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We characterized electrical slow wave activity in human stomach using intracellular electrical recording and suggested that previous studies may have under-estimated slow wave frequency (Rhee et al. 2011), because of the reliance on extracellular recording to monitor electrical activity in patients (e.g. Hinder & Kelly, 1977; O'Grady et al. 2010). We had shown previously that biopotentials recorded from gastric muscles with extracellular electrodes are largely movement artifacts due to muscle contractions and not reflective of authentic slow wave activity (Bayguinov et al. 2011). In his Letter to the Editor, Dr O'Grady and colleagues discount our results as artifacts and suggests that prostaglandin synthesis in gastric muscles caused abnormally high slow wave frequency in vitro (O'Grady, 2012). While no paper addresses all potential criticisms, our study controlled for the chronotropic effects of prostaglandins. We believe our paper makes an important contribution and raises questions about whether current evaluations of electrical rhythmicity in human patients are accurate when assessments are made with extracellular recording. Our findings also raise important questions about electrogastrography (EGG), which is used to measure gastric slow waves and diagnose electrical arrhythmias in patients. The value of these tests is questionable if this technique does not accurately record gastric electrophysiological events. O'Grady et al. (2012) suggest that slow wave frequencies are elevated abnormally by positive chronotropic effects imposed by prostaglandins in gastric muscles in vitro. In support of this hypothesis they cite a study of feline stomach in which in vivo recordings were compared with recordings from gastric muscles after excision and dicing of the stomach into many pieces (Xue et al. 1995). O'Gradyet al. misstate the findings of this study when they say that indomethacin blocked the increase in slow wave frequency only when applied immediately after tissue dissection. In fact, Xue et al. report (on p. 161): “Krebs solution containing indomethacin (10 μm) was added to the water bath after the frequency had increased to a maximum (2 h after isolating the segments). The high slow wave frequency returned to the in vivo level (17.3 to 4, and 12.2 to 4 cpm, respectively) after 60 min” (Xue et al. 1995). We performed equivalent tests on human muscles (Rhee et al. 2011, see Supplemental Fig. 1C). Actually, the issue of prostaglandins was the first question we addressed after observing the frequency of slow waves in human muscles. We were aware of the chronotropic effects of prostaglandins in vitro because one of us (K. M. Sanders) published the first study documenting this phenomenon in canine muscles 30 years ago (Sanders & Szurszewski, 1981), and we have investigated the chronotropic actions of prostglandins in several subsequent studies of gastric muscles (e.g. Sanders, 1984; Forrest et al. 2009). In our recent study, indomethacin had only minor effects on slow wave frequency (Rhee et al. 2011) and did not ‘restore’ slow wave frequencies to values considered acceptable by O'Grady and Colleagues. We also tested other chronotropic factors that might have influenced slow wave frequency but our observations did not conform to the dogma that O'Grady et al. seem to view as unassailable. There are problems using studies such as Xue et al. 1995 to refute our findings because that study employed extracellular recording to monitor slow wave activity. Extracellular electrical recordings are likely to be contaminated by muscle contractions (Bayguinov et al. 2011). When contractions are inhibited downstream of electrical slow waves, the biopotentials, typically claimed to be ‘slow waves’, disappear. Xue et al. 1995 made no attempt to suppress mechanical activity in excised muscles, so their conclusions about slow waves and slow wave frequency must be viewed with caution. Prostaglandins have dramatic effects on contractions of gastric muscles, and endogenous prostaglandins exert opposite effects in the proximal and distal stomach. In hundreds of hours of intracellular recording (e.g. Sanders & Szurszewski, 1981; Sanders, 1984; Forrest et al. 2009; Rhee et al. 2011) slow waves didn't ‘disappear’ and ‘reappear’ in response to changes in prostaglandin synthesis as was reported in various gastric regions and during different recording circumstances by Xue et al. 1995. Swings in contractile force and inhibitory effects in distal stomach and excitatory effects in corpus, rather than appearance and disappearance of slow waves, are likely to explain the mysterious occurrences and evanescence of ‘slow waves’ in various parts of the stomach during the course of their recordings. We recorded electrical slow waves in human gastric fundus (Rhee et al. 2011), but no one seems to find these events in the fundus with extracellular recording techniques (although small events are apparent in human gastric fundus in recordings of Hinder & Kelly, 1977). O'Grady et al. suggests that prostaglandins are responsible for the ‘appearance’ of slow waves in human fundus in vitro. They again misquote Xue and coworkers (1995) looking for support for this claim, but in fact the question of slow waves in the fundus in vitro was not addressed in that study. In reading the historical motility literature, we note that few investigators have questioned whether muscle movement is a significant contaminant in extracellular recordings. O'Grady and colleagues assume that since extracellular recording has been used for so long by so many luminaries in motility research, controls, such as movement suppression, are unnecessary and they want us to accept the findings of these studies (and his; O'Grady et al. 2010) on faith. We do not discount all findings of previous investigations of gastric electrical rhythmicity, as O'Grady et al. claim, but our observations raise questions about the ‘standard model’ of gastric electrophysiology that has been developed from extracellular electrical recording and/or observation of movements. O'Grady et al. point to MRI studies as proof of the 3 cpm electrical rhythm in human stomach, but MRI measures movements and movements may be the source of the 3 cpm biopotentials recorded with extracellular electrodes (Hinder & Kelly, 1977; O'Grady et al. 2010). Movement artifacts may also be a factor in magnetic field measurements of gastric activity because the source of the magnetic field might move during contraction in relation to the SQUID device (Bradshaw et al. 2006). Our recent findings challenge a bedrock technique of gastrointestinal (GI) motility research (Bayguinov et al. 2011) and current dogma on human gastric electrophysiology (Rhee et al. 2011). In the majority of previous studies, important control experiments were either not possible when classical studies were performed or ignored. Techniques now exist to isolate the electrical and mechanical components of smooth muscle activity; muscle contraction can be inhibited without affecting electrical slow waves (Bayguinov et al. 2011). We also understand the limitations on resolving field potentials imposed by: (i) the morphology of GI muscles, (ii) the limited mass of regenerative tissue within GI muscles (

