Assessment of co-localization of hpChAT and nNOS in the preparations shown in Figure 9. The distance between each center of two different color spots was measured, using Imaris 9.5 “center point” mode, to identify co-labeled neurons being close in distance (<10.5 μm). Co-labeled spots (yellow), extracted using the ImarisColoc tool, were superimposed in hpChAT-ir (blue, A-C) or nNOS-ir (purple, D-F) spot images. A, D, G, inner submucosal plexus (ISP). B, E, H: outer submucosal plexus OSP). C, F, I, myenteric plexus (MP). The bottom images (G-I) show co-labeled neurons intermingled with both hpChAT and nNOS ones. Bars =100 μm. J, The co-localization was calculated in 4–10 ganglia of each plexus from a patient and expressed as the percentage of co-labeled neurons in hpChAT-ir or nNOS-ir neurons. Error bars: mean ±SEM of 3–4 patients (3 for ISP and OSP). Significant difference in number: *** or < 0.001 or p < 0.01 compared with hpChAT-ir neurons in ISP or OSP, ###p < 0.001 compared with hpChAT-ir neurons in OSP, respectively; +++p < 0.001 compared with nNOS-ir neuorns in ISP; &&p < 0.01 compared with nNOS-ir neurons in OSP