A commentary on the morphological identification of interstitial cells of Cajal in the gut

Journal of the Autonomic Nervous System, 37 (1992) 75-88 75 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00

JANS 01236

Review

A commentary on the morphological identification of interstitial cells of Cajal in the gut

J ames Chr i s t ensen

Department of lnternal Medicine, University of Iowa, Iowa City, Iowa, U.S.A.

Introduction

How do nerve cells relate to other ceils? Do nerve cells fuse with one another and with the cells of the tissues they supply, or do they remain separate? Some still remember that old contro- versy.

The nature of the contact of nerve cells with other cells remained an open question in the peripheral autonomic nervous system long after it was closed elsewhere. The question centered especially upon the enteric innervation and upon a population of associated small cells, the interstitial ce.lls of Cajal. They were the focus of intense interest within a coterie of neuroanatomists for over a half-century after their first description. They long remained enigmatic but the recent revival of an old idea as to their function has prompted a re-examination that may resolve some of the old questions about these ceils.

Neither the microscopes nor the histologic methods of the early days made it easy to trace the processes of the enteric nervous system but it was clear that in many places these nerve processes encounter a population of fibroblast-like cells before they reached the effector ceils to which the nerve fibers are directed. The great Spanish neuroanatomist, Santiago Ramon y Ca- jal, was not the first to see these cells but he was

Correspondence: J. Christensen, Room 4548 JCP, Department of Internal Medicine, University of Iowa Hospital, Iowa City, IA 52242, U.S.A.

the first to make them widely known [7,8], and that justifies the eponym. Cajal's original description is the reference point for work on these cells and so it is useful to summarize his original description:

(i) He saw the cells in preparations stained with silver chromate and with methylene blue.

(ii) He found the cells between the acini of salivary glands, in the connective tissue in the pancreas, between the glands of Lieberkuhn, in the intestinal villi, on the internal surface of the circular muscle layer of the small intestine and within the meshes of Auerbach's plexus.

(iii) He described the cells as smaller than nerve cells, variable in shape (fusiform, triangular and stellate), poor in cytoplasmic mass and giving off several varicose processes which extended for long distances and branched at right angles.

(iv) He suggested that the processes anasto- mose to form a tight web, reserving the possibility that they only appear anastomotic because of frequent crossings.

(v) He described the smallest or terminal branches as ending in a close relationship to smooth muscle fibers or close to gland cells.

(vi) He emphasized that these cells lie close to but remain separate from postganglionic nerve fibers.

Various investigators developed the concept of a reticulum of fibers or cell processes which en- velop the cells of innervated tissue [6,31,39,40,44, 49,52]. This came to be called variously the 'autonomic ground plexus', the 'sympathetic ground plexus', and the ' terminal reticulum'. It was con-

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ceived as a network that allowed the integration of function over relatively great distances. Some considered it to be a free-standing network, while others thought that it was made up of processes from postganglionic nerve cells with a contribution from the interstitial cells of Cajal (hereafter to be called 'interstitial cells') which were known to lie in the same region. The uncertainties arose from the fact that both nerves and interstitial cells stain with methylene blue, and so the reticu- lure of fine processes could not easily be distinguished as arising from the one or the other source. Indeed, some [34] suggest that Cajal himself presented drawings of interstitial cells which, in fact, depicted a chimera of interstitial cells and axons.

The voluminous literature on interstitial cells centered on the myenteric plexus of the small intestine of rodents and upon the regulation of intestinal motility. But many doubts persisted about the interstitial cells so that they remained lodged in the realm of the uncertain for a long time. Standard teaching in anatomy gave them only passing mention.

Agreement was reached as to the existence of the interstitial cells but there was little agreement on other questions, especially the following:

(i) Are the interstitial cells a single clearly-defined set of cells?

(ii) Are interstitial cells universally distributed with the terminal axonal networks of enteric nerves?

(iii) Do interstitial cells have a synaptic relationship with nerves and with effector cells?

(iv) Are interstitial cells intercalated between nerve and muscle?

(v) Are interstitial cells neural in nature, as Cajal believed?

(vi) 'Do interstitial cells establish the rhythmicity of contractions in the gut?

Cajal implied that the interstitial cells are involved in the communication between nerves and effector cells. The modern theory of transmission in autonomic nerves, developed since Cajal's time, does not require any elements other than nerve terminals and the effector tissues which they supply. This may account in part for the relative neglect of the interstitial cells.

A modern revival in interest in the interstitial cells was prompted by the recent revival [52] of an old suggestion [53,54,56] that they account for the rhythmicity of contractions in the intestine. The suggestion was first made before the physio- logical basis of rhythmicity was clear. We now know that the rhythmicity of contractions arises from the existence of pacemaking electrical signals, called slow waves, which are detectable in the walls of the gut in the distal part of the stomach, the small intestine and the colon. Slow waves are not detected in those regions where rhythmicity of contractions is absent, the esophagus, the proximal stomach and the gallbladder.

