## 2 Materials and Methods  
### EAE Induction  
For the analysis of neurotransmitter signatures, female C57 BL/6J mice (\(n=15\)) were obtained from Envigo (Horst, Netherlands) and kept under specific pathogen-free (SPF) conditions at the animal facility of the Center for Dental, Oral and Jaw Health (Zentrum fur Zahn-, Mund- und Kiefergesundheit), University of Wurzburg, Wurzburg, Germany. For the analysis of ICCs, female C57 BL/6J mice (\(n=6\)) were obtained from Charles River (Sulzfeld, Germany) and kept under SPF conditions at the House for Experimental Therapy 4 (Haus fur Experimentelle Therapie 4, \"Maushaus\") at the University Hospital Bonn, Bonn, Germany. Female mice were used for all experiments to account for the gender bias in MS [2] and because female mice could be housed more easily in groups compared to male mice. All mice were between seven and nine weeks of age at the time point of EAE induction.  
In preparation for EAE induction, complete Freund's adjuvant (CFA) was obtained by combining nine parts paraffin oil (Sigma-Aldrich, St. Louis, MI, USA) and one part mannide monooleate (Sigma-Aldrich) with 5 mg/mL _Mycobacterium tuberculosis_ H37 Ra (BD Difco Laboratories, Franklin Lakes, NJ, USA).  
EAE was induced by MP4, which has previously been shown to induce ENS pathology in the MP [29]. All EAE mice (\(n=13\); \(n=10\) for the analysis of neurotransmitters and \(n=3\) for the analysis of ICCs) were immunized subcutaneously in both flanks with a total dose of 200 \(\upmu\)g MP4 (Alexion Pharmaceuticals, New Haven, CT, USA) emulsified in 200 \(\upmu\)L CFA. On the day of immunization and 48 h later, mice received an intraperitoneal injection of 200 ng pertussis toxin from List Biological Laboratories (Campbell, CA, USA; experiments in Wurzburg) or 120 ng pertussis toxin from Hooke Laboratories (Lawrence, MA, USA; experiments in Bonn), respectively, in 250 \(\upmu\)L sterile phosphate-buffered saline (PBS; Thermo Fisher, Waltham, MA, USA).  
For the analysis of neurotransmitter signatures, an additional cohort of mice (\(n=5\)) was immunized with 200 \(\upmu\)g hen egg lysozyme (HEL) (Sigma-Aldrich) in CFA as a control group, following the same protocol as for MP4 immunization, while for ICC analysis, the control group (\(n=3\)) remained non-immunized. In order to assess the neurotransmitter signature of the ENS over the course of the disease, two groups of EAE mice were analyzed: (i) five mice were analyzed 12 days after immunization, before the clinical onset of disease; (ii) five mice were euthanized during late-stage EAE, i.e., 25 days after the onset of clinical symptoms, corresponding to 40 days after immunization. To assess alterations of ICCs, only late-stage EAE mice were analyzed.  
All animal experiments were performed according to protocols that were approved by the Government of Lower Franconia (Regierung von Unterfranken; file nos. 55.2.2531.01-114/13 and 55.2.2532.01-91/14) or by the State Office for Nature, Environment and Consumer Protection North Rhine-Westphalia (LANUV; file no. 81-02.04.2021.A146) and complied with the German Law on the Protection of Animals, the \"Principles of Laboratory Animal Care\" (NIH publication no. 86-23, revised 1985), and the ARRIVE guidelines for reporting animal research [33].