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 Ca

L-type CaA multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine CaCaThese results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species.

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.

The peristaltic reflex is a fundamental behavior of the gastrointestinal (GI) tract in which mucosal stimulation activates propulsive contractions. The reflex occurs by stimulation of intrinsic primary afferent neurons with cell bodies in the myenteric plexus and projections to the lamina propria, distribution of information by interneurons, and activation of muscle motor neurons. The current concept is that excitatory cholinergic motor neurons are activated proximal to and inhibitory neurons are activated distal to the stimulus site. We found that atropine reduced, but did not block, colonic migrating motor complexes (CMMCs) in mouse, monkey, and human colons, suggesting a mechanism other than one activated by cholinergic neurons is involved in the generation/propagation of CMMCs. CMMCs were activated after a period of nerve stimulation in colons of each species, suggesting that the propulsive contractions of CMMCs may be due to the poststimulus excitation that follows inhibitory neural responses. Blocking nitrergic neurotransmission inhibited poststimulus excitation in muscle strips and blocked CMMCs in intact colons. Our data demonstrate that poststimulus excitation is due to increased Ca2+ transients in colonic interstitial cells of Cajal (ICC) following cessation of nitrergic, cyclic guanosine monophosphate (cGMP)-dependent inhibitory responses. The increase in Ca2+ transients after nitrergic responses activates a Ca2+-activated Cl− conductance, encoded by Ano1, in ICC. Antagonists of ANO1 channels inhibit poststimulus depolarizations in colonic muscles and CMMCs in intact colons. The poststimulus excitatory responses in ICC are linked to cGMP-inhibited cyclic adenosine monophosphate (cAMP) phosphodiesterase 3a and cAMP-dependent effects. These data suggest alternative mechanisms for generation and propagation of CMMCs in the colon.

