Gastrointestinal (GI) motility disorders represent a major medical challenge, with few effective therapies available. These disorders often result from dysfunction of inhibitory nitric oxide (NO)-producing motor neurons in the enteric nervous system, which are essential for regulating gut motility. Loss or dysfunction of NO neurons is linked to severe conditions, including achalasia, gastroparesis, intestinal pseudo-obstruction and chronic constipation1,2. Here we introduce a platform based on human pluripotent stem cells (hPSCs) for therapeutic development targeting GI motility disorders. Using an unbiased screen, we identified drug candidates that modulate NO neuron activity and enhance motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define developmental programs driving the specification of NO neurons and found that inhibition of platelet-derived growth factor receptors (PDGFRs) promotes their differentiation from precursors of the enteric nervous system. Transplantation of these neurons into NO-neuron-deficient mice led to robust engraftment and improved GI motility, offering a promising cell-based therapy for neurodegenerative GI disorders. These studies provide a new framework for understanding and treating enteric neuropathies.

The movement of intestinal smooth muscle is regulated by the external autonomic nervous system (ANS) and its internal enteric nervous system (ENS). Previous studies have shown that acupuncture has a bidirectional regulating effect on intestinal motility through the sympathetic and vagal ANSs. ENS can independently regulate the sensory, secretory, and motor functions of the intestine. The interstitial cells of Cajal (ICC), the pacemaker cells in ENS, play a key role in maintaining gastrointestinal motility. However, studies on the role and mechanism of ICC in the regulation of intestinal function by acupuncture are still unclear.

To investigate the effect of ICC on the regulation of intestinal motility by manual acupuncture (MA), we recorded the pressure of warm water-filled manometric balloons in duodenum, jejunum, and distal colon in ICC deficiency WsWs-/- rats and wild-type littermates WsRC+/+ rats, and performed MA at ST25 (Tianshu), ST37 (Shangjuxu), LI11 (Quchi), and BL25 (Danchangshu) acupoints. Furthermore, the excretion of phenol red in feces before and after MA at ST37 or ST25 was assessed.

In WsRC+/+ rats, MA at ST37, LI11, and BL25 promoted duodenal, jejunal, and distal colon motility, whereas MA at ST25 significantly inhibited duodenal and jejunal motility and promoted distal colon motility. ICC deficiency in WsWs-/- rats led to a reduction in the promoting effect of LI11 on duodenal motility, a decrease in the promoting effect of ST37 on jejunal motility, and a significant reduction in the promoting effect of BL25 on distal colonic motility in those rats. Additionally, ICC absence significantly attenuated the inhibitory effect of ST25 on duodenal motility. MA at ST37 or ST25 did not change the content of phenol red in the feces in WsRC+/+ and WsWs-/- rats.

Our results suggest that the absence of ICC impairs the bidirectional regulatory effect of MA on intestinal function. It reveals the important role of ICC in the treatment of intestinal dysfunction diseases by acupuncture and provides a new theoretical basis for the treatment of such diseases by MA.

Interstitial cells of Cajal (ICC) are distributed through the gastrointestinal (GI) tract, but the functional role of these cells comes primarily from studies of mice. Whether the functions of ICC are similar in larger animals is largely speculative. We investigated whether the Kit mutation in Ws/Ws rats had consequences on ICC populations in the stomach, small intestine, and colon and whether loss of ICC resulted in functional defects similar to Kit mutations in mice. Immunohistochemical labeling with c-KIT or ANO1 antibodies revealed loss of intramuscular ICC (ICC-IM) and reduced myenteric ICC (ICC-MY) in the stomachs of Ws/Ws mutants. Disruption of ICC-MY networks but not ICC within the deep muscular plexus (ICC-DMP) was observed in the small intestine. ICC in the proximal colon was reduced, but no population was absent. ICC loss in the stomach caused loss of spontaneous transient depolarizations, reduced pacemaker activity, and reduced responses to cholinergic and nitrergic nerve stimulation. Loss of ICC-MY in the small intestine resulted in abnormal pacemaker activity, but neural responses appeared to be normal. In the proximal colon, tonic inhibition due to ongoing nitrergic neural inputs was reduced, spontaneous spike complexes were less rhythmic, and nitrergic neural responses were reduced. Apamin-sensitive inhibitory neural responses were retained throughout the GI tract. In summary, Ws/Ws rats have lesions in ICC and functional deficits similar to, but not identical to, Kit mutant mice. These larger animals with more robust GI muscles may be useful for investigations into the role of ICC in normal and abnormal GI motility.NEW & NOTEWORTHY The physiological roles of interstitial cells of Cajal (ICC) throughout the gastrointestinal (GI) tract have been derived predominantly from studies of mice. We sought to determine whether reduction in ICC in the rat, a commonly used animal for studies of GI motor functions, leads to functional deficits. Ws/Ws rats display reduced ICC leading to a disruption in pacemaker activity and neuroeffector responses. Our results provide additional evidence for the functions of ICC in the GI tract.

