Interstitial cells of the sip syncytium regulate basal membrane potential in murine gastric corpus.

Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.

Mapping the rat gastric slow-wave conduction pathway: bridging in vitro and in vivo methods, revealing a loosely coupled region in the distal stomach.

Rhythmic electrical events, termed slow waves, govern the timing and amplitude of phasic contractions of the gastric musculature. Extracellular multielectrode measurement of gastric slow waves can be a biomarker for phenotypes of motility dysfunction. However, a gastric slow-wave conduction pathway for the rat, a common animal model, is unestablished. In this study, the validity of extracellular recording was demonstrated in vitro with simultaneous intracellular and extracellular recordings and by pharmacological inhibition of slow waves. The conduction pathway was determined by in vivo extracellular recordings while considering the effect of motion. Slow-wave characteristics [means (SD)] varied regionally having higher amplitude in the antrum than the distal corpus [1.03 (0.12) mV vs. 0.75 (0.31) mV; n = 7; P = 0.025 paired t test] and faster propagation near the greater curvature than the lesser curvature [1.00 (0.14) mm·s-1 vs. 0.74 (0.14) mm·s-1; n = 9 GC, 7 LC; P = 0.003 unpaired t test]. Notably, in some subjects, separate wavefronts propagated near the lesser and greater curvatures with a loosely coupled region occurring in the area near the distal corpus midline at the interface of the two wavefronts. This region had either the greater or lesser curvature wavefront propagating through it in a time-varying manner. The conduction pattern suggests that slow waves in the rat stomach form annular wavefronts in the antrum and not the corpus. This study has implications for interpretation of the relationship between slow waves, the interstitial cells of Cajal network structure, smooth muscles, and gastric motility.NEW & NOTEWORTHY Mapping of rat gastric slow waves showed regional variations in their organization. In some subjects, separate wavefronts propagated near the lesser and greater curvatures with a loosely coupled region near the midline, between the wavefronts, having a varying slow-wave origin. Furthermore, simultaneous intracellular and extracellular recordings were concordant and independent of movement artifacts, indicating that extracellular recordings can be interpreted in terms of their intracellular counterparts when intracellular recording is not possible.

Electromechanical coupling and anatomy of the in vivo gastroduodenal junction.

Few biomarkers support the diagnosis and treatment of disorders of gut-brain interaction (DGBI), although gastroduodenal junction (GDJ) electromechanical coupling is a target for novel interventions. Rhythmic "slow waves," generated by interstitial cells of Cajal (ICC), and myogenic "spikes" are bioelectrical mechanisms underpinning motility. In this study, simultaneous in vivo high-resolution electrophysiological and impedance planimetry measurements were paired with immunohistochemistry to elucidate GDJ electromechanical coupling. Following ethical approval, the GDJ of anaesthetized pigs (n = 12) was exposed. Anatomically specific, high-resolution electrode arrays (256 electrodes) were applied to the serosa. EndoFLIP catheters (16 electrodes; Medtronic, MN) were positioned luminally to estimate diameter. Postmortem tissue samples were stained with Masson's trichrome and Ano1 to quantify musculature and ICC. Electrical mapping captured slow waves (n = 512) and spikes (n = 1,071). Contractions paralleled electrical patterns. Localized slow waves and spikes preceded rhythmic contractions of the antrum and nonrhythmic contractions of the duodenum. Slow-wave and spike amplitudes were correlated in the antrum (r = 0.74, P < 0.001) and duodenum (r = 0.42, P < 0.001). Slow-wave and contractile amplitudes were correlated in the antrum (r = 0.48, P < 0.001) and duodenum (r = 0.35, P < 0.001). Distinct longitudinal and circular muscle layers of the antrum and duodenum had a total thickness of (2.8 ± 0.9) mm and (0.4 ± 0.1) mm, respectively. At the pylorus, muscle layers merged and thickened to (3.5 ± 1.6) mm. Pyloric myenteric ICC covered less area (1.5 ± 1.1%) compared with the antrum (4.2 ± 3.0%) and duodenum (5.3 ± 2.8%). Further characterization of electromechanical coupling and ICC biopsies may generate DGBI biomarkers.NEW & NOTEWORTHY This study applies electrical mapping, impedance planimetry, and histological techniques to the gastroduodenal junction to elucidate electromechanical coupling in vivo. Contractions of the terminal antrum and pyloric sphincter were associated with gastric slow waves. In the duodenum, bursts of spike activity triggered oscillating contractions. The relative sparsity of myenteric interstitial cells of Cajal in the pylorus, compared with the adjacent antrum and duodenum, is hypothesized to prevent coupling between antral and duodenal slow waves.