Postjunctional M2Rs on airway smooth muscle (ASM) outnumber M3Rs by a ratio of 4:1 in most species, however, it is the M3Rs that are thought to mediate the bronchoconstrictor effects of acetylcholine. In this study, we describe a novel and profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM at low stimulus frequencies.. Contractions induced by 2Hz EFS were augmented by 2.5-fold when the stimulus interval was reduced from 100 to 10 s. This effect was reversed by the M2R antagonists, methoctramine, and AFDX116, and was absent in M2R null mice. The M3R antagonist 4-DAMP abolished the entire response in both WT and M2R KO mice. The M2R-mediated potentiation of EFS-induced contractions was not observed when the stimulus frequency was increased to 20 Hz. A subthreshold concentration of carbachol enhanced the amplitude of EFS-evoked contractions in WT, but not M2R null mice. These data highlight a significant M2R-mediated potentiation of M3R-dependent contractions of ASM at low frequency stimulation that could be relevant in diseases such as asthma and COPD.

Dysfunction of embryo transport causes ectopic pregnancy which affects approximately 2% of conceptions in the US and Europe, and is the most common cause of pregnancy-related death in the first trimester. Embryo transit involves a valve-like tubal-locking phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilisation occurs, but the mechanisms involved are unknown. Here we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they do not relieve the AIJ restraining mechanism, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. Using a large-scale extracellular protein interaction screen, we identified Plxdc2 as an activating ligand for Adgrd1 displayed on cumulus cells. Our findings demonstrate that regulating oviductal fluid flow by Adgrd1 controls embryo transit and we present a model where embryo arrest at the AIJ is due to the balance of abovarial ciliary action and the force of adovarial tubal fluid flow, and in wild-type oviducts, fluid flow is gradually attenuated through Adgrd1 activation to enable embryo release. Our findings provide important insights into the molecular mechanisms involved in embryo transport in mice., Lack of correct embryo transport can cause ectopic pregnancy. Here, the authors show that female mice lacking the adhesion G-protein coupled receptor Adgrd1 are infertile, due to embryos being trapped in the ampulla as the result of dysregulated oviductal fluid flow.

Key points Colonic intramuscular interstitial cells of Cajal (ICC-IM) exhibit spontaneous Ca2+ transients manifesting as stochastic events from multiple firing sites with propagating Ca2+ waves occasionally observed. Firing of Ca2+ transients in ICC-IM is not coordinated with adjacent ICC-IM in a field of view or even with events from other firing sites within a single cell. Ca2+ transients, through activation of Ano1 channels and generation of inward current, cause net depolarization of colonic muscles. Ca2+ transients in ICC-IM rely on Ca2+ release from the endoplasmic reticulum via IP3 receptors, spatial amplification from RyRs and ongoing refilling of ER via the sarcoplasmic/endoplasmic-reticulum-Ca2+ -ATPase. ICC-IM are sustained by voltage-independent Ca2+ influx via store-operated Ca2+ entry. Some of the properties of Ca2+ in ICC-IM in the colon are similar to the behaviour of ICC located in the deep muscular plexus region of the small intestine, suggesting there are functional similarities between these classes of ICC. Abstract A component of the SIP syncytium that regulates smooth muscle excitability in the colon is the intramuscular class of interstitial cells of Cajal (ICC-IM). All classes of ICC (including ICC-IM) express Ca2+ -activated Cl- channels, encoded by Ano1, and rely upon this conductance for physiological functions. Thus, Ca2+ handling in ICC is fundamental to colonic motility. We examined Ca2+ handling mechanisms in ICC-IM of murine proximal colon expressing GCaMP6f in ICC. Several Ca2+ firing sites were detected in each cell. While individual sites displayed rhythmic Ca2+ events, the overall pattern of Ca2+ transients was stochastic. No correlation was found between discrete Ca2+ firing sites in the same cell or in adjacent cells. Ca2+ transients in some cells initiated Ca2+ waves that spread along the cell at ∼100 µm s-1 . Ca2+ transients were caused by release from intracellular stores, but depended strongly on store-operated Ca2+ entry mechanisms. ICC Ca2+ transient firing regulated the resting membrane potential of colonic tissues as a specific Ano1 antagonist hyperpolarized colonic muscles by ∼10 mV. Ca2+ transient firing was independent of membrane potential and not affected by blockade of L- or T-type Ca2+ channels. Mechanisms regulating Ca2+ transients in the proximal colon displayed both similarities to and differences from the intramuscular type of ICC in the small intestine. Similarities and differences in Ca2+ release patterns might determine how ICC respond to neurotransmission in these two regions of the gastrointestinal tract.

