Calcium transients in intramuscular interstitial cells of Cajal of the murine gastric fundus and their regulation by neuroeffector transmission

Abstract

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell-specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC-IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high-speed video-imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca²⁺ and post-junctional electrical responses to neural stimulation. ICC-IM were highly active, generating intracellular Ca²⁺-transients that occurred stochastically, from multiple independent sites in single ICC-IM. Ca²⁺-transients were not entrained in single ICC-IM or between neighbouring ICC-IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca²⁺-transients in ICC-IM. This inhibitory response was often preceded by a summation of Ca²⁺-transients that led to a global rise in Ca²⁺. Individual ICC-IM responded to nerve stimulation by a global rise in Ca²⁺ followed by inhibition of Ca²⁺-transients. The inhibition of Ca²⁺-transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in intracellular Ca²⁺ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video-imaging revealed that the rise in Ca²⁺ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca²⁺-transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Graphical Abstract

Intramuscular interstitial cells of Cajal (ICC-IM) of the gastric fundus receive nitrergic inhibitory and cholinergic excitatory neuroeffector motor inputs. Using a genetically encoded calcium sensor we demonstrate that ICC-IM are highly active cells generating stochastic intracellular Ca²⁺-transients. Stimulation of enteric motor nerves abolished Ca²⁺-transients in ICC-IM, produced an inhibitory junction potential (IJP) and muscle relaxation that was mediated by nitric oxide (left hand side of figure). This inhibitory response was often preceded by a global rise in intracellular Ca²⁺ in ICC-IM, a rapid excitatory junction potential (EJP) and muscle contraction that was mediated by acetylcholine (right hand side of figure). Individual ICC-IM could respond to both excitatory and inhibitory neural inputs. These data support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Introduction

The proximal stomach plays an important role in digestion by actively relaxing to accommodate ingestion of meals and then contracting to slowly empty the stored food into the distal stomach where larger masses of food are broken down into smaller, digestible particles (Desai et al., 1991; Tack et al., 2002). Fundus tone is modulated by contractions and relaxations of the tunica muscularis, and the state of fundus muscle excitability is regulated by responses that develop in an integrated, electrically coupled network of cells consisting of smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α positive (PDGFRα⁺), termed the SIP syncytium (Sanders 2019; Sanders & Ward 2019; Sanders et al, 2012; 2014). In the stomach, neural inputs to the SIP syncytium come from enteric motor neurons; excitatory inputs are largely cholinergic and inhibitory inputs are predominantly nitrergic; however, purinergic neurotransmission is also apparent (Kurahashi et al., 2014; Shaylor et al., 2016). Post-junctional neural responses are transduced by the cellular components of the SIP syncytium (Sanders et al., 2014).

Previous studies testing the role of ICC in the gastric fundus utilized W/W^v mice, that were shown to have developmental defects in specific populations of ICC (Burns et al., 1996; Ward et al., 1994). Intramuscular interstitial cells of Cajal (ICC-IM), the only population of ICC in the fundus, are significantly depleted in W/W^v mice (Burns et al., 1996), and nitrergic and cholinergic neural inputs are reduced or compromised in these mice (Beckett et al., 2017; Bhetwal et al., 2013; Burns et al., 1996; Ward et al., 2000). It was also shown that ICC-IM in the stomach make close, synapse-like contacts with varicosities of enteric motor neurons and form gap junctions with neighbouring SMCs (Beckett et al., 2005; Horiguchi et al., 2003; Sanders et al., 2014). Thus it was postulated that post-junctional motor responses developing in ICC-IM conduct to smooth muscle cells of the SIP syncytium (Sanders et al., 2012). However, this concept has been controversial, and others have concluded that ICC are not necessary for enteric motor neurotransmission (Goyal & Chaudhury, 2010; Huizinga et al., 2008; Sarna, 2008; Zhang et al., 2010).

ICC in GI muscles, including those of humans, express Ca²⁺-activated Cl⁻ channels, encoded by Anoctamin 1 (Ano1; Blair et al., 2012; Gomez-Pinilla et al., 2009; Hwang et al., 2009; Rhee et al., 2011; Zhu et al., 2009; 2011). For example, ICC-IM in the murine gastric fundus display robust expression of Ano1 transcripts and Ano1 protein, but Ano1 is not detectible in SMCs or PDGFRα⁺ cells (Sung et al., 2018). It was recently demonstrated that knockdown of Ano1 and Ano1 antagonists inhibited fundus cholinergic excitatory post-junctional neural responses (EJPs) and contractions, which is consistent with the hypothesis that ICC-IM are utilized in transduction of neuroeffector responses in the gastric fundus (Sung et al., 2018).

In the present study, we used mice with cell-specific expression of GCaMP6f to record responses to stimulation of intrinsic motor neurons in fundus ICC-IM. Simultaneous video imaging and intracellular electrical recordings were performed on intact fundus muscles to characterize the relationship between Ca²⁺-transients in ICC-IM and post-junctional electrical responses to nerve stimulation. The results show that ICC-IM are spontaneously active, generating robust Ca²⁺-transients, and changes in Ca²⁺-transients correlated with inhibitory and excitatory junction potentials mediated by nitrergic and cholinergic motor nerves, respectively. Individual ICC-IM responded to both excitatory and inhibitory neural inputs. These data support the concept that ICC-IM in the gastric fundus are serially innervated and play a critical role in post-junctional cholinergic excitatory and nitrergic inhibitory neuroeffector motor responses in the stomach.

Methods

Mouse generation, genotyping and tissue preparation

Mice were obtained either from The Jackson Laboratory (Bar Harbor, ME, USA) or generated in house at the University of Nevada, Reno. Ai95(RCL-GCaMP6f)-D (GCaMP6f mice) were purchased from The Jackson Laboratory.). Kit^{CreERT2Ejb1/+} (Kit-Cre) mice were obtained from Dr Dieter Saur (Technical University Munich, Germany; Klein et al., 2013). Kit^CreERT2+/− mice were bred with GCaMP6f mice and the resulting offspring are referred to as Kit-GCaMP6f throughout the manuscript. To induce Cre recombinase, 6- to 8-week-old Kit-GCaMP6f mice were treated with tamoxifen (Sigma-Aldrich, St Louis, MO, USA) by i.p. injection (2.0 mg i.p. made up as 20 mg ml⁻¹ solution in safflower oil). Each animal received three consecutive daily doses of tamoxifen and the experiments were performed 10 days following the last treatment. The expression of GCaMP6f was confirmed by geno- and phenotyping as previously described (Drumm, Hwang et al., 2019; Drumm, Rembetski et al., 2019).

The Institutional Animal Use and Care Committee at the University of Nevada approved procedures used on mice. Animals were fed ad libitum and had free access to water. Animals were humanely killed by isoflurane sedation followed by cervical dislocation and exsanguination. The investigators involved in the present study are aware of the ethical principles under which The Journal of Physiology operates and confirm that the use of animals presented here complies with the check list in Grundy (2015).

Entire stomachs, from the oesophagus to the pyloric sphincter, were removed and placed in oxygenated Krebs-Ringer buffer (KRB) for further dissection. Stomachs were opened along the lesser curvature and gastric contents were washed away with KRB. The fundus was isolated from the corpus along a line of demarcation indicated by gross changes in the structure of the mucosa. Gastric muscles were subsequently processed for morphological and physiological experiments.

Morphological studies

Whole mounts were prepared after removing the mucosa from the fundus by sharp dissection. The remaining strips of tunica muscularis were pinned to the base of a dish containing Sylgard elastomer (Dow Corning Corp., Midland, MI, USA) with the circular muscle layer facing upward and stretched to 110% of their resting length. Tissues were fixed in either paraformaldehyde (4% w/v in 0.1 m phosphate buffer (PB)) or Zamboni’s fixative (2% formaldehyde plus 0.2% picric acid in 0.1 m PB for 15 min at 4°C). Tissues were washed three times (10 min) with dimethyl sulfoxide (DMSO) and three times (10 min) with phosphate buffered saline (PBS; 0.01 m, pH 7.4). Following fixation, preparations were washed overnight in PBS. Incubation of tissues in BSA (1%) for 1 h at room temperature containing Triton X-100 (0.3%) was used to reduce non-specific antibody binding.

To confirm tamoxifen induced cell-specific (ICC-IM) expression of GCaMP6f in the gastric fundus double labelling immunohistochemistry was performed. Tissues were incubated sequentially in a combination of primary antibodies. Fundus muscles were labelled with an anti-eGFP antibody (1:1000; Abcam, Cambridge, MA, USA) and anti-mSCFR (a.k.a. Kit; 1:1000; R&D Systems, Minneapolis, MN, USA) or anti-Anoctamin 1 (anti-Ano1; 1:1000; Abcam, Cambridge, MA, USA). The combinations of antibodies used were chicken/goat and chicken/donkey. The first incubation was carried out for 48 h at 4°C; tissues were subsequently washed in PBS before being incubated in a second antibody for an additional 48 h at 4°C. Following incubation in primary antibodies, tissues were washed and incubated separately in appropriate secondary antibodies (Alexa Flor 488 and 594; Thermo Fisher Scientific Inc., Waltham, MA, USA, diluted to 1:1000 in PBS for 1 h at room temperature). Control tissues were prepared by either omitting primary or secondary antibodies from the incubation solutions. Tissues were examined with a Zeiss LSM 510 Meta (Jena, Germany) or a Nikon A1R (Melville, NY, USA) confocal microscope with appropriate excitation wavelengths. Confocal micrographs were digital composites of Z-series scans of 10–15 optical sections through a depth of 2–40 μm. Final images were constructed, and montages were assembled using Zeiss LSM 5 Image Examiner or Nikon NIS Elements and converted to Tiff files for final processing in Adobe Photoshop CS5 software (Adobe Co., Mountain View, CA, USA) and Photoshop 7.0 and Corel Draw X8 (Corel Corp. Ontario, Canada).

