Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine

Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 7m. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.     

Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence

The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.     

Subunit b of cholera toxin labels interstitial cells of Cajal in the gut of rat and mouse

Cholera toxin subunit b was found in vivo and in vitro to label interstitial cells of Cajal in the intestine of rat and mouse. Cholera toxin-labelled interstitial cells were present in the subserosa, the myenteric plexus and the deep muscular plexus of mouse small intestine, and the deep muscular plexus only of the rat small intestine. In the large intestine of the mouse, interstitial cells were present in the subserosa and in a plexus associated with the inner surface of the circular muscle, while in the rat they were only present in the latter location. Macrophages, which were present in many of the same locations as interstitial cells, were also labelled by cholera toxin but could be distinguished from interstitial cells by their ability to take-up fluorescein isothiocyanate-labelled dextran. Labelling with subunit b of cholera toxin is a simple way of labelling interstitial cells of Cajal and which is compatible with a range of physiological and histological procedures.     

Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium

Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duodenum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce perinuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular contacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium showing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-Kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells.     

Cytodifferentiation of the interstitial cells of Cajal of mouse colonic circular muscle layer. An EM study from fetal to adult life

Cytodifferentiation of the interstitial cells of Cajal related to the plexus entericus extremus and located on the inner face of the circular muscle layer of the mouse colon was studied in fetuses at term, unfed neonates, suckling and weaning young animals. In fetuses at term, the interstitial cells of Cajal are not found and their precursor cells are not identifiable among the cells present in the submucosal area facing the circular muscle layer, i.e., undifferentiated cells (mainly close to nerve fibers) and fibroblast-like cells (contacting each other and the developing smooth-muscle cells). Cells putatively considered as precursors of interstitial cells of Cajal, fibroblast-like cells rich in mitochondria, are present in unfed neonates (in small numbers) and in suckling animals (in large numbers). Differentiating interstitial cells of Cajal are undoubtedly recognizable during the second week of postnatal life (suckling period). Some of them have mixed fibroblastic- and interstitial-cell features and some have many interstitial-cell features. Both these cell types are already in contact with each other and differentiating and differentiated smooth-muscle cells. The undifferentiated cells, still present in unfed neonates, envelop the nerve fibers and differentiate as Schwann cells during the suckling period. During this period, as the nerve endings of the plexus entericus extremus develop, they immediately contact the differentiating interstitial cells of Cajal. During the weaning period, these cells are quite well differentiated although they do not have the same morphology as in adult animals before 30 days of age. Concomitantly, the nerve endings of the plexus entericus extremus contain an increasing number of synaptic vesicles.     

胚胎小肠Cajal细胞的发育研究

目的研究人胚胎小肠cajal细胞的发育变化规律。方法采用全层铺片结合切片的免疫细胞化学技术。结果Cajal细胞呈酪氨酸激酶受体(Kit)和波形蛋白(vinlentin)免疫反应阳性。在胚胎发育早期,cajal细胞较少,为单层,稀疏分布于肌间神经丛周围,细胞为梭形,可见两个短而小的突起,未见分支;随着胎龄的增加,Cajal细胞数量增多,胞体增大,突起伸长,并出现分支。此时,肌间神经丛周围的Cajal细胞出现两层,其长轴彼此垂直,分别平行于环行肌和纵行肌。与此同时环行肌层内亦可见少许Cajal细胞;出生前,肌间神经丛部位的Cajal细胞接近成熟,两层细胞的突起进一步增多、伸长,彼此间形成与成人相似的完整的细胞网络。此时深肌丛附近亦可见少量Cajal细胞。结论人的小肠Cajal细胞发育有一定的时间顺序,即肌间神经丛周围最先出现,肌内次之,深肌丛较晚,出生前肌间神经丛周围的Cajal细胞已经接近成熟。这种发育演变若发生异常,可能导致某些胃肠动力障碍性疾病。     

Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.     

Innervation of interstitial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon.