Mammalian oviducts play an essential role in female fertility by picking up ovulated oocytes and transporting and nurturing gametes (sperm/oocytes) and early embryos. However, the relative contributions to these functions from various cell types within the oviduct remain controversial. The oviduct in mice deficient in two microRNA (miRNA) clusters (miR-34b/c and miR-449) lacks cilia, thus allowing us to define the physiological role of oviductal motile cilia. Here, we report that the infundibulum without functional motile cilia failed to pick up the ovulated oocytes. In the absence of functional motile cilia, sperm could still reach the ampulla region, and early embryos managed to migrate to the uterus, but the efficiency was reduced. Further transcriptomic analyses revealed that the five messenger ribonucleic acids (mRNAs) encoded by miR-34b/c and miR-449 function to stabilize a large number of mRNAs involved in cilium organization and assembly and that Tubb4b was one of their target genes. Our data demonstrate that motile cilia in the infundibulum are essential for oocyte pickup and thus, female fertility, whereas motile cilia in other parts of the oviduct facilitate gamete and embryo transport but are not absolutely required for female fertility.

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.

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing 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 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.SynopsisHow Ca2+signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca2+signaling and defines Ca2+transients’ sources using optogenetic techniques.

Patients with diabetes often develop gastrointestinal motor problems, including gastroparesis. Previous studies have suggested this gastric motor disorder was a consequence of an enteric neuropathy. Disruptions in interstitial cells of Cajal (ICC) have also been reported. A thorough examination of functional changes in gastric motor activity during diabetes has not yet been performed. We comprehensively examined the gastric antrums of Lepob mice using functional, morphological, and molecular techniques to determine the pathophysiological consequences in this type 2 diabetic animal model. Video analysis and isometric force measurements revealed higher frequency and less robust antral contractions in Lepob mice compared with controls. Electrical pacemaker activity was reduced in amplitude and increased in frequency. Populations of enteric neurons, ICC, and platelet-derived growth factor receptor α+ cells were unchanged. Analysis of components of the prostaglandin pathway revealed upregulation of multiple enzymes and receptors. Prostaglandin-endoperoxide synthase-2 inhibition increased slow wave amplitudes and reduced frequency of diabetic antrums. In conclusion, gastric pacemaker and contractile activity is disordered in type 2 diabetic mice, and this appears to be a consequence of excessive prostaglandin signaling. Inhibition of prostaglandin synthesis may provide a novel treatment for diabetic gastric motility disorders.

Gastric endocrine cell hormones contribute to the control of the stomach and to signalling to the brain. In other gut regions, enteroendocrine cells (EECs) exhibit extensive patterns of colocalisation of hormones. In the current study, we characterise EECs in the human gastric fundus and corpus. We utilise immunohistochemistry to investigate EECs with antibodies to ghrelin, serotonin (5-HT), somatostatin, peptide YY (PYY), glucagon-like peptide 1, calbindin, gastrin and pancreastatin, the latter as a marker of enterochromaffin-like (ECL) cells. EECs were mainly located in regions of the gastric glands populated by parietal cells. Gastrin cells were absent and PYY cells were very rare. Except for about 25% of 5-HT cells being a subpopulation of ECL cells marked by pancreastatin, colocalisation of hormones in gastric EECs was infrequent. Ghrelin cells were distributed throughout the fundus and corpus; most were basally located in the glands, often very close to parietal cells and were closed cells i.e., not in contact with the lumen. A small proportion had long processes located close to the base of the mucosal epithelium. The 5-HT cells were of at least three types: small, round, closed cells; cells with multiple, often very long, processes; and a subgroup of ECL cells. Processes were in contact with their surrounding cells, including parietal cells. Mast cells had very weak or no 5-HT immunoreactivity. Somatostatin cells were a closed type with long processes. In conclusion, four major chemically defined EEC types occurred in the human oxyntic mucosa. Within each group were cells with distinct morphologies and relationships to other mucosal cells.

Key points Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.

