Updating Old Ideas and Recent Advances Regarding the Interstitial Cells of Cajal

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Review

Updating old ideas and recent advances regarding theInterstitial Cells of Cajal

P. Garcia-Lopez1, V. Garcia-Marin⁎,1, R. Martínez-Murillo, M. FreireCajal Institute, CSIC, Avda Doctor Arce 37, 28002 – Madrid, Spain

A R T I C L E I N F O

⁎ Corresponding author. Fax: +34 91 585 47 54E-mail address: [email protected] (V.

1 Both authors contributed equally to this a

0165-0173/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainresrev.2009.06.001

Please cite this article as: Garcia-Lopez,Cajal, Brain Res. Rev. (2009), doi:10.1016/

A B S T R A C T

Article history:Accepted 1 June 2009

Since their discovery by Cajal in 1889, the Interstitial Cells of Cajal (ICC) have generatedmuch controversy in the scientific community. Indeed, the nervous, muscle or fibroblasticnature of the ICC has remained under debate for more than a century, as has their possiblephysiological function. Cajal and his colleagues considered them to be neurons, whilecontemporary histologists like Kölliker and Dogiel categorized these cells as fibroblasts.More recently, the role of ICC in the origin of slow-wave peristaltism has been elucidated,and several studies have shown that they participate in neurotransmission (intercalationtheory). The fact that ICC assemble in the circular muscular layer and that they originatefrom cells which emerge from the ventral neural tube (VENT cells), a source of neurons, gliaand ICC precursors other than the neural crest, suggests a neural origin for this particularsubset of ICC. The discovery that ICC express the Kit protein, a type III tyrosine kinasereceptor encoded by the proto-oncogene c-kit, has helped better understand theirphysiological role and implication in pathological conditions. Gleevec, a novel moleculedesigned to inhibit the mutant activated version of c-Kit receptors, is the drug of choice totreat the so-called gastrointestinal stromal tumours (GIST), the most common non-epithelial neoplasm of the gastrointestinal tract. Here we review Cajal's originalcontributions with the aid of unique images taken from Cajal's histological slides(preserved at the Cajal Museum, Cajal Institute, CSIC). In addition, we present a historicalreview of the concepts associated with this particular cell type, emphasizing current datathat has advanced our understanding of the role these intriguing cells fulfil.

© 2009 Elsevier B.V. All rights reserved.

Keywords:CajalInterstitial Cells of Cajal

Contents

1. Introduction: the controversial origin of ICC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Location and morphology of ICC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Intravillous and periglandular plexi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Auerbach plexus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3. Deep muscular plexus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4. Intramuscular plexus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.5. Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

.Garcia-Marin).rticle.

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P., et al., Updating old ideas and recent advances regarding the Interstitial Cells ofj.brainresrev.2009.06.001

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2.6. Other locations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. The physiological function of ICC from Cajal to the present day . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. ICC in neurotransmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. ICC as pacemakers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.3. Stretch sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Pathological ICC and GIST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction: the controversial origin of ICC

Between1889and1893,Cajal publishedsomearticlesdescribinga new type of cell that he classified as a primitive neuron. Thistype of cell was located in the stroma of the villi (Figs. 1A, 2), inthe Auerbach's plexus (Figs. 1A, 3), deep muscular plexus (Figs.1A, 4A), circular muscular layer of the intestine (Figs. 1A, 4B–D),and around the acini and blood vessels of the pancreas (Fig. 5).Following Cajal's first descriptions, these cells were identifiedusing different names, including: células simpáticas intersticiales(sympathetic interstitial cells, 1891); células simpáticas (sympa-thetic cells, 1892); neuronas simpáticas intersticiales (sympatheticinterstitial neurons); or células intersticiales (interstitial cells,1899–1904). However, Dogiel subsequently called them –Ca-jal'sche zellen – andmore than 100 years after their discovery thename of Interstitial Cells of Cajal (ICC) is still used.

At that time, there was considerable controversy about thenatureof these cells andwhilesomeresearchers, includingCajaland his colleagues (LaVilla, 1897), thought they were neurons,others, such as Kölliker and Dogiel, classified them as fibro-blasts. This debate was somewhat clouded by the ongoingdiscussion as to whether neurons were individual structures orif they simply formed a syncitium. According to the reticularistpoint of view, neurons were thought to form an interconnectedcontinuous network built up of either axons and dendrites(Gerlach's), or exclusively of axons (Golgi's). Cajal's first paper onthe nervous system (Cajal, 1888) proposed that neurons endfreely, and that they connect with each other by contiguity andnot by continuity. This relevant observation was the firstdescription of the neuronal doctrine: neurons are independentunits from a morphological and even physiological point of view.However, Cajal's description of the ICC as neurons was incontradiction with his neuron doctrine as he was unable torecognize theaxonalprocess in thesecells that characteristicallyestablishes the typical network. In this respect, he recognized:

“I am neither exclusive nor dogmatic. I am proud ofretaining a mental flexibility which is not afraid ofcorrection. Neuronal discontinuity, extremely evident ininnumerable examples, could sustain some exceptions. Imyself have mentioned some of them, for example thoseprobably existing in the glands, vessels and intestines (myinterstitial neurons)” (Cajal, 1933).

During the twentieth century, there has been a longstandingdogmatic division regarding the nature of these cells (Jabonero,1960;Thuneberg, 1990; for a reviewseeThuneberg (1999)).When

Please cite this article as: Garcia-Lopez, P., et al., Updating oldCajal, Brain Res. Rev. (2009), doi:10.1016/j.brainresrev.2009.06.00

analysed by light and electron microscopy, Taxi referred thesecells as neuronoids in an attempt to distinguish them fromneurons, Schwann cells, smooth muscle cells, fibroblasts andmacrophages, although he recognised their tendency to co-stain with nerves (Taxi, 1952, 1965; for a review see Thuneberg,(1999)). Following these findings, ultrastructural studies on theICC suggested that they were primitive muscle cells (Imaizumiand Hama, 1969; Faussone-Pellegrini et al., 1977) or fibroblast-like cells (Richardson, 1958; Komuro, 1989).