Pyloric dysfunction is defined as hypertonia or spasm of the pyloric sphincter. The pylorus plays a key role in gastric emptying, but its function remains incompletely understood. Most studies have focused on gastroparesis regardless of the underlying pathophysiology. Few studies have reported pyloric dysfunction in patients with gastroparesis, and the diagnostic and treatment modalities for pyloric dysfunction are not well established. Recently developed diagnostic modalities assessing pyloric function, such as high-resolution antroduodenal manometry and endoluminal functional lumen imaging, are currently being evaluated. A variety of therapeutic interventions targeting the pylorus, including pharmacologic agents, intrapyloric botulinum injection, endoscopic balloon dilation, stent insertion, surgical pyloroplasty, and gastric peroral endoscopic pyloromyotomy, have been proposed. Among these, gastric peroral endoscopic pyloromyotomy has emerged as a novel, minimally invasive therapy with demonstrated efficacy and safety for refractory gastroparesis. This article reviews the pathophysiology of pyloric dysfunction and the potential diagnostic and therapeutic modalities based on the latest literature.

Gastroparesis is a neurogastrointestinal disorder of motility in which patients experience symptoms of nausea, vomiting, bloating, early satiety, postprandial fullness, upper abdominal discomfort or pain, and delayed gastric emptying of solids based on scintigraphy or stable isotope breath test when mechanical obstruction has been excluded. Symptoms of gastroparesis may result from diverse pathophysiological mechanisms, including antroduodenal hypomotility, pylorospasm, increased gastric accommodation, and visceral hypersensitivity. The most common etiologies of gastroparesis are idiopathic, diabetic, and postsurgical, and less frequent causes are neurodegenerative disorders (Parkinson's disease), myopathies (scleroderma, amyloidosis), medication-induced (glucagon-like peptide-1 agonists and opioid agents), and paraneoplastic syndrome. This review addresses pharmacologic management of gastroparesis including prokinetic and antiemetic agents, pharmacologic agents targeting the pylorus, and effects of neuromodulators. SIGNIFICANCE STATEMENT: Gastroparesis is a neurogastrointestinal motility disorder characterized by delayed gastric emptying without mechanical obstruction with numerous upper gastrointestinal symptoms, including nausea and vomiting. The management of gastroparesis involves nutritional support, medications, and procedures. The only Food and Drug Administration-approved medication for gastroparesis is metoclopramide. This article reviews the pharmacology and efficacy of all classes of antiemetics or prokinetic effects used in gastroparesis. There is still a considerable unmet need for efficacious medications specifically for the treatment of gastroparesis, especially in refractory cases.

Gastrointestinal motility persists when peripheral cholinergic signaling is blocked genetically or pharmacologically, and a recent study suggests nitric oxide drives propagating neurogenic contractions.

To determine the neuronal substrates that underlie these contractions, we measured contractile-associated movements together with calcium responses of cholinergic or nitrergic myenteric neurons in unparalyzed ex vivo preparations of whole mouse colon. We chose to look at these 2 subpopulations because they encompass nearly all myenteric neurons.

Many, but not all, cholinergic neurons of the middle colon exhibited contractile-associated calcium responses with distinct features. By contrast, a large population of nitrergic neurons of the middle colon shut their activity off just before contraction onset, whereas another population of nitrergic neurons initiated a response just after contraction onset. When contractions were evoked by a variety of stimuli to the proximal and distal colon, the same neuronal subtypes exhibited the same activity patterns during the contraction. However, stimulation of proximal colon produced a transient, stimulation-locked response before the ensuing contraction in a subpopulation of cholinergic neurons and in nearly all nitrergic neurons, suggesting that distinct neuronal activity patterns underlie specific stimuli. Finally, although blockade of nitric oxide failed to arrest the generation or propagation of neurogenic contractions, chemogenetic elimination of nitrergic activity impaired their propagation to middle and distal colon.

Genetic approaches were used to study the activity patterns of enteric neurons underlying spontaneous and evoked neurogenic contractions in unparalyzed colon. These approaches can be combined with a variety of other approaches to identify the neuronal subtypes and subclasses that coordinate colonic motility.

Aging is linked to a gradual decline in the gastric motor function, contributing to reduced food intake, and its association with frailty and sarcopenia. A key cellular change in the gastric neuromuscular apparatus is the loss of interstitial cells of Cajal (ICC), pacemaker cells of the gut. The ICC function as pacemakers that generate electrical slow waves and mediate enteric neurotransmission, playing a critical role in gastric motility. Aging-related ICC depletion leads to impaired gastric compliance and reduced slow wave activity, which contributes to early satiety and reduced food intake. Recent studies have elucidated the molecular and epigenetic mechanisms underlying aging-related ICC decline, highlighting the roles of ICC stem/precursor cells (ICC-SCs), transformation-related protein 53 (TRP53), extracellular signal-regulated kinase (ERK), and insulin-like growth factor 1 (IGF1) pathways, and epigenetic regulation mediated by the histone methyltransferase enhancer of zeste 2 (EZH2). By synthesizing the current findings, this review aims to provide a comprehensive understanding of the mechanisms driving ICC decline and to explore potential therapeutic strategies for preserving gastric motility in aging populations. Future research should aim to translate these discoveries into clinical applications to improve the gastric motor function and overall health in the aging population. Identifying effective interventions targeting ICC maintenance may ultimately help to alleviate age-related gastric motor dysfunction and its associated health burdens, including frailty, malnutrition, and impaired quality of life.

Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract, yet their function in the esophagus remains unknown. The mouse esophagus has been described as primarily skeletal muscle; however, ICC  have been identified in this region. This study characterizes the distribution of skeletal and smooth muscle cells (SMCs) and their spatial relationship to ICC, PDGFRα+ cells, and intramuscular motor neurons in the mouse esophagus. SMCs occupied approximately 30% of the distal esophagus, but their density declined in more proximal regions. Similarly, ANO1+ intramuscular ICC (ICC-IM) were distributed along the esophagus, with density decreasing proximally. While ICC-IM were closely associated with SMCs, they were also present in regions of skeletal muscle. Intramuscular, submucosal, and myenteric PDGFRα+ cells were densely distributed throughout the esophagus, yet only intramuscular PDGFRα+ cells in the lower esophageal sphincter (LES) and distal esophagus expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with intramuscular nNOS+, VIP+, VAChT+, and TH+ neurons and GFAP+ cells resembling intramuscular enteric glia. These findings suggest that ICC-IM and PDGFRα+ cells may have roles in regulating esophageal motility due to their close proximity to each other and to skeletal muscle and SMCs, although further functional studies are needed to explore their role in this region. The mixed muscular composition and presence of interstitial cells in the mouse distal esophagus is anatomically similar to the transitional zone found in the human esophagus, and therefore, motility studies in the mouse may be translatable to humans.

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.

Smooth muscle organs of the lower urinary tract comprise the bladder detrusor and urethral wall, which have a reciprocal contractile relationship during urine storage and micturition. As the bladder fills with urine, detrusor smooth muscle cells (DSMCs) remain relaxed to accommodate increases in intravesical pressure while urethral smooth muscle cells (USMCs) sustain tone to occlude the urethral orifice, preventing leakage. While neither organ displays coordinated regular contractions as occurs in small intestine, lymphatics or renal pelvis, they do exhibit patterns of rhythmicity at cellular and tissue levels. In rabbit and guinea-pig urethra, electrical slow waves are recorded from USMCs. This activity is linked to cells expressing vimentin, c-kit and Ca2+-activated Cl- channels, like interstitial cells of Cajal in the gastrointestinal tract. In mouse, USMCs are rhythmically active (firing propagating Ca2+ waves linked to contraction), and this cellular rhythmicity is asynchronous across tissues and summates to form tone. Experiments in mice have failed to demonstrate a voltage-dependent mechanism for regulating this rhythmicity or contractions in vitro, suggesting that urethral tone results from an intrinsic ability of USMCs to 'pace' their own Ca2+ mobilization pathways required for contraction. DSMCs exhibit spontaneous transient contractions, increases in intracellular Ca2+ and action potentials. Consistent across numerous species, including humans, this activity relies on voltage-dependent Ca2+ influx in DSMCs. While interstitial cells are present in the bladder, they do not 'pace' the organ in an excitatory manner. Instead, specialized cells (PDGFRα+ interstitial cells) may 'negatively pace' DSMCs to prevent bladder overexcitability.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

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.

Restricted gastric motor functions contribute to aging-associated undernutrition, sarcopenia, and frailty. We previously identified a decline in interstitial cells of Cajal (ICC; gastrointestinal pacemaker and neuromodulator cells) and their stem cells (ICC-SC) as a key factor of gastric aging. Altered functionality of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is central to organismal aging. Here, we investigated the role of EZH2 in the aging-related loss of ICC/ICC-SC.

klotho mice, a model of accelerated aging, were treated with the most clinically advanced EZH2 inhibitor, EPZ6438 (tazemetostat; 160 mg/kg intraperitoneally twice a day for 3 weeks). Gastric ICC were analyzed by Western blotting and immunohistochemistry. ICC and ICC-SC were quantified by flow cytometry. Gastric slow wave activity was assessed by intracellular electrophysiology. Ezh2 was deactivated in ICC by treating KitcreERT2/+;Ezh2fl/fl mice with tamoxifen. TRP53, a key mediator of aging-related ICC loss, was induced with nutlin 3a in gastric muscle organotypic cultures and an ICC-SC line.

In klotho mice, EPZ6438 treatment mitigated the decline in the ICC growth factor KIT ligand/stem cell factor and gastric ICC. EPZ6438 also improved gastric slow wave activity and mitigated the reduced food intake and impaired body weight gain characteristic of this strain. Conditional genomic deletion of Ezh2 in Kit-expressing cells also prevented ICC loss. In organotypic cultures and ICC-SC, EZH2 inhibition prevented the aging-like effects of TRP53 stabilization on ICC/ICC-SC.

Inhibition of EZH2 with EPZ6438 mitigates aging-related ICC/ICC-SC loss and gastric motor dysfunction, improving slow wave activity and food intake in klotho mice.

Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα+ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1+ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα+ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα+ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with nNOS+, VIP+, VAChT+ and TH+ neurons throughout the LES and distal esophagus. GFAP+ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα+ cells, occupying a similar location to c. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.

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.