Calcium imaging of ICC-IM

Gastric fundus muscles were prepared as described for morphological studies and pinned to a thin Sylgard sheet (35 mm diameter × 1 mm thick) with a central rectangular hole (5 × 7 mm). The Sylgard sheet was attached to a nylon mesh for stability and placed into a Corning dish with a hole (20 mm diameter) cut in it and a 25 mm No. 1 glass coverslip glued to the inside base of the Corning dish. The fundus muscles were placed so that tissues were adjacent to the glass coverslip with the longitudinal layer closest to the coverslip. The preparation was perfused with KRB solution at 37°C. After an equilibration period of 1 h, preparations were visualized and imaged using a spinning-disk confocal microscope (QLC100; Yokogawa Electric Corporation, Tokyo, Japan) mounted to an inverted Olympus IX-70 microscope equipped with a Uplan FLN 40× lens (Olympus America, Centre Valley, PA, USA). The GCaMPf Ca²⁺ indicator expressed in ICC-IM was excited at 488 nm using a Spectra Physics laser. The fluorescence emission (>515 nm) was captured using a high-speed EMCCD Camera (Andor iXon^em+; ANDOR Technology, Belfast, Ireland). Pixel size using this acquisition system was 0.225 μm. Image sequences were collected at 33 fps using Andor SOLIS software.

Calcium transient analysis

Movies of Ca²⁺ activity in fundus ICC-IM were converted to a stack of Tiff images and were imported into either Image J, version 1.40 (National Institutes of Health, MD, USA) for Ca²⁺-transient analysis or custom software (Volumetry G8d; GW Hennig, Department of Pharmacology, The University of Vermont) for further analysis. Where necessary, tissue movement was stabilized to ensure accurate measurements of Ca²⁺-transients from identified ICC-IM.

Ca²⁺-transients recorded from ICC-IM in situ were quantified using spatio-temporal maps (STMs) as described previously (Drumm, Hwang et al., 2019; Drumm, Rembetski et al., 2019). Briefly, movies of ICC-IM Ca²⁺ signals were converted to a stack of tagged image file format (TIFF) images and imported into Volumetry G8d for initial processing. Whole-cell regions of interest (ROIs) were created to produce STMs of Ca²⁺-transients in individual ICC-IM within a field of view (FOV). These STMs were imported into Image J (version1.52a, National Institutes of Health, MD, USA, http://rsbweb.nih.gov/ij) for post hoc analysis. Basal fluorescence was acquired from regions of ICC-IM that displayed the most uniform and least intense fluorescence (F₀). Then fluorescence values throughout the rest of the ICC-IM were divided by the F₀ value to calibrate the STM for the amplitudes of Ca²⁺-transients as F/F₀. Ca²⁺ transient amplitude, duration and spread were then calculated from the STM. Ca²⁺-transient frequency was expressed as the number of events fired per cell per second (s⁻¹). The amplitude of Ca²⁺-transients was expressed as ΔF/F₀, the duration of Ca²⁺-transients was expressed as full duration at half-maximum amplitude (FDHM) and the spatial spread of Ca²⁺-transients was expressed as micrometres of ICC-IM propagated per Ca²⁺-transient.

In some examples Ca²⁺-transients observed in ICC-IM cells were quantified using particle (PTCL) analysis as described previously (Drumm et al., 2017; Drumm et al., 2018; Drumm, Hennig et al., 2019; Drumm, Hwang et al., 2019). Briefly, movies were firstly imported into Volumetry G8d and motion stabilized to minimize residual motion artifacts. A differential (Δt = ±66–70 ms) and Gaussian filter (1.5 × 1.5 μm, StdDev 1.0) was applied to accurately distinguish Ca²⁺-transients in ICC-IM from the background. A particle analysis routine was applied by using a flood-fill algorithm, which marked the structure of all adjoining pixels that had intensities above the threshold. Ca²⁺-transient PTCLs in ICC-IM were brighter and larger than noise particles. The threshold at which noise particles emerged and reduced the average particle size was thresholded and then valid Ca²⁺ PTCLs, which were above this threshold, were then saved as a coordinate based PTCL movie. To identify Ca²⁺ firing sites in ICC-IM, only those particles that did not overlap with any particles in the previous frame but overlapped with particles in the next 70 ms were considered Ca²⁺ firing sites. Effects of EFS on Ca²⁺-transients were tested using the same stimulus parameters as described below. Analysis of Ca²⁺-transients was performed for 10 s before EFS stimulation was delivered to obtain baseline activity.

Electrophysiological experiments

Gastric fundus muscles were prepared as described above for morphological studies. Intracellular microelectrode recordings were performed as previously described (Burns et al., 1996; Ward et al., 1994). Briefly, impalements of circular muscle cells were made with glass microelectrodes having resistances of 80–120 MΩ. Transmembrane potentials were recorded with a high impedance amplifier (Axon Instruments, Union City, CA, USA). Data were recorded on a PC running AxoScope 10.4 data acquisition software (Axon Instruments) and hard copies were made using Clampfit analysis software (Axon Instruments). The microelectrode was placed in the centre of the FOV using a crosshair reticle in an eyepiece. To record neurally evoked responses to electrical field stimulation (EFS), parallel platinum electrodes were placed on either side of the muscle strips and to maintain close contact of the tissue with the cover glass. Neural responses were elicited with square wave pulses of EFS (0.3 ms pulse duration, 1–20 Hz, train durations of 1 s, 10–15 V) delivered by a Grass S48 stimulator (Grass Instrument Company, Quincy, MA, USA). EFS parameters were chosen to match previously published findings using the same pulse durations and frequencies (Sung et al., 2018) and to minimize movement artifacts. To ensure that electrical and video recordings were synchronous, activation of the Andor iXon^em+ camera by the SOLIS software simultaneously triggered the Axoscope acquisition software. In some experiments nifedipine (1 μm) was added to the bathing solution. It has been previously shown that nifedipine does not affect spontaneous transient depolarizations (STDs) in the gastric fundus (Beckett et al., 2004).

Data analysis of electrophysiological experiments

In experiments where intracellular electrical recordings were made, several electrical parameters were analysed: (i) resting membrane potential (RMP; mV), (ii) amplitude of STDs (mV), and (iii) post-junctional neural responses to EFS (mV, s). To evaluate the effects of the Ano1 inhibitor CaCC_inh-A01 on STDs, all values of membrane potential (up to 2000 s⁻¹) acquired during a recording period up to 3 min were used (i.e. up to 80,000–100,000 events) and plotted as ‘number of events vs. membrane potential (mV). RMP was defined as the membrane potential associated with the greatest number of events. Figures displayed were made from digitized data (Clampfit, Axon Instruments) using Corel Draw X8.

Solutions and drugs

Tissues were constantly perfused with oxygenated KRB of the following composition (mm): 118.5 NaCl, 4.5 KCl, 1.2. MgCl₂, 23.8 NaHCO₃, 1.2. KH₂PO₄, 11.0 dextrose, and 2.4 CaCl₂. The pH of the KRB was maintained at 7.3–7.4 when bubbled with 97% O₂–3% CO₂ at 37 ± 0.5°C. Muscles were equilibrated for at least 1 h before the experiments were initiated.

Nifedipine, cyclopiazonic acid (CPA), ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), caffeine, carbachol and tamoxifen were purchased from Sigma-Aldrich (Saint Louis, MO, USA), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- 1-one (ODQ; Cayman Chemical, Ann Arbor, MI, USA) and 6-(1,1-dimethylethyl)-2-[(2-furanylcarbonyl)amino]-4,5, 6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (CaCC_inh-A01; Tocris Bioscience, Minneapolis, MN, USA). Drugs were dissolved in appropriate solvents as stock solutions and to final concentrations in KRB. Tamoxifen was firstly dissolved in ethanol before addition of safflower oil to make a final concentration of 20 mg ml⁻¹.

Statistical analysis

Statistical analysis was performed using either a Student’s t test or a Mann-Whitney non-parametric test where appropriate and complies with The Journal of Physiology statistics policy. Differences between the means of measured parameters were evaluated using repeated measures of one-way analysis of variance (ANOVA) where appropriate in conjunction with the Dunnett’s multiple comparison test. Data are expressed as means ± standard deviation of the mean (SD) and bar graphs are plotted with all data points overlaid in box or whisker plot format. Exact P values are stated in text, figures and figure legends. Statistical tests were performed using Prism 9.03 (GraphPad Software, Inc., La Jolla, CA, USA). When describing data, ‘N’ refers to the number of animals used in that dataset, and ‘n’ refers to the numbers of ICC-IM or tissues where applicable used in that same data set.

