Rabies-Infected Neurons Can Be Transcriptomically Characterized Despite Changes in Gene Expression

Cortical circuit tracing using modified rabies virus can identify input neurons making direct monosynaptic connections onto neurons of interest. However, challenges remain in our ability to establish the cell type identity of rabies-labeled input neurons. While transcriptomics may offer an avenue to characterize inputs, the extent of rabies-induced transcriptional changes in distinct neuronal cell types remains unclear and whether these changes preclude characterization of rabies-infected neurons according to established transcriptomic cell types is unknown. We used single-nucleus RNA sequencing to survey the gene expression profiles of rabies-infected neurons and assessed their correspondence with established transcriptomic cell types. We demonstrated that when using transcriptome-wide RNA profiles, rabies-infected cortical neurons can be transcriptomically characterized despite global and cell-type-specific rabies-induced transcriptional changes. Notably, we found differential modulation of neuronal marker gene expression, suggesting that caution should be taken when attempting to characterize rabies-infected cells with single genes or small gene sets. Rabies-infected nuclei were collected from V1 of wild-type C57BL/6 mice (n=3, 2 females and 1 male) injected with unpseudotyped SAD-B19 G-deleted rabies virus expressing nuclear-localized mCherry (G+RVdG.H2B.mCherry). To enrich for rabies-infected inhibitory neurons, Gad2-Cre mice were crossed to R26-LSL-TVA-LacZ mice to express TVA in inhibitory neurons. These transgenic mice (n=8, 5 females and 3 males) were then injected with EnvA-pseudotyped G-deleted rabies virus expressing nuclear-localized mCherry (EnvA+RVdG.H2BmCherry), which resulted in direct and selective rabies infection of the targeted inhibitory cell class. 10 days after injection, V1 containing mCherry+ rabies-labeled nuclei was dissected and single-nucleus suspensions were prepared from dissected tissue for fluorescence-activated nuclei sorting (FANS) to collect mCherry+ nuclei. snRNA-seq of FANS sorted rabies-infected nuclei was performed using the 10X Genomics 3’ Kit v3.1.