Distribution and ultrastructure of interstitial cells of Cajal in the gastric antrum of wild-type and Ws/Ws rats

Interstitial cells of Cajal (ICC) in the stomach of wild-type and Ws/Ws mutant rats that are deficient in c-kit were studied by immunohistochemistry and electron microscopy to elucidate their regional specialization in the gastric antrum. Immunohistochemistry for Kit protein demonstrated that in wild-type rats ICC were located at the submucosal border of the circular muscle layer (ICC-SM) in a limited extension of the antrum from the pyloric sphincter towards the corpus, as well as within both the circular (ICC-CM) and longitudinal (ICC-LM) muscle layers and in the myenteric plexus region (ICC-AP). In c-kit mutant Ws/Ws rats while ICC-CM and ICC-LM were not observed, but unexpectedly, a few ICC-SM and ICC-AP were found. By electron microscopy, ICC-SM and ICC-AP were characterized by abundant mitochondria, many caveolae, a distinct basal lamina and formed gap junctions with other ICC or with smooth muscle cells and make close contacts with nerves. Thus, ICC-SM and ICC-AP of the rat antrum were classified as Type 3 ICC, the type most similar to smooth muscle cells. The functional significance of ICC-SM and their survival in the c-kit mutant animals is discussed in reference to the role of the c-kit/stem cell factor system for their cellular maturation.     

Toward a concept of stretch-coupling in smooth muscle. I. Anatomy of intestinal segmentation and sleeve contractions

Motility patterns and their structural basis were studied by video analysis, light and electron microscopy on the physiologically distended gut from normal and W/W(v) suckling mice and normal adult mice. Empty or diltiazem-relaxed intestine were used as references. In contrast to conventional primary aldehyde fixation, a brief primary fixation with osmic acid before aldehydes preserved the visible contraction patterns and revealed dynamic increases in the number of peg-and-socket junctions coupling muscle cells mutually and with interstitial cells of Cajal (ICC). In tissue engaged in segmentation, the major increase was in the circular muscle and involved the ICC-DMP (integrated in the circular muscle layer at the site of the deep muscular plexus), whereas the increase during sleeve contractions was in the longitudinal muscle and involved the ICC-AP (located at the site of Auerbach's plexus). The number and distribution of gap junctions were unaffected. Area analysis of cell profiles supported the involvement of circular muscle in segmentation, but longitudinal muscle alone in sleeve contractions. The gut of both normal and W/W(v) sucklings (and adults) contracted during segmentation at frequencies close to reported slow-wave frequencies. In W/W(v) sucklings, ICC-AP were absent whereas ICC-DMP were present in adult configuration. Before Day 8 pp gap junctions were seen only between ICC-DMP. In the sucklings ICC-DMP may be responsible for rapid circumferential coordination and pacemaking of ring contractions. The geometry, organization, and dynamic regulation of peg-and-socket junctions strongly suggest a crucial role in coordination of smooth muscle and pacemakers, probably as stretch sensors, mediating a 'stretch-coupling' in the system.     

Interstitial cells of Cajal associated with the submucosal plexus of the Guinea-pig stomach

Interstitial cells of Cajal (ICC) form specialized networks in the gastrointestinal tract that coordinate cellular communications between nerves and smooth muscle cells. However, little is known about ICC in the gut mucosa or submucosa. Here, we report for the first time that Kit-immunoreactive ICC are associated with the submucosal (Meissner's) plexus of the Guinea-pig stomach. In longitudinal sections along the greater curvature of the gastric corpus, short spindle-shaped ICC of the submucosal plexus (ICC-SP) were located around the PGP9.5-immunoreactive nerve elements in the submucosa. Observations of whole-mount preparations clearly demonstrated Kit-immunoreactive bipolar or multipolar cells with long cytoplasmic processes about 100 microm in length. Such cells had typical characteristics of ICC, confirming that they were not mast cells, which are also Kit-immunoreactive residents of the submucosal connective tissue space. Although some ICC-SP surrounded parts of the submucosal plexus, they did not appear to form wide extensions of the cellular network, suggesting that they acted locally. The demonstration of ICC-SP in the submucosal connective tissue space suggests that they may contribute to the regulation of secretion, absorption and transportation of fluids in the mucosa.     