Results

Determination of recombination efficiency and expression of GCaMP6f

Cellular localization of GCaMP6f and efficiency of the iCre was investigated to determine whether the Ca²⁺ sensor was expressed in ICC-IM generally and specifically. The number of ICC-IM in the circular muscle (CM) expressing GCaMP6f was compared to cells labelled with Kit or Ano1 using double-labelling immunohistochemical analysis and antibodies against eGFP and Kit or eGFP and Ano1. Spindle-shaped cells with eGFP (GCaMP6f) expression were present at an average density of 5.3 ± 1.5 cells per random 100 μm transecting line within the circular muscle layer (15 fields of view; N = 4 animals), and there was an average separation between cell bodies of 18.8 ± 2.0 μm. Double labelling with eGFP/Kit or eGFP/Ano1 antibodies confirmed that the eGFP labelling was specific for Kit⁺ and Ano1⁺ ICC-IM in Kit-GCaMP6f mice (Fig. 1). Little labelling with eGFP antibody was resolved in cells other than ICC-IM. A few Kit⁺ small-rounded cells that were assumed to be mast cells were observed, as these cells also expresses Kit (Fig. 1D and F). These cells were not judged to be a significant contaminant for imaging studies, because their morphology was dramatically different from the spindle-shaped ICC-IM, and they were few in number. Areas of gastric fundus were found where resolution of eGFP-like immunoreactivity was low or below resolution, but normal distributions of Kit⁺/Ano1⁺ immunoreactivity were apparent in these areas. These data suggest that although GCaMP6f was expressed in the majority of ICC-IM, recombination was not successful quantitatively.

Figure 1. The efficiency of Cre expression and the cellular localization of GCaMP6f in gastric fundus ICC-IM.

A–C, the cell-specificity of GCaMP6f in ICC-IM was determined by double label immunohistochemical analysis with antibodies against eGFP and Kit following tamoxifen treatment. A, eGFP expression was resolved in spindle-shaped cells (arrows, green) within the circular muscle layer. B, double labelling with Kit (arrows, red) revealed Kit expression was localized to cells that were morphologically similar to eGFP⁺ cells. C, merged image of A and B reveal the cellular co-localization and cell-specificity of eGFP expression in ICC-IM (yellow, arrows). D–F, confirmation that eGFP were in ICC was performed by double labelling with antibodies against eGFP and Ano1. D, eGFP expression resolved in spindle-shaped cells (arrows, green). E, Ano1 expression in spindle-shaped ICC-IM (arrowheads, red). F, merged imaged reveal that eGFP was localized in Ano1⁺ spindle shaped ICC-IM (arrows, yellow). eGFP expression also occurred in small, rounded cells (arrowhead) that were Ano1⁻. Scale bars = 50 μm indicated in C and F are representative for their respective panels. [Colour figure can be viewed at wileyonlinelibrary.com]

Ca²⁺-transients in single and neighbouring ICC-IM

ICC-IM generated intracellular Ca²⁺-transients from discrete locations along the lengths of cells. Time-lapse images of 5 ICC-IM within a single FOV are shown in Fig. 2A, and the dynamic changes in the regions denoted by numbers and arrows in the 2nd panel are displayed as traces in Fig. 2B. We sought to further characterize the relationship between Ca²⁺-transients in neighbouring ICC-IM to determine if these events synchronized or were entrained across FOVs. The Ca²⁺-transients in the 5 ICC-IM identified in Fig. 2A were analysed with particle (PTCL) routines (see Methods). Figure 2C shows a summation of Ca²⁺ signals within these ICC-IM over a 30 s period of recording (far left panel). Ca²⁺-transients were then designated as Ca²⁺ PTCLs and thresholded to determine the first occurrence of PTCLs and to highlight Ca²⁺ firing sites in each location in the five cells (Fig. 2C). The individual Ca²⁺ firing sites were then colour coded (Fig. 2C, far right panel) and plotted as an occurrence map against time (Fig. 2D). In the occurrence map, individual Ca²⁺ firing sites occupied a separate horizontal coloured lane, with the width of the coloured bands indicating the duration that each firing site was active. These data demonstrate that while there was apparent periodicity in firing from individual Ca²⁺ firing sites, there was little or no entrainment of Ca²⁺-transients between neighbouring ICC-IM.

Figure 2. Intracellular Ca²⁺-transients imaged in GCaMP6f⁺ ICC-IM.

A, ICC-IM actively generated intracellular Ca²⁺-transients throughout the entire cell. Time-lapse imaging of five ICC-IM is shown (cells 1–5 identified by arrows in A, 2nd panel). B, the dynamics of these Ca²⁺-transients from the ICC-IM identified in A are shown in intensity plot profiles (1 to 5, respectively). Ca²⁺-transient events in ICC-IM occurred stochastically, often developing from several independent sites along ICC-IM. However, the frequency of some Ca²⁺-transients occurred regularly within a given ICC-IM. C, the Ca²⁺-transient activity of the ICC-IM identified in A was analysed with particle (PTCL) analysis routines. C shows the summated Ca²⁺ signal within these ICC-IM over a 30 s recording. PTCL analysis was used to identify Ca²⁺-transients as Ca²⁺ PTCLs, which were then thresholded to identify the first occurrence and to highlight Ca²⁺ firing sites in different cells. The initiating sites of Ca²⁺-transients in individual ICC-IM. D, individual Ca²⁺ firing sites in ICC-IM were colour coded and plotted as an occurrence map against time. Individual Ca²⁺ firing sites occupied a separate coloured lane, with the width of the coloured band indicated the duration that the Ca²⁺ firing site was active. These data demonstrate that although there was reasonably regular frequency in Ca²⁺ firing sites, there was little or no entrainment of Ca²⁺-transients between neighbouring ICC-IM in a given FOV. Scale bars are as indicated in each panel. [Colour figure can be viewed at wileyonlinelibrary.com]

Ca²⁺-firing sites in individual ICC-IM

Ca²⁺-transients observed in single ICC-IM were analysed using similar methods, as above. A raw FOV and a summation of all Ca²⁺-transients occurring in this FOV during a recording period is shown in Fig. 3A and B. From the FOV, a single ICC-IM was selected (lower left cell in Fig. 3A and B), and the initiating Ca²⁺-PTCLs and firing sites of all Ca²⁺-transients were determined (Fig. 3C–E) and plotted as separate traces Fig. 3F. In this example, six discrete firing sites were identified in the ICC-IM. The activity of one firing site did not seem to affect the activity of adjacent firing sites within an individual ICC-IM. Individual firing sites (except the one colour-coded in white in Fig. 3E) displayed relatively regular frequencies, but the frequencies varied between sites. Firing sites with the highest frequencies were in the peri-nuclear region of the ICC-IM (Fig. 3F), and firing was less frequent in more peripheral regions Fig. 3F.

Figure 3. Ca²⁺-firing sites in individual ICC-IM.

To further analyse Ca²⁺-firing sites, individual ICC-IM were examined. A, raw image of ICC-IM within a given FOV. B, summated Ca²⁺-signal of FOV in A. C, an ICC-IM was selected (lower left cell in A and B) and the summed Ca²⁺-signal, initiating Ca²⁺-PTCLs (D) and initiation sites of Ca²⁺-transients (E) identified. F, Ca²⁺-transients were plotted as separate traces. Six discrete firing sites were identified in this ICC-IM and the activity of one firing site did not appear to influence the activity of any adjacent site within this ICC-IM. Individual firing sites (except the most peripheral; white in E) displayed regular frequencies, but these were highly variable between sites. The sites with the highest frequencies were in the peri-nuclear region of the ICC-IM and less frequent towards the peripheral regions of ICC-IM. Scale bars are as indicated in each panel. [Colour figure can be viewed at wileyonlinelibrary.com]

Quantification of Ca²⁺-transients in ICC-IM was performed using spatio-temporal mapping (STMs) (Fig. 4). As described above, ICC-IM displayed Ca²⁺-transients that originated from numerous sites within cells. Another example is shown by the cell depicted in Fig. 4 in which five firing sites were identified in time lapse images (Fig. 4A) and colour coded in Fig. 4B (numbered along left edge of panel). The Ca²⁺-transients at each identified site are shown as traces in Fig. 4C. Again, although individual sites displayed periodicity, when the firing at multiple sites were superimposed on a temporal map, no apparent relationship between firing sites could be observed (Fig. 4C, All sites). From analysis of STMs, summaries of the frequency, amplitude, duration and spatial spread of Ca²⁺-transients are shown in Figure 4D. In spite of the appearance of periodicity of Ca²⁺-transients from individual firing sites, this analysis shows significant variability in frequency, amplitude, duration and spatial spread. The frequency of Ca²⁺-transients averaged 2.1 ± 0.68 s⁻¹, with an average amplitude (F/F₀) of 1.3 ± 0.82. The duration of these events was 248 ± 64.4 and the spatial spread of events was 15.3 ± 8.1 μm (n = 89 ICC-IM; N = 22 animals for frequency and n = 941 ICC-IM; N = 22 animals for other parameters). The analysis shows that ICC-IM in gastric fundus are highly dynamic in terms of generation of Ca²⁺-transients, but events originating from individual firing sites are independent and not entrained to other firing sites within FOVs or even within individual cells.

Figure 4. Quantification of Ca²⁺-transients in ICC-IM using spatio-temporal mapping (STMs).