KEY POINTS Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post-junctional responses are controversial. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC-IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC-IM. Conductances activated by cholinergic stimulation in isolated ICC-IM and SMCs were determined. A CaCC was activated by carbachol in ICC-IM and a non-selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild-type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC-IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC-IM and not SMCs. ABSTRACT Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl- conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.

Key points Platelet-derived growth factor receptor-α (PDGFRα) is a novel biomarker along with smooth myosin heavy chain for the pacemaker cells (previously termed 'atypical' smooth muscle cells) in the murine and cynomolgus monkey pelvis-kidney junction. PDGFRα+ cells present in adventitial and urothelial layers of murine renal pelvis do not express smooth muscle myosin heavy chain (smMHC) but are in close apposition to nerve fibres. Most c-Kit+ cells in the renal pelvis are mast cells. Mast cells (CD117+ /CD45+ ) are more abundant in the proximal renal pelvis and pelvis-kidney junction regions whereas c-Kit+ interstitial cells (CD117+ /CD45- ) are found predominantly in the distal renal pelvis and ureteropelvic junction. PDGFRα+ cells are distinct from c-Kit+ interstitial cells. A subset of PDGFRα+ cells express the Ca2+ -activated Cl- channel, anoctamin-1, across the entire renal pelvis. Spontaneous Ca2+ transients were observed in c-Kit+ interstitial cells, smMHC+ PDGFRα cells and smMHC- PDGFRα cells using mice expressing genetically encoded Ca2+ sensors. Abstract Rhythmic contractions of the renal pelvis transport urine from the kidneys into the ureter. Specialized pacemaker cells, termed atypical smooth muscle cells (ASMCs), are thought to drive the peristaltic contractions of typical smooth muscle cells (TSMCs) in the renal pelvis. Interstitial cells (ICs) in close proximity to ASMCs and TSMCs have been described, but the role of these cells is poorly understood. The presence and distributions of platelet-derived growth factor receptor-α+ (PDGFRα+ ) ICs in the pelvis-kidney junction (PKJ) and distal renal pelvis were evaluated. We found PDGFRα+ ICs in the adventitial layers of the pelvis, the muscle layer of the PKJ and the adventitia of the distal pelvis. PDGFRα+ ICs were distinct from c-Kit+ ICs in the renal pelvis. c-Kit+ ICs are a minor population of ICs in murine renal pelvis. The majority of c-Kit+ cells were mast cells. PDGFRα+ cells in the PKJ co-expressed smooth muscle myosin heavy chain (smMHC) and several other smooth muscle gene transcripts, indicating these cells are ASMCs, and PDGFRα is a novel biomarker for ASMCs. PDGFRα+ cells also express Ano1, which encodes a Ca2+ -activated Cl- conductance that serves as a primary pacemaker conductance in ICs of the GI tract. Spontaneous Ca2+ transients were observed in c-Kit+ ICs, smMHC+ PDGFRα cells and smMHC- PDGFRα cells using genetically encoded Ca2+ sensors. A reporter strain of mice with enhanced green fluorescent protein driven by the endogenous promotor for Pdgfra was shown to be a powerful new tool for isolating and characterizing the phenotype and functions of these cells in the renal pelvis.

A growing, robust body of evidence from both epidemiological and animal studies unveils harmful effects of maternal gestational caffeine exposure, even from doses previously considered 'safe.' Rodent studies revealed that caffeine exposure during specific stages of pregnancy may disrupt embryo transport, embryo development, embryo implantation, and placental function, leading to pregnancy complications. Notably, caffeine sensitivity is highly variable from individual to individual. Genetic variations and epigenetic regulation, intermingling with intrinsic and environmental factors, might play pivotal roles in shaping the complex phenotypic variability. Exploring the underlying mechanism(s) will be helpful, not only for improving the guidelines of gestational caffeine consumption, but also for personalized dosing of drugs that interact with caffeine at the pharmacokinetic or pharmacodynamic level. Evidence from rodent studies demonstrated that in utero caffeine exposure triggered cardiometabolic defects on both the immediate offspring and subsequent generations. Further studies are needed regarding caffeine’s long-term effects and multigenerational influence in humans.

