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.

Interstitial cell of Cajal-like cells (ICC-LC) exhibit dynamic spontaneous activity but are not functionally innervated in mouse urethra.

Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit+ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca2+ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca2+ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca2+ signalling. ICC-LC generated Ca2+ waves spontaneously that propagated on average 40.1 ± 0.7 µm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca2+ signalling between ICC-LC was obtained. Ca2+ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca2+ channels (GSK-7975A). Phenylephrine increased Ca2+ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca2+ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.

FA4SANS-GAN: A Novel Machine Learning Generative Adversarial Network to Further Understand Ophthalmic Changes in Spaceflight Associated Neuro-Ocular Syndrome (SANS).

Purpose: To provide an automated system for synthesizing fluorescein angiography (FA) images from color fundus photographs for averting risks associated with fluorescein dye and extend its future application to spaceflight associated neuro-ocular syndrome (SANS) detection in spaceflight where resources are limited. Design: Development and validation of a novel conditional generative adversarial network (GAN) trained on limited amount of FA and color fundus images with diabetic retinopathy and control cases. Participants: Color fundus and FA paired images for unique patients were collected from a publicly available study. Methods: FA4SANS-GAN was trained to generate FA images from color fundus photographs using 2 multiscale generators coupled with 2 patch-GAN discriminators. Eight hundred fifty color fundus and FA images were utilized for training by augmenting images from 17 unique patients. The model was evaluated on 56 fluorescein images collected from 14 unique patients. In addition, it was compared with 3 other GAN architectures trained on the same data set. Furthermore, we test the robustness of the models against acquisition noise and retaining structural information when introduced to artificially created biological markers. Main Outcome Measures: For GAN synthesis, metric Fréchet Inception Distance (FID) and Kernel Inception Distance (KID). Also, two 1-sided tests (TOST) based on Welch's t test for measuring statistical significance. Results: On test FA images, mean FID for FA4SANS-GAN was 39.8 (standard deviation, 9.9), which is better than GANgio model's mean of 43.2 (standard deviation, 13.7), Pix2PixHD's mean of 57.3 (standard deviation, 11.5) and Pix2Pix's mean of 67.5 (standard deviation, 11.7). Similarly for KID, FA4SANS-GAN achieved mean of 0.00278 (standard deviation, 0.00167) which is better than other 3 model's mean KID of 0.00303 (standard deviation, 0.00216), 0.00609 (standard deviation, 0.00238), 0.00784 (standard deviation, 0.00218). For TOST measurement, FA4SANS-GAN was proven to be statistically significant versus GANgio (P = 0.006); versus Pix2PixHD (P < 0.00001); and versus Pix2Pix (P < 0.00001). Conclusions: Our study has shown FA4SANS-GAN to be statistically significant for 2 GAN synthesis metrics. Moreover, it is robust against acquisition noise, and can retain clear biological markers compared with the other 3 GAN architectures. This deployment of this model can be crucial in the International Space Station for detecting SANS. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

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.

Interstitial cells of Cajal - pacemakers of the gastrointestinal tract.

Gastrointestinal (GI) organs display spontaneous, non-neurogenic electrical, and mechanical rhythmicity that underlies fundamental motility patterns, such as peristalsis and segmentation. Electrical rhythmicity (aka slow waves) results from pacemaker activity generated by interstitial cells of Cajal (ICC). ICC express a unique set of ionic conductances and Ca2+ handling mechanisms that generate and actively propagate slow waves. GI smooth muscle cells lack these conductances. Slow waves propagate actively within ICC networks and conduct electrotonically to smooth muscle cells via gap junctions. Slow waves depolarize smooth muscle cells and activate voltage-dependent Ca2+ channels (predominantly CaV1.2), causing Ca2+ influx and excitation-contraction coupling. The main conductances responsible for pacemaker activity in ICC are ANO1, a Ca2+ -activated Cl- conductance, and CaV3.2. The pacemaker cycle, as currently understood, begins with spontaneous, localized Ca2+ release events in ICC that activate spontaneous transient inward currents due to activation of ANO1 channels. Depolarization activates CaV 3.2 channels, causing the upstroke depolarization phase of slow waves. The upstroke is transient and followed by a long-duration plateau phase that can last for several seconds. The plateau phase results from Ca2+ -induced Ca2+ release and a temporal cluster of localized Ca2+ transients in ICC that sustains activation of ANO1 channels and clamps membrane potential near the equilibrium potential for Cl- ions. The plateau phase ends, and repolarization occurs, when Ca2+ stores are depleted, Ca2+ release ceases and ANO1 channels deactivate. This review summarizes key mechanisms responsible for electrical rhythmicity in gastrointestinal organs.