Ca2+ dynamics in interstitial cells: foundational mechanisms for the motor patterns in the gastrointestinal tract.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons, and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGF receptor (PDGFR)α+ cells. Together these cells form the SIP syncytium. ICC and PDGFRα+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRα+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transients occur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarizations (STDs). Neurotransmission increases or decreases Ca2+ transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract suggest that the term "myogenic" should be replaced by the term "SIPgenic," as this review discusses.

Three-dimensional multi-field modelling of gastric arrhythmias and their effects on antral contractions.

The contraction activation of smooth muscle in the stomach wall (SW) is coordinated by slow electrical waves. The interstitial cells of Cajal (ICC), specialised pacemaker cells, initiate and propagate these slow waves. By establishing an electrically coupled network, each ICC adjusts its intrinsic pacing frequency to a single dominant frequency, to be a key aspect in modelling the electrophysiology of gastric tissue. In terms of modelling, additional fields associated with electrical activation, such as voltage-dependent calcium influx and the resulting deformation, have hardly been considered so far. Here we present a three-dimensional model of the electro-chemomechanical activation of gastric smooth muscle contractions. To reduce computational costs, an adaptive multi-scale discretisation strategy for the temporal resolution of the electric field is used. The model incorporates a biophysically based model of gastric ICC pacemaker activity that aims to simulate stable entrainment and physiological conduction velocities of the electrical slow waves. Together with the simulation of concomitant gastric contractions and the inclusion of a mechanical feedback mechanism, the model is used to study dysrhythmias of gastric slow waves induced by abnormal stretching of the antral SW. The model is able to predict the formation of stretch-induced gastric arrhythmias, such as the emergence of an ectopic pacemaker in the gastric antrum. The results show that the ectopic event is accompanied by smooth muscle contraction and, although it disrupts the normal propagation pattern of gastric slow electrical waves, it can also catalyse the process of handling indigestible materials that might otherwise injure the gastric SW.

Recording and analysis of slow waves of the small intestine of mice with heart failure.

BACKGROUND: Gastrointestinal (GI) symptoms in heart failure (HF) patients are associated with increased morbidity and mortality. We hypothesized that HF reduces bioelectrical activity underlying peristalsis. In this study, we aimed to establish a method to capture and analyze slow waves (SW) in the small intestine in mice with HF. METHODS: We established a model of HF secondary to coronary artery disease in mice overexpressing tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells. The myoelectric activity was recorded from the small intestine in live animals under anesthesia. The low- and high-frequency components of SW were isolated in MATLAB and compared between the control (n = 12) and eTNAP groups (n = 8). C-kit-positive interstitial cells of Cajal (ICC) and Pgp9.5-positive myenteric neurons were detected by immunofluorescence. Myenteric ganglia were assessed by hematoxylin and eosin (H&E) staining. RESULTS: SW activity was successfully captured in vivo, with both high- and low-frequency components. Low-frequency component of SW was not different between endothelial TNAP (eTNAP) and control mice (mean[95% CI]: 0.032[0.025-0.039] vs. 0.040[0.028-0.052]). High-frequency component of SW showed a reduction eTNAP mice relative to controls (0.221[0.140-0.302] vs. 0.394[0.295-0.489], p < 0.01). Dysrhythmia was also apparent upon visual review of signals. The density of ICC and neuronal networks remained the same between the two groups. No significant reduction in the size of myenteric ganglia of eTNAP mice was observed. CONCLUSIONS: A method to acquire SW activity from small intestines in vivo and isolate low- and high-frequency components was established. The results indicate that HF might be associated with reduced high-frequency SW activity.

Embryonic Development of Motility: Lessons from the Chicken.

I outline here the development of intestinal motility in the chicken embryo. The first contractile events are circular smooth muscle driven calcium waves (E6), that gain a clock-like regularity when interstitial cells of Cajal become electrically active (E14). Soon after longitudinal smooth muscle contractions become prominent (E14), the enteric nervous system starts controlling motility (E16) by coupling the longitudinal and circular contractions via inhibitory neurotransmission. It gives rise to circular-longitudinal antagonism, to the migrating motor complex, and to the polarized ascending contraction-descending relaxation pressure response known as the "law of the intestine". The kinetics of gut development in the chicken appears to follow faithfully that of humans by simply converting embryonic days of chicken development into embryonic weeks of human development.

