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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE149527 | GEO | 2020/08/01

REPOSITORIES: GEO

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Project description:Purpose: Mounting evidence suggests that the spinal dorsal horn (SDH) contains multiple subpopulations of inhibitory interneurons that play distinct roles in somatosensory processing, as exemplified by the importance of spinal dynorphin-expressing neurons for the suppression of mechanical pain and chemical itch. While it is clear that GABAergic transmission in the SDH undergoes significant alterations during early postnatal development, little is known about the maturation of discrete inhibitory “microcircuits” within the region. As a result, the goal of the present study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life. Methods: We isolated nuclear RNA specifically from pDyn-lineage SDH interneurons at postnatal days 7, 21, and 80 using the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique in conjunction with Fluorescence-activated Nuclei Sorting (FANS), followed by RNAseq analysis. Results: Over 650 genes were ≥2-fold enriched in adult pDyn nuclei compared to non-pDyn spinal cord nuclei, including targets with known relevance to pain such as galanin (Gal), prepronociceptin (Pnoc), and nitric oxide synthase 1 (Nos1). In addition, the gene encoding a membrane-bound guanylate cyclase, Gucy2d, was identified as a novel and highly selective marker of the pDyn population within the SDH. Differential gene expression analysis comparing pDyn nuclei across the three ages revealed sets of genes that were significantly upregulated (such as Cartpt encoding cocaine- and amphetamine-regulated transcript peptide) or downregulated (including Npbwr1 encoding the receptor for neuropeptides B/W) during postnatal development. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

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

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