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.

New open-source software for subcellular segmentation and analysis of spatiotemporal fluorescence signals using deep learning.

Cellular imaging instrumentation advancements as well as readily available optogenetic and fluorescence sensors have yielded a profound need for fast, accurate, and standardized analysis. Deep-learning architectures have revolutionized the field of biomedical image analysis and have achieved state-of-the-art accuracy. Despite these advancements, deep learning architectures for the segmentation of subcellular fluorescence signals is lacking. Cellular dynamic fluorescence signals can be plotted and visualized using spatiotemporal maps (STMaps), and currently their segmentation and quantification are hindered by slow workflow speed and lack of accuracy, especially for large datasets. In this study, we provide a software tool that utilizes a deep-learning methodology to fundamentally overcome signal segmentation challenges. The software framework demonstrates highly optimized and accurate calcium signal segmentation and provides a fast analysis pipeline that can accommodate different patterns of signals across multiple cell types. The software allows seamless data accessibility, quantification, and graphical visualization and enables large dataset analysis throughput.

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.

PDGFRα+ Interstitial Cells are Effector Cells of PACAP Signaling in Mouse and Human Colon.

BACKGROUND & AIMS: Platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance Ca2+ activated K+ channels 3 (SK3 channels) and transduce inhibitory inputs from purinergic and sympathetic nerves in mouse and human colon. We investigated whether PIC also express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, PAC1 (PAC1R), and are involved in mediating inhibitory regulation of colonic contractions by PACAP in mouse and human colons. METHODS: Gene expression analysis, Ca2+ imaging, and contractile experiments were performed on mouse colonic muscles. Ca2+ imaging, intracellular electrical recordings, and contractile experiments were performed on human colonic muscles. RESULTS: Adcyap1r1 (encoding PAC1R) is highly expressed in mouse PIC. Interstitial cells of Cajal (ICC) and SMCs expressed far lower levels of Adcyap1r. Vipr1 and Vipr2 were expressed at low levels in PIC, ICC, and SMCs. PACAP elicited Ca2+ transients in mouse PIC and inhibited spontaneous phasic contractions via SK channels. In human colonic muscles, PAC1R agonists elicited Ca2+ transients in PIC, hyperpolarized SMCs through SK channels and inhibited spontaneous phasic contractions. CONCLUSIONS: PIC of mouse and human colon utilize PAC1R-SK channel signal pathway to inhibit colonic contractions in response to PACAP. Effects of PACAP are in addition to the previously described purinergic and sympathetic inputs to PIC. Thus, PIC integrate inhibitory inputs from at least 3 neurotransmitters and utilize several types of receptors to activate SK channels and regulate colonic contractile behaviors.

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.

Software for segmenting and quantifying calcium signals using multi-scale generative adversarial networks.

Cellular calcium fluorescence imaging utilized to study cellular behaviors typically results in large datasets and a profound need for standardized and accurate analysis methods. Here, we describe open-source software (4SM) to overcome these limitations using an automated machine learning pipeline for subcellular calcium signal segmentation of spatiotemporal maps. The primary use of 4SM is to analyze spatiotemporal maps of calcium activities within cells or across multiple cells. For complete details on the use and execution of this protocol, please refer to Kamran et al. (2022).1.

Ca2+ transients in ICC-MY define the basis for the dominance of the corpus in gastric pacemaking.

