DOI: 10.5061/dryad.7wm37pw71
title: Role of interstitial cells of Cajal in regulating tone and responses to enteric motor neurons in the murine pyloric sphincter

description: Gastric slow waves fail to propagate through the pyloric sphincter (PS),
 thus isolating the specialized motility patterns of the stomach and small
 intestine. We investigated the role of interstitial cells of Cajal (ICC)
 in PS of mice.  Ca2+ waves in ICC, events responsible for
 electrical slow waves, propagated along the gastric wall but failed to
 propagate into the PS.  ICC in PS fired localized Ca2+ transients
 and displayed low expression of voltage-dependent Ca2+
 conductances.  These are properties of intramuscular ICC (ICC-IM)
 that cannot regenerate and propagate slow waves.  A T-type Ca2+
 channel antagonist had no effect on Ca2+ transients, but these events were
 blocked by thapsigargin and CPA, suggesting they result from Ca2+ release.
  PS ICC expressed ANO1, a Ca2+-activated Cl-
 conductance.  Ca2+ released from stores actives ANO1 channels,
 thus exerting a depolarizing influence on PS.  Ani9, selective
 antagonist of ANO1 channels, hyperpolarized cells and reduced contractile
 tone.  Electrical field stimulation (EFS) of intrinsic neurons
 yielded inhibitory junction potentials (IJPs), and cessation of EFS
 resulted in post-stimulus depolarization and contraction. L-NNA abolished
 relaxation responses to EFS and switched responses to contractions.
  Application of atropine or Ani9 (in the presence of L-NNA)
 abolished contraction during EFS.  Our results describe new and
 fundamental functions of ICC-IM in the PS.  The inability of
 these cells to propagate slow waves provides the insulator function of PS
 muscles and localized Ca2+ transients and activation of ANO1 regulates PS
 tone and mediates inputs from enteric neurons. 
description: # Role of interstitial cells of Cajal in regulating tone and responses to
 enteric motor neurons in the murine pyloric sphincter Dataset DOI:
 [10.5061/dryad.7wm37pw71](https://doi.org/10.5061/dryad.7wm37pw71) ## PS
 & ICC Ca²⁺ Imaging and Electrophysiology Dataset This dataset contains
 summary data analyses of calcium imaging, gene expression, and
 electrophysiological experiments in interstitial cells of Cajal (ICC) and
 pyloric sphincter (PS) muscles in mice. All data files are provided as
 GraphPad Prism (.prism) files. Supplemental movies illustrating calcium
 imaging experiments are also included. ### Prism Data Files
 Fig_1ICC_Ca2__imaging_in_the_whole_murine_stomach._Calcium_waves_freq_and_propagation.prism Calcium waves across the whole stomach (corpus to antrum) in ICC GCaMP mice. ST maps were generated using Fiji/ImageJ; duration and propagation velocity of each wave were measured and analyzed with GraphPad Prism 10. Fig_2-ICC_Ca2__signals_pyloric_sphincter._Frequency_and_duration_of_PS_imaging.prism ICC cells in the PS were imaged to measure calcium transients, and the frequency and duration were quantified using Fiji/ImageJ and normalized to control wells before analysis with GraphPad Prism. Fig_3-Effects_of_Ca2_-free_solution_and_SERCA_inhibitors_on_the_spatio-temporal_(ST)_analysis_of_Ca2__transients_in_the_PS.prism Calcium activation signals in PS ICC were imaged. Spatio-temporal (ST) maps were generated using Fiji/ImageJ, and the frequency of each wave was measured and analyzed with GraphPad Prism. Fig_4-Effects_of_T-type_Ca2__channel_blocker_(Z944)*on_the_spatio-temporal*(ST)_analysis_of_Ca2__transients_in_the_PS..prism PS ICC cells were treated with the T-type Ca²⁺ channel blocker Z944. Calcium transients were analyzed using spatio-temporal (ST) maps generated with Fiji/ImageJ, normalized to control conditions, and the frequency, duration, and propagation velocity of each wave were quantified using GraphPad Prism. Fig_5_The_quantitative_transcriptional_analysis_of_possible_Ca2__influx_gene_candidates_and_immunohistochemistry_of_ICC_and_ANO1_in_the_PS.__.prism Transcriptional analysis of Ano1 and voltage-gated Ca²⁺ channel genes was performed in isolated ICC of the PS. Expression of c-kit and ANO1 was confirmed by immunohistochemistry. Fig_6-Fig_6._Effects_of_Ani9_on_mechanical_tone_and_membrane_potential_of_the_PS_muscle_strips..prism PS muscle strips were treated with Ani9 to assess contractile tone and resting membrane potential. Contractile responses, including AUC and resting membrane potential, were measured. Additionally, in the presence of Ani9, electrically evoked contractions were recorded, and both the on-response and post-stimulus contraction (PSC) were quantified. All data were analyzed using GraphPad Prism.   Fig_7-LNNA_Ani9_and_atropine_on___off_response-of_muscle_contraction.prism Ongoing contractile activity of PS muscle strips was recorded in the presence of TTX. Additionally, contractile responses and EFS under LNNA + Ani9 and LNNA + atropine treatments were quantified. The area under the curve (AUC) of these responses was analyzed using Clampfit, and all data were further analyzed with GraphPad Prism. ### Supplemental Movies #### movie1.avi Supplemental Movie 1. Use of Ca^2+^ imaging to monitor slow wave propagation in mouse stomach. Calcium imaging to monitor slow wave propagation in mouse stomach was performed, fluorescence intensity measured across stomach regions, and data are included for visualization. #### movie2.avi Supplemental Movie 2. Ca^2+^ transients in ICC within the pyloric sphincter (PS).  Calcium transients in ICC within the pyloric sphincter were recorded, fluorescence intensity measured across ICC clusters, and data are included for visualization.