The most important advance in this area came with thediscovery that ICC express the tyrosine kinase receptor (c-Kit:Maeda et al., 1992), which permitted them to be chemicallydifferentiated from other cell types sharing similar morpholo-gical characteristics in the tunica muscularis (Thuneberg, 1982;Ward and Sanders, 2001a). The use of this specific markerallowed the mesenchyma to be identified as the source of ICC(Lecoin et al., 1996) and indeed, the detection of c-Kit expressionin aneural explants confirmed that ICC are not of neural crestorigin inmammals (Young et al., 1996). Moreover, it was shownthat both smooth muscle cells and ICC have a commonmesodermal origin (Torihashi et al., 1997; Kluppel et al., 1998).However, it is noteworthy that a subset of ICCmight be ofneuralorigin, since the ICCof thecircularmuscular layeroriginate fromcells that emerge fromtheventral neural tube (VENTcells: Sohalet al., 2002), a source of neurons, glia and ICC precursors thatdiffer from the neural crest cells. Hence, the neuronal origin ofICC suggested byCajal could be at least partially true. VENT cellsoriginate in the ventral part of the hindbrain neural tube andthey migrate through the site of attachment of the cranialnerves to colonize the gastrointestinal tract, particularly theduodenumand stomach (Sohal et al., 1996; Bockman and Sohal,1998). Significantly, not all ICC express c-Kit, such as the ICC-DMP (deep muscular plexus, see Fig. 1A) in the human smallintestine (Torihashi et al., 1999; Wang et al., 2003). Moreover,there are many different cell types besides ICC that express c-Kit, such as mast cells, melanocytes, neurons and glia (Zhangand Fedoroff, 1997). Thus, ultrastructural analysis has proved tobe essential to finally determine whether a particular cellbelongs to the ICC family. The ultrastructural characteristics ofICChavebeenwelldefined (Faussone-Pellegrini andThuneberg,1999; Rumessen and Vanderwinden, 2003; Komuro, 1999) andthey have been summarized as a gold standard (Huizinga et al.,1997) for ICC identification. ICC are generally characterized by anumber of morphological aspects including the presence of: i)numerous mitochondria and caveolae; ii) a basal lamina,although discontinuous; iii) abundant intermediate filaments;iv) moderately developed Golgi apparatus, few ribosomes and a

ideas and recent advances regarding the Interstitial Cells of1


Fig. 1 – (A) Location of the different ICC subtypes according to Cajal and to the present nomenclature based on a semi-schematicdrawingofCajal (Cajal, 1899–1904) ofa longitudinal sectionof theguineapig small intestinestainedby theGolgimethod.Accordingto Cajal: A, longitudinal muscle fibres; B, circular muscle fibres; C, submucose connective tissue in the Meissner plexus; D,Lieberkuhn glandules; E, intestinal villi; a, Auerbach plexus; g, Auerbach ganglion; b, deep muscular plexus; c, fibres from theMeissnerplexus; e, periglandularplexus; f, intravillousplexus.According to thepresentnomenclature: ICCof themyentericplexus(ICC-MPor ICC-MY); ICC of the circularmuscle (ICC-CM) and ICC of the longitudinalmuscle (ICC-LM), both referred to collectively asintramuscular ICC (ICC-IM); ICC of the deep muscular plexus (ICC-DMP); ICC of submucosa and submucosal plexus (ICC-SM andSMP). (B) Multipolar ICC-MP (asterisk) from the guinea pig small intestine evident through c-Kit immunohistochemistry. Thecytoplasmic processes undergo repeated dichotomous branching and they makemany contacts with those of the neighbours(Hanani et al., 2005). (C) Multipolar ICC-DMP of the guinea pig small intestine stained by c-Kit immunohistochemistry, with theirsecondary and tertiary slender processes mainly parallel to the axis of the circular muscle fibres (arrows: Hanani et al., 2005).(D) Bipolar ICC-CM from guinea pig small intestine demonstrated by c-Kit immunohistochemistry that emit only a few processes(Hanani et al., 2005). (E) Human exocrine pancreas. In the insterstitium, amongst the acini (a), note some spindle-shaped ortriangular cells (arrows)with very long cytoplasmic processes (several tens ofμm), indicated bydashed lines. The acinimarked byasterisks appear to be surrounded by periacinar pICC processes. Methylene blue staining (Popescu et al., 2005).

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rough and smooth endoplasmic reticula; and v) close contactsestablished with nerve varicosities and the formation ofnumerous gap junctions, bothwith eachother andwith smoothmuscle cells.