Chloride activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney

Muscle strips were excised from the circular and longitudinal layers of the fundus, corpus and antrum of canine stomach, and from the inner portion of the pyloric ring (inner pylorus). Substance P (SP) induced strong contractions, with the greatest sensitivity in fundus and circular corpus preparations (threshold near 10 -10 mol/l). The sensitivity to SP decreased in the sequence circular corpus - longitudinal antrum - circular antrum (threshold near 10(-7) mol/l); it re-increased towards the pylorus, and in the inner pylorus was nearly as high as in the fundus. The SP responses of fundus and longitudinal corpus were purely tonic, similar to acetylcholine (ACh) and histamine (H) responses. In circular corpus, SP induced a combined phasic-tonic response. SP induced in antrum strips purely phasic-rhythmical contractions of low frequency (similar to the H response), whereas ACh induced phasic contractions of high frequency, and in addition an increase of tone in the longitudinal antrum strips. The SP responses of the inner pylorus were not uniform; in some preparations purely tonic contractions were observed, and large phasic fluctuations of low frequency occurred in others. The phasic components of all the responses were completely suppressed by nifedipine (10(-6) mol/l). Tetrodotoxin as well as blockade of adrenoceptors, of ACh and of H receptors had no effect on the SP responses. Comparative studies with preparations from guinea-pig showed that species differences exist in the SP responses of gastric muscle.     

Interstitial cells of Cajal in the gastrointestinal musculature of W mutant mice

Interstitial cells of Cajal (ICC) are important regulatory cells generating electrical rhythmicity and transducing neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and can be examined morphologically using the c-Kit antibody. The c-kit gene is allelic with the murine white-spotting locus W, and the c-kit mutation (W mutation) affects various aspects of hematopoietic cells, germ cells, melanocytes, mast cells, and ICC. Heterozygous W/W( v) mutant mice lack a specific type of ICC and have been used to reveal its function. To search for a new model that lacks a specific type of ICC, we examined homozygous W( v)/W( v) black-eyed-white mice that are viable with anemia. Results showed the principal patterns of ICC deficiency were the same between the W/W( v) and W( v)/W( v) mutants. In the stomach of both mice, intramuscular ICC (ICC-IM) were missing and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced in spite of a normal distribution of deep muscular plexus ICC (ICC-DMP). The cecum also exhibited fewer reduced. ICC-IM in the colon were almost entirely missing, whereas ICC-MY were reduced only in the distal colon. In the small intestine and colon, the number of remaining ICC-MY in W( v)/W( v) mice was greater than that in W/W( v) mice. The enteric nervous system of the two mutant mice showed normal characteristics. From these findings, we conclude that W( v)/W( v) mice represent a new genotype that lacks a part of the ICC in its gastrointestinal musculature.     

Direct effects of motilin on isolated smooth muscle from various regions of the human stomach

The effects of motilin on gastrointestinal muscles show great variations in different organs and different species. For a precise regional differentiation, we recorded the mechanical activity of longitudinal and circular strips from fundus, corpus and antrum and of circular preparations from the inner and outer layer of the pyloric sphincter and from the duodenum (20 human stomachs). Motilin produced excitatory effects on the mechanical activity of the circular muscle strips from all regions of the human stomach including the pylorus. The effects on longitudinal preparations and on duodenal strips were weak. The most striking effect was an increase of phasic activity (amplitude) in circular antrum preparations, which exceeded the acetylcholine- and bombesin-induced activity. In pylorus preparations, a strong stimulation of phasic activity was observed with a transition to tonic activity in the inner layer of the pyloric ring at high motilin concentrations. The motilin-induced activity of the pyloric preparations was greater than the acetylcholine-induced contractions and even exceeded the bombesin-induced responses in the outer pylorus. The responses of the muscle strips of the proximal stomach (fundus and corpus) were weaker and did not exceed the acetylcholine-induced activity. All effects remained unaltered by atropine and tetrodotoxin application. The study confirms that motilin can interact directly with the smooth muscle of human stomach.     

