (GO) terms enriched in up- and downregulated transcripts following ΔG-Rabies-N2c injection compared to PBS-injected controls organized by biological process (b), reactome pathway (c) and molecular function (d) (Fisher’s exact test with Benjamini and Hochberg multiple testing correction (n = 3 per condition).
Extended Data Fig. 4 Hierarchy of GO terms of ‘immune system process’ enriched in upregulated RNA transcripts from ΔG-Rabies-N2c virus-infected hippocampi.
Enrichment analysis using GOrilla web-based tool (exact mHG p-value computation with p-value threshold 10−3) (ref. 53).
Extended Data Fig. 5 SiR-N2c displays increased peripheral neurotropism and retains transsynaptic spreading capabilities.
(a) CRE-reporter pups (P3-P5) were injected with either SiR-N2c-CRE or oG-coated SiR-B19-CRE in the quadriceps muscle area to target motor neuron terminals. (b) Quantification of total motor neurons infected post-injection, normalized by relative titer of the two viruses. (mean ± SEM, n = 3, two-tailed, unpaired Student’s T-test; P = ***0.0002) (c) Scheme of the strategy for transsynaptic labeling of premotor neurons. tdTomato reporter pups (P3-P5) were injected with a mixture of SiR-N2c-CRE and AAV-G-nucFLAG in the quadriceps muscle area to target motor neuron terminals. (d) Representative images of the spinal cord of mice injected with SiR-N2c-CRE in absence of AAV-G-nucFLAG displaying Chat+ motor neurons (cyan) labeled with tdTomato and FLAG (n = 3) (e) Zoomed images of tdTomato+ motor neurons. (f) Representative images of tdTomato+ SiR-N2c-CRE infected neurons in the spinal cord of injected mice at 1 week p.i. stained for Chat (cyan) and FLAG (gray) (n = 3). (g) Zoomed images of tdTomato +/FLAG+ starter motor neurons. Scale bars: 200 µm for entire spinal cord, 50 µm for zoomed images.
Extended Data Fig. 6 SiR-N2c infection induces lower glial immune response in the spinal cord compared to ΔG-Rabies-N2c.
(a) tdTomato CRE-reporter pups were injected in the quadriceps muscle area with a mixture of either ΔG-Rabies- N2c-CRE or SiR-N2c-CRE and AAV-G-nucFLAG and killed up to 2 weeks p.i. for immunofluorescence studies (b–e). Comparison of GFAP+ staining for activated astrocytes in spinal cords from pups injected with ΔG-Rabies- N2c (B) or SiR-N2c (C), with close up panel showing regions with tdTomato+ neurons. Comparison of Iba1+ staining for activated microglia in spinal cords from pups injected with ΔG-Rabies-N2c (D) or SiR-N2c (E), with close up panel showing regions with tdTomato+ neurons. Scale bars 200 μm for entire spinal cord images and 50 μm for zoomed images.
Extended Data Fig. 7 SiR-N2c EnvA-pseudotyped specificity.
(a) CRE-reporter mice were either not injected or injected in the NAc with AAV-TVA or AAV-TVA-G. 3 weeks later NAc was injected with SiR-N2c-CRE (EnvA). (b-d) Representative images of whole brains of Rosa-LoxPSTOPLoxP-tdTomato mice. (e–g) Representative images of the NAc of injected (nuclear GFP in F and TVA-GFP fusion in G). (h-j) Zoomed images of starter neurons in the NAc. Scale bars: 1 mm for whole brains (B-D), 100 μm for NAc images (E–G), 15 μm for zoomed panels (H-J).
Extended Data Fig. 8 SiR-N2c traced GFP+ neurons show enrichment for specific neuronal subtypes within the spinal premotor circuit.
A stacked tile plot where the horizontal length of rectangles on the x axis represents the relative frequency of neuron subtypes in SiR-N2c traced GFP+ neurons versus non-virally traced GFP- control spinal cord neurons. An oval represents 0% frequency. Each rectangle and circle is colored to represent the Pearson residual from a two-sided chi-squared test. X is placed adjacent to neuronal subtypes that were not identified in ΔG-Rabies-N2c-infected GFP+ neurons.
Extended Data Fig. 9 SiR-N2c-infected neurons show less disruption of transcriptional regulation compared to ΔG-Rabies-N2c-infected neurons.
(a) Enrichment of Biological Process Gene Ontology terms related to neuronal structure, function and RNA synthesis in differentially downregulated genes (gene set enrichment analysis, cumulative hypergeometric test with g:Set Counts and Sizes correction method)57, in ΔG-Rabies-N2c-infected neurons compared to non-infected neurons (blue) and in SiR-N2c-infected neurons compared to non-infected neurons (orange, p > 0.05). (b) Enrichment of Cell Component Gene Ontology terms related to neuronal structures in differentially downregulated genes in ΔG-Rabies-N2c-infected neurons compared to non-infected neurons (blue)and in SiR-N2c-infected neurons compared to non-infected neurons (orange). Gene set enrichment analysis using cumulative hypergeometric test with g:Set Counts and Sizes correction method57. (c) Gene ontology terms enriched in genes upregulated in SiR-N2c-infected neurons compared to ΔG-Rabies-N2c-infected neurons (blue) or upregulated in ΔG-Rabies-N2c-infected neurons compared to SiR-N2c-infected neurons (red). Enrichment analysis using GOrilla web-based tool, exact mHG p-value computation with p-value threshold 10−3 53. (d-g) Gene expression levels of canonical neuronal genes (Kirrel3, Grik3, Igf1r) and vesicle membrane protein Sec61a1 in uninfected neurons (green), SiR-N2c-infected neurons (pink), and ΔG-Rabies-N2c-infected neurons (blue). (mean ± SEM, differential expression analyses, two-sided chi-squared test of differential proportion genes; n = 224 for GFP-, 628 for SiR GFP+, and 580 for WT GFP+. Each experimental condition consists of neurons from 3 biologically independent samples). From left to right: P = ****3.2e-11, ****1.6e-06, ****4.1e-07, ****3.7e-09.
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Lee, H., Ciabatti, E., González-Rueda, A. et al. Combining long-term circuit mapping and network transcriptomics with SiR-N2c. Nat Methods 20, 580–589 (2023). https://doi.org/10.1038/s41592-023-01787-1
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DOI: https://doi.org/10.1038/s41592-023-01787-1
