Single-cell RNA-sequencing of mouse periaqueductal gray (PAG) neurons using Smart-seq2.

We optimized Voltage-Seq which combines, all-optical physiology, spatial mapping, on-site classification, and RNA-transcriptomics to robustly increase the throughput of synaptic connectivity testing and targeted molecular classification of postsynaptic neurons. Single-cell RNA-sequencing was performed on spatial- and voltage-recorded neurons in the mouse PAG. Uniform Manifold Approximation and Projection (UMAP) clustered cells as excitatory or inhibitory, and further differential expression analyses highlighted putative marker genes of GABAergic neurons. These sequencing results were in agreement with in-situ hybridization (ISH) and neuronal activity recordings.