The pyloric sphincter receives parasympathetic vagal innervation from the dorsal motor nucleus of the vagus (DMV). However, little is known about its higher-order neurons and the nuclei that engage the DMV neurons controlling the pylorus. The purpose of the present study was twofold. First, to identify neuroanatomical connections between higher-order neurons and the DMV. This was carried out by using the transneuronal pseudorabies virus PRV-152 injected into rat pylorus torus and examining the brains of these animals for PRV labeling. Second, to identify the specific sites within the DMV that functionally control the motility and tone of the pyloric sphincter. For these studies, experiments were performed to assess the effect of DMV stimulation on pylorus activity in urethane-anesthetized male rats. A strain gauge force transducer was sutured onto the pyloric tonus to monitor tone and motility. L-glutamate (500 pmol/30 nL) was microinjected unilaterally into the rostral and caudal areas of the DMV. Data from the first study indicated that neurons labeled with PRV occurred in the DMV, hindbrain raphe nuclei, midbrain Edinger-Westphal nucleus, ventral tegmental area, lateral habenula, and arcuate nucleus. Data from the second study indicated that microinjected L-glutamate into the rostral DMV results in contraction of the pylorus blocked by intravenously administered atropine and ipsilateral vagotomy. L-glutamate injected into the caudal DMV relaxed the pylorus. This response was abolished by ipsilateral vagotomy but not by intravenously administered atropine or L-NG-nitroarginine methyl ester (L-NAME). These findings identify the anatomical and functional brain neurocircuitry involved in controlling the pyloric sphincter. Our results also show that site-specific stimulation of the DMV can differentially influence the activity of the pyloric sphincter by separate vagal nerve pathways.

Propulsion of contents in the gastrointestinal tract requires coordinated functions of the extrinsic nerves to the gut from the brain and spinal cord, as well as the neuromuscular apparatus within the gut. The latter includes excitatory and inhibitory neurons, pacemaker cells such as the interstitial cells of Cajal and fibroblast-like cells, and smooth muscle cells. Coordination between these extrinsic and enteric neurons results in propulsive functions which include peristaltic reflexes, migrating motor complexes in the small intestine which serve as the housekeeper propelling to the colon the residual content after digestion, and mass movements in the colon which lead to defecation.

Impaired gastric motor function in the elderly causes reduced food intake leading to frailty and sarcopenia. We previously found that aging-related impaired gastric compliance was mainly owing to depletion of interstitial cells of Cajal (ICC), pacemaker cells, and neuromodulator cells. These changes were associated with reduced food intake. Transformation-related protein 53-induced suppression of extracellular signal-regulated protein kinase (ERK)1/2 in ICC stem cell (ICC-SC) cell-cycle arrest is a key process for ICC depletion and gastric dysfunction during aging. Here, we investigated whether insulin-like growth factor 1 (IGF1), which can activate ERK in gastric smooth muscles and invariably is reduced with age, could mitigate ICC-SC/ICC loss and gastric dysfunction in klotho mice, a model of accelerated aging.

Klotho mice were treated with the stable IGF1 analog LONG R3 recombinant human (rh) IGF1 (150 μg/kg intraperitoneally twice daily for 3 weeks). Gastric ICC/ICC-SC and signaling pathways were studied by flow cytometry, Western blot, and immunohistochemistry. Gastric compliance was assessed in ex vivo systems. Transformation-related protein 53 was induced with nutlin 3a and ERK1/2 signaling was activated by rhIGF-1 in the ICC-SC line.

LONG R3 rhIGF1 treatment prevented reduced ERK1/2 phosphorylation and gastric ICC/ICC-SC decrease. LONG R3 rhIGF1 also mitigated the reduced food intake and impaired body weight gain. Improved gastric function by LONG R3 rhIGF1 was verified by in vivo systems. In ICC-SC cultures, rhIGF1 mitigated nutlin 3a-induced reduced ERK1/2 phosphorylation and cell growth arrest.

IGF1 can mitigate age-related ICC/ICC-SC loss by activating ERK1/2 signaling, leading to improved gastric compliance and increased food intake in klotho mice.

Gastrointestinal dysfunction seriously affects the prognosis and quality of life of patients with multiple fractures. However, experimental evidence of this relationship is lacking. Here we describe a newly developed mouse model of postoperative gastrointestinal dysfunction after multiple fractures.

Trauma severity was assessed using the injury severity score (ISS). Based on the ISS, a multiple fracture model was established in mice as follows: limb fractures with pelvic fractures and multiple rib fractures; limb fractures with multiple rib fractures; closed fracture of both forelegs with pelvic fracture and rib fractures; closed limb fractures; limb fracture with pelvic fracture; spinal fractures; hind leg fractures with pelvic fractures; pelvic fracture with multiple rib fractures; closed fracture of both fore legs with pelvic fracture; and closed fracture of both fore legs with multiple rib fractures. In each model group, gastrointestinal motility was assayed and the histopathology of the small intestine was examined. Western blot and immunohistochemical analyses of jejunal tissue were performed to detect c-kit protein expression, the level of which was compared with that of a control group. The results of ANOVA are expressed as mean ± standard deviation.

In mice with multiple fractures, food intake was greatly reduced, consistent with histopathological evidence of an injured intestinal epithelium. The jejunal tissue of mice in groups a, c, f, and h was characterized by extensively necrotic and exfoliated intestinal mucosal epithelium and inflammatory cell infiltration in the lamina propria. In the gastrointestinal function assay, gastrointestinal motility was significantly reduced in groups a, b, c, f, and g; these group also had a higher ISS (p < 0.01). The expression of c-kit protein in groups with gastrointestinal dysfunction was significantly up-regulated (p < 0.001) compared with the control group. The close correlation between c-kit expression and the ISS indicated an influence of trauma severity on gastrointestinal motility.

Gastrointestinal dysfunction after multiple fractures was successfully reproduced in a mouse model. In these mice, c-kit expression correlated with gastrointestinal tissue dysfunction and might serve as a therapeutic target.

