Stimulus-induced pacemaker activity in interstitial cells of Cajal associated with the deep muscular plexus of the small intestine.

BACKGROUND: The ICC-DMP have been proposed to generate stimulus-dependent pacemaker activity, rhythmic transient depolarizations, that take part in orchestrating segmentation and clustered propulsive motor patterns in the small intestine. However, little is known about the fundamental properties of ICC-DMP. METHODS: This study was undertaken to increase our understanding of intrinsic properties of the ICC-DMP through calcium imaging and intracellular electrical recordings. KEY RESULTS: Without stimulation, most ICC-DMP were quiescent. In some preparations ICC-DMP generated rhythmic low-frequency calcium oscillations (<10 cpm) with or without high frequency activity superimposed (>35 cpm). Immunohistochemistry proved the existence of NK1R on the ICC-DMP and close contacts between ICC-DMP and substance P-positive nerves. Substance P (25 nM) induced low-frequency calcium oscillations that were synchronized across the ICC-DMP network. Substance P also induced low frequency rhythmic transient depolarizations (<10cpm) in circular muscle cells close to the ICC-DMP. An intracellular recording from a positively identified ICC-DMP showed rhythmic transient depolarizations with superimposed high frequency activity. To investigate if quiescent ICC-DMP were chronically inhibited by nitrergic activity, nNOS was inhibited, but without effect. CONCLUSIONS & INFERENCES: Substance P changes non-synchronized high frequency flickering or quiescence in ICC-DMP into strong rhythmic calcium transients that are synchronized within the network; they are associated with rhythmic transient depolarizations within the same frequency range. We hypothesize that Substance P, released from nerves, can evoke rhythmicity in ICC-DMP, thereby providing it with potential pacemaker activity.

Involvement of 5-HT3 and 5-HT4 receptors in colonic motor patterns in rats.

BACKGROUND: Colonic migrating motor complexes in the rat constitute two distinct propulsive motor patterns, pan-colonic rhythmic long distance contractions (LDCs), and rhythmic propulsive motor complexes (RPMCs) occurring primarily in the mid/distal colon. Interstitial cells of Cajal govern their rhythmicity, but their occurrence is dependent on neural programs. Our aim was to investigate the involvement of 5-HT3 and 5-HT4 receptors in the generation and pharmacological control of the motor patterns. METHODS: Effects of 5-HT-related drugs on colonic motor patterns were analyzed through spatio-temporal maps created from video recordings of whole organ motility. KEY RESULTS: 5-HT3 antagonists abolished RPMCs and LDCs. 5-HT4 agonists inhibited LDCs; they promoted RPMCs, which was blocked by the 5-HT4 antagonist GR 125487. 5-HT and the 5-HT3 agonist m-CPBG strongly inhibited LDCs and RPMCs. CONCLUSIONS & INFERENCES: The generation of LDCs involves ongoing 5-HT release acting on 5-HT3 and 5-HT4 receptors. The spontaneous generation of RPMCs involves ongoing 5-HT release acting on 5-HT3 but not 5-HT4 receptors. Prucalopride and mosapride promote RPMCs, an effect that is inhibited by the 5-HT4 receptor antagonist GR 125487. A 5-HT3 agonist does not promote RPMCs. Segmentation, including a pattern of sequential segmental activity not previously described, can occur without significant involvement of 5-HT3 and 5-HT4 receptors. 5-HT and a 5-HT3 agonist are strongly inhibitory indicating that 5-HT receptors are present in inhibitory pathways which are normally not involved in the generation of spontaneous or distention-induced motor patterns.

Intestinal microbiota influence the early postnatal development of the enteric nervous system.

BACKGROUND: Normal gastrointestinal function depends on an intact and coordinated enteric nervous system (ENS). While the ENS is formed during fetal life, plasticity persists in the postnatal period during which the gastrointestinal tract is colonized by bacteria. We tested the hypothesis that colonization of the bowel by intestinal microbiota influences the postnatal development of the ENS. METHODS: The development of the ENS was studied in whole mount preparations of duodenum, jejunum, and ileum of specific pathogen-free (SPF), germ-free (GF), and altered Schaedler flora (ASF) NIH Swiss mice at postnatal day 3 (P3). The frequency and amplitude of circular muscle contractions were measured in intestinal segments using spatiotemporal mapping of video recorded spontaneous contractile activity with and without exposure to lidocaine and N-nitro-L-arginine (NOLA). KEY RESULTS: Immunolabeling with antibodies to PGP9.5 revealed significant abnormalities in the myenteric plexi of GF jejunum and ileum, but not duodenum, characterized by a decrease in nerve density, a decrease in the number of neurons per ganglion, and an increase in the proportion of myenteric nitrergic neurons. Frequency of amplitude of muscle contractions were significantly decreased in the jejunum and ileum of GF mice and were unaffected by exposure to lidocaine, while NOLA enhanced contractile frequency in the GF jejunum and ileum. CONCLUSIONS & INFERENCES: These findings suggest that early exposure to intestinal bacteria is essential for the postnatal development of the ENS in the mid to distal small intestine. Future studies are needed to investigate the mechanisms by which enteric microbiota interact with the developing ENS.