Generation of Gut Motor Patterns Through Interactions Between Interstitial Cells of Cajal and the Intrinsic and Extrinsic Autonomic Nervous Systems.

The musculature of the gastrointestinal tract is a vast network of collaborating excitable cell types. Embedded throughout are the interstitial cells of Cajal (ICC) intertwined with enteric nerves. ICC sense external stimuli such as distention, mediate nerve impulses to smooth muscle cells, and provide rhythmic excitation of the musculature. Neural circuitry involving both the intrinsic and extrinsic autonomic nervous systems, in collaboration with the ICC, orchestrate an array of motor patterns that serve to provide mixing of content to optimize digestion and absorption, microbiome homeostasis, storage, transit, and expulsion. ICC are specialized smooth muscle cells that generate rhythmic depolarization to the musculature and so provide the means for peristaltic and segmenting contractions. Some motor patterns are purely myogenic, but a neural stimulus initiates most, further depolarizing the primary pacemaker cells and the musculature and/or initiating transient pacemaker activity in stimulus-dependent secondary ICC pacemaker cells. From stomach to rectum, ICC networks rhythmically provide tracks along which contractions advance.

Ca2+ Signaling Is the Basis for Pacemaker Activity and Neurotransduction in Interstitial Cells of the GI Tract.

Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit+ (interstitial cells of Cajal or ICC), and PDGFRα+ cells (formerly "fibroblast-like" cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca2+ release from the endoplasmic reticulum (ER) and activation of Ca2+-activated ion channels in the plasma membrane (PM). ICC express Ca2+-activated Cl- channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα+ cells express Ca2+-activated K+ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca2+ transients in ICC and PDGFRα+ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca2+ conductances that organize Ca2+ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca2+ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca2+ dynamics in these cells in GI motility disorders.

Localized bioelectrical conduction block from radiofrequency gastric ablation persists after healing: safety and feasibility in a recovery model.

Gastric ablation has demonstrated potential to induce conduction blocks and correct abnormal electrical activity (i.e., ectopic slow-wave propagation) in acute, intraoperative in vivo studies. This study aimed to evaluate the safety and feasibility of gastric ablation to modulate slow-wave conduction after 2 wk of healing. Chronic in vivo experiments were performed in weaner pigs (n = 6). Animals were randomly divided into two groups: sham-ablation (n = 3, control group; no power delivery, room temperature, 5 s/point) and radiofrequency (RF) ablation (n = 3; temperature-control mode, 65°C, 5 s/point). In the initial surgery, high-resolution serosal electrical mapping (16 × 16 electrodes; 6 × 6 cm) was performed to define the baseline slow-wave activation profile. Ablation (sham/RF) was then performed in the mid-corpus, in a line around the circumferential axis of the stomach, followed by acute postablation mapping. All animals recovered from the procedure, with no sign of perforation or other complications. Two weeks later, intraoperative high-resolution mapping was repeated. High-resolution mapping showed that ablation successfully induced sustained conduction blocks in all cases in the RF-ablation group at both the acute and 2 wk time points, whereas all sham-controls had no conduction block. Histological and immunohistochemical evaluation showed that after 2 wk of healing, the lesions were in the inflammation and early proliferation phase, and interstitial cells of Cajal (ICC) were depleted and/or deformed within the ablation lesions. This safety and feasibility study demonstrates that gastric ablation can safely and effectively induce a sustained localized conduction block in the stomach without disrupting the surrounding slow-wave conduction capability.NEW & NOTEWORTHY Ablation has recently emerged as a tool for modulating gastric electrical activation and may hold interventional potential for disorders of gastric function. However, previous studies have been limited to the acute intraoperative setting. This study now presents the safety of gastric ablation after postsurgical recovery and healing. Localized electrical conduction blocks created by ablation remained after 2 wk of healing, and no perforation or other complications were observed over the postsurgical period.

Deciphering Stomach Myoelectrical Slow Wave Conduction Patterns via Confocal Imaging of Gastric Pacemaker Cells and Fractal Geometry.