The hypothesis was tested, through structural and functional studies, that interstitial cells of Cajal receive and can respond to direct innervation from nerves containing the vasoactive intestinal polypeptide neuromediator. The submucosal network of interstitial cells of Cajal has been postulated to provide pacemaking activity for the circular muscle and to be involved in neurotransmission from nonadrenergic, noncholinergic nerves for which vasoactive intestinal polypeptide is a putative mediator. The distribution of vasoactive intestinal polypeptide and substance P immunoreactive material in nerve profiles of the enteric nervous system of the canine colon was examined. In addition, electrophysiological studies were done on the interstitial cells bordering the submucosal side of the circular muscle layer after they were electrically isolated using heptanol. The vasoactive intestinal polypeptide immunoreactivity, located exclusively in nerve large granular vesicles, was found throughout the enteric nervous system (myenteric plexus, submucous plexus, and circular muscle--submucosa interface). The highest proportion (38% compared with 22-24%) of profiles of large granular vesicles with vasoactive intestinal polypeptide immunoreactivity was found in nerve profiles of the circular muscle--submucosa interface. In contrast, substance P immunoreactivity was found in nerve profiles of myenteric plexus (33% of large granular vesicles were positive) but not associated with submucosal interstitial cell nerve network. The vasoactive intestinal polypeptide hyperpolarized interstitial cells by 9 mV when electrically isolated by 1 mM heptanol and markedly reduced (about 50%) their input membrane resistance. We conclude that the distribution of vasoactive intestinal polypeptide immunoreactivity and its action are consistent with a postulated role of the interstitial cells as a major site of neurally mediated inhibition of colonic pacemaker activity.     

Distribution and ultrastructure of interstitial cells of Cajal in the mouse colon, using antibodies to Kit and Kit W-lacZ mice

The roles of the interstitial cells of Cajal in the stomach and intestine are becoming increasingly clear. Interstitial cells of Cajal in the colon are less well known, however. We studied the development and distribution of the interstitial cells of Cajal in the mouse colon, using the tyrosine kinase receptor Kit as a marker. Sections and whole mounts were studied by confocal microscopy after double immunofluorescence with specific antibodies. The ultrastructure of Kit-expressing cells was examined by electron microcopy in KitW-lacz/+ transgenic mice, which carry the lacz gene inserted in place of the first exon of the Kit gene. In the subserosa, the interstitial cells of Cajal formed a two-dimensional plexus. In the myenteric area, the interstitial cells of Cajal formed a dense plexus that gradually merged with the interstitial cells of Cajal in the outer half of the circular muscle. The inner half of the circular layer was devoid of interstitial cells of Cajal whereas in the submuscular region the interstitial cells of Cajal formed a two-dimensional plexus. Tertiary nerves with various chemical codings closely followed interstitial cell of Cajal processes. By electron microscopy, Kit-expressing cells in the outer parts of the musculature had scattered caveolae, inconspicuous basal lamina and numerous mitochondria, whereas in the submuscular region they had more pronounced myoid features. Kit-expressing cells in the mouse colon are identifiable as interstitial cells of Cajal by their ultrastructure. The interstitial cells of Cajal in the mouse colon mature postnatally. They are organized into a characteristic plexus, close to the nerves with various chemical codings.     

Structural characterization of interstitial cells of Cajal in myenteric plexus and muscle layers of canine colon

We have carried out a detailed ultrastructural study of the interstitial cells near the myenteric plexus of the canine colon and defined the structural characteristics which distinguish them from other resident non-neural cells. We have also examined the interconnections of these interstitial cells with nerves, the longitudinal muscle, and the circular muscle. In addition, we sought connections between interstitial cells of the myenteric plexus and those described earlier at the inner border of the circular muscle in proximal and distal colon. The interstitial cells of the myenteric plexus were structurally distinctive, and made gap junctions with one another and occasionally with smooth muscle. There seemed to be two subsets of these interstitial cells, one associated with the longitudinal muscle and the other with the circular muscle. Cells of both subsets were often close (less than or equal to 20 nm) to nerve profiles. The interstitial cells near the longitudinal muscle layer penetrated slightly into the muscle layer, but those near the circular muscle did not and neither set contacted the other. Moreover, interstitial cells of Cajal located near the myenteric plexus were never observed to contact those at the inner border of circular muscle. The interstitial cells of Cajal at the canine colon myenteric plexus are structurally organized to provide independent pacemaking activities for the longitudinal and adjacent circular muscle. Their dense innervation suggests that they mediate neural modulation of intestinal pacemaker activities. Moreover, they lack direct contacts with the interstitial cell network at the inner border of circular muscle, which is essential for the primary pacemaking activity of circular muscle. The structural organization of interstitial cells in canine colon is consistent with their proposed role in pacemaking activity of the two muscle layers.     