Bifidobacterium longum NCC3001 inhibits AH neuron excitability.

BACKGROUND: Bifidobacterium longum (B. longum) NCC3001 can affect behavior and brain biochemistry, but identification of the cellular targets needs further investigation. Our hypothesis was that the communication with the brain might start with action on enteric sensory neurons. METHODS: Ileal segments from adult mice were used to create a longitudinal muscle-myenteric-plexus preparation to expose sensory after-hyperpolarizing (AH) neurons in the myenteric plexus to allow access with microelectrodes. The intrinsic excitability of AH neurons was tested in response to the perfusion of conditioned media (B. longum culture supernatant) or unconditioned media (growth medium, MRS). KEY RESULTS: B. longum conditioned medium significantly reduced the excitability of AH neurons compared to perfusion with the unconditioned medium. Specifically, a reduction was seen in the number of action potentials fired per depolarizing test pulse, the instantaneous and time-dependent input resistances and the magnitude of the hyperpolarization-activated cationic current (Ih ). CONCLUSIONS & INFERENCES: The probiotic B. longum reduces excitability of AH sensory neurons likely via opening of potassium channels and closing of hyperpolarization-activated cation channels.

Test of normality for integrated change point detection and mixture modeling.

Single-molecule data often show step-like changes in the quantity measured between constant levels. Analysis of this data consists of detecting the steps, i.e., change point detection (CPD), and determining the levels, i.e., clustering. We describe a novel algorithm which integrates these two analyses, based on a statistical test of a normal distribution. The test of normality (TON) algorithm integrates statistical CPD with gaussian mixture model clustering. We used TON with both simulated data and ion channel patch-clamp recordings. It performed well with simulated data except at a high signal-to-noise ratio and when the frequency of steps was high compared to the sampling frequency. TON has advantages over separate CPD and mixture modeling algorithms, especially for complex single-molecule data. This was illustrated by its application to the maxichannel, an ion channel with multiple subconductance states.

Ca2+ sensitivity of the maxi chloride channel in interstitial cells of Cajal.

BACKGROUND: Interstitial cells of Cajal (ICC) associated with the myenteric plexus of the small intestine express maxi chloride channels. Our aim was to investigate whether or not these channels would be activated by increases in intracellular Ca(2+) , as that would strengthen evidence for their potential role in ICC pacemaking. A further aim was to examine whether inwardly and outwardly rectifying maxi chloride currents signify different channels. METHODS: We used Fluo-4 AM Ca(2+) imaging and patch clamp electrophysiology (cell-attached and inside-out) on isolated ICC in short term culture. KEY RESULTS: Increasing intracellular Ca(2+) by three functionally distinct mechanisms (blocking sarcoplasmic reticulum Ca(2+) refilling, creating membrane Ca(2+) pores and a solution designed to block plasmalemmal Ca(2+) extrusion) was followed by inwardly rectifying maxi chloride channel activation assessed in the cell-attached configuration. Furthermore, in the inside-out configuration, increased outwardly rectifying maxi-chloride channel activity followed an increase in Ca(2+) to 2 mmol L(-1) at the cytoplasmic face of the channel. CONCLUSIONS & INFERENCES: Increase in intracellular Ca(2+) will activate the maxi chloride channels. Maxi chloride currents are inwardly rectifying in the cell-attached patch clamp configuration under physiological conditions and are outwardly rectifying in the inside-out configuration. The same channel is responsible for both currents. Ca(2+) does not appear to regulate the rectification.

The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gut-brain communication.

