DOI: 10.5061/dryad.fttdz093m
title: Data from: Ca²⁺ signaling in myenteric interstitial cells of Cajal (ICC-MY) and their role as conditional pacemakers in the colon

description: Interstitial cells of Cajal in the plane of the myenteric plexus (ICC-MY)
 serve as electrical pacemakers in the stomach and small intestine. A
 similar population of cells is found in the colon, but these cells do not
 appear to generate regular slow wave potentials, as characteristic in more
 proximal gut regions. Ca2+ handling mechanisms in ICC-MY of the
 mouse proximal colon were studied using confocal imaging of muscles from
 animals expressing GCaMP6f exclusively in ICC. ICC-MY displayed
 stochastic, localized Ca2+ transients that seldom propagated
 between cells. Colonic ICC express ANO1 channels, so
 Ca2+ transients likely couple to activation of spontaneous
 transient inward currents (STICs) in these cells. The
 Ca2+ transients were due to Ca2+ release and blocked by
 cyclopiazonic acid (CPA), thapsigargin and caffeine, but unaffected by
 tetracaine. Antagonists of L- and T-type Ca2+ channels and
 reduction in extracellular Ca2+ had minimal effects on
 Ca2+ transients. We reasoned that STICs may not activate
 regenerative Ca2+ waves in ICC-MY because voltage-dependent
 Ca2+ conductances are largely inactivated at the relatively
 depolarized potentials of colonic muscles. We tested the effects of
 hyperpolarization with pinacidil, a KATP agonist.
 Ca2+ waves were initiated in some ICC-MY networks when muscles
 were hyperpolarized, and these events were blocked by a T-type
 Ca2+ channel antagonist, NNC 55-0396. Ca2+ waves activated by
 excitatory nerve stimulation were significantly enhanced by
 hyperpolarization. Our data suggest that colonic ICC-MY are conditional
 pacemaker cells that depend upon preparative hyperpolarization, produced
 physiologically by inputs from enteric inhibitory neurons and necessary
 for regenerative pacemaker activity.
description:   <strong>Ca2+
 imaging</strong> <br>Segments of
 proximal colon (1–2 cm or 2–3 cm in length) were removed from mice and
 placed in Krebs-Ringer bicarbonate solution (KRB). The colonic segments
 were opened along the mesenteric border and intraluminal contents were
 washed away with KRB. After removing the mucosa, the muscle sheets were
 pinned to a Sylgard coated dish and perfused with oxygenated KRB solution
 at 37 °C for a 60 min equilibration period peristaltic pump (minipuls 3,
 Gilson, WI, USA) with an electric in-line heater and controller (SH-27B
 and TC-344C, Warner instruments, CT, USA).
 Ca<sup>2+</sup> imaging was performed using a spinning-disk,
 confocal system (CSU-W1; spinning disk, Yokogawa Electric, Tokyo, Japan)
 mounted on an upright Nikon Eclipse FN1 microscope equipped with several
 water immersion Nikon CFI Fluor lenses (10 × 0.3 NA, 20 × 0.5 NA, 40 × 0.8
 NA, 60 × 0.8 NA and 100 × 1.1 NA) (Nikon Instruments, New York, USA). The
 system is equipped with two solid-state laser lines of 488 nm and 561 nm.
