Size of isolated DRG neurons and their expression of oxytocin and its related receptors
The diameters of single DRG neurons picked up from the culture dishes were measured with a micrometer (Fig. 1a). The diameters varied from 14.5 µm to 49.0 µm (n = 79). The amount of the mRNA for 5 molecules in relation to OXT, i.e., OXT, OXTR, V1aR, TRPV1, and Piezo2, was examined in single DRG neurons by using real-time PCR. When fluorescence signal strength above threshold was obtained by the 40th cycle (Ct ≦ 40), we judged that the single DRG neuron expresses the target molecule. Of 79 single DRG neurons, 58 neurons expressed Piezo2, 38 neurons TRPV1, 31 neurons OXTR, 23 neurons OXT, and 4 neurons V1aR (Fig. 1b).
Cluster Analysis Of Isolated Single Drg Neurons
A cluster analysis was performed by using the Morisita-Horn index and the Ward method (Fig. 2). Based on the gene expression pattern, DRG neurons were classified into 4 groups. Of 79 single DRG neurons, Cluster 1 contained 46 neurons, Cluster 2 contained 16 neurons, Cluster 3 contained 12 neurons, and Cluster 4 contained 5 neurons. Cluster 1 had the most distinctive gene expression pattern compared to other clusters.
Cell Body Size And Expression Of Oxytocin And Its Related Receptors In The 4 Clusters
The characteristics of the 4 clusters were examined (Fig. 3). The median diameters of DRG neurons in Clusters 1, 2, 3, and 4 were 34.5, 19.0, 24.0, and 20.0 µm, respectively (Fig. 3a). The cell sizes of Cluster 1 DRG neurons were significantly larger than those of Cluster 2 neurons (P < 0.01). When focusing on the percentage of cells expressing a particular target molecule in the cluster, we found that Cluster 1 was characterized mainly by the high expression of Piezo2, Cluster 2 by TRPV1, and Cluster 4 by OXTR, whereas cells in Cluster 3 did not express any of the target genes (Fig. 3b-e).
Comparison Of Characteristics Between Cluster 1 And Cluster 2
Comparing Cluster 1 and Cluster 2, the relative expression level of TRPV1 was significantly higher in Cluster 2 (Fig. 4a, P < 0.01). It is important to note that the smaller ΔCt is, the higher the expression level is. For Piezo2, the relative expression level in Cluster 1 was significantly higher compared to Cluster 2 (Fig. 4b, P < 0.01). For OXT and OXTR, there were no significant statistical differences between Cluster 1 and Cluster 2 (Fig. 4c, d). When we focused on OXT-expressing cells among DRG neurons, average diameter of OXT-expressing neurons in Cluster 1 was 40.5 µm, indicating that OXT-expression neurons are relatively large cells in Cluster 1. On the other hand, average diameter of OXT-expressing neurons in Cluster 2 was 18.1 µm, which was significantly smaller than that in Cluster 1 (Fig. 4e, P < 0.01).
Co-expression Of Oxt And Trpv1 And That Of Oxt And Piezo2 In Single Drg Neurons
Previous reports showed that OXT and TRPV1 were co-expressed in DRG neurons [10]. Our single-cell real-time PCR approach showed that about three fourths of DRG neurons expressing OXT also co-expressed TRPV1 (Fig. 5), reconfirming the co-expression of OXT and TRPV1 in DRG neurons [10]. Furthermore, most OXT-expressing DRG neurons, including those do not express TRPV1, were found to co-express Piezo2. As far as we know, the co-expression of OXT and Piezo2 was confirmed for the first time in the present study.