Interstitial Cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells that generate and actively propagate slow waves of depolarization (SWs) of the muscularis propria. SWs regulate the motility of the GI tract necessary for digestion, absorption of nutrients, and elimination of waste. Within the gastric wall, there are three main inter-connected layers of ICC networks: longitudinal muscle ICC (ICC-LM), myenteric plexus ICC (ICC-MP) & circumferential muscle (ICC-CM). Fractal structural parameters such as Fractal Dimension (FD), Lacunarity and Succolarity, have many advantages over physically-based parameters when it comes to characterizing the complex architectures of ICC networks. The analysis of networks of ICC throughout the proximal and distal murine gastric antrum with the FD and Lacunarity metrics was previously performed. Although the application of Succolarity is relatively nascent compared to the FD and Lacunarity; nevertheless, numerous studies have demonstrated the capability of this fractal measure to extract information from images associated with flow by which neither the FD nor Lacunarity are capable of discerning. In this study, Succolarity analysis of ICC-MP and ICC-CM networks were performed with confocal images taken across the proximal and distal murine antrum. Our findings demonstrated the Succolarity of ICC-MP and ICC-CM varied with directions and antral regions. The Succolarity of ICC-MP did not vary considerably with direction, however, Succolarity was higher in the aboral direction with 0.2113 ±0.1589, and 0.0637 ±0.0822 in the proximal and distal antrum, respectively. The overall Succolarity of ICC-MP was significantly higher than that of ICC-CM in the proximal antrum ( 0.1580±0.1325 vs [Formula: see text]) and in the distal antrum ( 0.0449 ±0.0409 vs [Formula: see text]). Clinical Relevance-Modeling SWs conduction patterns via image analysis of detailed ICC networks help to facilitate an improved understanding of the mechanisms underpinning GI myoelectric activity and the diseases associated with its dysfunction.

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

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. 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.

Insights on gastrointestinal motility through the use of optogenetic sensors and actuators.

The muscularis of the gastrointestinal (GI) tract consists of smooth muscle cells (SMCs) and various populations of interstitial cells of Cajal (ICC), platelet-derived growth factor receptor α+ (PDGFRα+ ) cells, as well as excitatory and inhibitory enteric motor nerves. SMCs, ICC and PDGFRα+ cells form an electrically coupled syncytium, which together with inputs from the enteric nervous system (ENS) regulates GI motility. Early studies evaluating Ca2+ signalling behaviours in the GI tract relied upon indiscriminate loading of tissues with Ca2+ dyes. These methods lacked the means to study activity in specific cells of interest without encountering contamination from other cells within the preparation. Development of mice expressing optogenetic sensors (green calmodulin fusion protein (GCaMP), red calmodulin fusion protein (RCaMP)) has allowed visualization of Ca2+ signalling behaviours in a cell specific manner. Additionally, availability of mice expressing optogenetic modulators (channelrhodopsins or halorhodospins) has allowed manipulation of specific signalling pathways using light. GCaMP-expressing animals have been used to characterize Ca2+ signalling behaviours of distinct classes of ICC and SMCs throughout the GI musculature. These findings illustrate how Ca2+ signalling in ICC is fundamental in GI muscles, contributing to tone in sphincters, pacemaker activity in rhythmic muscles and relaying enteric signals to SMCs. Animals that express channelrhodopsin in specific neuronal populations have been used to map neural circuitry and to examine post junctional neural effects on GI motility. Thus, optogenetic approaches provide a novel means to examine the contribution of specific cell types to the regulation of motility patterns within complex multi-cellular systems.

Ca2+ signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower oesophageal sphincters.

