Inhibitory interneurons in the adult spinal dorsal horn (DH) can be neurochemically classified into subpopulations that regulate distinct somatosensory modalities. Although inhibitory networks in the rodent DH undergo dramatic remodeling over the first weeks of life, little is known about the maturation of identified classes of GABAergic interneurons, or whether their role in somatosensation shifts during development. We investigated age-dependent changes in the connectivity and function of prodynorphin (DYN)-lineage neurons in the mouse DH that suppress mechanosensation and itch during adulthood. In vitro patch clamp recordings revealed a developmental increase in primary afferent drive to DYN interneurons and a transition from exclusive C-fiber monosynaptic input to mixed A-fiber and C-fiber innervation. Although most adult DYN interneurons exhibited tonic firing as expected from their inhibitory phenotype, neonatal and adolescent DYN cells were predominantly classified as phasic or single-spiking. Importantly, we also found that most of the inhibitory presynaptic terminals contacting lamina I spinoparabrachial projection neurons (PNs) originate from DYN neurons. Furthermore, inhibitory synaptic input from DYN interneurons onto PNs was weaker during the neonatal period, likely reflecting a lower number of GABAergic terminals and a reduced probability of GABA release compared to adults. Finally, spinal DYN interneurons attenuated mechanical sensitivity throughout development, but this population dampened acute nonhistaminergic itch only during adulthood. Collectively, these findings suggest that the spinal “gates” controlling sensory transmission to the brain may emerge in a modality-selective manner during early life due to the postnatal tuning of inhibitory synaptic circuits within the DH.
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. Although 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 this study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life. 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, followed by RNA-seq analysis. Over 650 genes were ≥2-fold enriched in adult pDyn nuclei compared with 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 3 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. Collectively, these results provide new insight into the potential molecular mechanisms underlying the known age-dependent changes in spinal nociceptive processing and pain sensitivity.
Neonatal tissue damage can have long-lasting effects on nociceptive processing in the central nervous system, which may reflect persistent injury-evoked alterations to 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 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 mouse spinal pDyn cells characterized this population in depth and then identified changes in gene expression evoked by neonatal hindpaw incision. The analysis revealed 11 transcriptionally distinct subpopulations (ie, clusters) of dynorphin-lineage cells, including both inhibitory and excitatory neurons. Investigation of injury-evoked differential gene expression identified 15 genes that were significantly upregulated or downregulated in adult pDyn neurons from neonatally incised mice compared with naive littermate controls, with both cluster-specific and pan-neuronal transcriptional changes observed. Several of the identified genes, such as Oxr1 and Fth1 (encoding ferritin), were related to the cellular stress response. However, the relatively low number of injury-evoked differentially expressed genes also suggests that posttranscriptional 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 insights 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.
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