 The laser lines are combined with a borealis system (ANDOR Technology,
 Belfast, UK) to increase laser intensity and uniformity throughout the
 imaging field of view (FOV). The system also has two high-speed electron
 multiplying charged coupled devices (EMCCD) cameras (Andor iXon-Ultra 897
 EMCCD Cameras; ANDOR Technology, Belfast, UK) to maintain sensitive and
 fast speed acquisition at full frame of 512 × 512 active pixels as
 previously described[15, 17]. Briefly, images were captured and image
 sequences were collected at 33 fps using MetaMorph software (MetaMorph
 Inc., TN, USA) using a 60x objective. Most FOVs contained 2–3 cells, as
 shown in supplemental movie 2. In some FOVs, clusters of cells were observed, as seen in supplemental movie 1. Cells located outside the focal plane were excluded from the analysis. For experiments with pharmacological agents, a control activity period (20–30 s) was recorded prior to drug application into the chamber for 20 min. Due to the contractile activity of the colon muscles during Ca<sup>2+</sup> imaging, post-acquisition analysis was often difficult to perform because of movement artifacts. Therefore, contractions of colonic muscle tissues were reduced by addition of ML7 hydrochloride (selective inhibitor of myosin light chain kinase; 10–50 mM). The muscles were exposed to ML7 hydrochloride throughout our experiments to minimize movement artifacts and allow high-resolution imaging.   <strong>Ca2+ imaging analysis</strong>   Movies of Ca<sup>2+</sup> transients in ICC-MY were imported, preprocessed and analyzed using Fiji/Image J (National Institutes of Health, MD, USA, http://rsbweb.nih.gov/ij); Briefly, movies of Ca<sup>2+</sup> transients (stacks of TIFF images) were imported into Fiji and background subtracted and smoothed (Gaussian filter: 1.5 × 1.5 µm, StdDev 1.0). The Ca<sup>2+</sup> signal was plotted in a 2D Ca<sup>2+</sup> occurrence maps (x axis= time, y axis= cell space) that gives information on their spatial and temporal activation. Signal segmentation and analysis were preformed using an automated machine learning plugin (STMapAuto), https://github.com/gdelvalle99/STMapAuto and 4SM software https://github.com/SharifAmit/CalciumGAN/tree/main as described previously [[21], [22], [23]]. To analyze ICC-MY Ca<sup>2+</sup> waves, we employed Fiji/ImageJ software and manually calculated the parameters of these waves. The Ca²⁺ event parameters include frequency (min<sup>-1</sup>), duration (s), area (squared pixels height and width μm*s), and spatial spread (mm). For accuracy, Ca²⁺ waves were identified and quantified only when they were synchronized with other cells within the field of view (FOV). This approach ensured that only relevant, coordinated Ca²⁺ wave events were included in the analysis, providing a more accurate representation of intercellular Ca²⁺ wave dynamics.
description: # Data from: Ca²⁺ signaling in myenteric interstitial cells of Cajal
 (ICC-MY) and their role as conditional pacemakers in the colon author
 links open overlay panel
 [https://doi.org/10.5061/dryad.fttdz093m](https://doi.org/10.5061/dryad.fttdz093m) ## Description of the data and file structure -These data files are generated using prisim software. It contains the calcium firing parameters (Frequency, duration, spatial spread and area events). ### Files and variables #### File: ICC-MY-ALLCONTROLS-NOFLASHES-COLON-CALCIUM-.pzfx **Description:** Dataset for stochastic calcium signals  #### File: ICC-MY-ALLCONTROLS-FLASHES-COLON-CALCIUM-.pzfx **Description:**  Dataset for rhythmic calcium signals  ## **-STMmaps datasets:  generated from cellular calcium transients**  -STMmaps datasets:  generated from cellular calcium transients  File: Paper1-Dryad.zip The file includes datasets from ICC-MY cellular calcium events under control and pharmacological agents This directory contains subfolders related to different compounds used in experiments. Each folder contains data corresponding to specific control subfolder and  treatments subfolder. Below is a brief description of each subfolder: * **Caffeine** – Contains experimental data related to caffeine and its effects on cellular activity. 2 subfolders: control and in the presence of caffeine * **CPA** – Includes data for Cyclopiazonic Acid (CPA), a SERCA inhibitor affecting calcium signaling. 2 subfolders: control and in the presence of CPA * **Isradipine** – Contains data for experiments using Isradipine, a calcium channel blocker. 2 subfolders: control and in the presence of Isradipine * **Nicardipine** – Stores experimental results related to Nicardipine, another calcium channel blocker. 2 subfolders: control and in the presence of Nicardipine * **Nicardipine (waves)** – A subset of Nicardipine experiments specifically analyzing wave-like calcium signaling patterns. 2 subfolders: control and in the presence of Nicardpine * **NNC (waves)** – Contains data related to NNC, with a focus on wave propagation in calcium signaling. 2 subfolders: control and in the presence of NNC * **Tetracaine** – Includes experimental data involving Tetracaine, a local anesthetic known to influence calcium dynamics. 2 subfolders: control and in the presence of Tetracaine * **Thapsigargin** – Contains results from experiments with Thapsigargin, a known SERCA inhibitor that disrupts calcium homeostasis. 2 subfolders: control and in the presence of Thapsigargin ## **Code/software** Prism software : [https://www.graphpad.com/features](https://www.graphpad.com/features)  STMapAuto software: [https://github.com/gdelvalle99/STMapAuto](https://github.com/gdelvalle99/STMapAuto)