长爪沙鼠胃肠道Cajal间质细胞的形态学

目的 探讨长爪沙鼠胃肠道Cajal间质细胞（ICCs）的形态和分布规律。 方法 采用10只成年长爪沙鼠,体重50~70g,取胃、小肠、结肠制作冷冻切片,结合全层铺片的c-Kit免疫荧光染色。结果 ICCs呈网络状分布于整个胃肠道,不同部位ICCs的分布及形态有所不同。在胃底部,仅见肌内ICCs(ICC-IM）,而在胃体和胃窦部除ICC-IM外,可见肌间ICCs(ICC-MY)分布在肌间神经丛周围;其细胞密度胃底ICC-IM最多,由胃底至胃窦逐渐减少,而ICC-MY由胃体至胃窦逐渐增多。在小肠可见ICC-IM, ICC-MY和深肌层ICCs(ICC-DMP)3个亚群,结肠管壁内也分布有ICC-IM、ICC-MY和黏膜下ICCs(ICC-SM)3个亚群。结论 沙鼠可用于有关ICCs正常形态、结构及功能的研究。     

Electrical events underlying organized myogenic contractions of the guinea pig stomach

The stomach generates a characteristic pattern of coordinated activity whereby rings of contraction regularly start in the corpus and migrate slowly down the stomach to the duodenum. This behaviour persists after isolating the stomach and after blocking nervous activity; hence the response is myogenic, resulting from organized contractions of smooth muscle cells lying in the stomach wall. Each ring of contraction is triggered by a long lasting wave of depolarization, termed a slow wave. Slow waves are now known to be generated by sets of interstitial cells of Cajal (ICC), which intermingle with gastric smooth muscle cells. This article describes some studies which identify the roles played by ICC in the on-going generation of coordinated gastric movements. Intramuscular ICC in the corpus generate slow waves and these provide the dominant pacemaker frequency in the stomach. Corporal slow waves, in turn, activate a network of myenteric ICC, which starts in the antrum and slowly conducts waves of depolarization down the stomach. As these waves pass over bundles of circularly orientated muscle cells, they activate a set of intramuscular ICC which lie in the circular muscle layer: these generate slow waves that rapidly spread radially, so triggering each ring of contraction.     

Structure and organization of interstitial cells of Cajal in the gastrointestinal tract

The morphological features of interstitial cells of Cajal (ICC) in the gastrointestinal (GI) tract are described based on observations of laboratory animals including mice, rats and guinea-pigs, using immunohistochemical staining for Kit and electron microscopy. ICC show a specific distribution, arrangement and cell shape depending on their location within various regions and tissue layers of the GI tract. Hence they are classified into several subtypes. The stomach shows distinct regional variations in the distribution of subtypes of ICC from the cardia to pylorus, whereas the small intestine and colon both seem to retain nearly the same distribution pattern of subtypes of ICC throughout each organ. All subtypes of ICC share common ultrastructural features, such as the presence of numerous mitochondria, abundant intermediate filaments, and formation of gap junctions with the same type of cells and with smooth muscle cells. In addition, depending on their species and anatomical location, some subtypes of ICC show some features typical of smooth muscle cells including a basal lamina, caveolae, subsurface cisterns and dense bodies. ICC are somewhat heterogeneous morphologically. A question is raised on a special relationship between their ultrastructural features and dependency on Kit/stem cell factor system. As the neuromediator function of ICC, reciprocal distribution of ICC and gap junctions in the muscle coat is demonstrated by the comparison of Kit immunoreactive cells and gap junction protein connexin 43 in both small intestine and colon.     