One of the most ubiquitous active learning modalities in the biological sciences at third level is the journal club. Journal club can promote several beneficial learning outcomes for students such as gaining critical reading skills to evaluate the scientific literature, improving scientific literacy, serving as an introduction to new concepts and techniques and improving communication skills. However, it can be difficult for instructors who facilitate journal club to gauge student audiences' understanding of topics being related by presenters. At the University of Nevada, Reno School of Medicine, international life sciences undergraduate students enrolled in our research program undergo a 12-month placement in selected research laboratories within the medical school in order to develop an understanding of basic medical scientific research and physiological concepts. As such, an integral component of this program is participation in regular journal club sessions which we had assumed helped students to develop such an understanding. However as we had never empirically assessed if this was the case or not, the aim of the current study was to determine if student understanding could be improved by complementing the standard journal club with peer-mentored workshop presentations. Data from this case study suggest that by allowing students to undergo peer-mentored learning in conjunction with journal club, student understanding of physiological concepts, as well as student confidence in presenting and communication, increases.

Oviducts (also called fallopian tubes) are smooth muscle-lined tubular organs that at one end extend in a trumpet bell-like fashion to surround the ovary, and at the other connect to the uterus. Contractions of the oviduct smooth muscle (myosalpinx) and the wafting motion of the ciliated epithelium that lines these tubes facilitate bidirectional transport of gametes so that newly released ovum(s) are transported in one direction (pro-uterus) while spermatozoa are transported in the opposite direction (pro-ovary). These transport processes must be temporally coordinated so that the ovum and spermatozoa meet in the ampulla, the site of fertilization. Once fertilized, the early embryo begins another precisely timed journey towards the uterus for implantation. Myosalpinx contractions facilitate this journey too, while luminal secretions from secretory epithelial cells aid early embryo maturation.The previous paradigm was that oviduct transport processes were primarily controlled by fluid currents generated by the incessant beat of the ciliated epithelium towards the uterus. More recently, video imaging and spatiotemporal mapping have suggested a novel paradigm in which ovum/embryo transport is highly dependent upon phasic and propulsive contractions of the myosalpinx. A specialized population of pacemaker cells, termed oviduct interstitial cells of Cajal (ICC-OVI), generate the electrical activity that drives these contractions. The ionic mechanisms underlying this pacemaker activity are dependent upon the calcium-activated chloride conductance, Ano1.This chapter discusses the basis of oviduct pacemaker activity, its hormonal regulation, and the underlying mechanisms and repercussions when this activity becomes disrupted during inflammatory responses to bacterial infections, such as Chlamydia.

KEY POINTS: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium‐activated chloride conductance anoctamin‐1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. ABSTRACT: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca(2+)‐activated Cl(−) conductance (CaCC), and constitutive knockout of the channel protein anoctamin‐1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin‐1 channels. Furthermore, the significance of anoctamin‐1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell‐specific knockdowns of anoctamin‐1 in ICC (Kit(CreERT2/+);Ano1(tm2jrr/+)) in GI muscles. The recombination efficiency of Kit(CreERT) was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin‐1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin‐1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin‐1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin‐1 knockdown and weak efficacy of anoctamin‐1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin‐1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.

Elevated glucose increases vascular reactivity by promoting L-type CaV1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a Gs-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y11, the only Gs-coupled P2Y receptor, was detected in nanometer proximity to CaV1.2 and PKA. FRET-based experiments revealed that the selective P2Y11 agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y11 inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y11-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y11 in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.

Key points The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+-activated Cl− channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL, encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. Abstract The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+-activated Cl− channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Electrical slow waves in the small intestine are generated by pacemaker cells called interstitial cells of Cajal. Drumm et al. record clusters of Ca2+ transients in these cells that are entrained by voltage-dependent Ca2+ entry and which define the duration of the electrical slow waves., Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl− channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves.

Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase ( Nos1 −/−) and P2Y1 receptors ( P2ry1 −/−) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1 −/− mice IJPs and relaxations persisted whereas in P2ry1 −/− mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species.

Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression.Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 ?m) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ.

Background and Purpose High-throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second-generation. low MW inhibitors of the calcium-activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors. Experimental Approach Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose-dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves. Key Results The efficacy of second-generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh-A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh-A01 in both preparations. Conclusions and Implications We have identified several second-generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.

