Relationship between interstitial cells of Cajal and enteric motor neurons in the murine proximal colon

Interstitial cells of Cajal (ICC) are interposed between enteric neurons and smooth muscle cells in gastrointestinal (GI) muscles. The specific relationships between these cells in the murine proximal colon were studied with conventional and immunoelectron microscopy and immunohistochemistry. Intramuscular interstitial cells (IC-IM) formed discrete networks within the circular muscle layer of the murine proximal colon. Nerve trunks ran in close association with IC-IM and individual nerve trunks came into close contact with multiple IC-IM. Conventional electron microscopy revealed very close (< or = 20 nm) associations between nerve fibers and IC-IM. Processes of IC-IM also formed close contacts with neighboring smooth muscle cells. At the points of close association between neurons and IC-IM, areas of membrane densification in both pre- and postjunctional cells were present, suggesting specialized contacts or synaptic-like structures. Similar points of contact between neurons and smooth muscle cells were extremely rare. Immunoelectron microscopy demonstrated that IC-IM formed close associations with neurons containing nitric oxide synthase-like immunoreactivity (NOS-LI) or vesicular acetylcholine transporter-like immunoreactivity (vAChT-LI), suggesting innervation by both inhibitory and excitatory motor neurons. IC-IM were also labeled with anti-NOS antibodies. These observations suggest that IC-IM are an integral part of the neuromuscular junction in the colon. These cells may be the primary site of innervation, and neural regulation of the musculature may occur via IC-IM.     

Survival dependency of intramuscular ICC on vagal afferent nerves in the cat esophagus

Interstitial cells of Cajal (ICC) have been proposed as stretch receptors for vagal afferent nerves in the stomach based on immunohistochemical studies. The aim of the present study was to use electron microscopy and the anterograde degeneration technique to investigate ultrastructural features and survival dependency of ICC associated with vagal afferent innervation of the cat esophagus. This is the first report on the ultrastructural characteristics of ICC in the cat esophagus. Intramuscular ICC (ICC-IM) were identified throughout the musculature, whereas ICC in the myenteric plexus were rare. ICC-IM were particularly numerous in septa aligned with smooth muscle bundles. They were in synapse-like contact with nerve varicosities and in gap junction contact with smooth muscle cells. Smooth muscle cells also made contact with ICC through peg and socket junctions. Precision damage through small-volume injection of saline in the center of the nodose ganglion from the lateral side, known to selectively affect sensory nerves, was followed within 24 h by degeneration of a subset of nerve varicosities associated with ICC-IM, as well as degeneration of the associated ICC-IM. Smooth muscle cells were not affected. Nerves of Auerbachs plexus and associated ICC were not affected. In summary, ICC-IM aligning the esophageal muscle bundles form specialized synapse-like contacts with vagal afferent nerves as well as gap junction and peg-and-socket contacts with smooth muscle cells. This is consistent with a role of ICC-IM as stretch receptors associated with vagal afferent nerves; the ICC-vagal nerve interaction appears essential for the survival of the ICC.     

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.     

Three-dimensional observation of the fibroblast-like cells associated with the rat myenteric plexus,with special reference to the interstitial cells of Cajal

Summary An extensive cellular network becomes visible over the myenteric plexus of the rat after removal of the overlying tissues under the scanning electron microscope. The cells are mainly stellate and have many slender processes via which they interconnect. They form a three-dimensional network and are closely associated with the ganglia and nerve bundles, and also extend over the smooth muscle cells. They are considered to correspond to the interstitial cells of Cajal because of their peculiar arrangement and their topography. Transmission electron-microscopic evidence demonstrates that the majority of those cells have features of fibroblasts. Gap junctions and intermediate junctions are observed between these fibroblast-like cells, and also between them and smooth muscle cells. Examination of serial thin sections reveals that single fibroblast-like interstitial cells connect to both circular and longitudinal muscle cells via gap junctions. It is suggested that the network of interstitial cells conducts electrical signals.     