Pathology of interstitial cells of Cajal in relation to inflammation revealed by ultrastructure but not immunohistochemistry

The role of interstitial cells of Cajal associated with Auerbach's plexus (ICC-AP) in the pathophysiology of inflammation-induced abnormalities in gut motor activity is poorly understood. Therefore we applied a well-described model of inflammation (infection by Trichinella spiralis) to the mouse small intestine where the structure and function of ICC-AP are best known. Electron microscopic evaluation revealed that 1 to 3 days after infection, selective and patchy damage to the ICC processes occurred, thereby disrupting contacts between these ICC and smooth muscle cells as well as ICC and nerves, which was associated with disordered electrical activity and abnormal peristalsis. Ten to 15 days after infection, damage to ICC-AP was maximal and now involving the cell body and major processes. Marked synthetic activity and regrowth of their processes occurred from day 3 onward and recovery was completed at day 40 after infection. No changes to the network of ICC-AP were seen with c-Kit immunohistochemistry. From day 1 after infection, macrophages infiltrated the AP area, making close contact including peg-and-socket-like junctions with smooth muscle cells and ICC-AP but up to day 6 after infection without any sign of phagocytosis. By day 6 after infection, lymphocytes entered the musculature forming close contacts with ICC-AP. This was not associated with damage to ICC-AP but with proliferation of rough endoplasmic reticulum. From day 23 onward, immune cells withdrew from the musculature except macrophages, resulting in a markedly increased population of macrophages in the AP area at day 60 after infection.     

Increased eNOS accounts for changes in connexin expression in renal arterioles during diabetes

Previous studies have shown that connexin (Cx) expression is considerably higher in the preglomerular compared to postglomerular vasculature and that these differences are accentuated during diabetes. Since nitric oxide (NO) has been reported to alter Cx expression in endothelial cells and muscle cells and NO bioavailability is altered in diabetes, we hypothesized that NO may be responsible for the changes during diabetes. Cx expression was studied using immunohistochemistry in mice in which eNOS expression was either upregulated (eNOS transgenic) or downregulated (eNOS knockout). Diabetes was induced intraperitoneally with a single dose of alloxan or multiple low doses of streptozotocin. Expression of Cx40 in smooth muscle cells of afferent arterioles was increased, while expression of Cx43 in endothelial cells of efferent arterioles was absent in eNOS transgenic mice, similar to the changes occurring in wild-type mice during diabetes. Expression of Cx40 and Cx43 in eNOS knockout mice was not different from control; however, induction of diabetes in eNOS knockout mice failed to produce any changes in Cx40 or Cx43 in either afferent or efferent arterioles. Immunohistochemistry showed that eNOS expression was increased in the endothelium of renal arterioles in wild-type diabetic and eNOS transgenic mice, but absent from arterioles of eNOS knockout mice. We conclude that changes occurring in Cx expression in afferent and efferent arterioles during diabetes may result from increased eNOS.     

Interstitial cells of Cajal: primary targets of enteric motor innervation

For many years morphologists have noted the close relationship between interstitial cells of Cajal (ICC) and nerve fibers within the tunica muscularis of gastrointestinal (GI) organs. These observations led to speculations about a role for ICC in mediating neural inputs to the GI tract. Immunohistochemical and functional studies demonstrated the presence of receptors for the neurotransmitters utilized by enteric motor neurons, and changes in second messengers in ICC after field stimulation of intrinsic enteric neurons showed that ICC were functionally innervated in GI muscles. Recent double labeling experiments have shown that both excitatory and inhibitory enteric motor neurons are closely associated with ICC in the deep muscular plexus (IC-DMP) of the small intestine and intramuscular ICC (IC-IM) of the proximal and distal GI tract. Enteric motor neurons form synaptic-like structures with IC-IM and IC-DMP. Far fewer close contacts are found between enteric motor neurons and smooth muscle cells. Experiments on W/W(V) mutants that lack IC-IM in the stomach, lower esophageal sphincter, and pylorus have shown that these ICC are critical components of the neuromuscular junction. Cholinergic excitatory and nitrergic inhibitory neurotransmission are severely decreased in tissues lacking IC-IM, yet there is no loss of cholinergic or nitrergic neurons in W/W(V) mutants. These data suggest that either the post-junctional mechanisms responsible for receiving and transducing neurotransmitter signals are specifically expressed by ICC, or that the large extracellular spaces typically between nerve terminals and smooth muscle cells may not allow effective concentrations of neurotransmitters to reach receptors expressed by smooth muscle cells. These findings indicate an important role for certain classes of ICC in enteric neurotransmission and predict that loss of ICC in human motor disturbances may significantly compromise neural regulation of GI motility.     

