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

Published: 5 February 2024| Version 2 | DOI: 10.17632/dctm2htrf5.2


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 anterograde tracing approaches have limited resolution at which connectivity can be mapped. 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 map connectivity in the mouse brain. We sequenced 391 cells expressing rabies barcodes across seven brains using single-cell RNA-seq, and 3,899,611 cells, including 7,338 neurons expressing rabies barcodes, across three mouse brains using in situ sequencing. 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.


Steps to reproduce

Please read the Read_me.txt file in each folder for detailed reproduction steps. NeMO_data.zip is scRNAseq data.


Allen Institute for Brain Science


New Sequencing Method


National Institutes of Health