Data from: Ca²⁺ signaling in myenteric interstitial cells of Cajal (ICC-MY) and their role as conditional pacemakers in the colon

Baker, Sal 1 ; Karwa, Manushri1; Lee, Ji Yeon1; Riar, Sarah1; Drumm, Bernard2; Sanders, Kenton1

Published Jan 31, 2025 on Dryad. https://doi.org/10.5061/dryad.fttdz093m

Data files

Jan 31, 2025 version files 23.72 MB

ICC-MY-ALLCONTROLS-FLASHES-COLON-CALCIUM-.pzfx
55.09 KB
ICC-MY-ALLCONTROLS-NOFLASHES-COLON-CALCIUM-.pzfx
68.19 KB
Paper_1-Dryad.zip
23.59 MB
README.md
2.81 KB

Abstract

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 K_ATP 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.

README: 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

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

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

STMapAuto software: https://github.com/gdelvalle99/STMapAuto

Methods

Ca2+ imaging

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). Ca²⁺ 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 Ca²⁺ 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.

Ca2+ imaging analysis

Movies of Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signal was plotted in a 2D Ca²⁺ 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 Ca²⁺ waves, we employed Fiji/ImageJ software and manually calculated the parameters of these waves. The Ca²⁺ event parameters include frequency (min^-1), 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.