A, ICC-IM displayed Ca²⁺-transients that originated from numerous sites within the cell. The five firing sites identified in time lapse images (A) are colour coded in B (1 to 5, arrows). Levels of Ca²⁺ fluorescence are represented from greatest to lowest by red>orange>yellow>green>blue>black. C, the firing of Ca²⁺-transients at discrete sites within this ICC-IM is shown as individual traces (1 to 5). Although many of the sites were rhythmic and highly periodic, when the sites were summed as traces on a temporal map, there was no apparent relationship between these sites (lower trace in C). D, from STMs, the summary of the frequency (Ca²⁺-transients s⁻¹), amplitude (ΔF/F₀), full duration at half-maximum amplitude (FDHM) and spatial spread of Ca²⁺-transients (μm) is shown. Although the firing of Ca²⁺-transients was often periodic, there was significant variability in frequency, amplitude, duration and spatial spread of these events. Scale bar in A is representative for all panels. [Colour figure can be viewed at wileyonlinelibrary.com]

Role of extracellular Ca²⁺ in the generation and/or spread of Ca²⁺-transients in ICC-IM

The role of extracellular Ca²⁺ (i.e. [Ca²⁺]_o) on generation and spread of Ca²⁺-transients in ICC-IM was tested by exposing cells to nominally [Ca²⁺]_o-free KRB during imaging (Fig. 5A and B). Spontaneous Ca²⁺-transients persisted after addition of [Ca²⁺]_o-free KRB (15 min); however, there was a slight but significant reduction in the frequency, duration and spatial spread of these events (Fig. 5C; n = 25 ICC-IM; N = 6 animals). Prior to replacement of normal [Ca²⁺]_o with [Ca²⁺]_o-free KRB, Ca²⁺-transient frequency occurred at 1.9 ± 0.5 s⁻¹ with an average amplitude (F/F₀) of 1.6 ± 0.78 s⁻¹, an average half-maximal duration of 261.0 ± 32.0 ms and a spatial spread of these Ca²⁺-transient events that averaged 16.0 ± 7.6 μm (n = 25 ICC-IM; N = 6 animals). After 15 min in nominally [Ca²⁺]_o-free KRB, the frequency of Ca²⁺-transients averaged 1.6 ± 0.6 s⁻¹, had an average amplitude (F/F₀) of 1.4 ± 0.6, an average half-maximal duration of 237.0 ± 26.4 ms and a spatial spread that averaged 12.5 ± 2.7 μm (P = 0.0042 for frequency, P = 0.0005 for duration, P = 0.0086 for spatial spread and P = 0.0692 for amplitude of Ca²⁺-transients; n = 25 ICC-IM; N = 6 animals).

Figure 5. Role of extracellular calcium in the generation and spread of Ca²⁺-transients in ICC-IM.

A, spontaneous Ca²⁺-transients under control condition (i.e. 2.5 mM [Ca²⁺_ext]). B, nominal [Ca²⁺_ext]-free KRB buffer after 15 min. C, summary of six experiments. Although Ca²⁺-transients still occurred in nominally [Ca²⁺_ext]-free KRB there was a significant reduction in the frequency (Ca²⁺-transients s⁻¹; **P = 0.0042), full duration at half-maximum amplitude (FDHM; ***P = 0.0001), and spatial spread (μm; **P = 0.0086) of these events. The amplitude of Ca²⁺-transients (ΔF/F₀) were slightly reduced but did not reach significance (P = 0.069, n.s.). D and E, the importance of [Ca²⁺_ext] was further examined where nominal Ca²⁺-free KRB was supplemented with ethylene glycol-bis (β-aminoethyl ether)-N,N,N′, N′-tetraacetic acid (EGTA) and Ca²⁺-transients recorded for 30 s every 5 min. D, as stated above, in nominally [Ca²⁺_ext]-free KRB, Ca²⁺-transients persisted. E, addition of EGTA (0.5 mm) to the nominal [Ca²⁺_ext]-free KRB caused a rapid disruption in the generation of Ca²⁺-transients and the loss in activity within 15 min. F, summary of the loss in Ca²⁺-transient activity (i.e. frequency of Ca²⁺-transients s⁻¹; ****P = 0.00001); amplitude (ΔF/F₀; ****P = 0.00001); full duration at half-maximum amplitude (FDHM; ****P = 0.00001); and spatial spread of Ca²⁺-transients (μm; ****P = 0.00001). [Colour figure can be viewed at wileyonlinelibrary.com]

Depending on the purity of salts used, nominally Ca²⁺-free buffers can contain [Ca²⁺]_o in the range of several micromolar (Bird et al., 2008), and therefore a positive gradient for Ca²⁺ entry may remain in solutions that are nominally [Ca²⁺]_o-free. Therefore, additional experiments were performed in which normal [Ca²⁺]_o KRB was replaced with a solution containing [Ca²⁺]_o buffered by addition of the Ca²⁺-chelator EGTA (0.5 mm) to ensure the extracellular KRB solution was [Ca²⁺]_o-free. Cells were imaged for 30 s every 5 min after addition of this solution. Addition of EGTA to nominally [Ca²⁺]_o-free KRB caused rapid disruption in the generation of Ca²⁺-transients and loss of activity within 15 min (Fig. 5D and E). In this series of experiments and as stated above, in nominally [Ca²⁺]_o-free KRB spontaneous Ca²⁺-transients persisted. Ca²⁺-transient frequency occurred at 2.3 ± 0.4 s⁻¹ with an average amplitude (F/F₀) of 1.1 ± 0.2 s⁻¹, an average half-maximal duration of 212 ± 19.2 ms and an average spatial spread of 15.1 ± 4.2 μm (Fig. 5D; n = 25 ICC-IM; N = 6 animals). After 15 min in nominally [Ca²⁺]_o-free KRB buffered with EGTA (0.5 mm) Ca²⁺-transients were abolished (Fig. 5E). A summary of these data is shown in Fig. 5F (n = 25 ICC-IM; N = 6 animals; P = 0.0001). Thus, [Ca²⁺]_o is necessary for the generation of Ca²⁺-transients but buffering of [Ca²⁺]_o is necessary to inhibit these events.

Dyhydropyridine-sensitive L-type Ca²⁺-channels do not contribute to Ca²⁺-transients in ICC-IM

The role of L-type Ca²⁺-channels in the generation and spread of Ca²⁺-transients in ICC-IM was tested by imaging Ca²⁺-transients before and after addition the L-type Ca²⁺-channel antagonist, nifedipine (1 μm; Fig. 6A and B). Control recordings were made for 30 s, and then the same FOV was imaged after 15 min exposure to nifedipine. Under control conditions, Ca²⁺-transient frequency averaged 2.3 ± 0.82 s⁻¹and had an average amplitude (F/F₀) of 1.2 ± 0.46 s⁻¹. The half-maximal duration of Ca²⁺-transients averaged 247 ± 48.4 ms and had a spatial spread that averaged 16.3 ± 4.2 μm (n = 42 ICC-IM; N = 10 animals). Nifedipine had no effect on the frequency, amplitude and spatial spread of Ca²⁺-transients in gastric fundus ICC-IM (i.e. 2.5 ± 0.86 s⁻¹; 1.2 ± 0.44 F/F₀ and 15.6 ± 3.9 μm, respectively; P = 0.1466; P = 0.988 and P = 0.3299 for each parameter). Nifedipine had a small but significant effect on the duration of these events (i.e. decreased to 231 ± 32.5 ms; P = 0.0253; Fig. 6C). The results show that Ca²⁺ entry via L-type Ca²⁺ channels are not important for Ca²⁺-transients in ICC-IM.

Figure 6. Dyhydropyridine-sensitive L-type Ca²⁺-channels do not contribute to Ca²⁺-transients in ICC-IM.

A, control video recordings performed before addition of nifedipine. B, recordings performed on the same FOV after the addition of nifedipine (1 μm) for 15 min. C, summary of the effects of nifedipine on frequency of Ca²⁺-transients (Ca²⁺ events s⁻¹, P = 0.14, n.s.), amplitude (ΔF/F₀, P = 098, n.s.)., full duration at half-maximum amplitude (FDHM, *P = 0.025) and spatial spread of Ca²⁺-transients (μm, P = 0.33, n.s.). [Colour figure can be viewed at wileyonlinelibrary.com]

Involvement of the SERCA pump in the generation of Ca²⁺-transients in ICC-IM

In the absence of effects of Ca²⁺ entry via L-type Ca²⁺ channels, except for an effect on Ca²⁺-transient duration, we investigated whether the Ca²⁺-transients were due to release from intracellular stores. We investigated the effects of the SERCA ATPase inhibitor, cyclopiazonic acid (CPA; 1–10 μm), on Ca²⁺-transients in ICC-IM. Under control conditions, the frequency of Ca²⁺-transients was 2.5 ± 0.35 s⁻¹ with an average amplitude of 1.3 ± 0.46 F/F₀, half-maximal duration of 211 ± 26.9 ms and spatial spread of 15.6 ± 5.8 μm (n = 19 ICC-IM; N = 5 animals; Fig. 7A and C). CPA (1–3 μm) caused dose-dependent disruption in the frequency, duration and spatial spread of Ca²⁺-transients. CPA (10 μm) abolished Ca²⁺-transients in ICC-IM (Fig. 7B; n = 19 ICC-IM; N = 5 animals). A summary of these data is shown in Fig. 7C (n = 19 ICC-IM; N = 5 animals; P = 0.0001). Thus, intracellular Ca²⁺-sources are essential for gastric fundus ICC-IM Ca²⁺-transient activity.