Distribution and morphological characteristics of the interstitial cells of Cajal in the ileocaecal junction of the guinea-pig

The guinea-pig ileocaecal junction including the valve was studied by immunohistochemistry to clarify the organization of the muscle bundles, the enteric nerves and the interstitial cells of Cajal (ICC). This region clearly exhibited characteristic features in the distribution patterns of ICC in a proximal to distal direction: (1) the thickened portion of the terminal ileum immediately adjacent to the ileocecal junction contained many ICC throughout the circular (ICC-CM) and longitudinal (ICC-LM) muscle layers, but ICC were few or absent in the rest of the ileum; (2) the ileal side of the valve contained ICC associated with the deep muscular plexus (ICC-DMP) as in the small intestine, whereas ICC-DMP were absent in the caecal side as in the caecum; (3) the valve contained many ICC-CM and ICC-LM in both the ileal and caecal sides; (4) many ICC associated with the myenteric plexus were observed in both the ileal and caecal sides of the valve, whereas they were only sparsely found in the caecum; (5) ICC were also observed around the submucosal plexus in a confined area of the terminal ileum and the ileocaecal valve. These observations provide morphological evidence that the terminal ileum and ileocaecal valve are specially equipped for their active involvement in the movement of the junctional area.     

Possible involvement of muscularis resident macrophages in impairment of interstitial cells of Cajal and myenteric nerve systems in rat models of TNBS-induced colitis

Resident macrophages are distributed in the network of interstitial cells of Cajal (ICC) and the myenteric nerve within the myenteric plexus. We evaluated changes in chemoattractant protein mRNA expression in macrophages and neutrophils, the ICC, nerve and macrophages in the myenteric plexus of model rats with TNBS-induced colitis. Chemoattractant proteins, MCP-1, GRO, MIP-2 and CINC-2α were upregulated in the colonic muscle layer after inflammation. Leukocyte infiltration and MPO activity were increased in the muscle layer. Electron microscopy indicated an irregular contour of the myenteric ganglia into which numerous macrophages had penetrated. Macrophages were also distributed near the ICC in the inflamed myenteric plexus. Immunohistochemistry showed that the ICC network and myenteric nerve system had disappeared from the inflamed region, whereas the number of resident macrophages was increased. TTX-insensitive, possibly ICC-mediated, rhythmic contractions of circular smooth muscle strips and enteric neuron-mediated TTX-sensitive peristalsis in the whole proximal colon tissue were significantly inhibited in the inflamed colon, indicating that the ICC-myenteric nerve system was dysfunctional in the inflamed muscle layer. Their accumulation around the myenteric nerve plexus and the ICC network suggests that macrophages play an important role in inducing intestinal dysmotility in gut inflammation.     

Development of the enteric nervous system,smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4–14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75^NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2–3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of -smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of -smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.A.S.W. is funded by a PhD studentship awarded to A.J.B. by the Child Health Research Appeal Trust.     

Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the presence of enterochromaffin cells

Summary The formaldehyde-induced fluorescence technique had shown 5-hydroxytryptamine-containing enteric neurons in the intestine of the teleost Platycephalus bassensis, but did not reveal such neurons in the intestine of Tetractenos glaber or Anguilla australis. Re-examination of these animals with 5-hydroxytryptamine immunohistochemistry showed immunoreactive enteric neurons in the intestine of all three teleost species. The 5-hydroxytryptamine-containing enteric neurons showed essentially the same morphology in all species examined: the somata were situated in the myenteric plexus, extending down into the circular muscle layer, but none were found in the submucosa; processes were found in the myenteric plexus, the circular muscle layer and the lamina propria. It was concluded that the neurons may innervate the muscle layers or the mucosal epithelium, but were unlikely to be interneurons. In a range of teleosts, enterochromaffin cells were found in the intestine of only those species in which the formaldehyde technique did not visualize neuronal 5-hydroxytryptamine. Available evidence suggests that, in vertebrates, 5-HT-containing enterochromaffin cells are lacking only where there is an innervation of the gut mucosa by nerve fibres containing high concentrations of 5-HT.     