From the time of the first observations of the pacemaking electrical slow waves, the question of their origin was paramount. The fact that electrodes detected them in specimens of gut wall in vitro excluded the central nervous system and the extrinsic innervation of the gut as sources. Within the gut wall itself only two sources were considered, the intrinsic nerves and the smooth muscle. Since manipulations that should alter nerve function failed to affect the pacemaking slow waves, it was concluded that the smooth muscle itself produced the signals. Furthermore, the slow waves were recorded readily from the smooth muscle itself with both intracellular and extracellular electrodes, even from specimens so small that they could reasonably be assumed to contain no nerve cell bodies.

Slow waves were found to arise at certain planes in the gut wall. The longitudinal muscle layer seemed to be the source in the intestine, while the circular muscle layer seemed to be responsible in the colon. Most recently, slow wave origination has been postulated to occur at more restricted planes. The best evidence comes from the colon where the source is at the submucosal surface of the circular muscle layer. The recent observation that interstitial cells are concentrated at this plane is consistent with the new idea that interstitial cells generate slow waves [11,20,38,47].

The six questions listed above arose in the era before electron microscopy. The ultrastructural description of interstitial cells began with the 1958 paper of Richardson [44] in which he said, as one of his aims: 'Secondly, further information

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is needed on the nature of the interstitial cells; whether they possess an ultrastructure confirming their classification either as neural or connective tissue elements. Do these ceils, in fact, forr/l part of a conducting plasmodium interposed between the nerve fibers and the effector ceils'? Richard- son posed this aim as one of his purposes in pursuing an ultrastructural description of the myenteric plexus of the rabbit intestine. Subse- quent electron microscopic studies now provide a considerable literature on the subject.

A morphological study to correlate electron microscopy with light microscopy requires a means to assure that the same structure is being examined in both cases. The intestinal wall contains many kinds of small polymorphic ceils. Lacking a distinctive marker for the interstitial cells, one visible with both light and electron microscopy, the microscopist must depend upon a set of predefined features, such as the plane of concentration, the adjacent kinds of ceils and cell dimensions, to assure himself that the cell he identifies with the electron microscope is the same that he identified with the light microscope.

Interstitial cells in the smooth muscle esophagus

The first report was from an electron microscopic study by Faussone-Pellegrini and her col- leagues [26]. They examined fragments removed from the human esophagus at 5 cm above the lower esophageal sphincter, at the level of the sphincter and 4 cm below it. They saw ceils within the muscle substance which were notably different from myocytes. These ceils, which they called interstitial cells, had an elongated body, many thin branches, an oval and indented nucleus, and pinocytotic vesicles (caveolae) along the cell membrane. The cytoplasm contained variable amounts of mitochondria, smooth endoplasmic reticulum, and Golgi membranes. Bundles of thin filaments at the periphery of the cytoplasm attached to electron-dense areas of the cell membrane. A discontinuous basal lamina surrounded the cells and they made close contacts with one another and with myocytes. They were often adja-

cent to vesicle-filled nerve fibers to which close approaches were made. The authors viewed them as specialized smooth muscle cells.

Seven years later Daniel and Posey-Daniel [17], working in apparent ignorance of the earlier pub- lication, reported an electron microscopic study in the esophagus of the American opossum, an animal possessing a distal esophagus composed of smooth muscle. In the circular muscle layer taken from the lower esophageal sphincter and from the esophageal body 1 cm above the sphincter, they found ceils, which they called interstitial cells, with an oval nucleus and many thin cytoplasmic extensions. The cytoplasm contained many free and attached ribosomes, small mitochondria, Golgi membranes and endoplasmic reticulum. There were a few structures that resembled microtubules or neurofilaments and there were numerous membrane caveolae. The cells formed gap junctions with one another and with muscle cells and they were commonly close to the profiles of nerve processes.

The only published light microscopic description of these cells [10] came from a study that was intended to visualize the total innervation of the esophagus with the zinc iodide-osmic acid stain and with intravital methylene blue. The circular layer of the smooth muscle segment contained numerous non-muscular bipolar cells with oval nuclei which were darkly stained by both techniques. The other layers of smooth muscle and the striated musculature of the proximal esophagus were devoid of such cells. These bipolar ceils had relatively unstained nuclei and a sparse cytoplasm which gave off broad flat processes that branched to form fine beaded extensions extend- ing along the muscle bundles. The bipolar cells were aligned in rows parallel to the muscle ceils with the tips of their processes intermingled. Varicose axons ran along the rows of the bipolar cells to make contact with other bipolar cell bodies and processes over many cell lengths. They were more numerous (8700-8800 cel ls /cm 2) at the lower esophageal sphincter than at higher levels. The cells themselves had a mean area of 148 izm 2 (excluding the fine processes) and their mean nuclear area was 76 /.Lm 2. They were considered to be a class of interstitial ceils.

