Barcoded Rabies In Situ Connectomics for high-throughput reconstruction of neural circuits
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ABSTRACT:
PROVIDER: PRJNA1292769 | ENA |
REPOSITORIES: ENA
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Project description:Sequencing of oligonucleotide barcodes holds promise as a high-throughput approach for reconstructing synaptic connectivity at scale. Rabies viruses can act as a vehicle for barcode transmission, thanks to their ability to spread between synaptically connected cells. However, applying barcoded rabies viruses to map synaptic connections in vivo has proved challenging. Here, we develop Barcoded Rabies In Situ Connectomics (BRISC) for high-throughput connectivity mapping in the mouse brain. To ensure that the majority of post-synaptic "starter" neurons are uniquely labeled with distinct barcode sequences, we first generated libraries of rabies viruses with sufficient diversity to label >1000 neurons uniquely. To minimize the probability of barcode transmission between starter neurons, we developed a strategy to tightly control their density. We then applied BRISC to map inputs of single neurons in the primary visual cortex (V1). Using in situ sequencing, we read out the expression of viral barcodes in rabies-infected neurons, while preserving spatial information. We then matched barcode sequences between starter and presynaptic neurons, mapping the inputs of 385 neurons and identifying 7,814 putative synaptic connections. The resulting connectivity matrix revealed layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs to V1. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons while preserving spatial information, enabling reconstruction of neural circuits at an unprecedented scale.
2025-07-18 | GSE302891 | GEO
Project description:We describe "SBARRO" a system in which synaptic networks of cells are reconstructed from scRNA-seq data using clonal infectivity paths of barcoded rabies virus RNAs
2022-10-06 | GSE214386 | GEO
Project description:We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In Situ analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POL2RA respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglia cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.
2021-02-07 | GSE166293 | GEO
Project description:Cortical circuit tracing using modified rabies virus can identify input neurons making direct monosynaptic connections onto neurons of interest. However, challenges remain in our ability to establish the cell type identity of rabies-labeled input neurons. While transcriptomics may offer an avenue to characterize inputs, the extent of rabies-induced transcriptional changes in distinct neuronal cell types remains unclear and whether these changes preclude characterization of rabies-infected neurons according to established transcriptomic cell types is unknown. We used single-nucleus RNA sequencing to survey the gene expression profiles of rabies-infected neurons and assessed their correspondence with established transcriptomic cell types. We demonstrated that when using transcriptome-wide RNA profiles, rabies-infected cortical neurons can be transcriptomically characterized despite global and cell-type-specific rabies-induced transcriptional changes. Notably, we found differential modulation of neuronal marker gene expression, suggesting that caution should be taken when attempting to characterize rabies-infected cells with single genes or small gene sets.
2022-05-25 | GSE196771 | GEO
Project description:Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses
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Project description:Gene regulatory network reconstruction using single-cell RNA sequencing of barcoded genotypes in diverse environments
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Project description:Rabies is an ancient infectious disease but still lacking efficient therapeutic approach despite of vaccine. In this study, we have identified a novel cytoplasmic lncRNA, namely rabies virus related lncRNA 1(RVRL1), whose expression in neuronal cells is up-regulated upon the infection of the causative agent of rabies, the neurotropic virus rabies virus (RABV). RVRL1 effectively inhibits RABV infection both in neuronal cells and in a mouse model. RVRL1 binds to EZH2 and disrupts the PRC2 complex, which is consistent with the inverse relationship between RVRL1 expression and the cellular H3K27me3 level. RVRL1 expression positively regulates the expression of PCP4L1 encoding a 10 kD peptide, which is shown to inhibit RABV replication. These findings highlight a novel mechanism for lncRNAs to upregulate the expression of antiviral genes, and define two potential anti-rabies reagents including an antiviral lncRNA and an antiviral peptide.
2020-09-08 | GSE107285 | GEO