Figure 7. SERCA pump involvement in the generation of Ca²⁺-transients in ICC-IM.

A, video recordings of Ca²⁺-transients in ICC-IM under control conditions, i.e. before the addition of any drugs. B, the SERCA ATPase inhibitor cyclopiazonic acid (CPA; 10 μm) rapidly abolished Ca²⁺-transients in ICC-IM. C, summary of the effects of CPA (10 μm) on frequency (Ca²⁺ events s⁻¹, ****P = 0.00001), amplitude (ΔF/F₀ P = 0.00001), full duration at half-maximum amplitude (FDHM, ****P = 0.00001) and spatial spread of Ca²⁺-transients (μm, ****P= 0.00001). [Colour figure can be viewed at wileyonlinelibrary.com]

Since removal of [Ca²⁺]_o with addition of EGTA and the SERCA-pump antagonist CPA both inhibited Ca²⁺-transients, we sought to resolve whether depletion of [Ca²⁺]_o affected intracellular Ca²⁺-stores. Therefore, fundus muscles were treated with caffeine or the muscarinic agonist carbachol to stimulate intracellular Ca²⁺ release following incubation in [Ca²⁺]_o-free KRB with EGTA (0.5 mm) for 15 min. Neither caffeine (1 mm; n = 16 ICC-IM; N = 4 animals) nor carbachol (10 μm; n = 16 ICC-IM; N = 4 animals) initiated Ca²⁺-transients after incubation with the [Ca²⁺]_o-free buffer. These results suggest that treatment with [Ca²⁺]_o-free KRB buffered with EGTA causes depletion of intracellular stores that are required for ICC-IM Ca²⁺-transients.

Effects of Ano1 inhibition on electrical activity and Ca²⁺-transients in ICC-IM

To further examine Ca²⁺-transients and electrical activity of the gastric fundus, a series of intracellular electrical recordings and Ca²⁺ imaging experiments was performed. Electrical slow waves are not generated in murine gastric fundus, but membrane potential was dynamic and characterized by small amplitude, noisy oscillations consisting of depolarizations and hyperpolarizations that have been termed unitary potentials or spontaneous transient depolarizations (STDs; Beckett et al., 2002, 2004; Burns et al., 1996; Edwards et al., 1999; Van Helden et al., 2000).

Experiments examining the effects of the Ano1 inhibitor CaCC_inh-A01were performed in the presence of the nitric oxide synthase inhibitor l-NNA (100 μm), atropine (1 μm) and nifedipine (1 μm ). l-NNA slightly but significantly depolarized membrane potential (−47.5 ± 3.4 mV before and −44.8 ± 3.7 after l-NNA; P = 0.03; n = 5 tissues; N = 5 animals). Addition of CaCC_inh-A01(1–10 μm) dose dependently hyperpolarized membrane potential (i.e. −51.2 ± 2.7 mV in 10 μm CaCC_inh-A01; P = 0.024) and inhibited STDs. Figure 8C shows a plot of the deviation in membrane potential before and after CaCC_inh-A01 (10 μm). Ca²⁺-transients were also recorded before and after CaCC_inh-A01 in the presence of l-NNA, atropine and nifedipine. CaCC_inh-A01 (1–10 μm) had little or no effect on Ca²⁺-transients in ICC-IM (n = 20 ICC-IM; N = 4 animals; Fig. 8D and E). A summary of the effects of CaCC_inh-A01 (10 μm) on Ca²⁺-transient frequency, amplitude, duration and spatial spread is shown in Fig. 8F. There was a slight but significant increase in calcium transient amplitude after CaCC_inh-A01 (P = 0.02). The results show that Ca²⁺-transients were not inhibited by an Ano1 inhibitor but associated STDs were.

Figure 8. Spontaneous transient depolarizations (STDs) are inhibited by the Ano1 inhibitor CaCC_inh-A01.

A and B, STDs recorded from gastric fundus under control conditions (L-NNA; 100 μM, atropine 1 μm and nifedipine 1 μm; A) and after the addition of CaCC_inh-A01 (10 μm; B). Dashed lines denote resting membrane potential (RMP). RMP hyperpolarized in the presence of CaCC_inh-A01 (P = 0.03,*). C, plot of the distribution of membrane potential (see Methods) recorded under control conditions (black bars) and after CaCC_inh-A01 (red bars) in the experiment in A and B. Continuous black line, summarized data under control conditions; continuous red line, summarized data after CaCC_inh-A01. Note that the membrane potential deviation (i.e. STDs) is reduced after the addition of the Ano1 inhibitor. D, control video recordings performed before addition of CaCC_inh-A01 (i.e. 100 μm L-NNA, 1 μm atropine and 1 μm nifedipine) E, recordings performed on the same FOV after the addition of CaCC_inh-A01 (10 μm; for 30 min). F, summary of the lack of effects of CaCC_inh-A01 (10 μm) on Ca²⁺-transient parameters. There was no statistical difference in any parameter except transient amplitude was slightly increased, i.e. frequency (Ca²⁺ events s⁻¹, P = 0.71, n.s.), amplitude (ΔF/F₀, *P = 0.02), full duration at half-maximum amplitude (FDHM, P = 0.2, n.s.) and spatial spread of Ca²⁺-transients (μm, P = 0.13, n.s.). [Colour figure can be viewed at wileyonlinelibrary.com]

Neural regulation of Ca²⁺-transients in gastric fundus ICC-IM

We investigated the effects of activating intrinsic neurons on Ca²⁺-transients in ICC-IM. Electric field stimulation (EFS 1–20 Hz; 0.3 ms pulse duration for 1 s) under control conditions (i.e. no drugs added to the bathing solution) produced two types of responses on Ca²⁺-transients in ICC-IM. The dominant response was a frequency-dependent reduction in Ca²⁺-transients and their inhibition at higher EFS frequencies (Fig. 9A and B). The duration of inhibition of the Ca²⁺-transients was also frequency dependent and greatest at 10 Hz where inhibition persisted for 3.2 ± 0.9 s (Fig. 9A and B; n = 8 tissues; N = 8 animals). The second type of response observed in ICC-IM was initial excitation of Ca²⁺-transients that summated into a global rise in intracellular Ca²⁺ that persisted for 0.65 ± 0.8 s (Fig. 9C; n = 8 tissues; N = 8 animals) followed by inhibition of Ca²⁺-transients (Fig. 9C). Since EFS evoked responses occurred immediately in ICC-IM, it is likely that these responses were due to direct neural innervation of these cells. A summary of the types of responses are shown in Fig. 9D.

Figure 9. Neurally evoked post-junctional responses of Ca²⁺-transients in ICC-IM.

A, under control conditions (i.e. no drugs), electrical field stimulation (EFS; 1–10 Hz, 0.3 ms for 1 s) produced little or no changes in Ca²⁺-transients in a few preparations. B, in the majority of fundus muscles, EFS produced an almost immediate inhibition of Ca²⁺-transients in ICC-IM that was dependent upon the frequency of stimulation. The inhibition of Ca²⁺-transients persisted for several seconds at higher EFS frequencies (5–10 Hz). C, in other fundus muscles ICC-IM displayed an immediate summation of Ca²⁺-transients into a global increase in intracellular Ca²⁺ in response to EFS. This global increase in intracellular Ca²⁺ was followed by a period of Ca²⁺-transient inhibition before these events returned to pre-stimulus control levels. Periods of EFS stimulation are indicated by arrows and a dashed white line or rectangular box. Analysis of Ca²⁺-transients was performed for 10 s before EFS stimulation was delivered to obtain basal activity. D, summary of the neuroeffector responses of Ca²⁺-transients of ICC-IM to EFS. [Colour figure can be viewed at wileyonlinelibrary.com]

The effects of specific classes of enteric motor neurons on Ca²⁺-transients were also tested. l-NNA (100 μm) did not affect spontaneous Ca²⁺-transients but abolished inhibitory effects of EFS on Ca²⁺-transients at all frequencies tested (Fig. 10A and B). A summary of frequency, amplitude, duration and spread of Ca²⁺-transient activity, pre-EFS, EFS (1–20 Hz), and EFS in the presence of l-NNA (100 μm) is shown in Fig. 11.

Figure 10. Inputs from nitrergic inhibitory motor nerves contribute to decreases in Ca²⁺-transients in ICC-IM.

A, EFS under control conditions (1–10 Hz, 0.3 ms for 1 s, indicated by arrows or dashed white lines or rectangular boxes) produced a frequency-dependent inhibition of Ca²⁺-transients in ICC-IM. The inhibition in Ca²⁺-transients was short at single pulses but persisted for several seconds at higher stimulation frequencies (5–10 Hz). B, in the presence of L-NNA (100 μM), the inhibitory responses to EFS was abolished and a slight increase in Ca²⁺-transient activity observed. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 11. Summary of the decreases in Ca²⁺-transients in ICC-IM in response to EFS.