Unique distribution of interstitial cells of Cajal in the feline pylorus

The feline gastrointestinal (GI) tract is an important model for GI physiology but no immunohistochemical assessment of interstitial cells of Cajal (ICC) has been performed because of the lack of suitable antibodies. The aim of the present study was to investigate the various types of ICC and associated nerve structures in the pyloric sphincter region, by using immunohistochemistry and electron microscopy to complement functional studies. In the sphincter, ICC associated with Auerbach’s plexus (ICC-AP) were markedly decreased within a region of 6–8 mm in length, thereby forming an interruption in this network of ICC-AP, which is otherwise continuous from corpus to distal ileum. In contrast, intramuscular ICC (ICC-IM) were abundant within the pylorus, especially at the inner edge of the circular muscle adjacent to the submucosa. Similar distribution patterns of nerves positive for vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS) and substance P (SP) were encountered. Quantification showed a significantly higher number of ICC-IM and the various types of nerves in the pylorus compared with the circular muscle layers in the adjacent antrum and duodenum. Electron-microscopic studies demonstrated that ICC-IM were closely associated with enteric nerves through synapse-like junctions and with smooth muscle cells through gap junctions. Thus, for the first time, immunohistochemical studies have been successful in documenting the unique distribution of ICC in the feline pylorus. A lack of ICC-AP guarantees the distinct properties of antral and duodenal pacemaker activities. ICC-IM are associated with enteric nerves, which are concentrated in the inner portion of the circular muscle layer, being part of a unique innervation pattern of the sphincter. This study was supported by operating grants from the Canadian Institutes of Health Research (to J.D.H. and N.E.D.) and from the Canadian Association of Gastroenterology (to L.W.C.L.).     

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.     

Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells

In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, beta-GA (10 muM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca(2+) current in a dose-dependent manner without affecting the voltage dependence of this current. The IC(50) for Ca(2+) currents was 1.9 muM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca(2+)-activated K(+) currents but decreased net delayed rectifier K(+) currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca(2+) currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K(+) conductances in smooth muscle cells.     

Direct and indirect innervation of smooth muscle cells of rat stomach,with special reference to the interstitial cells of Cajal

Interstitial cells of Cajal in the circular (ICC-CM) and longitudinal (ICC-LM) muscle layer of the rat gastric antrum and their innervation were studied ultrastructurally. Both ICC-CM and ICC-LM are characterized by many mitochondria, rough and smooth endoplasmic reticulum, caveolae, and formation of gap junctions with each other and with muscle cells, though ICC-LM tend to show more variable cytoplasmic features depending on section profiles. Close contacts between nerve terminals and both ICC-CM and ICC-LM are observed. These possible synaptic structures are characterized by: (1) accumulation of synaptic vesicles in nerve varicosities, (2) a narrow gap (about 20 nm) between pre- and postjunctional membranes, (3) lack of a basal lamina between pre- and postjunctional membranes, and (4) the presence of an electron-dense lining on the inner aspect of prejunctional membranes. Almost the same characteristics are observed between the nerve terminals and the muscle cells of both circular and longitudinal muscle layers of the same specimens. Therefore, we conclude that the smooth muscle cells of both circular and longitudinal layers of the rat antrum are directly and indirectly innervated via ICC. Their functional significance is discussed.     

Smooth muscle cells, interstitial cells of Cajal and myenteric plexus interrelationships in the human colon

The plane between longitudinal and circular muscle of human colon, as revealed on examination with light and electron microscopes, has no clear-cut border. Some groups of smooth muscle cells, obliquely oriented and with features similar to both circular and longitudinal ones--the connecting muscle bundles--run from one muscle layer to another. Other groups of smooth muscle cells, possessing their own specific ultrastructural features--the myenteric muscle sheaths--, make up envelopes of variable thickness around some myenteric ganglia and nerve strands, partially or completely embedding them in one or other muscle layer. Non-neuronal, non-muscular cells (interstitial cells of Cajal, covering cells, fibroblast-like and macrophage-like cells) complicate the texture of the myenteric muscle sheaths, creating an intricate, interconnected cellular network inside them, widespread among nerve bundles and smooth muscle cells; however, only interstitial cells have cell-to-cell junctions also with the smooth muscle cells and nerve endings. These data document the existence in this colonic area of two different types of muscle cell arrangements, one of which, the myenteric muscle sheath, only contains putative pacemaker cells.     

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

The network of interstitial cells of Cajal associated with Auerbach’s (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach’s plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach’s plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities. Received: 21 July 1995 / Accepted: 22 April 1998     

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.     

