Fig 2: Examples of overlap between MOR and DOR in different myenteric neuronal populations of distal colon. (A) All MOR-DOR expressing cells were positive for pan-neuronal marker Hu (arrows). (B) Both nNOS positive (arrows) and negative (arrowheads) neurons expressed MOR-DOR. Similarly, MOR and DOR were coexpressed in subset of both ChAT (C) and CalR (D) positive neuronal populations. Scale: 50 µm.

Fig 3: MORmCherry expression in the submucosal plexus. (A) MORmCherry labeling in the ileum was not detected in neurons of the submucosal plexus, as identified by NPY and Hu staining. (B) MORmCherry was expressed by small number of submucosal neurons of distal colon. These neurons were generally nNOS positive. (C) MOR and DOR were coexpressed in very limited number of submucosal neurons. Scale: 50 μm. Arrows represent coexpression of markers.

Fig 4: CYM51010 is a selective DOR agonist in the ENS. (A) CYM51010 evoked concentration-dependent contractions (A) that were effectively blocked by NLT (B) but not by CTOP (C). (D) Concentration-response curves of contractions evoked by CYM51010. Data are presented as mean ± standard error of the mean, n = 7–10 mice per group. Treatment groups were compared at each individual concentration by Kruskal-Wallis test, followed by Dunn’s post hoc analysis (**P < .01 and *P < .05 compared with vehicle-treated group). (E) DOR-eGFP was internalized from the cell surface to endosomes of myenteric neurons of the colon after treatment with SNC80 (data from Figure 9D) and CYM51010. CYM51010-evoked internalization was effectively blocked by NLT but not by CTOP. No redistribution of DOR-eGFP was detected on stimulation with MOR agonist DAMGO (taken from Figure 9D). (F) Both SNC80 and CYM51010, but not DAMGO, promoted DOR-eGFP internalization in submucosal neurons of the ileum, a site in which no MORmCherry expression was detected. Scale: 20 µm. Arrowheads, DOR-eGFP retained at plasma membrane; arrows, DOR-eGFP internalized. (G) Quantitative analysis of DOR-eGFP distribution in myenteric neurons. (H) Equivalent DOR-eGFP endocytosis in NOS positive and negative populations in response to CYM51010 (1 µmol/L). Selective inhibition of DOR (NLT) did not result in a difference in CYM51010-evoked DOR-eGFP internalization between the 2 populations. Similarly, there was no difference in DOR-eGFP internalization in NOS positive and negative populations after treatment with either CYM51010 in the presence of CTOP or MOR agonist DAMGO. (I) Quantitative analysis of DOR-eGFP distribution in submucosal neurons. Endocytosis data are presented as mean values ± standard error of the mean and were analyzed by one-way analysis of variance with Dunnett’s post hoc test. Individual data points are represented as triangles. ***P < .001 compared with vehicle treatment. (J) CYM51010 produced a concentration-dependent increase in G protein activation only in DOR expressing CHO cells. Data are presented as mean ± standard error of the mean from 4 independent experiments.

Fig 5: Examples of overlap between MOR and DOR in different myenteric neuronal populations of the ileum. (A) Both nNOS positive (arrows) and negative (arrowheads) neurons coexpressed MOR and DOR. Similarly, MOR and DOR were coexpressed in subset of both ChAT (B) and CalR (C) positive neuronal populations. Scale: 50 µm.