Myenteric interstitial cells of Cajal (ICC-MY) generate and actively propagate electrical slow waves in the stomach. Slow wave generation and propagation are altered in gastric motor disorders, such as gastroparesis, and the mechanism for the gradient in slow wave frequency that facilitates proximal to distal propagation of slow waves and normal gastric peristalsis is poorly understood.  Slow waves depend upon Ca2+-activated Cl- channels (encoded by Ano1). We characterized Ca2+ signaling in ICC-MY in situ using mice engineered to have cell-specific expression of GCaMP6f in ICC. Ca2+ signaling differed in ICC-MY in corpus and antrum. Localized Ca2+ transients were generated from multiple firing sites and were organized into Ca2+ transient clusters (CTCs). Ca2+ transient refractory periods occurred upon cessation of CTCs, but a relatively higher frequency of Ca2+ transients persisted during the inter-CTC interval in corpus than in antrum ICC-MY. The onset of Ca2+ transients after the refractory period was associated with initiation of the next CTC. Thus, CTCs were initiated at higher frequencies in corpus than in antrum ICC-MY. Initiation and propagation of CTCs (and electrical slow waves) depends upon T-type Ca2+ channels, and durations of CTCs relied upon L-type Ca2+ channels. The durations of CTCs mirrored the durations of slow waves. CTCs and Ca2+ transients between CTCs resulted from release of Ca2+ from intracellular stores and were maintained, in part, by store-operated Ca2+ entry. Our data suggest that Ca2+ release and activation of Ano1 channels both initiate and contribute to the plateau phase of slow waves.

Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon.

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

A high throughput machine-learning driven analysis of Ca2+ spatio-temporal maps.

High-resolution Ca2+ imaging to study cellular Ca2+ behaviors has led to the creation of large datasets with a profound need for standardized and accurate analysis. To analyze these datasets, spatio-temporal maps (STMaps) that allow for 2D visualization of Ca2+ signals as a function of time and space are often used. Methods of STMap analysis rely on a highly arduous process of user defined segmentation and event-based data retrieval. These methods are often time consuming, lack accuracy, and are extremely variable between users. We designed a novel automated machine-learning based plugin for the analysis of Ca2+ STMaps (STMapAuto). The plugin includes optimized tools for Ca2+ signal preprocessing, automated segmentation, and automated extraction of key Ca2+ event information such as duration, spatial spread, frequency, propagation angle, and intensity in a variety of cell types including the Interstitial cells of Cajal (ICC). The plugin is fully implemented in Fiji and able to accurately detect and expeditiously quantify Ca2+ transient parameters from ICC. The plugin's speed of analysis of large-datasets was 197-fold faster than the commonly used single pixel-line method of analysis. The automated machine-learning based plugin described dramatically reduces opportunities for user error and provides a consistent method to allow high-throughput analysis of STMap datasets.

A novel intramuscular Interstitial Cell of Cajal is a candidate for generating pacemaker activity in the mouse internal anal sphincter.

The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca2+ transients in mice expressing a genetically-encoded Ca2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.

Excitatory cholinergic responses in mouse colon intramuscular interstitial cells of Cajal are due to enhanced Ca2+ release via M3 receptor activation.

Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca2+ release in ICC-IM activates Ano1, a Ca2+ -activated Cl- conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca2+ imaging of colonic ICC-IM in situ, using mice expressing GCaMP6f in ICC to evaluate ICC-IM responses to excitatory neurotransmission. Expression of muscarinic type 2, 3 (M2 , M3 ), and NK1 receptors were enriched in ICC-IM. NK1 receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca2+ transients. These effects were reversed by DAU 5884 (M3 receptor antagonist) but not AF-DX 116 (M2 receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca2+ transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca2+ stores with cyclopiazonic acid and reduced by inhibiting Ca2+ influx via Orai channels. Contractions induced by EFS were reduced by an Ano1 channel antagonist, abolished by DAU 5884, and unaffected by AF-DX 116. Colonic ICC-IM receive excitatory inputs from cholinergic neurons via M3 receptor activation. Enhancing ICC-IM Ca2+ release and Ano1 activation contributes to excitatory responses of colonic muscles.

Corrigendum: Na+/Ca2+ Exchange and Pacemaker Activity of Interstitial Cells of Cajal.

[This corrects the article DOI: 10.3389/fphys.2020.00230.].

