Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse

Abstract

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.

Figure 1.. Selective GFP-tagging and fluorescent nuclei sorting effectively isolates mRNA from spinal pDyn neurons.

(A) Breeding pDyn-IRES-cre mice with R26-LSL-Sun1-GFP mice results in the selective tagging of pDyn-lineage spinal nuclei with GFP-Sun1 fusion protein. Scale bar = 50 μm. (B-D) In situ hybridization demonstrates substantial co-localization of Sun1-GFP fusion protein (B; green) and Pdyn mRNA (C; magenta). Filled arrowheads indicate co-localized cells, while asterisk indicates a GFP+ cell that does not express Pdyn mRNA and open arrowheads indicate cells expressing Pdyn mRNA but not Sun1-GFP (D). Scale bar = 20 μm. (E) Quantification of in situ results shows that the majority of Sun1-GFP+ cells express Pdyn mRNA (left; n = 12), and the majority of Pdyn mRNA-expressing cells are tagged with Sun1-GFP (right; n = 12). (F) After homogenization and filtering, intact nuclei are stained with propidium iodide (magenta), while pDyn nuclei also exhibit GFP fluorescence (green; arrowheads). Scale bar = 20 μm. (G) Fluorescence Activated Nuclei Sorting (FANS) separates PI+/GFP− nuclei (orange) from PI+/GFP+ nuclei (green). (H) qPCR validation shows significant enrichment for Pdyn mRNA in GFP+ nuclei compared to GFP− nuclei. Expression is normalized to Hprt1 reference gene (n = 6; t = 3.723; p = 0.0137; paired t-test). (I) qPCR validation shows GFP+ nuclei are enriched in known markers of pDyn neurons and exhibit lower relative expression of genes which mark excitatory neurons and glia (n = 4; p < 0.0001; RM two-way ANOVA; ** p < 0.001; ***p < 0.0001; Bonferroni post-test).

Figure 2.. Differential gene expression in adult spinal pDyn neurons.

(A) Hierarchical clustering of mRNA from spinal pDyn nuclei (GFP+) and non-pDyn (GFP−) nuclei. Color scale indicates z-score of normalized expression values. M = male; F = female. (B) GFP+ samples are depleted in non-neuronal markers and excitatory neuron markers, and enriched in inhibitory neuronal markers, compared to GFP− samples. Astro = astrocytes, Micro = microglia, Oligo = oligodendrocytes, Excit = excitatory neurons, Inhib = inhibitory neurons. Left heatmap shows each sample’s expression (Norm. Expression) normalized to the maximum expression value in each row. Adjacent right heat map reports the Log2 of mean fold-change (FC) relative to GFP− samples, −Log10 of the q-value (p-value adjusted for False Discovery Rate), and Log2 of the maximum normalized expression value of each gene (n = 4). (C) Top 50 enriched genes in mature spinal pDyn neurons, ranked in order of Log2(FC) from highest to lowest. Only genes which met inclusion criteria of Log2(FC) > 1, q-val < 0.01, CV of GFP+ samples < 65%, and mean normalized expression of GFP+ samples > 20 were considered in this list.

Figure 3.. Genes enriched in adult spinal pDyn neurons listed by functional class.

(A) Genes of selected functional classes which are at least 2-fold enriched in spinal GFP+ pDyn neuron nuclei compared to spinal GFP− nuclei. Left heat map shows each sample’s expression (Norm. Expression) normalized to the maximum expression value in each row. Right heat map reports the Log2 of mean fold-change relative to GFP− samples, −Log10 of the q-value, and Log2 of the maximum normalized expression value of each gene (n= 4). Only genes which met inclusion criteria of Log2(FC) > 1, q-val < 0.01, CV < 65%, and mean normalized expression of GFP+ samples > 20 were considered in these lists. (B) Intersection of same selected functional classes with genes of established relevance to pain.

Figure 4.. In situ hybridization validation of selected targets enriched in adult spinal pDyn neurons.

mRNA transcript expression of (A) Gucy2d, (B) Pnoc, (C) Adra2b, and (D) Crhr1 in the spinal dorsal horn (SDH). For all targets: (1) Low magnification (20x) of in situ hybridization for target (magenta). Square shows area magnified in (2-4), and solid line indicates boundary of spinal gray matter. Scale bar = 50 μm. Higher magnification (40x) of inset area in (1) shows target mRNA (2; magenta), Pdyn mRNA (3; green), and merged (4). DAPI (blue) was used to stain nuclei in panels 2-4. Scale bar = 20 μm. (E) Npbwr1 mRNA (magenta) co-localizes with pDyn-GFP nuclei (green). Arrowheads indicate co-localized cells. Scale bar = 50 μm. (F, G) Bath application of the selective neuropeptide B/W receptor agonist W-23 (10 nM) significantly hyperpolarized pDyn-tdTomato neurons in the SDH (n = 11; t = 3.005; *p = 0.0132; paired t-test).