Ultrastructural characterization of interstitial cells of Cajal in the rat small intestine using control and Ws/Ws mutant rats

Interstitial cells in the myenteric plexus and the deep muscular plexus of the small intestine of the c-kit mutant rats (Ws/Ws) and their normal siblings (+/+) were studied. c-Kit immunoreactivity was detected in two regions corresponding to the myenteric plexus and the deep muscular plexus in the jejunum of +/+ rats, while no immunoreactivity was detected in Ws/Ws rats. Using electron microscopy, two types of gap junction-forming interstitial cells were found in association with the myenteric plexus in +/+ rats: one type characterized by a typical fibroblastic ultrastructure, and the other characterized by numerous mitochondria and less electron-dense cytoplasm. Since the latter were greatly reduced in Ws/Ws rats, it was suggested that these cells correspond to c-kit-expressing cells, i.e. interstitial cells of Cajal in the myenteric plexus region. In contrast, two types of interstitial cells in the region of the deep muscular plexus were observed with no difference between +/+ and Ws/Ws rats. Probable interstitial cells of Cajal in this region were characterized by a basal lamina and numerous caveolae as well as large gap junctions that interconnect with each other and with the smooth muscle cells. We concluded that interstitial cells of Cajal in the rat intestine are heterogeneous in ultrastructure, c-kit dependency in the cell maturation, and functional role.     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.     

Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine

Specific functions of interstitial cells of Cajal (ICC) have been linked to distinct classes that differ by morphology and distribution. In the small intestine, slow wave-generating ICC are located in the myenteric region (ICC-MY), whereas ICC that mediate neuromuscular neurotransmission occur either throughout the circular muscle layer (intramuscular ICC, ICC-IM) or in association with the deep muscular plexus (ICC-DMP). Selective isolation of ICC to characterize specific properties has been difficult. Recently, neurokinin-1 receptors have been detected in murine ICC-DMP and neurons but not in ICC-MY. Here we identified and isolated ICC-DMP/IM by receptor-mediated internalization of fluorescent substance P and Kit immunofluorescence. Specificity of labeling was verified by confocal microscopy. Mouse and human ICC-DMP/IM were detected in suspension by fluorescent microscopy and harvested for RT-PCR with micropipettes. The isolated cells expressed Kit but not markers for neurons, smooth muscle, or antigen-presenting cells. ICC-DMP expressed neurokinin-1 receptor, M(2) and M(3) muscarinic receptors, P2Y(1) and P2Y(4) purinergic receptors, VIP receptor 2, soluble guanylate cyclase-1 subunits, and protein kinase G. L- or T-type Ca(2+) channels were not detected in these cells. ICC-MY and ICC-DMP were simultaneously detected and enumerated by flow cytometry and sorted to purity by fluorescence-activated cell sorting. In summary, functional classes of ICC have distinct molecular identities that can be used to selectively identify and harvest these cells with, for example, receptor-mediated uptake of substance P and Kit immunofluorescence. ICC-DMP express neurotransmitter receptors and signaling intermediate molecules that are consistent with their role in neuromuscular neurotransmission.     

Evidence for 5-hydroxytryptamine in neurones in the gut of the toad,Bufo marinus

Summary The stomach, small intestine and large intestine of the toad, Bufo marinus, were processed for formaldehyde-induced fluorescence histochemistry. After extrinsic denervation or pretreatment with 6-hydroxydopamine to remove catecholamine fluorescence, yellow fluorescence typical of 5-hydroxytryptamine was observed in neurones in the small intestine only. The cell bodies and their processes were confined to the myenteric plexus. Additional pretreatment with 5-hydroxytryptamine enhanced the fluorescence of neurones in the small intestine and revealed yellowfluorescent nerve fibres, but not cell bodies, in the longitudinal and circular muscle layers and myenteric plexus of the large intestine. No fluorescent neurones were observed in the stomach. Following reserpine treatment, which removed native yellow fluorescence in the small intestine, exposure to 5-hydroxytryptophan produced yellow fluorescence in axons in both small and large intestine; exposure to tryptophan never restored fluorescence. The neurotoxin, 5,7-dihydroxytryptamine had no effect on the distribution of yellow-fluorescent neurones in the small and large intestine. No 5-HT-containing mast cells were present in either the small or large intestine. Thin layer chromatography with three different mobile phases showed a 5-hydroxytryptamine-like compound in extracts of mucosa-free small and large intestine but not of stomach.     

Interstitial cells in the primate gastrointestinal tract

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine⁺ mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.