The lower oesophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Cav 1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC-IM), through activation of Ca2+ -activated Cl- channels (ANO1), set membrane potentials of SMCs favourable for activation of Cav 1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC-IM are activated by Ca2+ transients, but no studies have examined Ca2+ dynamics in ICC-IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC-IM and SMCs. These studies revealed that Ano1 and its gene product, ANO1, are expressed in c-Kit+ cells (ICC-IM) in mouse and monkey LES clasp muscles. Ca2+ signalling was imaged in situ, using mice expressing GCaMP6f specifically in ICC (Kit-KI-GCaMP6f). ICC-IM exhibited spontaneous Ca2+ transients from multiple firing sites. Ca2+ transients were abolished by cyclopiazonic acid or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca2+ transients depended on Ca2+ influx and store reloading, as Ca2+ transient frequency was reduced in Ca2+ free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced ∼80% either by Ani9, an ANO1 antagonist or by the Cav 1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca2+ activates ANO1 channels in ICC-IM in the LES. Coupling of ICC-IM to SMCs drives depolarisation, activation of Cav 1.2 channels, Ca2+ entry and contractile tone. KEY POINTS: The lower oesophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the oesophagus. LES smooth muscle cells (SMCs) display depolarised membrane potentials facilitating activation of L-type Ca2+ channels. Interstitial cells of Cajal (ICC) express Ca2+ -activated Cl- channels encoded by Ano1 in mouse and monkey LES. Ca2+ signalling in ICC activates ANO1 currents in ICC. ICC displayed spontaneous Ca2+ transients in mice from multiple firing sites in each cell and no entrainment of Ca2+ firing between sites or between cells. Inhibition of ANO1 channels with a specific antagonist caused hyperpolarisation of mouse LES and inhibition of tone in monkey and mouse LES muscles. Our data suggest a novel mechanism for LES tone in which Ca2+ transient activation of ANO1 channels in ICC generates depolarising inward currents that conduct to SMCs to activate L-type Ca2+ currents, Ca2+ entry and contractile tone.

Targeted ablation of gastric pacemaker sites to modulate patterns of bioelectrical slow wave activation and propagation in an anesthetized pig model.

Gastric motility is coordinated by underlying bioelectrical slow waves. Gastric dysrhythmias occur in gastrointestinal (GI) motility disorders, but there are no validated methods for eliminating dysrhythmias. We hypothesized that targeted ablation could eliminate pacemaker sites in the stomach, including dysrhythmic ectopic pacemaker sites. In vivo high-resolution serosal electrical mapping (16 × 16 electrodes; 6 × 6 cm) was applied to localize normal and ectopic gastric pacemaker sites in 13 anesthetized pigs. Radiofrequency ablation was performed in a square formation surrounding the pacemaker site. Postablation high-resolution mapping revealed that ablation successfully induced localized conduction blocks after 18 min (SD 5). Normal gastric pacemaker sites were eliminated by ablation (n = 6), resulting in the emergence of a new pacemaker site immediately distal to the original site in all cases. Ectopic pacemaker sites were similarly eliminated by ablation in all cases (n = 7), and the surrounding mapped area was then entrained by normal antegrade activity in five of those cases. Histological analysis showed that ablation lesions extended through the entire depth of the muscle layer. Immunohistochemical staining confirmed localized interruption of the interstitial cell of Cajal (ICC) network through the ablation lesions. This study demonstrates that targeted gastric ablation can effectively modulate gastric electrical activation, including eliminating ectopic sites of slow wave activation underlying gastric dysrhythmias, without disrupting surrounding conduction capability or tissue structure. Gastric ablation presents a powerful new research tool for modulating gastric electrical activation and may likely hold therapeutic potential for disorders of gastric function.NEW & NOTEWORTHY This study presents gastric ablation as a novel tool for modulating gastric bioelectrical activation, including eliminating the normal gastric pacemaker site as well as abnormal ectopic pacemaker sites underlying gastric dysrhythmias. Targeted application of radiofrequency ablation was able to eliminate these pacemaker sites without disrupting surrounding conduction capability or tissue structure. Gastric ablation presents a powerful new research tool for modulating gastric electrical activation and may likely hold therapeutic potential for disorders of gastric function.

A framework for the design of a closed-loop gastric pacemaker for treating conduction block.

BACKGROUND AND OBJECTIVE: Gastrointestinal (GI) motility disorders can be significantly detrimental to the quality of life. Pacing, or long pulse gastric electrical stimulation, is a potential treatment option for treating GI motility disorders by modulating the slow wave activity. Open-loop pacing of the GI tract is the current standard for modulating dysrhythmic patterns, but it is known to be suboptimal and inefficient. Recent work on sensing intracellular potentials and pacing accordingly in a closed-loop has been shown to be effective at modulating dysrhythmic patterns. However, capturing intracellular potentials in an in-vivo setting is not viable. Therefore a closed-loop gastric electrical stimulation that can sense extracellular potentials and pace accordingly to modulate dysrhythmic patterns is required. This paper presents a closed-loop Gastric Electrical Stimulator (GES) design framework, which comprises of extracellular potential generation, sensing, and closed-loop actuation. METHODS: This work leverages a pre-existing high-fidelity two-dimensional Interstitial Cells of Cajal (ICC) network modeling framework to mimic several normal and dysrhythmic patterns observed in experimental recordings of patients suffering from GI tract diseases. The activation patterns of the of the ICC network are captured by an extracellular potential generation model and is integrated with the GES in a closed-loop to validate the efficacy of the developed pacing algorithms. The proposed GES pacing algorithms extend existing offline filtering and activation detection methods to process the sensed extracellular potentials in real time. The GES detects bradygastric rhythms based on the sensed extracellular potentials and actuates the ICC network via pacing to rectify dysrhythmic patterns. RESULTS: The proposed GES model is able to sense and process the generated noisy extracellular potentials, detect the bradygastric patterns, and modulate the slow wave activities to normal propagation effectively. CONCLUSIONS: A closed-loop GES design, which can be applied in an experimental and clinical setting is developed and validated through the ICC network model. The proposed GES model has the ability to modulate a variety of bradygastric patterns, including conduction block effectively in a closed-loop.

