Single-nucleus characterization of spinal dynorphin-lineage cells and identification of persistent transcriptional effects of neonatal hindpaw incision

Neonatal tissue damage can have long-lasting effects on nociceptive processing in the central nervous system, which may be partially due to persistent injury-evoked alteration of the normal balance between synaptic inhibition and excitation in the spinal dorsal horn. Spinal dynorphin-lineage (pDyn) neurons are part of an inhibitory circuit which limits the flow of nociceptive input from the periphery to the brain and is disrupted by neonatal tissue damage. To identify the potential molecular underpinnings of this disruption, an unbiased single-nucleus RNAseq analysis of adult spinal pDyn cells characterized this population in depth, then identified changes in gene expression evoked by neonatal hindpaw incision. The analysis revealed twelve transcriptionally distinct subpopulations (i.e., clusters) of dynorphin-lineage cells, including both inhibitory and excitatory neurons as well as oligodendrocytes and astrocytes. Investigation of injury-evoked differential gene expression identified genes which were significantly up- or downregulated in adult pDyn neurons from neonatally incised mice compared to naïve littermate controls. Some of these injury-evoked changes appeared to be cluster-specific while others were similar across all pDyn neuron clusters, and many of the identified genes were related to the cellular stress response. However, the relatively low number of injury-evoked differentially expressed genes also suggests that post-transcriptional regulation within pDyn neurons may play a key role in the priming of developing nociceptive circuits by early life injury. Overall, the findings reveal novel insight into the molecular heterogeneity of a key population of dorsal horn interneurons that has previously been implicated in the suppression of mechanical pain and itch. Overall design: Prodynorphin (Pdyn)-lineage spinal nuclei were collected from 8 naïve adult mice and 8 adult mice with prior neonatal (P3) hindpaw incision. Single-cell RNA sequencing data was obtained from each injured and naïve sample using 10X Chromium 3v3.