High-Complexity Barcoded Rabies Virus for Scalable Circuit Mapping Using Single-Cell and Single-Nucleus Sequencing.

Area of Science:

• Neuroscience
• Genomics
• Molecular Biology

Background:

• Single-cell genomics has advanced neuronal cell type identification.
• Scalable methods for mapping single-cell connectivity are needed to understand functional brain circuits.
• Current understanding of how molecularly defined cell types form circuits is limited.

Purpose of the Study:

• To develop a scalable protocol for mapping neuronal connectivity using barcoded rabies virus (RV).
• To enable the readout of connectivity information using single-nucleus RNA sequencing (snRNA-seq).
• To investigate the emergence of cell type-specific circuit motifs in the developing human brain.

Main Methods:

• Generation of high-complexity barcoded rabies virus (RV) for parallel circuit tracing.
• Targeting RV-encoded barcode transcripts to the nucleus for snRNA-seq analysis.
• Application of the protocol in organotypic slice cultures of the developing human cerebral cortex.

Main Results:

• Demonstration of a scalable protocol for mapping synaptic connectivity from tens of thousands of starter cells.
• Identification of cell type-specific circuit motifs in the midgestation human cerebral cortex.
• Successful integration of barcoded RV tracing with snRNA-seq for circuit analysis.

Conclusions:

• The developed protocol offers a scalable approach to map synaptic connectivity of molecularly defined cell types.
• This technology provides a path forward for circuit mapping in both healthy and diseased states.
• Reveals insights into the developmental emergence of functional brain circuits.