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

The gut peristaltic movement, a wave-like propagation of a local contraction, is important for the transportation and digestion of ingested materials. Among three types of cells, the enteric nervous system (ENS), smooth muscle cells, and interstitial cells of Cajal (ICCs), the ICCs have been thought to act as a pacemaker, and therefore it is important to decipher the cellular functions of ICCs for the understanding of gut peristalsis. c-Kit, a tyrosine kinase receptor, has widely been used as a marker for ICCs. Most studies with ICCs have been conducted in mammals using commercially available anti-c-Kit antibody. Recently, the chicken embryonic gut has emerged as a powerful model to study the gut peristalsis. However, since the anti-c-Kit antibody for mammals does not work for chickens, cellular mechanisms by which ICCs are regulated have largely been unexplored. Here, we report a newly raised polyclonal antibody against the chicken c-Kit protein. The specificity of the antibody was validated by both Western blotting analyses and immunocytochemistry. Co-immunostaining with the new antibody and anti-α smooth muscle actin (αSMA) antibody successfully visualized ICCs in the chicken developing hindgut in the circular muscle- and longitudinal muscle layers: as previously shown in mice, common progenitors of ICCs and smooth muscle cells at early stages were double positive for αSMA and c-Kit, and at later stages, differentiated ICCs and smooth muscle cells exhibited only c-Kit and αSMA, respectively. A novel ICC population was also found that radially extended from the submucosal layer to circular muscle layer. Furthermore, the new antibody delineated individual ICCs in a cleared hindgut. The antibody newly developed in this study will facilitate the study of peristaltic movement in chicken embryos.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adult central nervous system (CNS) synapses, but it excites immature CNS neurons as well as neurons in the myenteric plexus. The present work aimed to determine whether GABA-induced nonadrenergic, noncholinergic (NANC) neuronal-mediated relaxation of the rat duodenum is dependent on the activity of Na⁺ K⁺ Cl^- cotransporters (NKCC) and requires calcium influx. In the presence of guanethidine (3 µmol/L), atropine (3 µmol/L), and indomethacin (1 µmol/L), relaxations induced by GABA (100 µmol/L), KCl (5-10 mmol/L) and electrical field stimulation (1-8 Hz, 2 ms, 60 V), but not those induced by bradykinin (10-100 nmol/L) were abolished by lidocaine (300 µmol/L). However, only GABA-induced relaxations were reduced in a concentration-dependent manner by the NKCC1/2 inhibitors bumetanide (0.1-1 µmol/L) and furosemide (1-10 µmol/L). GABA-induced NANC neuronal relaxation was abolished by bicuculline (30 µmol/L) and inhibited by N-nitroarginine methyl ester (l-NAME, 300 µmol/L). The ω-conotoxin GVIA (1 µmol/L), which acts exclusively on neuronal Ca_V2 channels, but not on smooth muscle voltage-gated Ca²⁺ Ca_V1 channels, and nonselective blockers of these channels (verapamil 100 nmol/L and ruthenium red 10 µmol/L), reduced GABA-induced relaxations. These results showed that the activation of GABA_A receptors induces NANC nitrergic neuronal relaxations in the rat duodenum, which depend on NKCC activity and Ca_V2 channel activation, suggesting that this phenomenon results from neuronal depolarization promoted by Cl^- efflux through GABA_A receptors, with subsequent Ca²⁺ influx and nitric oxide release.

Obesity is a critical risk factor of several life-threatening diseases and the prevalence in adults has dramatically increased over the past ten years. In the USA the age-adjusted prevalence of obesity in adults was 42.4%, i.e., with a body mass index (BMI, weight (kg)/height (m)2) that exceeds 30 kg/m2. Obese individuals are at the higher risk of obesity-related diseases, co-morbid conditions, lower quality of life, and increased mortality more than those in the normal BMI range i.e., 18.5-24.9 kg/m2. Surgical treatment continues to be the most efficient and scientifically successful treatment for obese patients. Sleeve gastrectomy or vertical sleeve gastrectomy (VSG) is a relatively new gastric procedure to reduce body weight but is now the most popular bariatric operation. To date there have been few studies examining the changes in the cellular components and pacemaker activity that occur in the gastric wall following VSG and whether normal gastric activity recovers following VSG. In the present study we used a murine model to investigate the chronological changes of gastric excitability including electrophysiological, molecular and morphological changes in the gastric musculature following VSG. There is a significant disruption in specialized interstitial cells of Cajal in the gastric antrum following sleeve gastrectomy. This is associated with a loss of gastric pacemaker activity and post-junctional neuroeffector responses. Over a 4-month recovery period there was a gradual return in interstitial cells of Cajal networks, pacemaker activity and neural responses. These data describe for the first time the changes in gastric interstitial cells of Cajal networks, pacemaker activity and neuroeffector responses and the time-dependent recovery of ICC networks and normalization of motor activity and neural responses following VSG.

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) regulate 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 (GCaMP, 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. Abstract Figure Legends Optogenetic activators and sensors can be used to investigate the complex multi-cellular nature of the gastrointestinal (GI tract). Optogenetic activators that are activated by light such as channelrhodopsins (ChR2), OptoXR and halorhodopsinss (HR) proteins can be genetically encoded into specific cell types. This can be used to directly activate or silence specific GI cells such as various classes of enteric neurons, smooth muscle cells (SMC) or interstitial cells, such as interstitial cells of Cajal (ICC). Optogenetic sensors that are activated by different wavelengths of light such as green calmodulin fusion protein (GCaMP) and red CaMP (RCaMP) make high resolution of sub-cellular Ca²⁺ signalling possible within intact tissues of specific cell types. These tools can provide unparalleled insight into mechanisms underlying GI motility and innervation. This article is protected by copyright. All rights reserved.