PDGFRα+ subepithelial interstitial cells act as a pacemaker to drive smooth muscle of the guinea pig seminal vesicle.

Smooth muscle cells (SMCs) of the guinea pig seminal vesicle (SV) develop spontaneous phasic contractions, Ca2+ flashes and electrical slow waves in a mucosa-dependent manner, and thus it was envisaged that pacemaker cells reside in the mucosa. Here, we aimed to identify the pacemaker cells in SV mucosa using intracellular microelectrode and fluorescence Ca2+ imaging techniques. Morphological characteristics of the mucosal pacemaker cells were also investigated using focused ion beam/scanning electron microscopy tomography and fluorescence immunohistochemistry. Two populations of mucosal cells developed spontaneous Ca2+ transients and electrical activity, namely basal epithelial cells (BECs) and subepithelial interstitial cells (SICs). Pancytokeratin-immunoreactive BECs were located on the apical side of the basement membrane (BM) and generated asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). The spontaneous Ca2+ transients and STDs were not diminished by 10 µM nifedipine but abolished by 10 µM cyclopiazonic acid (CPA). Platelet-derived growth factor receptor α (PDGFRα)-immunoreactive SICs were distributed just beneath the basal side of the BM and developed synchronous Ca2+ oscillations and electrical slow waves, which were suppressed by 3 µM nifedipine and abolished by 10 µM CPA. In SV mucosal preparations in which some smooth muscle bundles remained attached, SICs and residual SMCs developed temporally correlated spontaneous Ca2+ transients. Neurobiotin injected into SICs spread not only to neighbouring SICs but also to neighbouring SMCs or vice versa. These results suggest that PDGFRα+ SICs electrotonically drive the spontaneous contractions of SV smooth muscle. KEY POINTS: In many visceral smooth muscle organs, spontaneous contractions are electrically driven by non-muscular pacemaker cells. In guinea pig seminal vesicles (SVs), as yet unidentified mucosal cells appear to drive neighbouring smooth muscle cells (SMCs). Two populations of spontaneously active cells are distributed in the SV mucosa. Basal epithelial cells (BECs) generate asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). In contrast, subepithelial interstitial cells (SICs) develop synchronous Ca2+ oscillations and electrical slow waves. Pancytokeratin-immunoreactive (IR) BECs are located on the apical side of the basement membrane (BM), while platelet-derived growth factor receptor α (PDGFRα)-IR SICs are located on the basal side of the BM. Spontaneous Ca2+ transients in SICs are synchronised with those in SV SMCs. Dye-coupling between SICs and SMCs suggests that SICs act as pacemaker cells to drive the spontaneous contractions of SV smooth muscle.

Hyperpolarization-activated cyclic nucleotide-gated channels working as pacemaker channels in colonic interstitial cells of Cajal.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels function as pacemaker channels in spontaneously active cells. We studied the existence of HCN channels and their functional roles in the interstitial cells of Cajal (ICC) from the mouse colon using electrophysiological, immunohistochemical and molecular techniques. HCN1 and HCN3 channels were detected in anoctamin-1 (Ca2+ -activated Cl- channel; ANO1)-positive cells within the muscular and myenteric layers in colonic tissues. The mRNA transcripts of HCN1 and HCN3 channels were expressed in ANO1-positive ICC. In the deletion of HCN1 and HCN3 channels in colonic ICC, the pacemaking potential frequency was reduced. Basal cellular adenylate cyclase activity was decreased by adenylate cyclase inhibitor in colonic ICC, whereas cAMP-specific phosphodiesterase inhibitors increased it. 8-Bromo-cyclic AMP and rolipram increased spontaneous intracellular Ca2+ oscillations. In addition, Ca2+ -dependent adenylate cyclase 1 (AC1) mRNA was detected in colonic ICC. Sulprostone, a PGE2 -EP3 agonist, increased the pacemaking potential frequency, maximum rate of rise of resting membrane in pacemaker potentials and basal cellular adenylate cyclase activity in colonic ICC. These results indicate that HCN channels exist in colonic ICC and participate in generating pacemaking potentials. Thus, HCN channels may be therapeutic targets in disturbed colonic motility disorders.