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These three principal reports are supple- mented by six others. Daniel et al. [15] gave a brief electron microscopic description of these cells in the lower esophageal sphincter of the opossum in a preliminary report. Daniel and Posey-Daniel [16] described them again in the opossum by electronmicroscopy in a study designed to compare them to nerves in respect to the damage done by scorpion venom: the interstitial ceils were found to be relatively resistant to the venom, as judged ultrastructurally. Daniel et al. [18] observed, again in an ultrastructural study, that the interstitial cells in the circular muscle layer of the opossum esophagus are destroyed after exposure to 160 mM KCi for 1-2 h. That study was meant to assess the damage that occurs in muscle strips in the sucrose gap apparatus, a device for electrophysiological studies. Berezin et al. [3] observed that the axons investing interstitial cells in the circular muscle layer of the most distal part of the canine esophagus contained large granular vesicles which are immunoreactive to vasoactive intestinal polypeptide. Allescher et al. [1] described the interstitial cells of the lower esophageal sphincter of the dog in an ultrastructural study designed to establish a basis for the use of that tissue as an experimental model for the study of sphincter function. And Wong et al. [58] described cells in the monkey (Macaca fascicularis) which were similar except that there was a paucity of smooth endoplasmic reticulum, caveolae, and filaments. These authors saw some changes in ultrastructure after vagotomy and suggested that the interstitial ceils are vagally innervated.

Interstitial cells in the stomach

The electrical slow waves which control rhythmicity of contractions have been closely studied in the mammalian stomach, especially in the dog, but the gastric interstitial cells have not.

In fact, the first modern description of gastric interstitial cells came from a memorably exotic

species (from the point of view of gastric physiol- ogists), the love-bird, in which Imaizumi and Hama [32] examined the gizzard with the electron microscope. In all three muscle layers of the gizzard they saw interstitial cells lying among the muscle cells. They filled the spaces between muscle cells giving off many slender processes. A basal lamina surrounded these cells and they lay very close to nerves. Interstitial cells formed gap junctions with smooth muscle cells and with one another. The cells had a central, round and smooth nucleus. A scanty perinuclear cytoplasm contained granular endoplasmic reticulum, free ribosomes, vacuoles, mitochondria, glycogen, pinocytotic vesicles and a small number of filaments. Interstitial cells of similar appearance were subsequently identified in the chicken gizzard by Gabella [30].

The first study of the mammalian stomach by Cook and Burnstock [12,13] was part of an ultrastructural study of the entire myenteric plexus in the guinea pig stomach, ileum and colon. The authors did not distinguish between organs in this study. Two kinds of cells considered to be interstitial cells were found in the myenteric plexus. One had an elongated nucleus and a sparse perinuclear cytoplasm and gave off attenuated processes. The cytoplasm contained an extensive and swollen rough endoplasmic reticulum containing a granular matrix, and it contained many ribosomes, microtubules, lysosomes and multivesicu- lar bodies, and a few lipid droplets. Large and elongated mitochondria were numerous. No mention was made of caveolae, pinocytotic vesicles or a basal lamina. The other type of interstitial cell was similar except that the cell body was more rounded, and it contained large numbers of glycogen granules.

Another early report in mammals by Daniel et al. [19] came from an electron microscopic study of the circular muscle layer of the canine gastric body. The cells had a lobulated nucleus with condensed chromatin and a cytoplasm that con~ rained numerous ribosomes, mitochondria and endoplasmic reticulum membranes. There were many cytoplasmic extensions or processes. The cells formed gap junctions with muscle cells and with one another. Nerve profiles did not make

close contact with the cells. These interstitial cells occurred with an estimated ratio to muscle cells of 1 : 900.

Daniel et al. [14] gave a further brief description of gastric interstitial ceils in an ultrastructural study of the pylorus in the dog. Here they described two kinds of interstitial cells, one being more fibroblast-like than the other in having fewer caveolae and more granules. The cell which they considered to be the more characteristic interstitial cell contained many mitochondria, filaments, granules, caveolae and endoplasmic reticulum. The basal lamina was not mentioned. Neither type of cell was closely associated with nerves. The interstitial ceils were described as sparse.

A more comprehensive report on the mammalian stomach is that of Faussone-Pellegrini et al. [27]. They examined the ultrastructure of the muscle coats of the human stomach in surgically- resected specimens from fundus, corpus and antrum. Interstitial cells were concentrated in different planes in the three regions. They were sparse in the fundus where they lay only within the circular muscle layer. They were more numerous in the corpus, where they were concentrated within the circular muscle layer and on the surface of that layer facing the intermuscular space. They were most numerous in the antrum where they lay within the circular muscle layer, on the surface of the circular muscle layer facing the intermuscular space, and on the submucosal surface of the circular muscle layer.