BACKGROUND: The probiotic Bifidobacterium longum NCC3001 normalizes anxiety-like behavior and hippocampal brain derived neurotrophic factor (BDNF) in mice with infectious colitis. Using a model of chemical colitis we test whether the anxiolytic effect of B. longum involves vagal integrity, and changes in neural cell function. Methods Mice received dextran sodium sulfate (DSS, 3%) in drinking water during three 1-week cycles. Bifidobacterium longum or placebo were gavaged daily during the last cycle. Some mice underwent subdiaphragmatic vagotomy. Behavior was assessed by step-down test, inflammation by myeloperoxidase (MPO) activity and histology. BDNF mRNA was measured in neuroblastoma SH-SY5Y cells after incubation with sera from B. longum- or placebo-treated mice. The effect of B. longum on myenteric neuron excitability was measured using intracellular microelectrodes. KEY RESULTS: Chronic colitis was associated with anxiety-like behavior, which was absent in previously vagotomized mice. B. longum normalized behavior but had no effect on MPO activity or histological scores. Its anxiolytic effect was absent in mice with established anxiety that were vagotomized before the third DSS cycle. B. longum metabolites did not affect BDNF mRNA expression in SH-SY5Y cells but decreased excitability of enteric neurons. CONCLUSIONS & INFERENCES: In this colitis model, anxiety-like behavior is vagally mediated. The anxiolytic effect of B. longum requires vagal integrity but does not involve gut immuno-modulation or production of BDNF by neuronal cells. As B. longum decreases excitability of enteric neurons, it may signal to the central nervous system by activating vagal pathways at the level of the enteric nervous system.

Role of interstitial cells of Cajal in the generation and modulation of motor activity induced by cholinergic neurotransmission in the stomach.

BACKGROUND: Interstitial cells of Cajal (ICC) are intimately linked to the enteric nervous system and a better understanding of the interactions between the two systems is going to advance our understanding of gut motor control. The objective of the present study was to investigate the role of ICC in the generation of gastric motor activity induced by cholinergic neurotransmission. METHODS: Gastric motor activity was evoked through activation of intrinsic cholinergic neural activity, in in vitro muscle strips by electrical field stimulation, in the in vitro whole stomach by distension and in vivo by fluoroscopy after gavaging the stomach with barium sulfate. The cholinergic activity was assessed as that component of the effect of the stimulus that was sensitive to atropine. These experiments were carried out in wild-type and Ws/Ws rats that have few intramuscular ICC (ICC-IM) in the stomach. KEY RESULTS: Under all three experimental conditions, cholinergic activity was prominent in both wild-type and W mutant rats providing evidence against the hypothesis that cholinergic neurotransmission to smooth muscle is primarily mediated by ICC-IM. Strong cholinergic activity in Ws/Ws rats was not due to upregulation of muscarinic receptors in ICC but possibly in smooth muscle of the antrum. CONCLUSIONS & INFERENCES: Pacemaker ICC play a prominent role in the expression of motor activity induced by cholinergic activity and our data suggest that cholinergic neurotransmission to ICC affects the pacemaker frequency.

Measurement of intracellular chloride ion concentration in ICC in situ and in explant culture.

BACKGROUND: Chloride channels are proposed to play a central role in the electrical pacemaking mechanism of interstitial cells of Cajal (ICC). A key unknown factor in the consideration of this role is the chloride equilibrium potential (E(Cl)), as determined by the relative concentrations of intra- ([Cl(-)](i)) and extracellular ([Cl(-)](o)) chloride ions. METHODS: To calculate the E(Cl) of ICC, [Cl(-)](i) was measured with the fluorescent chloride indicator N-(6-methoxyquinolyl) acetoethyl ester (MQAE). Pacemaker ICC in explant cultures or in situ, i.e. ICC associated with the myenteric plexus of the small intestine, were loaded with MQAE and fluorescence was measured by laser scanning confocal microscopy. The dye fluorescence was then calibrated against known [Cl(-)](i) by treating the explants or in situ preparations with chloride ionophore and varying bath chloride concentrations. KEY RESULTS: In explants, ICC [Cl(-)](i) was measured as 13 mmol L(-1) with [Cl(-)](o) of 100 mmol L(-1), giving an E(Cl) of -52 mV [corrected]. With [Cl(-)](o) at 166 mmol L(-1), [Cl(-)](i) was 26 mmol L(-1), giving an E(Cl) of -47 mV[corrected]. In situ, ICC [Cl(-)](i) was measured as 26 mmol L(-1) with [Cl(-)](o) of 130 mmol L(-1), giving an E(Cl) of -41 mV [corrected]. Importantly ICC compensate for changes in extracellular chloride by changing [Cl(-)](i) and thus maintain E(Cl). In ICC explant clusters, [Cl(-)](i) was seen to fluctuate, possibly evoked by rhythmic changes in intracellular calcium. CONCLUSIONS & INFERENCES: The intracellular chloride concentration in ICC fluctuates to keep its equilibrium potential constant. The identification of E(Cl) as positive to the resting membrane potential of ICC indicates that opening of chloride channels will depolarize ICC.