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.

Complete surgical resection, accessible therapeutic targets and effective tyrosine kinase inhibitors (TKIs) have not completely cured gastrointestinal stromal tumours (GISTs), with most patients suffering from residual tumours and recurrence. The existence of nerve infiltration in GIST provides a way for tumour cells to escape local resection and systemic targeted therapy, which may challenge the previous understanding of its behaviour patterns and inspire the development of more radical excision and more precise targeted therapy. Moreover, tumour dormancy has emerged as a major cause of drug resistance and tumour relapse. Among these pathways, the nerve-tumour regulatory axis GDNF-GFRA1 is activated in GISTs, assists tumour cells in achieving dormancy and protects them from apoptosis under environmental stress by enhancing autophagic flux. The concrete mechanism is that the GDNF-regulating interaction between GFRA1 and the lysosomal calcium channel MCOLN1 activates Ca²⁺-dependent TFEB signalling. Activated TFEB transcriptionally regulates intracellular lysosome levels, which could achieve feedback upregulation of cellular autophagy flux during TKI treatment. This dormancy-transition axis fills parts of the mechanistic vacancy before the onset of secondary mutations, and strategies for TKIs combined with targeting GFRA1-dependent autophagy have distinct promise as prospective clinical therapies.

The gut contains the largest macrophage pool in the body, with populations of macrophages residing in the mucosa and muscularis propria of the gastrointestinal (GI) tract. Muscularis macrophages (MMs), which are located within the muscularis propria, interact with cells essential for GI function, such as interstitial cells of Cajal, enteric neurons, smooth muscle cells, enteric glia, and fibroblast-like cells, suggesting that these immune cells contribute to several aspects of GI function. This review focuses on the latest insights on the factors contributing to MM heterogeneity and the functional interaction of MMs with other cell types essential for GI function. This review integrates the latest findings on macrophages in other organs with increasing knowledge of MMs to better understand their role in a healthy and diseased gut. We describe the factors that contribute to (muscularis macrophage) MM heterogeneity, and the nature of MM interactions with cells regulating GI function. Finally, we also describe the increasing evidence suggesting a critical role of another immune cell type, the mast cell, in normal and diseased GI physiology.

The network of Interstitial Cells of Cajal (ICC) plays a plethora of key roles in maintaining, coordinating, and regulating the contractions of the gastrointestinal (GI) smooth muscles. Several GI functional motility disorders have been associated with ICC degradation. This study extended a previously reported 2D morphological analysis and applied it to 3D spatial quantification of three different types of ICC networks in the distal stomach guided by confocal imaging and machine learning methods. The characterization of the complex changes in spatial structure of the ICC network architecture contributes to our understanding of the roles that different types of ICC may play in post-prandial physiology, pathogenesis, and/or amelioration of GI dsymotility- bridging structure and function.

A validated classification method using Trainable Weka Segmentation was applied to segment the ICC from a confocal dataset of the gastric antrum of a transgenic mouse, followed by structural analysis of the segmented images.

The machine learning model performance was compared to manually segmented subfields, achieving an area under the receiver-operating characteristic (AUROC) of 0.973 and 0.995 for myenteric ICC (ICC-MP; n = 6) and intramuscular ICC (ICC-IM; n = 17). The myenteric layer in the distal antrum increased in thickness (from 14.5 to 34 μm) towards the lesser curvature, whereas the thickness decreased towards the lesser curvature in the proximal antrum (17.7 to 9 μm). There was an increase in ICC-MP volume from proximal to distal antrum (406,960 ± 140,040 vs. 559,990 ± 281,000 μm³; p = 0.000145). The % of ICC volume was similar for ICC-LM and for ICC-CM between proximal (3.6 ± 2.3% vs. 3.1 ± 1.2%; p = 0.185) and distal antrum (3.2 ± 3.9% vs. 2.5 ± 2.8%; p = 0.309). The average % volume of ICC-MP was significantly higher than ICC-IM at all points throughout sample (p < 0.0001).

The segmentation and analysis methods provide a high-throughput framework of investigating the structural changes in extended ICC networks and their associated physiological functions in animal models.

Gastroparesis is characterized by symptoms suggestive of, and objective evidence of, delayed gastric emptying in the absence of mechanical obstruction. This review addresses the normal emptying of solids and liquids from the stomach and details the myogenic and neuromuscular control mechanisms, including the specialized function of the pyloric sphincter, that result in normal emptying, based predominantly on animal research. A clear understanding of fundamental mechanisms is necessary to comprehend derangements leading to gastroparesis, and additional research on human gastric muscles is needed. The section on pathophysiology of gastroparesis considers neuromuscular diseases that affect nonsphincteric gastric muscle, disorders of the extrinsic neural control, and pyloric dysfunction that lead to gastroparesis. The potential cellular basis for gastroparesis is attributed to the effects of oxidative stress and inflammation, with increased pro-inflammatory and decreased resident macrophages, as observed in full-thickness biopsies from patients with gastroparesis. Predominant diagnostic tests involving measurements of gastric emptying, the use of a functional luminal imaging probe, and high-resolution antral duodenal manometry in characterizing the abnormal motor functions at the gastroduodenal junction are discussed. Management is based on supporting nutrition; dietary interventions, including the physical reduction in particle size of solid foods; pharmacological agents, including prokinetics and anti-emetics; and interventions such as gastric electrical stimulation and pyloromyotomy. These are discussed briefly, and comment is added on the potential for individualized treatments in the future, based on optimal gastric emptying measurement and objective documentation of the underlying pathophysiology causing the gastroparesis.

