High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling

Area of Science:

• Neuroscience
• Molecular Biology
• Genomics

Background:

• Understanding neural connectivity is crucial for brain function.
• Current tracing methods have limitations in throughput and joint analysis of transcriptomics.
• Barcode-based connectomics faces challenges in integrating single-cell transcriptomics.

Purpose of the Study:

• To develop a multiplexed tracing method for simultaneous projectome and transcriptome analysis at the single neuron level.
• To investigate projection patterns and molecular heterogeneity of ventromedial prefrontal cortex (vmPFC) neurons.
• To identify transcriptional signatures of projection-specific neurons.

Main Methods:

• Established a retrograde adeno-associated virus (rAAV2-retro) barcode-based multiplexed tracing technique.
• Simultaneously characterized single-neuron projectome and transcriptome.
• Analyzed projection patterns of vmPFC neurons to downstream targets.

Main Results:

• Uncovered dedicated and collateral projection patterns of vmPFC neurons to five targets.
• Demonstrated molecular heterogeneity among projection-defined vmPFC neurons.
• Identified transcriptional signatures, including Pou3f1, for projection-specific neurons, particularly those targeting the lateral hypothalamus.

Conclusions:

• Developed a novel multiplexed technique for paired connectome and gene expression data.
• The method aids in revealing organizational principles of neural circuits and information processing.
• Identified Pou3f1 as a marker for a distinct subset of vmPFC neurons with collateral projections.