2. Location and morphology of ICC

Cajal described ICC at different sites in the intestinal tube. Apartfrom the classical Meissner plexus (located in the submuscular

Please cite this article as: Garcia-Lopez, P., et al., Updating old iCajal, Brain Res. Rev. (2009), doi:10.1016/j.brainresrev.2009.06.001

connective tissue) and the Auerbach plexus (located betweenthe longitudinal and circular smoothmuscle fibres), Cajal foundthem in three more plexi: the deep muscular plexus, theperiglandular and the intravillous plexi (summarized inFig. 1A). At these sites, he found small fusiform and triangularcells with little protoplasm that had a number of varicoseanastomosed processes, often ramifying at a right angle. Thesegeneral characteristics of ICC varied at their different locations(Figs. 1B–E) and thus, it is worthwhile describing the characte-ristics of the ICC at the locations where Cajal observed them.

deas and recent advances regarding the Interstitial Cells of


Fig. 2 – (A) Drawing of the periglandular and intravillous plexi in the guinea pig intestine stained by the Golgi method (Cajal,1899–1904): a, triangular cell; b, fusiform cell whose inner process result in fascicles of fibrils; c, triangular cell with asimilar pattern; d, fusiform cell of the periglandular plexus; e, f, fusiform cell of the villi; g, layer of subglandular nerve fibrefascicles receiving processes of cells of the periglandular plexus. (B–D) Cajal's original histological slide from the intestine ofthe guinea pig impregnated by the Golgi technique. (B) Periglandular and fusiform cell of the villous plexi. (C) Fusiform cellsof the villous plexus. (D) Triangular cell of the periglandular plexus. Scale bar: 100 μm (B) and 50 μm (C, D).


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2.1. Intravillous and periglandular plexi

Cajal described this new type of cell for the first time in theintestinal villi (Cajal, 1889), situated either at the basal or theapical portion of the villi and forming the periglandular plexus(Figs. 2A–B). In the basal portion of the intestinal villi, the cellswere long and fusiform,with twoprocesses emanating in oppo-


site directions, up and down (Fig. 2C). By contrast, the cells wereround, triangular or stellate in the apical portion, close to thelumen of the intestinal tract (Fig. 2A). The periglandular plexushas triangular or stellate cells (Fig. 2D), and these cells hadnumerous processes that ramified in a complex way. Cajalrealized that the numerous processes anastomosed and hecould not differentiate any axonal process (Cajal, 1893). In



Fig. 3 – (A) Drawing of ICC from the frog Auerbach plexus stained by Ehrlich's method (Cajal, 1892): A, long cells; B, star shapecell; a, intercellular anastomosis; b, small terminal branches with varicosities; d, nucleiform protuberances; c, protoplasmaticgranules]. (B) Drawing of ICC from the frog Auerbach plexus stained by Ehrlich's method: a, nerve cells; b, nerve fibres;c, bundle of nerve fibres; d, cellular processes ending in a bundle of nerve fibres; e, fusiform cell along a bundle of nerve fibres;f, ganglion formed by three cells; g, unstained cells in a ganglion; h, nuclei. (C) Drawing of ICC in the adult rabbit Auerbachplexus stained by Ehrlich's method (LaVilla, 1898): A, cells in the interganglionar mesh; B, anastomosis between two of thesecells; C, marginal or periganglionar cells. D, ICC of the Auerbach plexus from one of Cajal's original histological preparation(Ehrlich's method). Scale bar: 50 μm.


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Cajal's original histological preparations preserved in the CajalMuseum, we have found these cells in the basal portion of thevilli and in theperiglandular plexusbutnot in theapical portion.In this regard, Cajal said (1899–1904), that they ICC in the apicalportion of the intestina villi are very difficult to stain.

Besides these first observations, Güldner also found aconnected system of fibroblasts in the villi of the duodenum(Güldner et al., 1972; for a review see Thuneberg (1999);Huizinga and Faussone-Pellegrini (2005)). These cells formeda cellular network establishing close contact with axons,smooth muscle cells and partially embracing the capillariesand terminal arteriole. Later, it was observed that the fibro-


blasts in the villi are connected through gap junctions(Komuro, 1990; Komuro and Hashimoto, 1990). In contrast totypical ICC, these myofibroblasts do not bear c-Kit (Vannucchiet al., 2002) but they do express NK1r. Moreover, since they arein close contact with one another and with nerve fibres(Vannucchi and Faussone-Pellegrini, 2000), they can still beconsidered as a class of ICC. Concerning the possiblephysiological role of these cells, it is thought that they mayserve as a barrier/sieve, a flexible mechanical frame, mechan-osensors and signal transduction machinery in the intestinalvilli, regulated locally and dynamically by rapid changes in cellshape (Furuya et al., 2005; Furuya and Furuya, 2007).



Fig. 4 – (A) Drawing of ICC in deep muscle plexus from theguinea pig seen in a section parallel to the muscle layer andstained by the Golgi method, according to Cajal (1893):A, nerve cells. a, b, nerve cells of the interstitial plexus;e, arborization of one interstitial cell process; f, interstitial cellwith long processes. (B) ICC under the layer of circularmusclein the rabbit stained by Ehrlichs' method (Cajal, 1893)(C–D) ICC-CM stained by Ehrlichs' method (C) and the Golgimethod (D) from Cajal's original histological preparations.Scale bar: 25 μm (C, D).


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2.2. Auerbach plexus

Cajal studied the Auerbach's plexus in the frogwith the help ofmethylene blue staining. He identified a network of stainedthick, flexible and ramified fibres parallel to the circular mus-cular fibres (Cajal, 1892). This plexus is endowed with gang-lions (2 to 12 cells) joined into anastomotic bundles. Besides


the nervous cells, Cajal also described his interstitial cells assmall fusiform or triangular shaped cells with little proto-plasm, a long and thick nucleus, and with very long andvaricose processes (Fig. 3: Cajal, 1892; 1899–1904). The fusiformshaped cells give off processes from opposing poles while themultipolar ones had several cytoplasmic processes (Cajal,1892; 1899–1904). With regard their connections, Cajal statedthat these cells could establish associations either withAuerbach's plexus, with other ICC or with the muscle cells(Cajal, 1892). Nowadays, these cells are referred to asmyenteric interstitial cells (ICC-MY). The distribution of ICC-MY varies greatly between different parts of the gastrointes-tinal tract, and they are fewer in number and their cellularnetworks are relatively looser in the gastric corpus and colonthan in the small intestine (see Komuro, 2006).