The interstitial cells of the intermuscular space were long fusiform ceils with many lateral branches, an oval clear nucleus, a large Golgi apparatus, many mitochondria, a small rough endoplasmic reticulum, an abundant smooth endoplasmic reticulum and both 6-7 nm and 4-5 nm filaments. A few caveolae lay along the membrane. Their many processes extended at right angles to the cell body and branched to contact muscle cells and processes of other interstitial cells. Gap junctions were infrequent. Nerves always lay near these cells in close but not intimate contact. The interstitial cells were partially covered by a basal lamina. A thicker amorphous (or faintly fibrillar) material formed an incomplete capsule about these cells, linking the cells to the

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adjacent muscle. Also, elastic fibers linked interstitial cells to nerve and muscle cells.

The interstitial cells within the circular muscle layer were somewhat different in the three regions of the stomach. In the fundus, they were few (about 2.2% of all cells), they were compara- tively poor in processes and in cytoplasmic filaments, the amorphousff ibr i l la r capsule was sparse and the basal lamina was incomplete. In the corpus, they were more frequent (about 8.3% of all cells) and arranged in a 3-dimensional network. These cells were elongated and highly branched. The basal lamina was absent and the amorphous/f ibr i l lar capsule was sparse. Cyto- plasmic filaments were abundant. Contacts to muscle and nerve profiles were more numerous. In the antrum, the cells were much like those of the corpus, the chief difference being a concentration of cells on the submucosal surface which were connected, through their processes, to the interstitial cells within the circular muscle substance.

Interstitial cells in the small intestine

The first electron microscopic study of interstitial cells in the small intestine is that of Richard- son [44]. He saw interstitial cells especially well in the connective tissue spaces on each side of the intermuscular space adjacent to the muscle coats. The irregular cells gave off branching processes. The cytoplasm contained vacuoles, endoplasmic reticulum and mitochondria. A basement membrane was lacking. The processes overlapped and intermingled, and they came into close proximity to bundles of nerve processes. Interstitial cells also extended into the muscle layers along with axons, but the axons reached further than the interstitial cells. Richardson was convinced that interstitial cells are not Schwann cells and he believed they were different from fibroblasts.

Taxi [50] performed a similar study in the mouse intestine. He described two sorts of interstitial-type cells (which he distinguished from Schwann cells), and he showed with trypan red that one of them was a histiocyte, leaving the

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other as a ' true' interstitial cell. The latter cells contained fewer ribosomes than the histiocytes, and they possessed a well-developed endoplasmic reticulum, mitochondria and large vacuoles which Taxi considered characteristic but probably partly artifactual.

Rogers and Burnstock [45] made a similar study in the toad small intestine. By light microscopy they saw small stellate cells with anastomosing processes in the meshes of the myenteric plexus. They considered them to be interstitial cells. At electron microscopy these stellate cells resembled those described above by Richardson [44] and by Taxi [50]. The cells, often closely related to nerves, sent processes into the muscle layers (mainly the circular layer) and whole cells were occasionally found in the circular layer. They had an oval, sometimes indented nucleus. The cytoplasm contained numerous agranular vesicles, tubules, sparse mitochondria, lysosomes and vacuoles. There was no basal lamina. The authors called them 'connective tissue cells', believing that they encompassed several cell types including fibroblasts and histiocytes.

Gabella [28,29] described interstitial cells in the guinea pig ileum. They were elongated along small nerve bundles and they partly surrounded ganglia and strands of the myenteric plexus. They sent processes into the circular muscle layer but not the longitudinal. An oval lobulated nucleus with chromatin aggregated along the nuclear envelope was surrounded by a sparse cytoplasm in which mitochondria and a smooth endoplasmic reticulum were conspicuous. Centrioles, rough endoplasmic reticulum and ribosomes were less prominent, and there were occasional large dense-cored vesicles and microtubules. The cytoplasmic processes contained many small (4-8 nm) filaments and a few microtubules. A basal lamina was absent. Processes made close contact with both nerve profiles and muscle profiles. Other similar cells, identified as fibroblasts, contained mainly dilated sacs of rough endoplasmic reticulum with rare filaments.

Yamamoto [59] described interstitial ceils in the small intestine of the bat and mouse by electron microscopy. The cells had a large indented and oval nucleus, a conspicuous nucleo-

[us, a thin perinuclear cytoplasm and many processes. The cytoplasm contained free ribosomes, rough endoplasmic reticulum and dense bodies. There were many invaginations of the cell membrane. The processes contained many smooth vesicles. The basal lamina was only occasionally complete. Some other cells were distinguished by abundant marginal smooth vesicles, abundant free ribosomes and abundant mitochondria. These cells were considered to be of a different sort. The interstitial cells made close contacts with both nerve and muscle. Yamamoto considered them to be immature muscle cells.

Taylor et al. [51] described three types of connective tissue cells in the intestinal intermuscular space in the cat, considering the first two as interstitial cells and the third as a fibrocyte. The first type was elongated with long tapering processes and a large nucleus. Its cytoplasm contained many organelles, mitochondria, endoplasmic reticulum, vesicles, ribosomes and filaments. The other types had shorter fusiform processes and different proportions of organelles. The three types of cell were not always readily distinguished because of intermediate forms, which led the authors to propose that they represented either stages of differentiation or different functional states in a single type of cell. They proposed that these cells (or this cell) is responsible for electrical coupling between the two muscle layers because the processes made close approaches to muscle cells.