Oviducts (also called fallopian tubes) are smooth muscle-lined tubular organs that at one end extend in a trumpet bell-like fashion to surround the ovary, and at the other connect to the uterus. Contractions of the oviduct smooth muscle (myosalpinx) and the wafting motion of the ciliated epithelium that lines these tubes facilitate bidirectional transport of gametes so that newly released ovum(s) are transported in one direction (pro-uterus) while spermatozoa are transported in the opposite direction (pro-ovary). These transport processes must be temporally coordinated so that the ovum and spermatozoa meet in the ampulla, the site of fertilization. Once fertilized, the early embryo begins another precisely timed journey towards the uterus for implantation. Myosalpinx contractions facilitate this journey too, while luminal secretions from secretory epithelial cells aid early embryo maturation.The previous paradigm was that oviduct transport processes were primarily controlled by fluid currents generated by the incessant beat of the ciliated epithelium towards the uterus. More recently, video imaging and spatiotemporal mapping have suggested a novel paradigm in which ovum/embryo transport is highly dependent upon phasic and propulsive contractions of the myosalpinx. A specialized population of pacemaker cells, termed oviduct interstitial cells of Cajal (ICC-OVI), generate the electrical activity that drives these contractions. The ionic mechanisms underlying this pacemaker activity are dependent upon the calcium-activated chloride conductance, Ano1.This chapter discusses the basis of oviduct pacemaker activity, its hormonal regulation, and the underlying mechanisms and repercussions when this activity becomes disrupted during inflammatory responses to bacterial infections, such as Chlamydia.

Caffeine consumption has been widely used as a central nervous system stimulant. Epidemiological studies, however, have suggested that maternal caffeine exposure during pregnancy is associated with increased abnormalities, including decreased fertility, delayed conception, early spontaneous abortions, and low birth weight. The mechanisms underlying the negative outcomes of caffeine consumption, particularly during early pregnancy, remain unclear. In present study, we found that pregnant mice treated with moderate (5 mg/kg) or high (30 mg/kg) dosage of caffeine (intraperitoneally or orally) during preimplantation resulted in retention of early embryos in the oviduct, defective embryonic development, and impaired embryo implantation. Transferring normal blastocysts into the uteri of caffeine-treated pseudopregnant females also showed abnormal embryo implantation, thus indicating impaired uterine receptivity by caffeine administration. The remaining embryos that managed to implant after caffeine treatment also showed increased embryo resorption rate and abnormal development at mid-term stage, and decreased weight at birth. In addition to a dose-dependent effect, significant variations between individual mice under the same caffeine dosage were also observed, suggesting different sensitivities to caffeine, similar to that observed in human populations. Collectively, our data revealed that caffeine exposure during early pregnancy impaired oviductal embryo transport, embryonic development, and uterine receptivity, which are responsible for abnormal implantation and pregnancy loss. The study raises the concern of caffeine consumption during early stages of pregnancy.

Background/Aims Gastric peristalsis begins in the orad corpus and propagates to the pylorus. Directionality of peristalsis depends upon orderly generation and propagation of electrical slow waves and a frequency gradient between proximal and distal pacemakers. We sought to understand how chronotropic agonists affect coupling between corpus and antrum. Methods Electrophysiological and imaging techniques were used to investigate regulation of gastric slow wave frequency by muscarinic agonists in mice. We also investigated the expression and role of cholinesterases in regulating slow wave frequency and motor patterns in the stomach. Results Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to varicose processes of motor neurons. Inhibition of AChE in the absence of stimulation increased slow wave frequency in corpus and throughout muscle strips containing corpus and antrum. CCh caused depolarization and increased slow wave frequency. Stimulation of cholinergic neurons increased slow wave frequency but did not cause depolarization. Neostigmine (1 μM) increased slow wave frequency, but uncoupling between corpus and antrum was not detected. Motility mapping of contractile activity in gastric muscles showed similar effects of enteric nerve stimulation on the frequency and propagation of slow waves, but neostigmine (＞ 1 μM) caused aberrant contractile frequency and propagation and ectopic pacemaking. Conclusions Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and suggest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns. (J Neurogastroenterol Motil 2015;21:200-216)