Neuron density and distribution of NADPH-diaphorase positive neurons in the human stomach

Neuron density and distribution of the NADPH-diaphorase positive neurons were studied in the fundus, corpus and antrum of adult human stomach using cresyl violet staining and NADPH-diaphorase histochemistry. The submucous plexus contained significantly less neurons than the myenteric plexus. Submucous NADPH-d positive neurons were mostly located in ganglia close to the circular muscle layer. Myenteric NADPH-d positive neurons represented 50–60% of the neurons in all the three regions; their density, however, was significantly lower in the fundus. NADPH-d positive fibers formed a rich plexus in the innermost portion of the circular muscle layer of the corpus.     

Spontaneous and inducible ventricular arrhythmias after myocardial infarction in mice.

INTRODUCTION: Remodeling of gap junctions has been implicated in development of ventricular arrhythmias following myocardial infarction (MI) but the specific contribution of reduced electrical coupling is not known. We addressed this question using hearts from mice heterozygous for a connexin43 null allele (Cx43(+/-)). METHODS: To determine whether Cx43-deficient mice exhibit increased spontaneous ventricular arrhythmias in the setting of chronic ischemic heart disease, radiofrequency transmitters were implanted in wild-type and Cx43(+/-) mice 2 days or 9 weeks after left anterior descending coronary artery ligation or sham operations. ECGs were recorded from unanesthetized, unrestrained mice 1 and 10 weeks after MI. Isolated, perfused hearts excised 1 and 10 weeks after MI were subjected to programmed electrical stimulation to induce arrhythmias. RESULTS AND CONCLUSIONS: Hearts with infarcts exhibited more spontaneous and inducible arrhythmias, but there was no significant difference between wild-type and Cx43-deficient mice. Fewer hearts exhibited spontaneous ventricular tachycardia (VT) in vivo than were inducible in vitro, suggesting that structural and functional substrates for inducible VT in isolated hearts may not be sufficient for initiation and maintenance of sustained VT in vivo. Previous studies have shown that Cx43-deficient mice exhibit more VT than wild-type mice during acute regional ischemia. Mice with MI exhibit increased arrhythmias. However, reduced coupling in Cx43-deficient mice does not significantly enhance spontaneous or inducible VT after MI.     

Migrating Motor Complexes do not Require Electrical Slow Waves in the Mouse Small Intestine

We have investigated whether migrating motor complexes (MMCs) are impaired or absent in the small intestine of W/W^v mutant mice, which lack pacemaker interstitial cells of Cajal (ICC) and electrical slow waves. The intracellular electrical and mechanical activities of the small intestines of wild-type (+/+) and W/W^v mutant mice were recorded. Electrical recordings from circular muscle cells confirmed the absence of slow waves in W/W^v mice, whereas slow waves were always recorded from +/+ muscle cells. Spontaneous phasic contractions were recorded from W/W^v muscles in the absence of slow waves, but these events occurred at a lower frequency than in +/+ tissues. MMC activity was recorded consistently from the ileum of +/+ mice, and normal MMCs were also recorded from W/W^v mice. MMCs in both +/+ and W/W^v mice were abolished by tetrodotoxin (1 μ m ), hexamethonium (300 μ m ) or atropine (1 μ m ), suggesting that the neural control mechanisms responsible for MMCs in +/+ mice are intact and are responsible for MMCs in W/W^v mice. Transmural nerve stimulation demonstrated intact inhibitory and excitatory neural regulation of W/W^v intestinal muscles. Prolonged trains of cholinergic motor nerve stimulation failed to activate slow waves in the intestinal muscles of W/W^v mice. Our findings show that the generation and directional propagation of MMC activity in mouse small intestine does not require slow-wave activity or an intact network of myenteric ICC. The generation and propagation of MMCs appear to be an intrinsic capability of the enteric nervous system and are not related to slow waves or the gradient in slow-wave frequency.     