Vajda and Feher [55] described in the cat intestine stellate cells with round or oval nuclei within the circular muscle layer. Their long branching processes ramified among the muscle cells. The cytoplasm contained numerous mitochondria, abundant rough endoplasmic reticulum, many free ribosomes and a medium Golgi apparatus. Many granular or secretory vesicles (100-300 nm diameter) contained a dense and finely granular material. There was no basal lamina. The processes made close contacts with muscle and nerve profiles.

A broad picture of interstitial cells in the intestine was provided by Thuneberg's monograph [52] which included a complete survey of previous work, presented the results of his own extensive

light- and electron-microscopic studies in the mouse intestine, and advanced his support for the idea that these cells are pacemakers for intestinal contractions. He distinguished four classes of interstitial cells on the basis of location. Type I was that associated with the myenteric plexus, type II was subserosal, type III was associated with the plexus muscularis profundus (an axonal plexus between the outer thicker component of the circular muscle layer and the inner thinner part of that muscle layer), and type IV cells lay within the substance of the outer thicker part of the circular muscle layer. He distinguished these four types on ultrastructural grounds. Type I had caveolae, abundant smooth endoplasmic reticulum, filaments (both intermediate and thin), a sparse rough endoplasmic reticulum, numerous large mitochondria and no 'true basal lamina'. Types II and IV were more fibroblast-like cells. Type III ceils were like type I cells except that they had more prominent rough endoplasmic reticulum, fewer mitochondria, no filaments and a distinct basal lamina. They formed gap junctions with circular muscle cells and with one another. This description of type III cells was later amplified by the same group [46]. Further studies from Thuneberg's laboratory distinguished the interstitial cells from the macrophage-like cells of the myenteric plexus in the mouse intestine [41,42] and emphasized the close relationship between the interstitial cells and the macrophage-like ceils of the myenteric plexus.

Faussone-Pellegrini and Cortesini [24] examined the human small intestine. They described interstitial cells of Cajal at the level of the deep muscular plexus and within the inner lamina of the circular muscle layer. These were elongated cells bearing many processes. The ovoid nucleus had dispersed chromatin and one or two nucleoli. The cytoplasm contained mitochondria, smooth endoplasmic reticulum, dense bodies and many thick and thin filaments. Glycogen particles, ribosomes and rough endoplasmic reticulum were rare. The membrane contained caveolae and there was an incomplete basal lamina. The cells made frequent contact with one another and they infre- quently contacted adjacent muscle cells. They were closely related to nerve profiles. The au-

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thors considered these to be like Thuneberg's type III interstitial cells.

These same authors described another set of interstitial cells in man which they considered to be the same as Thuneberg's type I cells. These lay alongside the myenteric plexus. Their cytoplasm prominently contained 8-10 nm and 5 nm filaments, smooth endoplasmic reticulum and mitochondria. The Golgi apparatus, ribosomes, glycogen particles and rough endoplasmic reticulum were less prominent. There was no basal lamina and caveolae were rare. Contacts with muscle were uncommon and there were no gap junctions. The authors proposed that intestinal interstitial cells in man are uniquely different from those of other species in their ultrastruc- rural features, having more thin filaments, less smooth endoplasmic reticulum and a paucity of gap junctions with muscle.

Faussone-Pellegrini [21,22] examined the his- togenesis of type III interstitial ceils in the mouse intestine. She concluded that they develop from blast precursors within the myenteric plexus in the fetal animals but she could not distinguish whether these were ectodermal or mesenchymal blast ceils.

Scanning electronmicroscopy has been used to examine interstitial cells. In the guinea pig small intestine Baluk and Gabella [2] , observed 'fibroblast-like cells' to be concentrated at several levels: in the serosa, around elements of the myenteric plexus, in the circular muscle layer, on the submucosal surface of the circular muscle layer and in the submucosa. They assumed that these cells included interstitial cells but they pointed out the absence of a certain means to distinguish fibroblasts from interstitial cells by this method. They proposed that all fibroblast-like elements in the gut wall may be a single population, whose morphological features differ from one level to another because of different mi- croenvironments.

Komuro [37] scanned the myenteric plexus of the rat small intestine and saw 'fibroblast-like' cells. They were stellate, their extensive processes interconnecting to form a 3-dimensional network. They were closely associated with both neural and muscular tissues. There were two types of

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cells. One had a flatter cell body and more processes, and it made up most of the cells. The other was more round with fewer processes. Ko- muro distinguished three kinds of cells by transmission electron microscopy. The first type was like a fibroblast. It had long processes, an elongated smooth nucleus with peripherally-condensed chromatin and a thin perinuclear cytoplasm. The cytoplasm contained conspicuous dilated rough endoplasmic reticulum, a conspicuous Golgi apparatus, mitochondria and free ribosomes. Microtubules, filaments and smooth endoplasmic reticulum were sparse. There was no basal lamina. These cells formed gap junctions with one another and with muscle cells. The second type had an oval nucleus, less chromatin than the first type and long processes. Its cytoplasm contained rough endoplasmic reticulum in flattened form, smooth endoplasmic reticulum, Golgi complexes and no basal lamina. The third type had an oval nucleus, large granules or vacuoles, coated vesicles, some free ribosomes, sparse rough endoplasmic reticulum, inconspicuous Golgi complexes, few mitochondria and no basal lamina. Komuro tentatively identified the third type as a macrophage.

