Profile of dorsal root ganglion neurons: study of oxytocin expression

Abstract

Although dorsal root ganglion (DRG) neurons have been so far classified according to the difference in their fibers (Aβ, Aδ, and C), this classification should be further subdivided according to gene expression patterns. We focused on oxytocin (OXT) and its related receptors, because OXT plays a local role in DRG neurons. We measured the mRNA levels of OXT, OXT receptor (OXTR), vasopressin V1a receptor (V1aR), transient receptor potential cation channel subfamily V member 1 (TRPV1), and piezo-type mechanosensitive ion channel component 2 (Piezo2) in single DRG neurons by using real-time PCR, and then performed a cluster analysis. According to the gene expression patterns, DRG neurons were classified into 4 clusters: Cluster 1 was characterized mainly by Piezo2, Cluster 2 by TRPV1, Cluster 4 by OXTR, and neurons in Cluster 3 did not express any of the target genes. The cell body diameter of OXT-expressing neurons was significantly larger in Cluster 1 than in Cluster 2. These results suggest that OXT-expressing DRG neurons with small cell bodies (Cluster 2) and large cell bodies (Cluster 1) probably correspond to C-fiber neurons and Aβ-fiber neurons, respectively. Furthermore, the OXT-expressing neurons contained not only TRPV1 but also Piezo2, suggesting that OXT may be released by mechanical stimulation regardless of nociception. Thus, mechanoreception and nociception themselves may induce the autocrine/paracrine function of OXT in the DRG, contributing to alleviation of pain.

Fig. 1

Size of isolated DRG neurons and gene expression in the cells. a Diameters of DRG neurons varied from 14.5 to 49.0 μm. The total number of the isolated cells was 79. b Percentage of DRG neurons expressing each target molecule. OXT oxytocin, OXTR OXT receptor, V1aR vasopressin V1a receptor, TRPV1 transient receptor potential cation channel subfamily V member 1, Piezo2 piezo-type mechanosensitive ion channel component 2

Fig. 2

Cluster analysis for isolated DRG neurons. The neurons were classified into 4 clusters by cluster analysis of gene expression patterns using the Morisita-Horn index. Cluster 1, Cluster 2, Cluster 3, and Cluster 4 contained 46, 16, 12, and 5 cells, respectively. The height was obtained by the Ward method

Fig. 3

Characterization of the 4 clusters. a Diameters of DRG neurons in each cluster are expressed in a box plot. **P < 0.01. b–e Percentage of isolated DRG neurons expressing target molecules in Cluster 1, Cluster 2, Cluster 3, and Cluster 4 are shown. The abbreviations are the same as those in Fig. 1

Fig. 4

Comparison between Cluster 1 and Cluster 2. a Comparison of ΔCt value for TRPV1. The smaller the ΔCt value is, the larger the expression level is. **P < 0.01. b Comparison of ΔCt value for V1aR. c Comparison of ΔCt value for OXT. There is no significant difference between the 2 clusters. d Comparison of ΔCt value for OXTR. There is no significant difference between the 2 clusters. e Diameters of OXT-expressing neurons in Cluster 1 and Cluster 2. **P < 0.01

Fig. 5

Co-expression of OXT and TRPV1 and that of OXT and Piezo2 in DRG neurons. When assuming OXT-expressing neurons was 100%, RPV1(+) and Pizo2(+) in OXT-expressing neurons was 65%; TRPV1(+) and Pizo2(−) was 9%; and TRPV1(−) and Pizo2(+) was 26%