Deficiency of intramuscular ICC increases fundic muscle excitability but does not impede nitrergic innervation

The motility of the gastrointestinal tract is generated by smooth muscle cells and is controlled to a large extent by an intrinsic neural network. A gap of approximately 200 nm usually separates nerve varicosities from smooth muscle cells, which suggests that direct innervation of the smooth muscle by synapses does not occur. Enteric nerves do make synapse-like contact with proposed regulatory cells, the interstitial cells of Cajal (ICC), which in turn may be in gap junction contact with smooth muscle cells. The role played by ICC in enteric innervation is controversial. Experimental evidence has been presented in vitro for the hypothesis that nitrergic inhibitory innervation is strongly reduced in the absence of ICC. However, in vivo data appear to dispute that. The present report provides evidence that explains the discrepancy between in vivo and in vitro data and provides evidence that inhibitory neurotransmitters can reach smooth muscle cells without hindrance when ICC are absent. The fundic musculature shows increased responses to substance P-mediated innervation and shows marked spontaneous activity, which is consistent with increased muscle excitability.     

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.     

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.     

Enteric plexus and interstitial cells of Cajal: interrelationship in the stomach of Podarcis hispanica (Reptilia). An ultrastructural study

The ultrastructure organization of the stomach enteric plexus was examined in the lizard Podarcis hispanica. The ganglions of the myenteric plexus present a low number of nerve cell bodies with a peculiar nucleus, which occasionally establish direct contacts with cells of the circular muscle layer. Glial cells are smaller than the neurones, and their nucleus is very electron-dense. They surround the axons that constitute the fibres of the myenteric plexus. Four main types of axon profile are described in a morphological consideration of the vesicle population. In the interstice of the circular muscle layer we describe two types of interstitial cells that, due to their ultrastructural characteristics, may be equivalent to the interstitial cells of Cajal which have been described in mammalians. These cells shows parallel distribution to the stomach nerve plexuses, establishing close contacts with them through their long cytoplasmic prolongations. By means of small gap-like unions, they contact both each other and the smooth muscle cells near them. We describe a submucous plexus, where neuronal bodies are scattered among bundles of nervous fibres, some of which are myelinated. A mucous plexus with isolated neurones is located in the lamina propria. Axonal varicosities containing vesicles contact with the cells of the mucous. Interconnected interstitial cells may also be found in this plexus.     

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.     

Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.     

Morphology of the canine pyloric sphincter in relation to function

The ultrastructure and immunocytochemistry of the canine distal pyloric muscle loop, the pyloric sphincter, were studied. Cells in this muscle were connected by gap junctions, fewer than in the antrum or corpus. The sphincter had a dense innervation and a sparse population of interstitial cells of Cajal. Most such cells were of the circular muscle type but a few were of the type in the myenteric plexus. Nerves were sometimes associated with interstitial cell profiles, but most nerves were neither close to nor associated with interstitial cells nor close to smooth muscle cells. Nerve profiles were characterized by an unusually high proportion of varicosities with a majority or a high proportion of large granular vesicles. Many of these were shown to contain material immunoreactive for vasoactive intestinal polypeptide (VIP) and some had substance P (SP) immunoreactive material. All were presumed to be peptidergic. VIP was present in a higher concentration in this muscle than in adjacent antral or duodenal circular muscle. Interstitial cells of Cajal made gap junctions to smooth muscle and to one another and might provide myogenic pacemaking activity for this muscle, but there was no evidence of a close or special relationship between nerves with VIP or SP and these cells. The absence of close relationships between nerves and either interstitial cells or smooth muscle cells leaves unanswered questions about the structural basis for previous observations of discrete excitatory responses or pyloric sphincter to single stimuli or nerves up to one per second. In conclusion, the structural observations suggest that this muscle has special neural and myogenic control systems and that interstitial cells may function to control myogenic activity of this muscle but not to mediate neural signals.     

Identification of interstitial cells in canine proximal colon using NADH diaphorase histochemistry

In gastrointestinal muscles special cells, referred to as interstitial cells, may be involved in pacemaking and transduction of inputs from the enteric nervous system. We have used a modification of the NADH diaphorase method to characterize the distribution of interstitial cells in the muscularis externa of the canine colon. The staining product of the NADH diaphorase reaction is useful because it allows light and electron microscopic studies to be performed with the same marker. Therefore rigorous identification of the cells observed at the light microscopic level could be made by electron microscopy. We were able to lable at least three classes of interstitial cells: (1) at the submucosal surface of the circular muscle layer; (2) within the thickness of the circular and longitudinal muscle layers; and (3) in the region of the myenteric plexus. This technique also labeled cell bodies and initial segments of processes of Dogiel type II neurones in enteric ganglia. Nerve fibres within the muscle layers did not exhibit NADH diaphorase activity. This study has identified the interstitial cells within the circular and longitudinal muscle layers and shows the arrangement of these cells in a three-dimensional network.