Absence of peristalsis in the ileum of W/W(V) mutant mice that are selectively deficient in myenteric interstitial cells of Cajal.

It is well known that the enteric nervous system plays a key role in the generation of gastrointestinal peristaltic movements. Recently, the networks of interstitial cells of Cajal (ICC) have been found to be essential in the generation of spontaneous gastrointestinal movements. However, the role of ICC in the mechanisms involved in the generation of peristaltic movements is still controversial. The aim of the present study was to reveal how pacemaker myenteric ICC (ICC-MY) and the enteric nervous system contribute to the mechanisms involved in the generation of intestinal peristalsis. We compared spontaneous peristaltic movements of the ileum in wild type (WT) mice with those in W/W(V) mutant mice which are selectively deficient in ICC-MY. Simultaneous recordings were made from both the circular and longitudinal muscle of a 4-cm long segment of ileum under hydrostatic pressure of 0--0.5 cm H(2)O. Mechanical activity and continuous video-images of the ileum were compared between WT and W/W(V) mutant mice under control conditions, in the presence of N-nitro-L-arginine methyl ester (L-NAME) and after tetrodotoxin (TTX). In the WT mouse ileum, peristaltic waves to propagate from the oral to the anal end were frequently observed. The frequency of these peristaltic waves and their associated synchronous longitudinal and circular muscle contractions was increased by L-NAME. The peristaltic waves were abolished by TTX. In the W/W(V) mutant mouse ileum, no peristaltic waves to propagate from the oral to the anal end were observed in control and even after L-NAME, although the local spontaneously generated longitudinal and circular muscle contractions were enhanced by L-NAME. These local contractions were not abolished by TTX. The results presented here suggested that ICC-MY are essential for the generation of spontaneous intestinal peristaltic movements. It is conceivable that ICC-MY may determine the polarity of the excitation of the intestine such that longitudinal and circular muscle contractions propagate from the oral to the anal end of the intestinal segments, although the question of why ICC-MY are necessary for the neural pathways remains unresolved.     

Regional variation in ICC distribution, pacemaking activity and neural responses in the longitudinal muscle of the murine stomach

Intramuscular interstitial cells of Cajal (ICC-IM) play a critical role in enteric neural regulation of the circular muscle layer in the stomach, but no studies have been performed on the longitudinal layer. Kit immunohistochemistry was used to examine ICC-IM in the longitudinal muscle layer of the murine corpus and antrum, and it revealed marked heterogeneity in the distribution of ICC-IM in longitudinal muscles. In the corpus, ICC-IM were found along the greater curvature near the fundus. ICC-IM decreased in density in the circumferential axis toward the lesser curvature and in the longitudinal axis toward the antrum. ICC-IM were absent from the longitudinal layer of the antrum. Double labelling with markers for specific classes of enteric motor neurones revealed that cholinergic and nitrergic motor neurones formed close contacts with ICC-IM in the corpus but not in the antrum. Enteric nerve stimulation evoked prominent cholinergic excitatory and nitrergic inhibitory responses in longitudinal muscles of the corpus, but not in the antrum of wild-type animals. Cholinergic and nitrergic nerves were also present in W/W^V mice, but functional innervation of the longitudinal muscle layer by these nerves in the corpus and antrum were absent. The data show that cholinergic and nitrergic neurotransmission only occurs in the gastric longitudinal layer in regions where ICC-IM are present. In regions, such as the corpus, where ICC-IM are common, robust neural responses are present, but the reduced density of ICC-IM near the lesser curvature and in the distal stomach leads to reduced neural regulation in these gastric regions.     

