Transcriptomic Cell Type Specificity of Local Cortical Circuits

The complex functions of the neocortex rely on networks of interconnected excitatory and inhibitory interneurons, each of which are composed of multiple neuron types. Prior studies have established rules of local connectivity between major subclasses of inhibitory and excitatory cortical neurons, but the advent of single-cell transcriptomic technologies has revealed a remarkable diversity in transcriptomic neuronal subtypes. Is there specificity of synaptic connections between cortical neurons classified at the level of transcriptomic subtypes, as might be expected if the different types mediate different functions? Here we present a novel method that links transcriptomic cell type to anatomical connectivity, “Single Transcriptome Assisted Rabies Tracing” (START). START combines monosynaptic rabies tracing and single-nuclei RNA sequencing (snRNA-seq) to identify the transcriptomic cell types providing monosynaptic inputs to defined populations of neurons. We employed START in conjunction with Cre driver mouse lines to transcriptomically characterize 35,717 neurons providing monosynaptic input to 5 different layer-specific excitatory cortical neuron populations in mouse V1. At the subclass level, we observed results consistent with findings from prior studies that resolve neuronal subclasses using antibody staining, Cre-expressing mouse lines, or morphological characterization. With improved neuronal subtype granularity achieved with START, we demonstrate transcriptomic subtype specificity of inhibitory inputs to various subclasses of excitatory neurons. These results establish local connectivity rules at the resolution of transcriptomic inhibitory cell types. Overall design: Nuclei were collected from V1 of Cre-dependent mouse lines (Sepw1-Cre, Scnn1a-Cre, Tlx3-Cre, Npr3-Cre, and Ntsr1-Cre; n=4 per mouse for all lines except Npr3-Cre where n=5) following monosynaptic rabies tracing. 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.