Figure 5.. Genes enriched in spinal pDyn neurons at three developmental time points.

(A) Top 50 enriched genes in spinal pDyn neurons at postnatal days (P) 7, 21, and 80. Right heat map reports the Log2 of mean fold-change relative to GFP− samples, −Log10 of the q-value, and Log2 of the maximum normalized expression value of each gene. P7: n = 4, P21: n = 3, P80: n = 4. (B) Venn diagram of enriched genes at each age. Only genes which met inclusion criteria of Log2(FC) > 1, q-val < 0.01, CV < 65%, and mean normalized expression of GFP+ samples > 20 were considered in the lists shown in panels A and B.

Figure 6.. Developmental changes in gene expression within spinal pDyn neurons.

(A) Clustering of genes which significantly change over time (q < 0.05; likelihood ratio test) within GFP+ nuclei. Genes were mathematically grouped based on sub-patterns of relative change in expression across time, resulting in four clusters: A, B, C, and D. Color scale indicates z-score of normalized expression values. (B) Hierarchical analysis of all GFP+ samples. (C) Representative examples of changes in gene expression across time for each cluster (P7: n = 4, P21: n = 3, P80: n = 4).

Figure 7. Relative changes in spinal pDyn neuronal gene expression across the lifespan listed by functional class.

(A) Heat maps depicting genes from select functional classes which significantly increase or decrease by at least two-fold between any two time points. Left heat map (Mean Norm. Expression) shows z-score of the mean normalized expression value at each age. Right heat map reports the Log2 of mean enrichment compared to GFP− samples across all ages, −Log10 of the q-value, and Log2 of the mean normalized expression value of each gene at the time point of highest expression (P7: n = 4, P21: n = 3, P80: n = 4). Only genes which met inclusion criteria of q-val ≤ 0.05, CV < 65% (for at least one age), and mean normalized expression of GFP+ samples > 20 (for at least one age) were considered in these lists.

Fig 8.. Quantitative in situ hybridization confirms developmental shifts in gene expression.

(A) Normalized gene expression of Npbwr1 at P7, P21, and P80 in GFP+ and GFP− populations (P7: n = 4, P21: n = 3, P80: n = 4). (B-C) Comparison of Npbwr1 mRNA transcript expression in the P7 (B) and P74 (C) spinal dorsal horn (SDH). For both B and C: (1) In situ hybridization shows Npbwr1 mRNA (white) co-localized with Sun1-GFP tagged nuclei (green), (2) Pdyn mRNA (magenta) co-localized with GFP, and (3) merged. Arrowheads indicate Sun1-GFP+ nuclei in which Npbwr1 and Pdyn mRNA are co-expressed. Scale bar = 20 μm. (D) Comparison of percentage of Sun1-GFP+ nuclei which co-express both Pdyn and Npbwr1 mRNA transcripts detected by in situ hybridization at P7 and P74 (n = 9 at each age; t = 3.747; **p = 0.0018; unpaired t-test). (E) Comparison of Npbwr1 transcript abundance per Sun1-GFP+ nucleus, as determined by the mean number of in situ hybridization signal dots per Sun1-GFP+ nucleus. Only Sun1-GFP+ nuclei which co-expressed both Pdyn and Npbwr1 mRNA transcripts were included in this analysis (n = 9 at each age; t = 4.205; ***p = 0.0007; unpaired t-test). (F) Normalized gene expression of Cartpt at P7, P21, and P80 in GFP+ and GFP− populations (P7: n = 4, P21: n = 3, P80: n = 4). (G-H) Comparison of Cartpt mRNA transcript expression in the P7 (G) and P74 (H) spinal dorsal horn (SDH). For both G and H: (1) In situ hybridization shows Cartpt mRNA (white) co-localized with Sun1-GFP tagged nuclei (green), (2) Pdyn mRNA (magenta) co-localized with GFP, and (3) merged. Arrowheads indicate Sun1-GFP+ nuclei in which Cartpt and Pdyn mRNA are co-expressed. Scale bar = 20 μm. (I) Comparison of percentage of Sun1-GFP+ nuclei which co-express both Pdyn and Cartpt mRNA transcripts detected by in situ hybridization at P7 and P74 (P7: n = 8, P74: n = 9; t = 9.464; ***p < 0.0001; unpaired t-test). (J) Comparison of Cartpt transcript abundance per Sun1-GFP+ nucleus, as determined by the mean number of in situ hybridization signal dots per Sun1-GFP+ nucleus. Only Sun1-GFP+ nuclei which co-expressed both Pdyn and Cartpt mRNA transcripts were included in this analysis (P7: n = 8, P74: n = 9; t = 4.497; ***p = 0.0004; unpaired t-test).