Na+/Ca2 + Exchange and Pacemaker Activity of Interstitial Cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca2+-activated Cl- channels (CaCC) encoded by Ano1. Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca2+ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na+/Ca2+ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca2+ exit mode, helps to regulate basal [Ca2+] i in these cells. Shifting NCX into Ca2+ entry mode by replacing extracellular Na+ with Li+ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from -80 to -40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca2+ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca2+ during the inter-slow wave interval and providing Ca2+ for sustained activation of ANO1 during the slow wave plateau phase.

A novel postsynaptic signal pathway of sympathetic neural regulation of murine colonic motility.

Transcriptome data revealed α1 adrenoceptors (ARs) expression in platelet-derived growth factor receptor α+ cells (PDGFRα+ cells) in murine colonic musculature. The role of PDGFRα+ cells in sympathetic neural regulation of murine colonic motility was investigated. Norepinephrine (NE), via α1A ARs, activated a small conductance Ca2+ -activated K+ (SK) conductance, evoked outward currents and hyperpolarized PDGFRα+ cells (the α1A AR-SK channel signal pathway). α1 AR agonists increased intracellular Ca2+ transients in PDGFRα+ cells and inhibited spontaneous phasic contractions (SPCs) of colonic muscle through activation of a SK conductance. Sympathetic nerve stimulation inhibited both contractions of distal colon and propulsive contractions represented by the colonic migrating motor complexes (CMMCs) via the α1A AR-SK channel signal pathway. Postsynaptic signaling through α1A ARs in PDGFRα+ cells is a novel mechanism that conveys part of stress responses in the colon. PDGFRα+ cells appear to be a primary effector of sympathetic neural regulation of murine colonic motility.

Pacemaker function and neural responsiveness of subserosal interstitial cells of Cajal in the mouse colon.

KEY POINTS: Rhythmic action potentials and intercellular Ca2+ waves are generated in smooth muscle cells of colonic longitudinal muscles (LSMC). Longitudinal muscle excitability is tuned by input from subserosal ICC (ICC-SS), a population of ICC with previously unknown function. ICC-SS express Ano1 channels and generate spontaneous Ca2+ transients in a stochastic manner. Release of Ca2+ and activation of Ano1 channels causes depolarization of ICC-SS and LSMC, leading to activation of L-type Ca2+ channels, action potentials, intercellular Ca2+ waves and contractions in LSMC. Nitrergic neural inputs regulate the Ca2+ events in ICC-SS. Pacemaker activity in longitudinal muscle is an emergent property as a result of integrated processes in ICC-SS and LSMC. ABSTRACT: Much is known about myogenic mechanisms in circular muscle (CM) in the gastrointestinal tract, although less is known about longitudinal muscle (LM). Two Ca2+ signalling behaviours occur in LM: localized intracellular waves not causing contractions and intercellular waves leading to excitation-contraction coupling. An Ano1 channel antagonist inhibited intercellular Ca2+ waves and LM contractions. Ano1 channels are expressed by interstitial cells of Cajal (ICC) but not by smooth muscle cells (SMCs). We investigated Ca2+ signalling in a novel population of ICC that lies along the subserosal surface of LM (ICC-SS) in mice expressing GCaMP6f in ICC. ICC-SS fired stochastic localized Ca2+ transients. Such events have been linked to activation of Ano1 channels in ICC. Ca2+ transients in ICC-SS occurred by release from stores most probably via inositol trisphosphate receptors. This activity relied on influx via store-operated Ca2+ entry and Orai channels. No voltage-dependent mechanism that synchronized Ca2+ transients in a single cell or between cells was found. Nitrergic agonists inhibited Ca2+ transients in ICC-SS, and stimulation of intrinsic nerves activated nitrergic responses in ICC-SS. Cessation of stimulation resulted in significant enhancement of Ca2+ transients compared to the pre-stimulus activity. No evidence of innervation by excitatory, cholinergic motor neurons was found. Our data suggest that ICC-SS contribute to regulation of LM motor activity. Spontaneous Ca2+ transients activate Ano1 channels in ICC-SS. Resulting depolarization conducts to SMCs, depolarizing membrane potential, activating L-type Ca2+ channels and initiating contraction. Rhythmic electrical and mechanical behaviours of LM are an emergent property of SMCs and ICC-SS.