2.3. Deep muscular plexus

Cajal also described ICC in the deep muscular plexus, locatedunder themuscle tunic of circular fibres, in which themajorityof fascicles course parallel to the contractile fibres (Cajal, 1893;1899–1904). Here, the somata of the ICC are small and fusiform,with a triangular or stellate morphology. Indeed, they aremultipolar cells, with secondary or tertiary branches orientedparallel to the axis of circular smooth muscle cells (Fig. 4A).These cells maintain a close relationship with both themuscles and the nerve fibres, and their processes often spanabout 200–300 μm. The network established by the processescovers the whole area of the intestinal wall in a large meshpredominantly composed of long parallel partially anastomo-sised lines (see Hanani et al., 2005). These cells are currentlyreferred to as interstitial cells of the deep muscular plexus(ICC-DMP).

2.4. Intramuscular plexus

Cajal observed bipolar ICC with small ramifications in thecircular muscle layer (ICC-CM), parallel to the axis of musclefibres (Figs. 4B–D: Cajal, 1892, 1893).

“We have also noticed or believe to note some fusiformnervous corpuscle between the circular muscular fibresdirected in the same direction as the muscular fibres”(Cajal, 1892).

Themorphology, distribution and density of ICC-CMdiffersconsiderably from organ to organ in a given species. ICC-CM ofthe small intestine often show secondary cytoplasmicbranches and they are sparsely distributed in associationwith rather thicker nerve bundles, without forming their owncellular network. By contrast, ICC-CM of the stomach andcolon have a simple elongated spindle shape and they aredensely distributed along nerve bundles (see Komuro, 2006).

Some other ICC are also found in the longitudinal musclelayer ICC-LM. These cells are similar to ICC-CM in shape butthere are usually fewer in nearly the whole gastrointestinaltract (i.e. in the stomach, small intestine and colon: Komuro,2004). ICC-CM located in the circular muscle layer and thoselocated in the longitudinal muscle layer (ICC-LM) are referredto collectively as intramuscular ICC (ICC-IM).



Fig. 5 – (A) Terminal axonal plexus of the rabbit pancreas stained by the Golgi method (Cajal and Sala, 1891): A–F: ICC.(B) Terminal axonal plexus of the rabbit pancreas stained by the Golgimethod. A, perivascular nerve cell; B, C, ICC; a, b, terminalbranches between epithelial cells; D, neural plexus of an arteriole (Cajal and Sala, 1891).


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2.5. Pancreas

Cajal studied the distribution of ICC in the pancreas of differentspecies with the aid of the Golgi method and he found that theywere independent elements throughout the organ. These cellswere fusiform, triangular or stellate, with 3 or more divergentprocesses (Fig. 5: Cajal and Sala, 1891), and processes from thesecells formed a strong plexus around the acini or the bloodvessels (Fig. 5B). Again, the ICCprocesses anastomized such thathe could not discriminate a typical axon emanating from them.Recently, the presence of ICC in pancreas (pICC) was confirmedusing non-conventional light microscopy, immunohistoche-mistry and transmission electron microscopy (Popescu et al.,2005). These studies suggested that pICC may play a role inneurotransmission/modulation through two possible strate-gies: a) by co-operatingwith acinar cells andwith small vessels;and b) to engagemutual contact with pancreatic stellate cells orwith Pacinian receptors. In addition, ICC could fulfil anotherhypothetical role in controlling normal mechanoreceptor phy-siology, since they were closely apposed to the Paciniancorpuscle. Interestingly, they also considered that stromalpancreatic tumours (SPT) might originate from a subset ofpICC by analogy with GIST (see below).

2.6. Other locations

ICC are also located outside the gastrointestinal tract and byapplying the Golgi technique, some histologists even foundthem in the serose glands of the tongue (Krause, Fusari andPanasci) or in the myocardium (Berkley, 1893; review in Cajal,1899–1904). With modern techniques, ICC have also beenfound in many other locations such as in the upper urinarytract (Lang and Klemm, 2005), urethra (Sergeant et al., 2006),myometrium (Ciontea et al., 2005), myocardium (Hinescu andPopescu, 2005; Popescu et al., 2006), uterus, fallopian tube(Popescu et al., 2007), human placenta (Suciu et al., 2007) andthe ciliary muscle in monkeys (Paula et al., 2009). Thus, thesefindings confirm Cajal's conclusions that:

“All or almost all glands have terminal neural plexi, similarto those of the intestine, made of Remak fibres originatedin special autonomic ganglia, as well as fusiform or stellatecells of the previously described interstitial type” (Cajal,1899–1904).


3. The physiological function of ICC from Cajalto the present day

The history of gastrointestinal motility extends deep into thepast. From the observations of gastric contractions (Beau-mont, 1833) through to the discovery of spontaneous coloncontractions in the cat tract using X-rays (Cannon, 1902), therehave been many advances in this field. The implication of theAuerbach and Meissner plexi in the motility of the gastro-intestinal tract was fully appreciated from the beginning oflast century:

“The presence of these centres (referring to the Meissnerand Auerbach plexi) explains the automatism of intestinalmovements” (Cajal, 1899–1904).

However, the role of the ICC was less clear. Nevertheless,Cajal proposed these cells to bemediators of enteric transmis-sion (Fig. 6: Cajal, 1899–1904):

“It is not a risky conjecture, however, that the interstitialcells are subordinated to fibres of the autonomic ganglia.The impulse brought by these fibres would elicit a sup-plementary discharge in the interstitial cells, able to addstrength to the contraction or increase its duration”. (Cajal,1899–1904).

Later in this text and in reference to the motor pathway ofthe autonomic system, Cajal stated:

“Here, in all probability, the motor chain has twoadditional neurons: that of myenteric and mucosal plexi,and the interstitial or terminal cell”. (Cajal, 1899–1904).

