## 2 Materials and Methods
### Image Analysis  
Murine whole mounts stained for neurotransmitters were analyzed using a confocal laser scanning microscope system (Nikon Eclipse E1000-M; Nikon Digital Eclipse C1; Nikon, Tokyo, Japan) fitted with 488 nm and 543 nm solid-state lasers (Sapphire 488 LP, Sapphire 561-50; both from Coherent, Santa Clara, CA, USA), a 405 nm diode laser (CUBE 405-100C; Coherent), and a 642 nm diode laser ([PERSON], Carlsbad, CA, USA). A BIO1 filter set (DAPI/Cy5 for C1-Detector; AHF Analysentechnik, Tubingen, Germany) reduced nonspecific background fluorescence. Images were taken as z-series (z-steps: 1.0 \(\upmu\)m). Only ganglia and neurons that could be fully captured were considered. During analysis, detailed attention was paid to neurons located in more than one z-position to avoid multiple false-positive counts. For each mouse, ten images were analyzed from both the SP and MP. Most images displayed more than one ganglion. The mean number of neurons per ganglion was calculated as was the percentage of neurons containing either ChAT, nNOS, or VIP in relation to the total number of HU\({}^{+}\) neurons. Exemplary single-optical sections were selected from the z-series for representation in the figures.  
Murine tissue stained for ANO1 was analyzed using a Leica DMI8 Thunder imager equipped with a K5-14401790 camera (Leica) and the Leica filter cube DFT51010 with DAPI (excitation filter (EX) 391/32, dichroic mirror (DC) 415, suppressor filter (SF) 435/30), Cy2 (EX 479/33, DC 500, SF 519/25), and Cy3 (EX 554/24, DC 572, SF 594/32). For each whole mount, six z-stacks (z-steps: 2.4 \(\upmu\)m) were acquired from different locations. For each z-stack, one single-optical section that displayed the MP best was selected and analyzed. GFAP was used as a marker for the MP because ICCs are located at the level of the MP and within the longitudinal and circular muscle layer [21].  
The ANO1-positive (ANO1\({}^{+}\)) area was determined by setting a grayscale threshold in the corresponding channel after images underwent instant computational clearing using the thunder function of the LAS X software (Leica).  
After IHC staining, sections of human colon were analyzed using a Leica DM6 B fluorescence microscope, equipped with a Leica DFC3000G camera and the following filters: DAP (EX 350/50, DC 400, SF 460/50), L5 (EX 480/40, DC 505, SF 527/30), RHO (EX 546/10, DC 560, SF 585/40), and Y5 (EX 620/60, DC 660, SF 700/76). For the determination of neurotransmitter signatures, tissue from five patients with MS and 14 CTRL patients (Table 3) was analyzed. All HU\({}^{+}\) ganglia were imaged for IHC analysis.  
Light microscopic ANO1 staining was analyzed using a Leica DM6 B fluorescence microscope, equipped with a Leica DMC2900 camera and the filter block ANT (Leica). Staining for ANO1\({}^{+}\) ICCs was performed on sections from five patients with MS and 10 CTRL patients (Table 3); only tissue containing MP was included. Owing to their small cell bodies and numerous processes [9], ICCs cannot be counted as easily as neurons. Instead, entire sections of colon were scanned and, using ImageJ version 1.53c (National Institutesof Health, Bethesda, MD, USA), the areas of longitudinal and circular muscle tissue, MP, and ANO1+ ICCs were measured and compared.  
Image analysis was performed using Fiji software version 2.0.0-rc-68/1.52 h (National Institutes of Health) [36]. Exemplary images were selected for representation in the figures, which were prepared using Inkscape 1.0 (Inkscape Project, Boston, MA, USA).