Jessen and Thuneberg [33], in a scanning electron microscopic study of the myenteric plexus of the guinea pig, specified the criteria that distinguish interstitial cells from fibroblasts and macrophages: (i) the cell body is ovoid, triangular, or more irregular, with 2-5 primary processes; (ii) the perinuclear cytoplasm is sparse; (iii) the cell surface is smooth; (iv) processes branch dichoto- mously, with long unbranched segments which often run in bundles parallel to others of the same kind; (v) processes are rounded in profile; and (vi) cell bodies and processes are intimately associated with nerves of the tertiary component of the myenteric plexus. The authors identified macrophage-like cells from their surface irregu- larity produced by short, veil-like, irregularly shaped and folded processes. They identified fibroblasts by exclusion of interstitial cells and macrophages, being left with a few unclassified smooth-surfaced cells which have more perinuclear cytoplasm than interstitial cells and broader and flatter processes.

Immunocytochemical staining might be ex- pected to prove useful to distinguish interstitial cells from other kinds of similarly-shaped cells. Kobayashi et al. [35] applied antibody staining for the S-100 protein to the guinea pig jejunum. They tentatively concluded that interstitial cells contain the S-100 protein. This has not been confirmed.

Prosser et al. [43] examined interstitial cells of the myenteric plexus in the rat intestine with antibodies to a battery of antigens which included neurofilament protein, glial fibrillary acidic protein, vimentin, desmin, neuron specific enolase, substance P and vasoactive intestinal polypeptide. The interstitial cells stained positively only for neuron-specific enolase.

Interstitial cells in the colon

Stach was the first to describe interstitial cells in the colon [48] as a part of a description of a hitherto-undescribed plexus, the plexus entericus (submucosus) extremus (externus) (the Latin name used for this plexus has varied in its literature). Stach used silver impregnation, the zinc iodide-osmic acid reaction and electron microscopy to examine the colon of dog, cat, guinea pig and rat. He found a dense bed of axons innervating a layer of highly branched flat cells lying on the submucosal surface of the circular muscle layer. He considered these cells to be interstitial cells on the grounds of their light microscopic appearance and ultrastructure. U1- trastructurally, they were not homogeneous but they generally had a prominent smooth endoplasmic reticulum, a basal lamina, and pinocytotic activity.

Interstitial cells of the myenteric plexus of the rabbit colon were examined by Komuro [36] in a scanning and transmission electron microscopic study. The stellate or fusiform cells lay over ganglia and nerve bundles and between muscle cells. Resembling fibroblasts, the cells (6-12 ~m in diameter) gave off many processes up to 20 ~m long which contacted one another to form a network. They also contacted nerve and muscle cells. They possessed a prominent Golgi apparatus, a granular endoplasmic reticulum which was often

engorged, mitochondria, lysosomes, fat droplets and large vacuoles. Microtubules and thin filaments were indistinct. Nuclei were elongated and indented. There was no basal lamina. All fibroblast-like cells were of this one kind. The author considered these ceils to be of the nature of fibroblasts, differing mainly in their formation of a network and in having many membrane contacts.

A light microscopic description of colonic interstitial cells in opossum, cat, rat, dog, rabbit, guinea pig, ferret and man was given in 1987 [9]. Interstitial cells in both the myenteric plexus and in the plexus entericus extremus were found to be stained by the zinc iodide-osmic acid and NADH-diaphorase stains, but not by silver. They were polymorphic cells with round to oval clear nuclei. Flat branches radiated in all directions from the cytoplasm and intersected with one another to form a mat. They were closely related to but separated from the plexus of axons at the submucosal surface of the circular muscle layer, lying closer to" the muscle surface. In the intermuscular space, they lay close to both muscle surfaces. Although axons were demonstrated in the longitudinal and circular muscle layers, interstitial cells were absent from these layers.

Faussone-Pellegrini [23] used transmission electron microscopy to examine the cytodifferentiation of interstitial cells of the plexus entericus extremus in the mouse. She examined fetuses at term, unfed neonates, and suckling and weanling animals. In the fetuses at term, there were no interstitial cells in the plexus. Their precursors could not be specifically identified among the undifferentiated and fibroblast-like cells. In more mature animals, fibroblast-like cells which were rich in mitochondria appeared. At 2 weeks post- partum, interstitial cells were clearly recognized but some of them retained an immature appearance. Cells with the morphology of the interstitial cells of the mature animal appeared at 30 days of age. The author concluded that interstitial cells are probably mesenchymal in origin. She suggested either that fibroblasts, interstitial cells, and smooth muscle ceils arise from a common mesenchymal blast cell, or that the blast cells may be committed to one of the three pathways of

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differentiation but that that commitment is not distinguishable in the fetus at term. She proposed that the differentiation of interstitial cells de- pends upon the establishment of axonal contacts and that differentiation may be related to the establishment of an adult diet. She related these studies to the earlier studies on interstitial cell differentiation which she had done in the mouse ileum [21,22].