A–D, responses to EFS at 1, 5, 10 and 20 Hz, respectively. EFS (0.3 ms duration for 1 s) inhibited (from left) frequency of Ca²⁺-transients (number of Ca²⁺ events s⁻¹), amplitude (ΔF/F₀), full duration at half-maximum amplitude (FDHM) and spatial spread of Ca²⁺-transients (μm) at all frequencies, compared to pre-stimulus conditions (Pre EFS). In the presence of L-NNA (100 μM), the inhibition of Ca²⁺-transients that was observed in response to EFS was abolished. *P = 0.01; **P = 0.001; ***P = 0.0001; ****P = 0.00001. Ca²⁺-transients were measured for 10 s before EFS stimulation was delivered to obtain basal activity. [Colour figure can be viewed at wileyonlinelibrary.com]

The receptor for NO is soluble guanylate cyclase (sGC; Groneberg et al., 2015; 2016; Gil et al., 2014; Lies, Lies et al., 2015), a cytosolic heterodimer of α and β subunits. ICC-IM express both isoforms of sGG, sGCβ1 and sGCα1 throughout the GI tract (Iino et al., 2008, 2009). The sGC inhibitor, ODQ (10 μm), did not affect spontaneous Ca²⁺-transients but abolished inhibition of Ca²⁺-transients evoked by EFS at all frequencies (Fig. 12A and B). A summary of the effects of ODQ on EFS-evoked (1–20 Hz; 0.3 ms; 1 s duration) inhibition of frequency, amplitude, duration and spread of Ca²⁺-transients is shown in Fig. 13 (n = 22 ICC-IM; N = 6 animals). These data demonstrate that activation of nitrergic inhibitory nerves mediates inhibition of Ca²⁺-transients via sGC.

Figure 12. Inhibition of guanylate cyclase blocks the decreases in Ca²⁺-transients in ICC-IM in response to EFS.

A, under control conditions EFS (1–10Hz, 0.3 ms for 1 s, indicated by arrows or dashed white lines or rectangular boxes) produced frequency-dependent inhibition of Ca²⁺-transients in ICC-IM compared to pre-stimulus Ca²⁺-transient activity. The inhibition in Ca²⁺-transients was frequency dependent, short lived at single pulses but persisted for several seconds at higher stimulation frequencies. B, addition of the guanylate cyclase inhibitor ODQ (10 μM), abolished the inhibitory responses to EFS observed under control conditions at all frequencies tested. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 13. Summary of the effects of ODQ on the inhibition of Ca²⁺-transients in ICC-IM in response to EFS.

A–D, responses to EFS at 1, 5, 10 and 20 Hz (0.3 ms duration for 1 s), respectively. EFS inhibited (from left) frequency of Ca²⁺-transients (number of Ca²⁺ events s⁻¹), amplitude (ΔF/F₀), full duration at half-maximum amplitude (FDHM) and spatial spread of Ca²⁺-transients (μm) at all frequencies, compared to pre-stimulus conditions (Pre EFS). Following incubation in ODQ (10 μM), the inhibition of Ca²⁺-transients that was observed in response to EFS was reversed. ***P= 0.0001, ****P = 0.00001. Ca²⁺-transients were measured for 10 s before EFS stimulation was delivered to obtain basal activity. [Colour figure can be viewed at wileyonlinelibrary.com]

We have previously reported that the dominant neurally mediated motor responses in the murine gastric fundus are cholinergic excitatory and nitrergic inhibitory at frequencies 1–20 Hz (Sung et al., 2018). Therefore, the contributions of cholinergic and nitrergic neurotransmission on ICC-IM were further examined by applying l-NNA (100 μm) and atropine (1 μm) sequentially. In the presence of l-NNA, ICC-IM displayed a rapid summation of Ca²⁺-transients that led to a global rise in intracellular Ca²⁺ in response to EFS at all frequencies (1–20 Hz; Fig. 14A and C, n = 6 tissues; N = 6 animals). Atropine (1 μm; in the continued presence of l-NNA) did not affect spontaneous Ca²⁺-transients but inhibited the global increase in Ca²⁺ at 1–5 Hz EFS (Fig. 14B and D). At higher frequencies (10 Hz), atropine decreased, but did not block, the global increase in Ca²⁺ observed in l-NNA alone (Fig. 14D). There was also a slight inhibition in Ca²⁺-transient firing following EFS at higher frequencies in L-NA and atropine (Fig. 14D). These data show that cholinergic neural inputs increase Ca²⁺-transients causing summation into a global rise in intracellular Ca²⁺ in ICC-IM.

Figure 14. Inputs from cholinergic motor nerves contribute to global increases in Ca²⁺ in ICC-IM.

A, in the presence of L-NNA (100 μM), EFS (0.3 ms, 1 and 5 Hz for 1 s) delivered at the arrow, white dashed line or during the rectangular box, caused a global rise in intracellular Ca²⁺ in ICC-IM. B, the global rise in intracellular Ca²⁺ was inhibited by atropine (1 μM). C, a similar rise in Ca²⁺-transients in ICC-IM was also observed at 10 and 20 Hz. D, atropine (1 μM), in the continued presence of L-NNA (100 μM), did not affect spontaneous Ca²⁺-transients, but inhibited the global increases in intracellular Ca²⁺ in response to EFS at all frequencies tested (1–20 Hz). [Colour figure can be viewed at wileyonlinelibrary.com]

Electrical activity and Ca²⁺-transients in ICC-IM

Simultaneous intracellular electrical recordings were made during imaging to clarify the associations between Ca²⁺-transients in ICC-IM and ongoing discharge of STDs. Cells in the centre of the FOV (40× objective) within the region imaged were impaled with microelectrodes. In this series of experiments. resting membrane potentials in muscles of Kit-GCaMP6f mice averaged −40.4 ± 3.2 mV (n = 15 tissues; N = 15 animals). Membrane potential oscillations were observed in fundus muscles of Kit-GCaMP6f mice, as previously observed in wild-type mice (Burns et al., 1996; Beckett et al., 2004; Sung et al., 2018). STMs revealed no obvious relationship between the STDs and Ca²⁺-transients in ICC-IM within a given FOV (Fig. 15A and B). This is likely because there is little entrainment of Ca²⁺-transients between neighbouring ICC-IM and STDs are likely to be the consequence of the summed Ca²⁺-transients in more ICC-IM than imaged within a single FOV.

Figure 15. Electrical activity and Ca²⁺-transients in ICC-IM.

A and B, colour coded Ca²⁺-transient firing sites in an ICC (A) and simultaneous intracellular microelectrode recording from the centre of the FOV, i.e. immediately adjacent to the ICC-IM (B). The electrical activity of the murine gastric fundus does not generate slow waves, but rather generates small amplitude, noisy oscillations in membrane potential termed unitary potentials or spontaneous transient depolarizations (STDs). There was no obvious relationship between membrane potential oscillations and Ca²⁺-transients in ICC-IM in a given FOV. [Colour figure can be viewed at wileyonlinelibrary.com]

Post-junctional Ca²⁺-transients and electrical responses to nerve stimulation

Activation of enteric motor nerves has been shown to produce bi-phasic intracellular electrical responses in the gastric fundus (Beckett et al., 2002; Sung et al., 2018; Ward et al., 2000). During nerve stimulation there is a dominant frequency-dependent hyperpolarization in membrane potential termed an inhibitory junction potential (IJP). In some muscles a transient depolarization in membrane potential termed an excitatory junction potential (EJP) often preceded the IJP. EJPs and IJPs lead to contraction and relaxation of gastric fundus muscles, respectively (Beckett et al., 2002). To determine the temporal correlation between Ca²⁺-transients and post-junctional electrical responses, simultaneous intracellular microelectrode recordings and video-imaging of Ca²⁺-transients in ICC-IM during activation of enteric motor nerves by electrical field stimulation (EFS) was performed. As described above, the dominant response to neural activation was frequency-dependent inhibition of Ca²⁺-transients that was associated with IJPs with a precise temporal association (Fig. 16; n = 15 tissues; N = 15 animals). Higher frequency stimulations (5–20 Hz) produced more prolonged inhibition of Ca²⁺-transients that was associated with larger and more sustained IJPs, i.e. at 5 Hz IJPs averaged 14.5 ± 3.0 mV in amplitude and 5.5 ± 1.9 s in duration (Fig. 16; n = 15 tissues; N = 15 animals). In several tissues, the inhibition of Ca²⁺-transients was preceded by a transient summation of Ca²⁺-transients to produce a global rise in intracellular Ca²⁺ in ICC-IM, as described above. Simultaneous intracellular electrical and Ca²⁺-transient recordings revealed that the transient global rise in intracellular Ca²⁺ was tightly coupled to a fast EJP averaging 2.5±1.2 mV that preceded inhibition of Ca²⁺-transients that was coupled to a larger and more sustained IJP (Fig. 17A–C). In experiments where a global rise in intracellular Ca²⁺ preceded Ca²⁺-transient inhibition, addition of l-NNA abolished the inhibition in Ca²⁺-transients and the associated IJP and potentiated the global rise in intracellular Ca²⁺ and an associated EJP (Fig. 17D–F). These data show that both cholinergic excitatory and nitrergic inhibitory neural inputs regulate Ca²⁺-transient firing and contribute to ICC-IM excitability of these cells and membrane potential in the gastric fundus.

Figure 16. EFS evoked inhibition of Ca²⁺-transient activity is associated with membrane hyperpolarization.

A, EFS (0.3 ms, 1 pulse; delivered at the arrows and dashed white lines) under control conditions produced a dominant inhibition of Ca²⁺-transients (dashed horizontal lines) that was associated with IJPs (lower panel) with a precise temporal association. B, addition of atropine (1 μM) potentiated the inhibition of Ca²⁺-transients and the amplitude of the IJP to EFS. There was a relaxation of the circular layer in response to inhibitory motor input that resulted in a downward deflection in the STMap. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 17. EFS causes a global rise in intracellular Ca²⁺ followed by inhibition of Ca²⁺-transients and evokes EJPs followed by IJPs.