Lactobacillus reuteri ingestion and IK(Ca) channel blockade have similar effects on rat colon motility and myenteric neurones.

BACKGROUND: We have previously shown that ingestion of Lactobacillus reuteri may modulate colonic enteric neuron activity but with unknown effects on colon motility. The aim of the present report was to elucidate the neuronal mechanisms of action of the probiotic by comparing the effects on motility of L. reuteri ingestion with blockade of a specific ionic current in enteric neurons. METHODS: We have used intraluminal pressure recordings from ex vivo rat colon segments and whole cell patch clamp recordings from neurons of rat longitudinal muscle myenteric plexus preparations to investigate the effects of L. reuteri and TRAM-34 on colon motility and neurophysiology. The effects of daily feeding of 10(9) L. reuteri bacteria or acute application of TRAM-34 on threshold fluid filling pressure or pulse pressure was measured. KEY RESULTS: Lactobacillus reuteri increased intraluminal fluid filling pressure thresholds for evoking pressure pulses by 51% from 0.47 +/- 0.17 hPa; the probiotic also decreased the pulse pressure amplitudes, but not frequency, by 18% from 3.91 +/- 0.52 hPa. The intermediate conductance calcium-dependent potassium (IK(Ca)) channel blocker TRAM-34 (3 micromol L(-1)) increased filling threshold pressure by 43% from 0.52 +/- 0.22 hPa and reduced pulse pressure amplitude by 40% from 2.63 +/- 1.11 hPa; contraction frequency was unaltered. TRAM-34 (3 micromol L(-1)) reduced membrane polarization, leak conductance and the slow afterhyperpolarization current in 16/16 myenteric rat colon AH cells but 19/19 S cells were unaffected. CONCLUSIONS & INFERENCES: The present results are consistent with L. reuteri enhancing tonic inhibition of colon contractile activity by acting via the IK(Ca) channel current in AH cells.

Neurotransmission in lower esophageal sphincter of W/Wv mutant mice.

To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/W(v) wild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired IJPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/W(v) mutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/W(v) mutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal Ca2+-dependent signaling initiated by spontaneous Ca2+ release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle.

Loss of intramuscular and submuscular interstitial cells of Cajal and associated enteric nerves is related to decreased gastric emptying in streptozotocin-induced diabetes.

Interstitial cells of Cajal (ICC) are associated with afferent innervation and peristalsis of the stomach suggestive of a key role in the pathophysiology of gastroparesis. We studied changes in the density and ultrastructure of ICC and enteric nerves in the streptozotocin-induced diabetes mellitus (STZ-DM) in Wistar rats using immunohistochemistry and electron microscopy. Gastric emptying was studied in vivo by single-photon emission computed tomography. In the STZ-DM antrum, a marked reduction was observed in the density of the intramuscular ICC (ICC-IM) and ICC located at the submucosal border of the circular muscle layer of the antrum (ICC-SM). The surviving ICC showed lamellar bodies and partial vacuolation of the cytoplasm content, loss of connections between ICC-IM and nerves; it appeared that injured ICC-IM developed into fibroblast-like ICC. ICC associated with Auerbach's plexus (ICC-AP) in the antrum and ICC in the fundus were not affected significantly except for a loss of connections with nerve structures. Marked reduction in nerve tissue (Protein Gene Product-9.5 positivity) was also restricted to the muscle layers including nitrergic nerves (neuronal nitric oxide synthase positivity). In vivo assessed gastric emptying was markedly reduced in STZ-DM rats. Our data demonstrate in the STZ-DM rat stomach a decreased density of ICC limited to the antrum and to ICC-IM and ICC-SM, and structural degeneration in ICC-IM and associated nerves with a special emphasis on loss of synaptic connections, accompanied by a decrease in gastric emptying. Hence, in this model of gastroparetic diabetes, regional injury to subsets of ICC and nerves are associated with gastric motor dysfunction.

Substance P activates a non-selective cation channel in murine pacemaker ICC.

