## 2 Materials and Methods
### Whole-Mount Staining  
For the analysis of neurotransmitter signatures, one approximately 1 cm long segment was immediately removed from the distal colon of each mouse, cleared of fecal matter, and a peripheral venous catheter was placed into the colonic lumen. After washing with phosphate-buffered saline (PBS) (Roth, Karlsruhe, Germany and Merck, Darmstadt, Germany), the colon was filled with 4% paraformaldehyde (PFA) solution (Roth), the ends tied, and then placed into 4% PFA for 30 min. Subsequently, the colon was cut open longitudinally, and the segments were then subjected to whole mount preparation for immunohistochemical (IHC) staining of the ENS. The whole mounts were preincubated at room temperature for 2 h with 0.05 M tris-buffered saline (TBS; pH 7.4) (Roth) containing 1% bovine serum albumin (BSA) (Roth), 0.5% Triton X-100 (Merck Millipore, Burlington, MA, USA), 0.05% thimerosal (Roth), and 5% normal donkey serum (Dianova, Hamburg, Germany). After rinsing the whole mounts in TBS, they were incubated at 4 \({}^{\circ}\)C for 96 h with the primary antibodies diluted in TBS containing 1% BSA, 0.5% Triton X, and 0.05% thimerosal. The four primary antibodies were an Alexa Fluor 555-conjugated anti-HuC/D pan-neuronal antibody (HU) (Thermo Fisher; dilution 1:200; labeled with an antibody-labeling kit from Thermo Fisher), and antibodies against ChAT (Sigma-Aldrich; 1:40), neuronal nitric oxide synthase (nNOS) (University of Graz, Austria; 1:1000), and VIP (Biomedicals, Augst, Switzerland; 1:200).  
Specificity of the nNOS antibody was confirmed by preabsorption control experiments in previous studies at the Institute of Anatomy and Cell Biology in Erlangen [34; 35].  
Afterwards, the whole mounts were washed in TBS and incubated at room temperature for 4 h with the following three secondary antibodies: donkey anti-guinea pig DyLight 488 (Dianova; dilution 1:500), donkey anti-goat Alexa Fluor 647 (Thermo Fisher; 1:1000), and donkey anti-rabbit DyLight 405 (Dianova; 1:200). After washing with TBS, all whole mounts were mounted with TBS-glycerol (Roth; 1:1; pH 8.6).  
For ANO1 staining, colonic tissue was rinsed for clearance of fecal matter and threaded onto a glass rod. The remaining mesentery was removed, and tissue was incised carefully lengthwise using tweezers. Using a cotton swab soaked in buffer, longitudinal muscle layer and attached myenteric plexus (LMMP) were separated from the remaining tissue by carefully rubbing the cotton swab around the tissue. LMMP was cut into approximately 1 \(\times\) 0.5 cm\({}^{2}\) pieces and transferred into a 96-well plate. Tissue was blocked with 10% donkey serum (Biozol, Eching, Germany) in a buffer consisting of TBS + 0.5% Triton-X 100 (MP Biomedicals, Irvine, CA, USA), 1% BSA (Merck) and 0.02% sodium azide (Merck) (whole-mount buffer) for 5 h at room temperature on a plate shaker.  
Primary antibodies against GFAP (chicken polyclonal to GFAP, Abcam, Cambridge, UK; 1:400) and ANO1 (rabbit polyclonal to TMEM16a, Abcam; 1:200) were diluted in whole-mount buffer and applied for 72 h at 4 \({}^{\circ}\)C on a plate shaker. The tissue was washed in TBS + 0.05% Tween 20 (VWR, Radnor, PA, USA) (TBST) before the secondary antibodies donkey anti-chicken Cy2 (Jackson ImmunoResearch, West Grove, PA, USA, 1:200) and donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 1:200) were applied and incubated for 2 h at room temperature on a plate shaker. After another washing step using TBST, DAPI (Roche, Basel, Switzerland) was diluted 1:1000 in TBST and applied for 15 min. After rinsing in TBS, whole mounts were mounted using a 1:1 mixture of TBS and glycerol (Merck).