The interstitial cells of Cajal (ICCs) play an important role in coordinated gastrointestinal motility. The present study aimed to elucidate whether or how ICCs are involved in the lower esophageal sphincter (LES) relaxation induced by stimulation of the nicotinic acetylcholine receptor. The application of 1,1-dimethyl-4-phenyl-piperazinium (DMPP; a nicotinic acetylcholine receptor agonist) induced a transient relaxation in the circular smooth muscle of the porcine LES. DMPP-induced relaxation was abolished by not only 1 μM tetrodotoxin but also the inhibition of ICC activity by pretreatment with 100 μM carbenoxolone (a gap junction inhibitor), pretreatment with 100 μM CaCCinh-A01 (an anoctamin-1 blocker acting as a calcium-activated chloride channel inhibitor), and pretreatment with Cl^--free solution. However, pretreatment with 100 μM N^ω-nitro-L-arginine methyl ester had little effect on DMPP-induced relaxation. Furthermore, DMPP-induced relaxation was inhibited by pretreatment with 1 mM suramin, a purinergic P2 receptor antagonist, but not by 1 μM VIP (6-28), a vasoactive intestinal peptide (VIP) receptor antagonist. Stimulation of the purinergic P2 receptor with adenosine triphosphate (ATP) induced relaxation, which was abolished by the inhibition of ICC activity by pretreatment with CaCCinh-A01. In conclusion, membrane hyperpolarization of the ICCs via the activation of anoctamin-1 plays a central role in DMPP-induced relaxation. ATP may be a neurotransmitter for inhibitory enteric neurons, which stimulate the ICCs. The ICCs act as the interface of neurotransmission of nicotinic acetylcholine receptor in order to induce LES relaxation.

The Interstitial Cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells that generate and actively propagate electrophysiological events called slow waves. Slow waves regulate the GI motility necessary for digestion. Several functional GI motility disorders have been associated with depletion in the ICC. In this study, a validated Fast Random Forest (FRF) classification method using Trainable WEKA Segmentation for segmenting the networks of ICC was applied to confocal microscopy images of a whole mount tissue from the distal antrum of a mouse stomach (583 × 3,376 × 133 μm³, parcellated into 24 equal image stacks). The FRF model performance was compared to 6 manually segmented subflelds and produced an area under the receiver-operating characteristic (AUROC) of 0.95. Structural variations of ICC network in the longitudinal muscle (ICC-LM) and myenteric plexus (ICC-MP) were quantified. The average volume of ICC-MP was significantly higher than ICC-LM at any point throughout the antral tissue sampled. There was a pronounced decline of up to 80% in ICC-LM (from 3,705 μm³ to 716 μm³) over a distance of 279.3 μm, that eventually diminished towards the distal antrum. However, an inverse relationship was observed in ICC-MP with an overall increase of up to 157% (from 59,100 μm³ to 151,830 μm³) over a distance of approximately 2 mm that proceeds towards the distal antrum.

Our understanding of human colonic motility, and autonomic reflexes that generate motor patterns, has increased markedly through high-resolution manometry. Details of the motor patterns are emerging related to frequency and propagation characteristics that allow linkage to interstitial cells of Cajal (ICC) networks. In studies on colonic motor dysfunction requiring surgery, ICC are almost always abnormal or significantly reduced. However, there are still gaps in our knowledge about the role of ICC in the control of colonic motility and there is little understanding of a mechanistic link between ICC abnormalities and colonic motor dysfunction. This review will outline the various ICC networks in the human colon and their proven and likely associations with the enteric and extrinsic autonomic nervous systems. Based on our extensive knowledge of the role of ICC in the control of gastrointestinal motility of animal models and the human stomach and small intestine, we propose how ICC networks are underlying the motor patterns of the human colon. The role of ICC will be reviewed in the autonomic neural reflexes that evoke essential motor patterns for transit and defecation. Mechanisms underlying ICC injury, maintenance, and repair will be discussed. Hypotheses are formulated as to how ICC dysfunction can lead to motor abnormalities in slow transit constipation, chronic idiopathic pseudo-obstruction, Hirschsprung's disease, fecal incontinence, diverticular disease, and inflammatory conditions. Recent studies on ICC repair after injury hold promise for future therapies.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Traditionally, the urethra has been considered a mere conduit to guide urine from the bladder to the external side of the body. Building evidence indicates that the urethra may directly influence bladder function via mechanisms restricted to the lower urinary tract (LUT).

Here, we discuss the tissue arrangement of the urethra and addressed the contribution of new paraneuronal cells to LUT function. We also briefly reviewed two frequent LUT pathologies associated with urethral dysfunction.

Continence depends on an intact and functional urethral sphincter, composed of smooth, and striated muscle fibers and regulated by somatic and autonomic fibers. Recent studies suggest the existence of an urethro-vesical reflex that also contributes to normal LUT function. Indeed, the urethral lumen is lined by a specialized epithelium, the urothelium, in the proximal urethra. In this region, recent evidence demonstrates the presence of specific paraneuronal cells, expressing the neurotransmitters acetylcholine and serotonin. These cells are in close proximity of nerve fibers coursing in the lamina propria and are able to release neurotransmitters and rapidly induce detrusor contractions, supporting the existence of an urethro-vesical crosstalk.