Interstitial cells of Cajal (ICC) associated with Auerbach's plexus in the small intestine, provide pacemaker activity to orchestrate peristalsis and mixing. Despite the close apposition between ICC and enteric nerves, little is known about the neural regulation of pacemaker activity. The present study pursues the hypothesis that substance P can affect pacemaker activity through action on non-selective cation channels. Cell-attached and inside-out patch clamp studies were performed on isolated ICC in short-term cultures that provided evidence that substance P increases open probability or initiates activity in non-selective cation channels in ICC. The single-channel conductance is approximately 25 pS and in the on-cell configuration the activity can occur in a rhythmic fashion. Patches contained 1-10 channels and were most often accompanied by a approximately 12 pS chloride channel that was also activated by substance P. In a recently developed preparation that allows patch clamping in ICC in their natural environment within tissue, i.e. in situ, the presence of the channel and substance P activation was confirmed. The non-selective cation channel is one of the channels that initiate intestinal pacemaker activity and the present study provides further single-channel data on this critical channel. Because of the close proximity of enteric motor and sensory nerves to ICC, these data provide a potential mechanism underlying neural regulation of pacemaker activity. The data also indicate that neurokinergic pharmacology is a promising avenue for excitation of the intestinal pacemaker system.

Igf1r+/CD34+ immature ICC are putative adult progenitor cells, identified ultrastructurally as fibroblast-like ICC in Ws/Ws rat colon.

The colon of Ws/Ws mutant rats shows impairment of pacemaker activity and altered inhibitory neurotransmission. The present study set out to find structural correlates to these findings to resolve mechanisms. In the colon of Ws/Ws rats, interstitial cells of Cajal associated with Auerbach's plexus (ICC-AP) were significantly decreased and ICC located at the submuscular plexus and intramuscular ICC were rarely observed based on immunohistochemistry and electron microscopy. Ultrastructural investigations revealed that there was no overall loss of all types of interstitial cells combined. Where loss of ICC was observed, a marked increase in fibroblast-like ICC (FL-ICC) was found at the level of AP. Immunoelectron microscopy proved FL-ICC to be c-Kit(-) but gap junction coupled to each other and to c-Kit(+) ICC; they were associated with enteric nerves and occupied space normally occupied by ICC in the wild-type rat colon, suggesting them to be immature ICC. In addition, a marked increase in immunoreactivity for insulin-like growth factor 1 receptor (Igf1r) occurred, co-localized with CD34 but not with c-Kit. A significantly higher number of Igf1r(+)/CD34(+) cells were found in Ws/Ws compared to wild-type rat colons. These CD34(+)/Igf1r(+) cells in the Ws/Ws colon occupied the same space as FL-ICC. Hence we propose that a subset of immature ICC (FL-ICC) consists of adult progenitor cells. Immunohistochemistry revealed a reduction of neurons positive for neuronal nitric oxide synthase. The functional capabilities of the immature ICC and the regenerative capabilities of the adult progenitor cells need further study. The morphological features described here show that the loss of pacemaker activity is not associated with failure to develop a network of interstitial cells around AP but a failure to develop this network into fully functional pacemaker cells. The reduction in nitrergic innervation associated with the Ws mutation may be the result of a reduction in nitrergic neurons.

Ultrastructural evidence for communication between intramuscular vagal mechanoreceptors and interstitial cells of Cajal in the rat fundus.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.

Interstitial cells of Cajal and neuromuscular transmission in the rat lower oesophageal sphincter.

The distribution of interstitial cells of Cajal (ICC) and neurotransmission were investigated in lower oesophageal sphincter (LES) circular muscle strips from Sprague-Dawley (SD) rats, Ws/Ws mutant rats and their wild-type (+/+) siblings. Intramuscular c-Kit-positive cells, confirmed to be ICC-IM by electron microscopy, were observed throughout both muscle layers from SD and +/+ rats. In contrast, c-Kit-positive, ultrastructurally typical ICC-IM were absent in Ws/Ws. LES strips from Ws/Ws rats showed increased spontaneous contractile activity. Strips from SD and +/+ rats, responded to electrical neuronal stimulation with a relaxation that was in part L-NNA and in part apamin sensitive, followed by a contraction which was decreased by atropine. In Ws/Ws rats, similar to +/+ rats, neurally mediated relaxation was L-NNA and apamin sensitive and the contraction was decreased by atropine. We conclude that in the rat LES, relaxation is mediated by NO and an apamin-sensitive mediator, and contraction primarily by acetylcholine. Despite the absence of c-Kit-positive ICC, nerve-muscle interaction can be accomplished likely by diffusion of neurotransmitters to the smooth muscle cells. The lack of c-Kit-positive ICC is related to an increase in the basal tone and spontaneous contractile activity. The presence of fibroblast-like ICC in Ws/Ws rats might represent immature ICC whose possible functions need further investigation.

Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn's disease.

Crohn's disease associated dysmotility has been attributed to fibrosis and damage to enteric nerves but injury to interstitial cells of Cajal (ICC) could also be involved. We assessed ICC in specimens obtained from patients with Crohn's disease and determined the relation between ICC and the inflammatory infiltrate, particularly mast cells (MC) using quantitative immunohistochemistry and electron microscopy. Ultrastructural injury to ICC was patchy in all ICC subtypes but ICC-Auerbach's plexus (AP) showed damage more frequently, i.e. swelling of mitochondria, decreased electron density, autophagosomes and partial depletion of the cytoplasm. Light microscopy confirmed a significant decrease in c-kit immunoreactivity for ICC-AP and an increased number of MC in the muscularis externa. Electron microscopy showed MC exhibiting piecemeal degranulation and making frequent and selective membrane-to-membrane contact with all types of injured ICC which suggests chronic release of granule content to affect ICC. Extent of ICC injury was not associated with duration of the disease. In conclusion, ultrastructural injury and loss of ICC-AP is evident in Crohn's disease. Epidemiological and morphological data suggest that ICC have the capacity to regenerate in spite of the chronic insult. The muscularis hosts a marked number of MC that exhibit piecemeal degranulation associated with ICC and may facilitate ICC maintenance.

Pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats.

The aim of this study was to characterize the pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats, which harbor a mutation in the c-kit gene that affects development of interstitial cells of Cajal (ICC). In Ws/Ws rats, the density of KIT-positive cells was markedly reduced. Wild-type, but not Ws/Ws, rats showed low- and high-frequency cyclic depolarization that were associated with highly regular myogenic motor patterns at the same frequencies. In Ws/Ws rats, irregular patterns of action potentials triggered irregular muscle contractions occurring within a bandwidth of 10-20 cycles/min. Spontaneous activity of nitrergic nerves caused sustained inhibition of muscle activity in both wild-type (+/+) and Ws/Ws rats. Electrical field stimulation of enteric nerves, after blockade of cholinergic and adrenergic activity, elicited inhibition of mechanical activity and biphasic inhibitory junction potentials both in wild-type and Ws/Ws rats. Apamin-sensitive, likely purinergic, inhibitory innervation was not affected by loss of ICC. Variable presence of nitrergic innervation likely reflects the presence of direct nitrergic innervation to smooth muscle cells as well as indirect innervation via ICC. In summary, loss of ICC markedly affects pacemaker and motor activities of the rat colon. Inhibitory innervation is largely maintained but nitrergic innervation is reduced possibly related to the loss of ICC-mediated relaxation.

Interstitial cells of Cajal in health and disease. Part I: normal ICC structure and function with associated motility disorders.

Ramon y Cajal (1852-1934) is considered to be one of the founders of the field of neuroscience. In 1911, he described interstitial neurons in the gut, noting that they were primitive accessory components that perhaps modify smooth muscle contraction, themselves subject to regulation from principle neurons. The accuracy of his description of their appearance and activities has led to these cells now being called the interstitial cells of Cajal (ICC). Thuneberg and Faussone-Pellegrini were instrumental in bringing these cells to the attention of gastroenterologists and pathologists in the early 1980s. Subsequently, the development of antibodies to c-kit has allowed routine identification of the ICC in pathology specimens. c-Kit is a transmembrane protein kinase which has as ligand stem cell factor and is involved in cell development in a variety of cell lineages. In the gut musculature, ICC and mast cells are the only cells that have prominent c-kit expression. The ICC are now known to play an important role in gut motility and absent or disordered ICC networks have been identified in a variety of motility disorders.

Interstitial cells of Cajal in health and disease. Part II: ICC and gastrointestinal stromal tumours.

Mesenchymal tumours in the gastrointestinal tract have long been problematic in terms of diagnosis, prognosis and therapy, but recent advances in immunohistochemistry and related therapies have allowed more specific diagnosis. In particular, the recognition that both the interstitial cells of Cajal (ICC) and many gastrointestinal stromal tumours (GISTs) are positive for c-kit and CD34 and have other features similar to those of ICC has led to the use of imatinib, a novel small molecule therapy that blocks the CD117/c-kit tyrosine kinase receptor, which shows remarkable efficacy in treatment of malignant and metastatic GISTs as well as other malignancies.