For Cajal, the ICC would function as mediators of neuronaltransmission and subsequent studies supported this theory(Imaizumi and Hama, 1969; Yamamoto, 1977; Oki and Daniel,1973; Daniel, 1977). Currently there is no doubt that inter-stitial cells serve as mediators of enteric transmission andindeed, interstitial cells were proposed as pacemakers byKeith in 1915 (Keith, 1915; for a review see Thuneberg (1999))and this role in gastrointestinal pacemaking activity hassince been further demonstrated. These different functions



Fig. 6 – Diagram of sensory and motor pathways of theautonomic system according to (Cajal, 1899–1904).A, sympathetic ganglion; B, ventral horn of the spinal cord;C, dorsal root ganglion; D, small intestine; E, pancreas;F, visceral ganglion; J, glandular interstitial cell;K, perivascular nerve cell; V, blood vessels; A, motorspino-gangliar fibres of preganglionic Langley fibres;b, another spino-sympathetic fibre terminating exclusivelyin a single ganglion; c, sympathetic axon coursing throughtwo ganglia; d, sympathetic axon incorporated into a spinalnerve through the gray ramus communicans; e, autonomicaxon ending on a blood vessel (postganglionic fibre ofLangley); f, autonomic axon ending in the myenteric plexusof the intestine; g, motor cell of a myenteric ganglion;h, sensory spinal fibre ending in the intestinal mucosa;m, ganglion of the myenteric plexus; I, motor fibre ending ina visceral ganglion.


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(nervous transmission and pacemaking activity) are to someextent carried out by different types of ICC. While nervoustransmission is mediated by ICC-IM, the pacemaking activityis preferentially mediated by ICC-MY even though ICC-IMmay also regulate the frequency of the pacemaker.


3.1. ICC in neurotransmission

Several studies have revealed a close and functional relation-ship between ICC and enteric nerve fibres, confirming that ICCparticipate in neurotransmission (Cajal, 1899–1904; Daniel andPosey-Daniel, 1984; see also Ward, 2000; Ward and Sanders,2001b). The knock-out animals available for c-kit have beenparticularly useful in defining the role of ICC inneurotransmis-sion (Burns et al., 1996), especiallyW/Wv mutant animals. Theabsence of ICC-IM in themurine fundusnot only led to reducedNO (nitric oxide) dependent post-junctional responses (Burnset al., 1996; Ward et al., 1998) but also, it resulted in the loss ofcholinergic excitatory responses (Ward et al., 2000).

Immunohistochemical studies have revealed that isolatedICC are responsive to a variety of enteric transmitters, bothexcitatory and inhibitory. Nerve fibres stained with theprimary transmitters of excitatory motor neurons, such asvesicular ACh transporter (VAChT) and substance P, areclosely associated with cell bodies and processes of ICC-IMin the stomach (Beckett et al., 2002; Horiguchi et al., 2003; Songet al., 2005; Wang et al., 1999; Ward et al., 2000), and with ICC-DMP in the small intestine (Faussone-Pellegrini, 2006; Iinoet al., 2004; Lavin et al., 1998; Wang et al., 1999). Manyinhibitory motor neurons that contain nitric oxide synthase(NOS), vasoactive intestinal polypeptide (VIP) or adenosinetriphosphate (ATP) are closely associated with both the cellbodies and processes of ICC-IM (Beckett et al., 2002; Horiguchiet al., 2003; Song et al., 2005; Wang et al., 1999; Ward et al.,2000) and ICC-DMP (Toma et al., 1999; Wang et al., 1999). Inaddition, different neurotransmitter receptors are expressedby the ICC (See Box 1). These data demonstrate that ICC aredensely innervated by excitatory and inhibitory enteric motorneurons.

It should be noted that the close apposition of enteric nervefibres (both excitatory and inhibitory) and ICC, as well as thepresence of neurotransmitter receptors on these cells, doesnot imply that functional synaptic contacts aremade betweenthese structures. With the aid of electron microscopy,abundant close contacts (<25 nm) between varicose nerveterminals and ICC were demonstrated that support theexistence of a specialized kind of transmission of neuralinformation between enteric nerves and ICC (Daniel andPosey-Daniel 1984). These contacts have been confirmed(Wang et al., 1999; Ward et al., 2000) and the existence ofthese close interactions led to the recovery of the intercalationtheory which favours the existence of nerve transmissionthrough ICC (Cajal, 1899–1904; Daniel and Posey-Daniel, 1984;see alsoWard, 2000;Ward and Sanders, 2001b). These synapticspecializations are functional, since the soluble N-ethylma-leimide-sensitive attachment protein receptors (SNARE)implicated in neurotransmitter release is located in varicos-ities associated with ICC-IM (Nirasawa et al., 1997; Aguado etal., 1999; Beckett et al., 2005; for a review see Ward andSanders, (2006)). The postsynaptic densities are also func-tional since they contain typical postsynaptic proteins. Thereis also a decrease in the expression of postsynaptic density(PSD-93 and PSD-95) in kit mutant mice (W/Wv), whereasimmunohistochemistry for the PDZ domain of the PSD-95family and for Kit revealed some degree of co-localization(Beckett et al., 2005; for a review see Sanders andWard (2006)).



Box 1ICC receptors.