Berezin et al. [4] reported an electronmicro- scopic study of the dog colon which expanded on earlier descriptions. The cells had smooth ovoid nuclei with one nucleolus and dispersed chromatin. The perinuclear cytoplasm contained prominent Golgi structures, rough endoplasmic reticulum, mitochondria, free ribosomes, microtubules, thin and intermediate filaments, dense bodies, centrioles, and occasional lysosomes and lipid inclusions. Glycogen granules were rare. The intermediate filaments were hollow and covered with tiny filaments that gave them a hairy appearance. The thin filaments were like actin filaments of muscle. The membranes showed numerous caveolae and an incomplete basal lamina. The cells formed gap junctions with one another and with muscle cells.

Berezin et al. [5] later examined the myenteric plexus of the dog colon. Here, they considered the interstitial cells to be distinguishably different in ultrastructure from those of the plexus entericus extremus. There appeared to be two sets of these cells, one associated with each of the major muscle layers.

Faussone-Pellegrini et al. [25] made a detailed study of the plexus entericus extremus of the human colon. They extended previous studies, finding that the submucosal face of the circular muscle layer is rich in interstitial ceils of Cajal in the right colon (cecum, ascending and right trans- verse colon) but is essentially devoid of the cells in the left colon: the axonal plexus continued in the left colon without the interstitial cells.

Several recent studies have directly addressed the postulated pacemaker functions of the interstitial cells at the submucosal surface of the circular muscle layer in the colon. Langton et al. [38] isolated interstitial cells and found that these cells generate spontaneous signals similar in wave

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form to the electrical slow waves of the colonic musculature. Du and Conklin [20] found that the removal of the plexus entericus extremus from the submucosal surface of the circular muscle layer in the cat colon eliminated the generation of slow waves by the circular muscle layer. Con- klin and Du [11] also showed that the regenera- tion of slow waves in the cat colon during their propagation in the longitudinal axis takes place at the submucosal interface of the circular muscle layer. Serio et al. [47] have confirmed that the removal of the submucosal surface of the circular muscle layer (in the dog colon) yielded circular muscle preparations that were either devoid of slow waves or possessed slow waves of a different wave form.

Discussion

At the outset, this overview of the recent literature posed six old questions about interstitial cells. What answers can one now provide to them?

(i) Are interstitial cells a single clearly-defined set o f cells? The answer is probably yes. The morphologic definition has troubled the matter from the outset. The fact that investigators used the term so variably indicates the uncertainty. Investigators have commonly modified the term ( 'Types I - I V ' ) or evaded it ( 'fibroblast-like cell').

As to shape, the cell bodies are polymorphic and irregular, bearing multiple broad and branching processes, resembling fibroblasts.

As to size, they are smaller than nerve cells and about the same dimensions as smooth muscle cells.

As to location, major questions remain. Cajal and his contemporaries found the cells with autonomic nerves and that remains a universally accepted criterion. The early investigators described the wide distribution of these cells in salivary glands, pancreas, villi and the myenteric plexus, but more recent studies focus upon these cells only in relation to the enteric nerves in the gut wall. The only exception to that statement is the study of Ushiki and Ide [57] who examined the rat pancreas by scanning and electron microscopy. They reported the absence of intersti-

tial cells, the cells which resembled them proving to be specialized glial cells. The other organs remain to be explored.

Staining affinities are not restrictive. Cajal and his contemporaries emphasized the affinity of the cells for methylene blue but one can no longer accept that stain as selective. It simply constitutes a conventional way to see them. The same can be said of the zinc iodide-osmic acid stain. Silver chromate was used by Cajal but it has been neglected since then. Other silver methods show the cells to a variable degree. All these stains are difficult to control and hence seem capricious. None is specific for a defining chemical entity. The NADH-diaphorase stain is selective for cells rich in mitochondria and it reveals the interstitial cells, but not very clearly. Modern histochemical techniques, including immunocytochemistry, can demonstrate the presence of specific proteins which might be defining, but immunocytochemistry has been neglected in respect to these cells except for the study of Prosser et al. [43].

One can now perceive a generally applicable ultrastructure. A round or oval, sometimes indented nucleus contains dispersed chromatin with aggregation at the nuclear envelope. A sparse per inuclear cytoplasm contains conspicuous smooth endoplasmic reticulum, Golgi structures and mitochondria. Rough endoplasmic reticulum is relatively sparse, there are few microtubules, filaments are not conspicuous and free ribosomes are not abundant. Vacuoles are variably present. Lysosomes, fat droplets and small vesicles are never abundant. The membrane contains caveolae. A basal lamina is either absent or incomplete. Nerves always lie close to the cell bodies. The cell processes closely approach smooth muscle cells with which they may form gap junctions.

