Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing

Abstract

Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to achieve circuit mapping at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 295 transsynaptically labeled cells using single-cell RNAseq, and 4,130 retrogradely labeled cells and 2,914 transsynaptically labeled cells in situ. We determined the transcriptomic identities of rabies virus-infected cells robustly using both single-cell RNA-seq and in situ sequencing. We then distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus thus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.

Molecular Connectomics Reveals a Glucagon-Like Peptide 1 Sensitive Neural Circuit for Satiety

“…Recent studies have profiled gene expression in rabies-infected cells by single-cell RNA-sequencing (scRNA-seq). Their results show that, while rabies infection alters the expression of some genes, infected cells can still be classified by comparing their gene expression profiles to those of uninfected cells 24,29,[73][74][75] . Our use of RAMPANT confirms that rabies-infected cells can be molecularly identified and extends these previous findings, demonstrating that most AgRP neuron marker genes are unaffected by rabies 5 days after infection and that their molecular response to fasting and feeding remains largely intact.…”