Receptors ICC location References

5-HT, 5-HT3, 5-HT4 subtypes ICC-MY Glatzle et al. (2002); Liu et al. (2005); Poole et al. (2006);ICC-DMP

Bombesin, subtype-3 Almost in all ICC Porcher et al. (2005)

Cholecystokinin A ICC Patterson et al. (2001)G protein-coupled, Protein Kinase A (PKA),Protein Kinase C (PKC)

ICC Southwell (2003); Poole et al. (2004)

Muscarinic acetylcholine, M2, M3 subtypes ICC-IM, ICC-MY Epperson et al. (2000); Iino and Nojyo (2006)ICC-DMP

Neurokinin, NK1, NK3 subtypes ICC-IM, ICC-MY Sternini et al. (1995); Grady et al. (1996); Portbury et al. (1996); Lavin et al. (1998);Epperson et al. (2000); Iino et al. (2004)ICC-DMP

Purinergic, P2Y1, P2Y4 subtypes P2X2, P2X5subtypes

ICC-IM, ICC-MY,ICC-DMP

Burnstock and Lavin (2002); Van Nassauw et al. (2006); Chen et al. (2007)

Somatostatin 2A ICC-DMP Sternini et al. (1997)VIP ICC-IM, ICC-MY Epperson et al. (2000)VPAC1 subtype


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However, there is still little data regarding how the nervousimpulse is transmitted from the ICC-IM to the smooth musclecells. Although gap junctions were proposed to be involved inthis process, recent research using gap junction blockers sug-gests that gap junctions are not necessary for pacing or nervetransmission to the circular muscle of the mouse intestine(Daniel, 2004; Daniel et al., 2007).

3.2. ICC as pacemakers

The first suggestion that ICC may act as physiological pace-makers was made by Arthur Keith, the discoverer of thecardiac sino-atrial pacemaker organization (Keith, 1915; for areview see Thuneberg (1999)).

“A series of sections through the ileo-caecal junction of therat's bowel revealed a collar of peculiar tissue…there wasalso present a third element -numerous branching cells, notconnective tissue in nature with processes which unitedwith muscle cells, on the one hand, and with the processesfromtrueganglionic cells on theother. I regarded these inter-mediate cells as a possible representation of the nodal tissueof the heart”. (Keith, 1915); taken from (Thuneberg, 1999)

Ambache was the first to show that electrical slow wavescontrol intestinal contractions and to relate these slow wavesto the ICC (Ambache, 1947; reviewed in Thuneberg (1999)).Several subsequent studies (using different approaches thatincluded chemical lesions and dissection experiments, intra-cellular electrophysiological recording or mutant animals)indicated that ICC generated these slow waves. Slow wavesare cyclic depolarizations of the membrane potential ofsmooth muscle cells. The cellular mechanisms involved inthe generation of pacemaker potentials are not completelyunderstood, although it seems clear that the release of Ca2+

from internal IP3-dependent stores plays a key part in theirgeneration (Suzuki, 2000; for a review see Nakayama et al.(2007)). When rhythmic electrical oscillations reach the open-ing threshold of calcium channels, calcium enters the cell andtriggers the contraction of smooth muscle cells.


Early evidence of the implication of ICC in the generation ofslowwaves came from photochemical ablation of ICC, wherebymethylene blue followed by illumination blocks slow-waveactivity (Thuneberg et al., 1983; Liu et al., 1993, 1994). Alter-natively, othersused rhodamine123, a cytotoxic fluorescentdyethat specifically accumulates in the ICC, although it may alsoaccumulate in enteric neurons (Ward et al., 1990).

Other studies have been critical to confirm the role of ICC ingenerating slow-wave activity. Intracellular electrode record-ing and methylene blue staining were used to confirm thatslow waves are only observed in smooth muscle cells whenthe ICC are attached to the muscle layer registered (Suzukiet al., 1986). At the same time, intracellular recording of thelongitudinal, inner and outer circular muscle layers of the dog,cat, rabbit, opossumand human small intestine demonstratedthat the myenteric region is the dominant source of slowwaves in the small intestine (Hara et al., 1986). By contrast, thepacemaker in the colon resides at the submucosal surface ofthe circular muscle layer, since removing ICC-SMP from thesubmucosal border blocks the generation of slow waves(Smith et al., 1987). In mice with c-kit mutations (W/Wv), thenetworks of ICC-MY are grossly underdeveloped in the smallintestine where pacemaker activity was lacking (Ward et al.,1994; Huizinga et al., 1995), even though ICC are present in theDMP. In the latter experimental model, ICC-MY are evident inthe stomach where slow-wave activity can be recorded (Burnset al., 1996; see also Huizinga, 2001). These data suggest thatICC-MY but not ICC-DMP are crucial for generating slow-waveactivity (Ward et al., 1994, 1995; Huizinga et al., 1995; Huizingaet al., 2001). Similar experiments in rat mutants (Horiguchiand Komuro, 1998) or in steel-Dickie mutant mice (Sl/Sld), inwhich the gene encoding the c-Kit ligand (stem cell factor, SCF)is defective (Ward et al., 1995) confirmed these data. However,the generation of slow waves is not only mediated by ICC-MY.In the gastric corpus of the guinea pig where ICC-MY areabsent, the dominant pacemaker activity that entrains activityin other regions of the stomach is provided by the ICC-IM(Hashitani et al., 2005). Furthermore, ICC-IM contribute to theamplification of pacemaker signals from ICC-MY by genera-ting rhythmic oscillations known as unitary potentials,



Fig. 7 – (A) Adultmouse duodenumphysiologically distended by the intestinal contents according (Thuneberg and Peters, 2001).The electron micrograph shows the longitudinal muscle layer with five peg and socket junctions (*). Note the uniform,narrow space between peg and socket membranes, in contrast to simple folds of the cell surface. (B) Diagram of the distributionof the peg and socket junctions and the gap junctions in the muscularis of adult mouse small intestine (Thuneberg, 1999).


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regenerating the depolarizing currents from ICC-MY or thosethat follow exposure to acetylcholine (Dickens et al., 2001;Hirst et al., 2002a,b; Edwards et al., 1999).