The mechanism underlying the fast communication between the urethra and thebladder are beginning to be understood and should involve the interaction between specificepithelial cells and fibres innervating the urethral wall. It is likely that this reflex should bealtered in pathological conditions, becoming an attractive therapeutic target.

Urinary continence is maintained in the lower urinary tract by the contracture of urethral sphincters, including smooth muscle of the internal urethral sphincter. These contractions occlude the urethral lumen, preventing urine leakage from the bladder to the exterior. Over the past 20 years, research on the ionic conductances that contribute to urethral smooth muscle contractility has greatly accelerated. A debate has emerged over the role of interstitial cell of Cajal (ICC)-like cells in the urethra and their expression of Ca²⁺-activated Cl^- channels encoded by anoctamin-1 [Ano1; transmembrane member 16 A (Tmem16a) gene]. It has been proposed that Ano1 channels expressed in urethral ICC serve as a source of depolarization for smooth muscle cells, increasing their excitability and contributing to tone. Although a clear role for Ano1 channels expressed in ICC is evident in other smooth muscle organs, such as the gastrointestinal tract, the role of these channels in the urethra is unclear, owing to differences in the species (rabbit, rat, guinea pig, sheep, and mouse) examined and experimental approaches by different groups. The importance of clarifying this situation is evident as effective targeting of Ano1 channels may lead to new treatments for urinary incontinence. In this review, we summarize the key findings from different species on the role of ICC and Ano1 channels in urethral contractility. Finally, we outline proposals for clarifying this controversial and important topic by addressing how cell-specific optogenetic and inducible cell-specific genetic deletion strategies coupled with advances in Ano1 channel pharmacology may clarify this area in future studies.NEW & NOTEWORTHY Studies from the rabbit have shown that anoctamin-1 (Ano1) channels expressed in urethral interstitial cells of Cajal (ICC) serve as a source of depolarization for smooth muscle cells, increasing excitability and tone. However, the role of urethral Ano1 channels is unclear, owing to differences in the species examined and experimental approaches. We summarize findings from different species on the role of urethral ICC and Ano1 channels in urethral contractility and outline proposals for clarifying this topic using cell-specific optogenetic approaches.

β-Nicotinamide adenine dinucleotide (β-NAD) is a key inhibitory neurotransmitter in the colon. The neuroeffector junction in the gut consists of enteric motor neurons and SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα⁺ cells). Measuring metabolism of 1,N⁶ -etheno-NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFRα⁺ cells with HPLC-FLD, we report that (1) in tissues, eNAD is degraded to eADP-ribose, eAMP and e-adenosine (eADO) by CD38, ENPP1 and NT5E, (2) with SMCs and PDGFRα⁺ cells, eNAD is metabolized to eADO by ENPP1 and NT5E, (3) eNAD is not metabolized by ICC, (4) NT5E is expressed chiefly by SMCs and moderately by PDGFRα⁺ cells, (5) SIP cells are not the primary location of CD38. These data argue that the duration and strength of purinergic neurotransmission can be modulated by targeting multiple enzymes with specialized cellular distribution in the colon.

Prior studies suggest that β-nicotinamide adenine dinucleotide (β-NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of β-NAD at the neuroeffector junction (NEJ) is likely to be necessary for terminating inhibitory neurotransmission and may also produce bioactive metabolites. The enteric NEJ consists of enteric neurons and postjunctional cells of the SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα⁺ cells). We examined possible specialized functions of the NEJ in β-NAD metabolism by determining the degradation of 1,N⁶ -etheno-NAD (eNAD) in colonic tunica muscularis of wild-type, Cd38^-/- , Nt5e^-/- , Enpp1^-/- and Cd38^-/- /Nt5e^-/- mice and in SIP cells from mice expressing cell-specific fluorescent reporters purified by fluorescence activated cell sorting (FACS). We measured eNAD and its metabolites eADP-ribose (eADPR), eAMP and e-adenosine (eADO) from tissues and sorted SIP cells using liquid chromatography. eNAD exposed to colonic muscularis of wild-type mice produced eADPR, eAMP and eADO. CD38 mediated the conversion of eNAD to eADPR, whereas ENPP1 mediated degradation of eNAD and eADPR to eAMP. NT5E (aka CD73) was the primary enzyme forming eADO from eAMP. PDGFRα⁺ cells and SMCs were involved in production of eADO from eNAD, and ICC were not involved in extracellular metabolism of eNAD. CD38 mediated the eNAD metabolism in whole tissues, but CD38 did not appear to be functionally expressed by SMCs or ICC. NT5E was expressed in SMCs > PDGFRα⁺ cells. Our data show that extracellular metabolism of β-NAD in the colon is mediated by multiple enzymes with cell-specific expression.

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.

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.

Gastrointestinal (GI) vagal afferents convey sensory signals from the GI tract to the brain. Numerous subtypes of GI vagal afferent have been identified but their individual roles in gut function and feeding regulation are unclear. In the past decade, technical approaches to selectively target vagal afferent subtypes and to assess their function has significantly progressed. This review examines the classification of GI vagal afferent subtypes and discusses the current available techniques to study vagal afferents. Investigating the distribution of GI vagal afferent subtypes and understanding how to access and modulate individual populations are essential to dissect their fundamental roles in the gut-brain axis.

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.

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.