A, EFS (0.3 ms, 1–10 Hz for 1 s) under control conditions produced a robust summation of Ca²⁺-transients into a global rise in intracellular Ca²⁺, followed by a more prolonged inhibition of Ca²⁺-transients at all frequencies. Points of EFS delivery are illustrated by arrows, dashed white line or rectangular boxes. B, simultaneous intracellular microelectrode recordings with microelectrode placed in the centre of and immediately adjacent to the FOV during image recordings. EFS, at all frequencies, produced post-junctional neural responses consisting of an initial membrane depolarization or EJP followed by a larger, more sustained membrane hyperpolarization or IJP. A comparison of the temporal responses reveal that the EJP was associated with the global rise in intracellular Ca²⁺ in ICC-IM and the IJP was associated with the inhibition of Ca²⁺-transients (dashed horizontal lines). C, expanded time scales of the post-junctional neural responses shown in B. Dashed lines represent areas that were electrical recordings were expanded. D, following the addition of L-NNA (100 μM), EFS produced a marked summation of Ca²⁺-transients into a global rise in intracellular Ca²⁺ following EFS. E, simultaneous microelectrode recordings revealed an enhanced EJP in response to EFS. The IJP that followed the EJP was abolished at all frequencies. F, expanded time scales of the post-junctional neural responses shown in E, again, dashed lines represent areas of where recordings were expanded. [Colour figure can be viewed at wileyonlinelibrary.com]

Discussion

The gastric fundus provides an important role in digestion by regulating the contractile state of its muscular wall to accommodate the increase in volume that occurs during ingestion of meals. Coordinated relaxation and contraction of the fundus allows storage of food and gradual passage of the contents into the distal stomach for initial digestion and reduction in particle size before emptying into the duodenum for further digestion and absorption. The process of relaxation, known as the gastric accommodation reflex, is mediated primarily by nitrergic neurons that innervate the muscle layers (Desai et al., 1991; Tack et al., 2002). Excitatory inputs come mainly via cholinergic neurons that increase fundic tone. Previous studies suggested a role for ICC-IM in transducing cholinergic and nitrergic neural responses and conveying these responses to SMCs by electrical coupling (Beckett et al., 2002; Burns et al., 1996; Ward et al., 2000). These findings have been controversial, and others have disputed a role for ICC in the neuromuscular responses of the fundus (Goyal & Chaudhury, 2010; Huizinga et al., 2008; Sarna, 2008; Zhang et al., 2010). The present study demonstrates the dynamics of spontaneous Ca²⁺-transients in ICC-IM. Using direct imaging of Ca²⁺ events in ICC-IM in situ and simultaneous intracellular recordings the consequences of spontaneous Ca²⁺-transients generated in ICC-IM on resting potentials and responses to intrinsic nerve stimulation were characterized. Our results are consistent with a role for ICC-IM in regulating the basal excitability of fundus SMCs and receiving and transducing neural inputs from cholinergic and nitrergic motor neurons.

We characterized spontaneous Ca²⁺-transient activity in ICC-IM and responses to enteric neurotransmission in fundus muscles of mice that expressed GCaMP6f in a cell-specific manner. The gastric fundus has only a single population of ICC, i.e. ICC-IM (Beckett et al., 2002, 2004; Burns et al., 1996; Ward et al., 2000), and therefore influences from other ICC populations, such as ICC at the level of the myenteric plexus (ICC-MY) that exist in the gastric corpus and antrum (Beckett et al., 2003; Hirst et al., 2002), were absent. Simultaneous intracellular microelectrode recordings during Ca²⁺-imaging failed to show a one-to-one correlation between the Ca²⁺-transients within the FOV and fluctuations in membrane potential, suggesting that the resting potential may be influenced by many more ICC-IM outside the FOV. The Ca²⁺-transients correlated better with membrane potential during EFS, suggesting that many if not all ICC are innervated and Ca²⁺ release mechanisms are regulated by enteric motor neurons.

Fundus ICC-IM generated Ca²⁺-transients from multiple firing sites throughout cells. Ca²⁺-transients from single firing sites often appeared to be rhythmic. However, no coordination between firing sites within single cells or in neighbouring cells was observed. Unlike the myenteric class of ICC (ICC-MY), which generate coordinated, propagating clusters of Ca²⁺-transients organized by voltage-dependent Ca²⁺ entry (Baker et al., 2021; Drumm et al., 2017), entrainment or coordination between firing sites was not observed in fundus ICC-IM. Summation of Ca²⁺-transients from all firing sites in single cells showed stochastic activity, which is typical of Ca²⁺-transient activity in ICC in other regions of the GI tract, including ICC at the level of the deep muscular plexus in the small intestine (ICC-DMP) (Baker et al., 2016; 2018) and ICC-IM (Drumm, Hwang et al., 2019) and ICC-SS in the proximal colon (Drumm et al., 2020). Thus, a picture is emerging that stochastic Ca²⁺-transient generation is a fundamental behaviour in ICC, and organization of Ca²⁺-transients, as observed in ICC-MY of the small intestine and stomach, is a specialization caused by expression and function of voltage-dependent Ca²⁺ channels in ICC that exhibit pacemaker activity (Baker et al., 2021; Drumm et al., 2017).

Membrane potentials in fundus muscles reflected the stochastic nature of Ca²⁺ release and generation of STDs due to activation of Ano1 channels in ICC. The ongoing discharge of STDs (also referred to as unitary potentials by some authors; Beckett et al., 2004; Edwards et al., 1999) can be resolved with greater precision in small muscle bundles due to their higher input resistance. Previous studies of fundus muscle bundles showed that the occurrence of STDs in ICC-IM of the fundus was not affected by injection of depolarizing or hyperpolarizing currents, and it was concluded that this activity was voltage insensitive (Beckett et al., 2004). In the present study we examined whether Ca²⁺-transients and STDs in fundus ICC-IM were affected by the Ano1 inhibitor CaCC_inh-A01 as we have previously shown that they are inhibited by buffering intracellular Ca²⁺ with BAPTA-AM. CaCC_inh-A01 did not affect Ca²⁺-transients in ICC-IM but inhibited STDs and revealed ongoing spontaneous membrane hyperpolarizations. The membrane hyperpolarizations are inhibited by the small conductance calcium-activated potassium channel (SK3) blocker apamin (Beckett et al., 2004) and are likely to originate in PDGFRα⁺ cells that form a cellular component of the gastric SIP syncytium (Blair et al., 2012; Sanders et al, 2012; 2014; Sanders & Ward 2019; Sanders 2019).

A role for ICC-IM in neuroeffector responses was demonstrated originally in experiments on the murine gastric fundus (Beckett et al., 2004; Burns et al., 1996; Ward et al., 2000). Morphological studies showed that ICC-IM form synapse-like contacts with enteric nerve varicosities containing neurovesicles and gap junctions with neighbouring smooth muscle cells (Beckett et al., 2005; Horiguchi et al., 2003; Ward et al., 2000; Ward et al., 2004). W/W^V and Sl/Sl^d mutant mice that lack most gastric fundus ICC-IM have greatly attenuated nitrergic and cholinergic post-junctional neural responses (Beckett et al., 2002; Burns et al., 1996; Ward et al., 2000), but were able to respond to exogenous NO donors by membrane hyperpolarization and relaxation (Beckett et al., 2002; Burns et al., 1996) and muscarinic receptor agonists by membrane depolarization and contraction (Ward et al., 2000). Post-junctional cholinergic responses in smooth muscle cells were also evaluated biochemically using phosphorylation of proteins known to be involved in Ca²⁺ sensitization of the contractile apparatus responses (Bhetwal et al., 2013). In wild-type mice, phosphorylation of MYPT1 was unchanged after EFS, but was enhanced by the exogenous muscarinic agonist carbachol. In W/W^V tissues MYPT1 phosphorylation was noted after EFS, suggesting that the tight synaptic-like relationship between ICC-IM and nerve varicosities might restrict overflow of ACh released from enteric motor neurons, possibly due to the expression of acetylcholine esterase by enteric neurons (Worth et al., 2015). Higher concentrations of ACh in the excluded volumes between nerve varicosities and ICC-IM may enhance the rate of metabolism of the neurotransmitter. Taken together these findings suggest that gastric ICC-IM are directly involved in mediation of nitrergic and cholinergic neurotransmission, and the current study expands this concept by showing direct responsiveness of ICC-IM to enteric motor neurotransmission.

The hypothesis that ICC are intermediaries in enteric motor transmission has been controversial. Other investigators have concluded that ICC are either not responsive to enteric neurotransmitters or not a necessary element for post-junctional responses. (Goyal & Chaudhury, 2010; Huizinga et al., 2008; Sarna, 2008; Zhang et al., 2010). One study proposed that, in spite of the synaptic-like contacts between nerve varicosities and ICC-IM (Horiguchi et al., 2003; Ward et al., 2000, 2004), expression of synaptic proteins pre-and post-junctionally in nerve varicosities and ICC-IM (Beckett et al., 2005), expression of appropriate receptors and ion channels by ICC-IM and ICC-DMP of the small intestine (Grady et al., 1996; Iino et al., 2004, 2008, 2009; Lavin et al., 1998; Salmhofer et al., 2001; Shuttleworth et al., 1993; Sternini et al., 1995; Sung et al., 2018) and deficiency in post-junctional neuroeffector responses in gastric tissues when ICC-IM were reduced, volume transmission is better suited to the regulation of GI motility (Sarna, 2008). Arguments were given, without actual mathematical support, that the total volume of ICC (~5–7%), and presumably membrane area, is a small fraction of the total volume of SMCs (Sarna, 2008). Thus, ICC-IM would not be able to influence membrane potentials or responses of SMCs (Sarna, 2008).