Muscarinic regulation of pacemaker frequency in murine gastric interstitial cells of Cajal

Peristaltic contractions in the stomach are regulated by the spread of electrical slow waves from the corpus to the pylorus. Gastric slow waves are generated and propagated by the interstitial cells of Cajal (ICC). All regions distal to the dominant pacemaker area in the corpus are capable of generating slow waves, but orderly gastric peristalsis depends upon a frequency gradient in which the corpus pacemaker frequency exceeds the antral frequency. Cholinergic, muscarinic stimulation enhances pacemaker frequency. We investigated this phenomenon using intact murine gastric muscles and cultured ICC. Acetylcholine (ACh) increased the frequency of slow waves in antrum and corpus muscles. The increase was significantly greater in the antrum. ACh and carbachol (CCh) increased the pacemaker currents in cultured ICC. At high doses of CCh, transient pacemaker currents fused into sustained inward currents that persisted for the duration of stimulation. The effects of CCh were blocked by low doses of the M₃ receptor antagonist 1-dimethyl-4-diphenylacetoxypiperidinium. Frequency enhancement by CCh was not affected by forskolin, but the phospholipase C inhibitor U-73122 inhibited both the increase in frequency and the development of tonic inward currents. 2-Aminoethyldiphenyl borate also blocked the chronotropic responses to CCh. Inhibitors of protein kinase C did not block responses to CCh. These studies show that mice are an excellent model for studying mechanisms that regulate gastric slow-wave frequency. CCh, apparently via production of inositol 1,4,5-trisphosphate, accelerates the frequency of pacemaker activity. High concentrations of CCh may block the entrainment of pacemaker currents, resulting in a tonic inward current.     

Cells of origin of vagal motor neurons projecting to different parts of the stomach in the rat: confocal laser scanning and electron microscopic study

Parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) innervate the stomach by way of the gastric and hepatic branches of the vagus nerve. To investigate whether single neurons of the DMV provide collateral innervations to various parts of the stomach, we injected the retrograde tracer Fluoro-Gold (FG) into the cardia and the retrograde tracer cholera toxin subunit b (CTb) into the antrum or the pylorus of the same animal. Both retrogradely FG-labeled and CTb-labeled neurons were found throughout the DMV. Almost all CTb-labeled neurons (97%) were double-labeled with FG after injection of FG into the cardia and CTb into the antrum, while only a few CTb-labeled neurons (11%) were double-labeled with FG after injection of FG into the cardia and CTb into the pylorus. Thus, the cardia and the antrum received collateral projections, but the pylorus received projections mainly from different neurons in the DMV. These results indicate that different neurons in the DMV activate either the cardia or the pyloric sphincter muscles. We also labeled, retrogradely, the neurons projecting to the cardia and the pylorus in the DMV with cholera toxin-conjugated horseradish peroxidase (CT-HRP) to examine their ultrastructural characteristics. Although the neurons projecting to the cardia (21.6×15.0 µm, 248.0 µm² per section) were significantly smaller than the neurons projecting to the pylorus (27.5×15.9 µm, 323.2 µm² per section), their ultrastructural appearances were similar. Both types of neurons were small-to-medium sized, round or oval in shape, and generally had a small amount of cytoplasm containing a few Nissl bodies and a round nucleus. The average number of axosomatic terminals per section was low in the neurons projecting to the cardia (2.3) and the neurons projecting to the pylorus (3.0). Almost all axon terminals contacting these motor neurons contained round synaptic vesicles and made asymmetric synaptic contacts (Grays type I).