Engaging biological oscillators through second messenger pathways permits emergence of a robust gastric slow-wave during peristalsis.

Peristalsis, the coordinated contraction-relaxation of the muscles of the stomach is important for normal gastric motility and is impaired in motility disorders. Coordinated electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICC) and smooth muscle (SM) cells of the stomach wall as a slow-wave, underly peristalsis. Normally, the gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. Understanding of the integrative role of neurotransmission and intercellular coupling in the propagation of an entrained gastric slow-wave, important for understanding motility disorders, however, remains incomplete. Using a computational framework constituted of a novel gastric motility network (GMN) model we address the hypothesis that engaging biological oscillators (i.e., ICCs) by constitutive gap junction coupling mechanisms and enteric neural innervation activated signals can confer a robust entrained gastric slow-wave. We demonstrate that while a decreasing enteric neural innervation gradient that modulates the intracellular IP3 concentration in the ICCs can guide the aboral slow-wave propagation essential for peristalsis, engaging ICCs by recruiting the exchange of second messengers (inositol trisphosphate (IP3) and Ca2+) ensures a robust entrained longitudinal slow-wave, even in the presence of biological variability in electrical coupling strengths. Our GMN with the distinct intercellular coupling in conjunction with the intracellular feedback pathways and a rostrocaudal enteric neural innervation gradient allows gastric slow waves to oscillate with a moderate range of frequencies and to propagate with a broad range of velocities, thus preventing decoupling observed in motility disorders. Overall, the findings provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach, offer directions for future experiments and theoretical work, and can potentially aid in the design of new interventional pharmacological and neuromodulation device treatments for addressing gastric motility disorders.

Localized gastric distension disrupts slow-wave entrainment leading to temporary ectopic propagation: a high-resolution electrical mapping study.

Gastric distension is known to affect normal slow-wave activity and gastric function, but links between slow-wave dysrhythmias and stomach function are poorly understood. Low-resolution mapping is unable to capture complex spatial properties of gastric dysrhythmias, necessitating the use of high-resolution mapping techniques. Characterizing the nature of these dysrhythmias has implications in the understanding of postprandial function and the development of new mapping devices. In this two-phase study, we developed and implemented a protocol for measuring electrophysiological responses to gastric distension in porcine experiments. In vivo, serosal high-resolution electrical mapping (256 electrodes; 36 cm2) was performed in anaesthetized pigs (n = 11), and slow-wave pattern, velocity, frequency, and amplitude were quantified before, during, and after intragastric distension. Phase I experiments (n = 6) focused on developing and refining the distension mapping methods using a surgically inserted intragastric balloon, with a variety of balloon types and distension protocols. Phase II experiments (n = 5) used barostat-controlled 500-mL isovolumetric distensions of an endoscopically introduced intragastric balloon. Dysrhythmias were consistently induced in all five gastric distensions, using refined distension protocols. Dysrhythmias appeared 23 s (SD = 5 s) after the distension and lasted 129 s (SD = 72 s), which consisted of ectopic propagation originating from the greater curvature in the region of distension. In summary, our results suggest that distension disrupts gastric entrainment, inducing temporary ectopic slow-wave propagation. These results may influence the understanding of the postprandial stomach and electrophysiological effects of gastric interventions.NEW & NOTEWORTHY This study presents the discovery of temporary dysrhythmic ectopic pacemakers in the distal stomach caused by localized gastric distension. Distension-induced dysrhythmias are an interesting physiological phenomenon that can inform the design of new interventional and electrophysiological protocols for both research and the clinic. The observation of distension-induced dysrhythmias also contributes to our understanding of stretch-sensitivity in the gut and may play an important role in normal and abnormal postprandial physiology.