Interstitial cells can be confused with fibroblasts. Fibroblasts have a more conspicuous rough endoplasmic reticulum, a greater abundance of free ribosomes, few or no membrane caveolae and no basal lamina. Several reports describe forms intermediate between fibroblasts and interstitial cells or point out the difficulty in assigning all cells to the one or the other class. The three ultrastructural features that seem to be accepted as the most rigorously distinguishing interstitial

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cells from fibroblasts are the presence of many caveolae, the presence of at least a partial basal lamina and the presence of a reasonable number of cytoplasmic filaments.

The descriptions summarized above vary to some degree. Authors have tended to attribute the variation to species or organ differences, but it could also reflect experimental variables like age, techniques of fixation and the functional state of the animal at the moment of sacrifice.

(ii) Are interstitial cells universally distributed with terminal axonal networks of enteric nerves? The early idea of a universal distribution of these cells as a part of an autonomic ground plexus is no longer tenable. Although salivary glands, pancreas and villi remain to be thoroughly examined, the work on the gut itself answers the question. Interstitial cells are not found in, for example, the longitudinal muscle layer of the esophagus, intestine and colon where axons are abundant.

(iii) Do interstitial cells have a synaptic relationship with nerves and with effector cells? Although nerves commofily lie very close to interstitial cells, a gap no smaller than about 20 nm is always present. A synapse with nerves, defined as a morphological specialization at a point of contact or close approach, has not been convincingly described. The presence of gap junctions between interstitial cells and muscle cells implies a specialized point of communication. The capacity of interstitial cells to form gap junctions with muscle seems to vary between locations and species.

(iv) Are interstitial cells intercalated between nerve and muscle? Light microscopists made much of the idea of intercalation, without always being specific about what they meant. The term could be interpreted as meaning that the interstitial cells constitute the only pathway of nerve- muscle communication. This is clearly not so. But the cells are intercalated in another sense: in lying alongside axons they are interposed between axons and cells of other tissues. Axons, however, proceed beyond the range of interstitial cells to extend over long distances in the muscle substance. Thus, at most interstitial cells provide an alternate pathway for nerve-muscle communication, not an exclusive one.

(v) Are interstitial cells neural in nature as Cajal

believed? Cajal based his idea of the neural nature of these cells on their stainability with methylene blue and with silver chromate, and on their intimate relationship to nerves. Neither of these is specific or defining. Much of the evidence reviewed here excludes the idea that the interstitial cells are glial. Their close resemblance to fibroblasts suggests that they are mesenchymal. The few developmental studies support this view. Perhaps the interstitial cell is a mesenchymal cell that evolved to provide pacemaking or other functions for smooth muscle. In that case, one might expect variations from one place to another depending on the functions required. Baluk and Gabella [2] may be correct in their view that the microenvironment is the determining factor in the differentiation of interstitial cells.

(vi) Are interstitial cells involved in establishing the rhythmicity of contractions in the gut? Van Esvald [56] and Tiegs [53,54] did not know of the pacemaking electrical slow waves of the gut when they first suggested that interstitial cells might be involved in the myogenic control of contractions, but recent history strongly supports their idea. Indeed, the hypothesis has so well withstood criti- cal tests that some current experts take it as established that the cells are essential to the genesis of slow waves.

Pacing of contractions, however, cannot be the only function of these cells. They are present in the smooth-muscled esophagus but that organ is devoid of repetitive or rhythmic activity. Further- more, if cells of this kind are found to be distributed with nerves in glands then other functions of these cells await discovery.

The cells may be involved in other ways in autonomic neuroeffector transmission. Our con- cepts of autonomic neuroeffector transmission rest mainly upon experiments that involve manipulations of the neurotransmitters, their synthesis, their release and their actions at receptor sites. Autonomic neurotransmission theory neither requires nor excludes a third cell, adjacent, interposed or intercalated at the nerve-effector junction. The addition of a third element to the nerve-muscle junction multiplies the possible hy- potheses about nerve-muscle interactions.

This review began with the question of how

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nerves communicate with the cells of the tissues they supply, and it ends with the same question. Our progenitors proposed a nerve-effector syn- cytium because their light microscopic methods allowed no closer look at the interface of nerves and effector cells. Now that we can better see that interface we can reject the idea of a syn- cytium, substituting ideas which are much more complicated. The interstitial cells of Cajal still might occupy a central place in that communication, at least in certain tissues. Cajal himself proposed that. As in so many other areas of neuroscience, it looks as though he may have been right.

Acknowledgements

This review was supported in part by Research Grant DK 11242 from the NIH.

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Documents

A commentary on the morphological identification of interstitial cells of Cajal in the gut