Additional support for the role of ICC in neuroeffector transmission was provided in studies using genetic approaches. In vivo ablation of ICC in adult mice was achieved by crossing Kit^CreERT2+/− mice, the same strain used in the present study, with LSL-R26^DTA/+mice. Cre-mediated activation of diphtheria toxin (DTA) caused cell-specific ablation of Cre-expressing cells (Ivanova et al., 2005), i.e. ICC. EJPs evoked by EFS were absent in colonic muscles of these animals, but fast and slow IJPs persisted (Klein et al., 2013). The persistence of the slow IJPs, which are mediated by NO, following depletion of ICC is on the surface perplexing, but it may be due to incomplete loss of ICC (e.g. see Fig. S2 in Klein et al., 2013). In previous studies on colons of W/W^v mice or Ws/Ws rats, where ICC are reduced but not absent (Albertí et al., 2007; Sanders et al., 2010), slow IJPs could still be observed in some regions of the muscle.

Downstream signalling responsible for ICC-NO-dependent inhibitory responses has also been investigated. When cGMP-dependent protein kinase I (Prkg1), a mediator of NO-dependent responses, was knocked down by tamoxifen treatment of Prkg1^f/f mice bred with Kit^CreERT2+/− mice, NO-dependent, slow IJPs were not recorded in GI muscles (Klein et al., 2013). This finding received further support from experiments in which soluble guanylate cyclase (sGC) was knocked down in studies where cell-specific knock-down of sGC was performed. Reduction of sGC in ICC also decreased IJPs; however, mixed effects have also been noted in some GI muscles where knockdown of sGC in both ICC and SMCs has effects on nitrergic inhibition (Groneberg et al., 2013, 2015; Lies, Gil et al., 2014; Lies, Groneberg et al., 2014; Lies et al., 2015).

Using a similar imaging approach to that described in the present study, post-junctional neural responses in ICC in the deep muscular plexus of the small intestine (ICC-DMP) were examined (Baker et al., 2018). In mice expressing GCaMP3 in ICC, EFS reduced Ca²⁺-transients, and the inhibition was due to nitrergic neural effects. The NO donor (DEA-NONOate) also inhibited Ca²⁺-transients in ICC-DMP and Nω-Nitro-l-arginine (l-NNA) or the guanylate cyclase inhibitor (ODQ) blocked the inhibition induced by EFS, suggesting that it was mediated by cGMP, but not purinergic nerves or membrane hyperpolarization as both the P2Y1 antagonist MRS2500 or the K_ATP agonist pinacidil had no effect on Ca²⁺-transients in ICC-DMP (Baker et al., 2018). Thus, ICC in different regions of the GI tract appear to respond to nitrergic inhibition of Ca²⁺-transients in a similar manner. Ca²⁺-transients in ICC-DMP were much less sensitive to the removal of extracellular Ca²⁺ than ICC-IM in the fundus, colon and LES (Drumm, Hwang et al., 2019; Drumm et al., 2022), suggesting that different populations of ICC throughout the GI tract have different Ca²⁺ handling properties.

Muscarinic agonists are known to activate a non-selective cation conductance in SMCs (Inoue & Isenberg, 1990; Inoue, 1991; So & Kim, 2003), but neurotransmitter released from intrinsic cholinergic neurons activated a CaCC and post-junctional cholinergic EJPs are absent in mice with genetic deactivation of Ano1 (Sung et al., 2018). Contractile responses to cholinergic nerve stimulation were also greatly attenuated in Ano1 knockdown animals (Sung et al., 2018). Pharmacological blockers of Ano1channels inhibited EJPs and cholinergic contractile responses to EFS evoked stimulation in fundus muscles but not their exogenous application, further supporting a role for ICC-IM in transducing and mediating cholinergic excitatory motor responses. The present study, using cell-specific expression of GCaMP6f in ICC-IM, provides further evidence supporting the idea that ICC-IM are innervated and possess an effective means of transducing cholinergic and nitrergic neurotransmission (i.e. regulation of Ca²⁺-transients).

Responses to EFS were predominantly inhibitory, and these responses consisted of a frequency-dependent reduction in Ca²⁺-transients for several seconds. Inhibition of Ca²⁺ transients would be expected to reduce activation of Ano1 channels that are normally activated by the Ca²⁺-transient local increases in [Ca²⁺]_i. In some muscles a transient rise in global intracellular Ca²⁺ occurred immediately after initiation of EFS. Simultaneous electrical recordings performed by impalement of cells in the centre of the FOVs revealed a relationship between Ca²⁺-transients and post-junctional electrical responses. In muscles in which the dominant response to EFS was inhibition of Ca²⁺-transients membrane hyperpolarization or IJPs were observed. Muscles with transient increases in global intracellular Ca²⁺ in response to EFS displayed transient membrane depolarizations or EJPs. The transient global increase in intracellular Ca²⁺ was inhibited by atropine, and this muscarinic antagonist also blocked EJPs. Inhibition of Ca²⁺-transients by EFS was blocked by l-NNA, and this antagonist also inhibited IJPs. Coupling between Ca²⁺-transients and changes in membrane potential is likely to involve activation or deactivation of Ano1 channels (Sung et al., 2018; Drumm et al., 2020).

An important question was whether there are different sub-populations of ICC-IM: one population responding to cholinergic excitatory and another responding to nitrergic inhibitory neural inputs. Analysis of STMs from single ICC-IM showed that the cells responded to cholinergic stimulation with summation of Ca²⁺-transients into a brief global increase of intracellular Ca²⁺ followed by a more sustained inhibition of Ca²⁺-transients. Thus, individual ICC-IM can transduce both cholinergic excitatory and nitrergic inhibitory inputs.

In summary, ICC-IM in gastric fundus are highly active, generating stochastic Ca²⁺-transients from multiple firing sites within cells. These events are not entrained, either between sites in individual cells or in adjacent cells. Ca²⁺-transients require replenishment of intracellular Ca²⁺ stores from influx of extracellular Ca²⁺, but this mechanism was not dependent on L-type Ca²⁺ channels. SERCA pump antagonists abolished Ca²⁺-transients, demonstrating that Ca²⁺ release from intracellular stores is responsible for the stochastic nature of Ca²⁺-transients. Ca²⁺-transients activate Ano1 channels in ICC-IM, providing transient depolarizing currents in the SIP syncytium. Thus, spontaneous Ca²⁺ transients in ICC-IM produce the noisy resting potentials of fundus muscles. EFS, with parameters known to activate intrinsic neurons, caused bi-phasic responses consisting of a transient summation of Ca²⁺-transients to produce a global rise in intracellular Ca²⁺ followed by sustained inhibition of Ca²⁺-transients. The transient global rise in intracellular Ca²⁺ was mediated by cholinergic inputs and inhibited by atropine. Inhibition of Ca²⁺-transients during EFS was mediated by nitric oxide and its receptor, sGC. Individual ICC-IM can transduce both excitatory and inhibitory neural inputs. Nerve-evoked regulation of Ca²⁺-transients in ICC-IM affects post-junctional electrical responses. These data show that ICC-IM in the fundus are innervated and transduce at least a portion of responses to enteric motor neurons. Responses developing in ICC-IM are conducted to SMCs and regulate membrane potentials and excitability of the SMCs of the gastric fundus.

Key points.

• The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC-IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs.

• Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC-IM were examined. ICC-IM were highly active, generating stochastic intracellular Ca²⁺-transients.

• Stimulation of enteric motor nerves abolished Ca²⁺-transients in ICC-IM. This inhibitory response was preceded by a global rise in intracellular Ca²⁺. Individual ICC-IM responded to nerve stimulation with a rise in Ca²⁺ followed by inhibition of Ca²⁺-transients.

• Inhibition of Ca²⁺-transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in Ca²⁺ was inhibited by the muscarinic antagonist atropine.

• Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca²⁺ and inhibition of Ca²⁺-transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials.

• The data presented support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Biographies

Sung Jin Hwang received his PhD from the Department of Biology in the Wonkwang University of South Korea in 2003. He spent 5 years as a post-doctoral fellow at the University of Nevada Reno, School of Medicine from 2003 to 2008 and is currently an Assistant Professor of Physiology and Cell Biology. He primary research interests are in examining the cells involved in pacemaker activity and neuroeffector transmission in the gastrointestinal and female reproductive tracts.

Bernard T. Drumm received his PhD from the Dundalk Institute of Technology (DKIT), Ireland and is currently a lecturer and research investigator within the Department of Life & Health Science at DKIT. His research within the Smooth Muscle Research Centre at DKIT focuses on Ca²⁺ signalling in visceral smooth muscle organs of the gastrointestinal and urinary tracts. Prior to DKIT, he spent 7 years at the University of Nevada, Reno as a Postdoctoral Research Fellow and Research Assistant Professor, using optogenetic imaging approaches to study neuroeffector and pacemaking mechanisms in gastrointestinal muscle systems.