Other studies have been carried out on isolated and/orcultured ICC. Single ICC are spontaneously active, generatingelectrical depolarization similar to slow waves recorded inintact smooth muscle cells, as demonstrated in patch clampexperiments on the canine colon (Langton et al., 1989). Indeed,single ICC generate a rhythmic inward current insensitive to L-type calcium channel blockers that is crucial for the genera-tion of slow waves in smooth muscle cells (Thomsen et al.,1998).

But how the slow waves spread from the ICC-MY to thesmooth muscle cells is still unclear. Although ICC-MY arecoupled through gap junctions, there are no gap junctionsbetween ICC-MY and smooth muscle cells of the longitudinalor circular muscle layer (Fig. 7). Thus, other mechanisms suchas peg and socket connections, have been proposed thatmightprovide electrical coupling through the accumulation ofpotassium in the narrow cleft between the peg and the socketunder appropriate conditions (Vigmond et al., 2000). These pegand socket connections have also been proposed as stretchsensors, warranting a description of these structures and theirpossible implications.

3.3. Stretch sensors

The hypothesis that ICC could act as stretch sensors has beenproposed repeatedly (Daniel, 1977; Thuneberg, 1989; Faus-sone-Pellegrini and Thuneberg, 1999), based on the existenceof special structures called peg-and-socket junctions that mayrepresent the part of muscle cells most vulnerable to suchtension (Fig. 7). These peg-and-socket junctions are thought toconsist of a peg of 0.5 to several-micrometers long, whichextends from one smooth muscle cell into a narrow pocket orinvagination of the plasma membrane of a neighbouringsmooth muscle cell or ICC, excluding the connective tissuecomponents from the space between the tightly apposedmembranes. The morphology and fixed orientation of thesestructures suggests that they could serve as mechanicalstretch sensors, regulating smooth muscle/interstitial cell


coupling and the internal sensitivity of a muscular layer tostretch (Faussone-Pellegrini and Thuneberg, 1999; Thunebergand Peters, 2001).

However, the most solid physiological evidence supportingthe functioning of ICC as stretch sensors was obtained byapplying length ramps to the murine antral muscles, whilerecording intracellular electrical activity and isometric force(Won et al., 2005). Increasing the length caused membranedepolarization and an increase in the slow-wave frequency.The response was mediated by ICC-IM because no responsewas observed in antral muscles of c-kit mutants, W/Wv mice,which lack ICC-IM. The stretch sensor mechanism associatedwith the ICC is mediated by the cyclooxygenase enzymeCOX-II, since stretch-dependent responses were inhibited bythe COX-II inhibitor indomethacin and they were absent inCOX-II deficient mice (Won et al., 2005). COX-II is constitu-tively expressed by ICC-IM (Porcher et al., 2002) and it isimplicated in the prostaglandin cascade. Therefore, productsof arachidonic acid metabolism, such as prostaglandin E2(PGE2), are likely to mediate stretch-dependent responses(Won et al., 2005). Thus, ICC-IM seem to coordinate differentneural and mechanical inputs to regulate gastric motility.

4. Pathological ICC and GIST

There is evidence of a correlationbetweenalterations to ICCandsome digestive pathologies. Indeed, ICC are lacking, reduced ordamaged in some of the following digestive pathologies:idiopathic gastric perforation, hypertrophic pyloric stenosis,transient neonatal pseudo-obstruction, neonatal meoconiumileus, Hirshprung's disease, total colonic aganglionosis, intest-inal neuronal dysplasia, hypoganglionosis, internal sphincterachalasia or congenital uretopelvic junction obstruction inchildren and achalasia of oesophagus, gastroparesis, chronicidiopathic intestinal pseudo-obstruction, diabetic gastroentero-pathy, paraneoplastic dismotility, afferent loop syndrome,Chagas disease, or inflammatory bowel diseases such as ulce-rative colitis or Crohn's disease in adults (for a review seeStreutker et al. (2007)). While in the paediatric population someof these alterations may be caused by developmental delay, in



Fig. 8 – (A) Scheme of Kit structure and of Kit activation bySCF binding: EC, extracellular; TM, transmembrane;JM, juxtamembrane; and TK, Tyrosine Kinase. The growthfactor, SCF, causes dimerization of KIT, resulting in celldifferentiation and proliferation via MAP kinase and theJAK/STAT signal transduction pathway. Modified from(Isozaki and Hirota, 2006). (B) Location and type of c-kitgene mutation in sporadic GIST according to Isozaki andHirota (2006).


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the case of adults the causesare lesswell understood.Moreover,it is necessary to differentiate whether these alterations are theprimary cause of the disease or just an outcome of it.

ICC are also involved in the formation of GIST, the mostcommon mesenchymal tumours of the gastrointestinal tract.Most GIST (50–60%) arise in the stomach, yet 20–30% arisein the small bowel, less than 10% develop in the colon and1% in the oesophagus, while a small proportion are extra-gastrointestinal (5%). Prior to the availability of c-Kit immu-nohistochemistry, GIST were pathologically diagnosed asleiomyomas, leiomyoblastomas or leiomyosarcomas whenthey were considered to be of smooth muscle origin, or asschwannomas if they were considered to be of neural origin.For those tumours without smooth muscle or Schwann cellsfeatures (Mazur and Clark, 1983), the term GIST was intro-duced on the basis of immunohistochemical light and electronmicroscopy studies. It was not until 1998 that the relationshipbetween GISTs and ICC was demonstrated (Hirota et al., 1998).

