Significance Monosynaptic rabies tracing using glycoprotein (G)-deleted rabies virus is widely applied to study cortical circuit connectivity. However, connectivity tracing using rabies virus could benefit from higher throughput methods of assigning rabies-labeled inputs to neuronal cell types and the ability to assign cells to finer genetically defined subtypes. Using single-nucleus RNA sequencing, we demonstrate that rabies-infected cortical neurons can be transcriptomically characterized according to established cell types when using transcriptome-wide analysis. Furthermore, we find that rabies infection differentially affects distinct host genes, suggesting that some genes may be more vulnerable to transcriptional modulation than others, which may impede the classification of rabies-infected cells when using methods that rely on the detection of single or few genes.

Single-cell genetic and epigenetic analyses parse the brain′s billions of neurons into thousands of ″cell type″ clusters, each residing in different brain structures. Many of these cell types mediate their unique functions by virtue of targeted long-distance axonal projections to allow interactions between specific cell types. Here we have used Epi-Retro-Seq to link single cell epigenomes and associated cell types to their long-distance projections for 33,034 neurons dissected from 32 different source regions projecting to 24 different targets (225 source→target combinations) across the whole mouse brain. We highlight uses of this large data set for interrogating both overarching principles relating projection cell types to their transcriptomic and epigenomic properties and for addressing and developing specific hypotheses about cell types and connections as they relate to genetics. We provide an overall synthesis of the data set with 926 statistical comparisons of the discriminability of neurons projecting to each target for every dissected source region. We integrate this dataset into the larger, annotated BICCN cell type atlas composed of millions of neurons to link projection cell types to consensus clusters. Integration with spatial transcriptomic data further assigns projection-enriched clusters to much smaller source regions than afforded by the original dissections. We exemplify these capabilities by presenting in-depth analyses of neurons with identified projections from the hypothalamus, thalamus, hindbrain, amygdala, and midbrain to provide new insights into the properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription factor binding motifs, and neurotransmitter usage.