Based on the evidence that loss of function mutations ofthe c-kit gene led to deficiencies in ICC andmast cells, and thatgain-of-function c-kit mutations provoked the formation ofmast cell tumours (Furitsu et al., 1993), it was speculated thatICC tumours could be induced by c-kit gain-of-function (Hirotaet al., 1998; for a review see Hirota and Isozaki (2006)). WhileKit expression was undetectable by immunohistochemistry inauthentic leiomyoma and schwannomas, it was present in94% of GIST analyzed. These immunohistochemical charac-teristics of GIST were also shared by ICC that express Kit(Maeda et al., 1992) and CD34 (Nishida et al., 1998). Accor-dingly, the complete coding region c-kit (from human chromo-some 4) was sequenced from six GIST and in five cases therewere mutations in the region between the transmembraneand the tyrosine kinase domains (the juxtamembrane domainencoded primarily by exon 11 and rarely by exons 9 and 13).The mutations led to the constitutive activation of Kit protein,even in the absence of the c-Kit SCF ligand. This mutant c-kitinduced malignant transformation of Ba/F3 murine lymphoidcells, suggesting that GIST might originate from the ICC withmutations in the c-Kit receptor (Hirota et al., 1998), as laterconfirmed in other studies (Kindblom et al., 1998; Sarlomo-Rikala et al., 1998; Sircar et al., 1999; Robinson et al., 2000).Nevertheless, it is still not clear whether GIST originate fromICC or from a precursor of these cells that differentiates intoICC during the pathological process (this later mechanismsfollows Cajal's so-called “general doctrine of tissue composition”,for a review see Martinez et al. (2005)).

Although most GIST bear mutations in kit, a subset (10–15%)express wild type kit (Taniguchi et al., 1999; Rubin et al., 2001).Indeed, 35% of the GIST that lack mutations in kit have intra-genic activating mutations in the related receptor tyrosinekinase (Heinrich et al., 2003), platelet-derived growth factorreceptor α (PDGFRA), and this proportion reached 65% in a laterstudy (Hirota et al., 2003). These mutations in PDGFRAalso produce constitutively active proteins, these receptorsfreely activating their Receptor Tyrosine Kinase and Mitogen-Activated Protein kinase (RTKs andMAP) targets independentlyof their ligands (Fig. 8A). When the specific locations of themutations in kit were studied in mutational analysis: 66% arefound in the intracellular/juxtamembranous region of exon 11that fulfils physiological regulatory/autoinhibitory roles


(Nishida et al., 1998; Lasota et al., 1999; Moskaluk et al., 1999;Taniguchi et al., 1999); 13% lie in exon 9 corresponding to theextracellular domain (Lux et al., 2000; Lasota et al., 2000; Hirotaet al., 2001); less than 4% are located in the Tyrosine Kinase I(TKI) domain encoded by exon 13 (Lux et al., 2000; Lasota et al.,2000; Kinoshita et al., 2003); and less than 4% are in the TKIIdomain encoded by exon 17 (Lux et al., 2000; Lasota et al., 2000;Kinoshita et al., 2003: Fig. 8B). Themutations in the PDGFA genewere located at the JM domain encoded by exon 12 and the TKIIdomain encoded by exon 18 (Heinrich et al., 2003; Hirota et al.,2003), whereas mutations in exon 14 that encodes the TKIdomain have only rarely been reported (Corless et al., 2005;Lasota et al., 2006).

At least, 12 families with multiple GISTs and germ-line kitactivating mutations have been reported (Nishida et al., 1998;O'Brien et al., 1999; Hirota et al., 2000; Isozaki et al., 2000;Maeyama et al., 2001; Beghini et al., 2001; Hirota et al., 2002;Robson et al., 2004; Carballo et al., 2005; Li et al., 2005; Kimet al.,2005; Hartmann et al., 2005; O'Riain et al., 2005; for a review seeIsozaki and Hirota, (2006); Streutker et al., (2007)). Themutations of kit or PDGFRA in GIST tumours have permittednovel treatments to be developed that aim to inhibit theconstitutively activated receptors in these tumours. Imatinibmesylate or Gleevec was developed as a specific inhibitor ofPDGFRA and the chimeric fusion protein Bcrl–Abl, the activityof which is uncontrolled in myeologenus leukemia (CML). Inpreclinical models, imatinib mesylate was also shown to beefficient in inhibiting the mutant kit in GIST and the firstpatient to be treatedwith this oral treatment in 2001 displayeda strong response to the treatment (Joensuu et al., 2001). Sincethen, several trials have been carried outwith excellent results(Demetri et al., 2002; Verweij et al., 2004; Scaife et al., 2003).More recently (Demetri et al., 2006;Goodmanet al., 2007), a newmultitargeted kinase inhibitor has been tested in patientswith




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GIST resistant to Imatinib with promising results, whileanother novel inhibitor of Kit, PKC412 (Debiec-Rychter et al.,2005; Growney et al., 2005), also showed promising preclinicalactivity against certain imatinib-resistant mutations.

5. Conclusion

The observations and interpretations of Cajal on the ICCremain valid today, especially his intercalation theory of ICCasmediators of nervous activity. Following Cajal's discoveries,other important physiological functions for ICC have beendemonstrated, such as their activity as pacemakers or stretchsensors. The discovery of the c-Kit receptor in the ICC has notonly permitted the development of strategic tools to studytheir physiological functions, but it also serves as a target forthe development of accurate therapies to treat GIST.

Acknowledgments

We would like to acknowledge the inheritors of SantiagoRamón y Cajal © P. G-L. is supported by the Fundación CaixaGalicia. This work was supported by the Spanish Ministry ofScience and Education (Grant SAF2007-60010) and the Insti-tuto de Salud Carlos III (Grant RD06/0026/1001).

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Updating Old Ideas and Recent Advances Regarding the Interstitial Cells of Cajal