The formation of the chick ileal muscle layers as revealed by α-smooth muscle actin immunohistochemistry

The genesis of intestinal smooth muscle layers was immunohistochemically investigated by use of an antibody to α-smooth muscle actin (α-SMA) in the developing chick ileum. Myoblast cells positive for α-SMA were already found in the presumptive circular muscle layer on E 8.5. On E 11.5 radially oriented muscle fibers were protruded from the outermost layer of the developing circular musculature and then formed a tuft-like aggregates. These radial muscle bundles were bent into an L-shape. The long distal extension of muscle bundles run parallel to the long axis of the ileal loop and developed into the longitudinal muscle layer. The obliquely oriented muscle fibers, locating at the intermuscular space of the muscularis propria, probably are to be considered a remnant of the short extension of radial muscle bundles. The muscularis mucosae was formed by the processes equivalent to the genesis of longitudinal muscle layer. On E 14.5 centripetally oriented muscle fibers emerged from the innermost layer of circular musculature. The long distal extension of centripetal fibers lay along the inner surface of developing circular musculature. On E 19.5 the longitudinal muscle layer of the muscularis mucosae was newly formed by separating from the circular musculature. The villous myoblast cells initially developed from the innermost layer of the muscularis mucosae on E 18.5, and were widely distributed in the lamina propria mucosae on E 20.5. Temporal and chronological pattern in expression of α-SMA was observed during the development of the chick intestinal smooth muscle. By E 14.5 the entire layer of the muscularis propria was intensely immunostained for α-SMA, but from E 15.5 onward the staining intensity gradually began to decrease from the outer half of the circular musculature. Finally, the immunoreactivity was localized in the inner layer of circular muscle and the longitudinal muscle layer. A possible functional role of this inner layer of circular muscle is discussed.     

The formation of the chick ileal muscle layers as revealed by alpha-smooth muscle actin immunohistochemistry

The genesis of intestinal smooth muscle layers was immunohistochemically investigated by use of an antibody to α-smooth muscle actin (α-SMA) in the developing chick ileum. Myoblast cells positive for α-SMA were already found in the presumptive circular muscle layer on E 8.5. On E 11.5 radially oriented muscle fibers were protruded from the outermost layer of the developing circular musculature and then formed a tuft-like aggregates. These radial muscle bundles were bent into an L-shape. The long distal extension of muscle bundles run parallel to the long axis of the ileal loop and developed into the longitudinal muscle layer. The obliquely oriented muscle fibers, locating at the intermuscular space of the muscularis propria, probably are to be considered a remnant of the short extension of radial muscle bundles. The muscularis mucosae was formed by the processes equivalent to the genesis of longitudinal muscle layer. On E 14.5 centripetally oriented muscle fibers emerged from the innermost layer of circular musculature. The long distal extension of centripetal fibers lay along the inner surface of developing circular musculature. On E 19.5 the longitudinal muscle layer of the muscularis mucosae was newly formed by separating from the circular musculature. The villous myoblast cells initially developed from the innermost layer of the muscularis mucosae on E 18.5, and were widely distributed in the lamina propria mucosae on E 20.5. Temporal and chronological pattern in expression of α-SMA was observed during the development of the chick intestinal smooth muscle. By E 14.5 the entire layer of the muscularis propria was intensely immunostained for α-SMA, but from E 15.5 onward the staining intensity gradually began to decrease from the outer half of the circular musculature. Finally, the immunoreactivity was localized in the inner layer of circular muscle and the longitudinal muscle layer. A possible functional role of this inner layer of circular muscle is discussed. Accepted: 16 July 1999     

Structure of the esophagus in the adult opossum, Didelphis virginiana.

The structure of the esophagus has been studied in the adult opossum, Didelphis virginiana. A thickening of both layers of the muscularis externa occurs at the origin of the esophagus and may represent the upper esophageal sphincter; a massive expansion of the muscularis mucosae occurs in the region of the lower esophageal sphincter. The distribution of striated, mixed and smooth muscle in the muscularis externa differs in the inner and outer layers and elements of the myenteric plexus are found to occur even in the region of striated muscle; however, the ganglia of this plexus become much more prominent as smooth muscle makes its appearance. Esophageal glands are found in the lamina propria where they are confined to the 2 ends. They are especially prominent at the distal end where they are responsible for the formation of permanent transverse folds. Similar glands are found in the submucosa, scattered throughout the length of the esophagus but distally, in the region of the transverse folds, the submucous glands disappear. In both of these layers, the glands contain mucous, serous and myoepithelial cells.     

Deficient alpha smooth muscle actin expression as a cause of intestinal pseudo-obstruction: fact or fiction?

AIMS: To test the hypothesis that deficient alpha smooth muscle actin (ASMA) expression in intestinal smooth muscle, as assessed by immunohistochemistry, is specifically associated with clinical evidence of intestinal pseudo-obstruction. METHODS: Seventeen archival, formalin fixed, paraffin wax embedded samples of small intestine and 12 samples of large intestine were studied. Two of the small bowel samples and one large bowel sample were from patients with symptoms of intestinal pseudo-obstruction. The controls were longitudinal surgical margins from hemicolectomies performed for carcinoma. Immunohistochemistry was performed using primary antibodies to ASMA, smooth muscle myosin heavy chain (SMMHC), and desmin. The relative intensities of immunohistochemical expression in the circular and longitudinal muscle layers of the muscularis propria were assessed in each sample, for all three markers. RESULTS: All samples showed strong SMMHC and desmin expression in the inner circular and outer longitudinal layers of the muscularis propria. Both small intestinal samples from the cases and 11 of 15 controls showed no or minimal ASMA expression in the inner circular layer, with the remaining four controls also showing ASMA labelling in this layer that was weaker than within the longitudinal muscle. In contrast, intense ASMA expression was seen in both muscle layers within the large intestine in the remaining case, and in the controls. CONCLUSIONS: There is insufficient evidence from this study to support the hypothesis that ASMA deficiency in intestinal smooth muscle, as determined by immunohistochemistry on archival tissues, is specifically associated with intestinal pseudo-obstruction.     

Innervation of the intestinal muscular coat

Summary Nerve bundles have an uneven distribution in the muscularis externa of the guinea-pig ileum. They are absent in the longitudinal muscle layer, and are mainly concentrated between the two muscle layers and between the bulk of the circular layer and its innermost portion. The latter is formed by muscle cells which are smaller and more electron dense than the ordinary smooth muscle cells. In cross sections of the circular layer there are 5425 intramuscular axons (of which 921 contain vesicles) per 10000 smooth muscle cells, and they are grouped in bundles, mainly formed by 10–40 axons. The gap between axons and smooth muscle cells is generally hundreds of nanometres; only a few junctions with a gap smaller than 20 nm occur. Interstitial cells have a cytoplasm occupied mainly by smooth reticulum and ribosomes, and processes containing filaments. These processes are closely associated with smooth muscle cells and nerve bundles. Interstitial cells have no basal lamina. Nexuses are found in the circular but not in the longitudinal muscle layer. Attachment plaques and invaginating processes are found in both muscle layers. More complex interdigitations appear between contacting muscle cells. There are 84 intramuscular blood vessels per 10 000 cross-sectioned smooth muscle cells (or 1700 per mm²).     

Muscle system of Diplodiscus subclavatus (Trematoda: Paramphistomida) cercariae,pre-ovigerous,and ovigerous adults

The musculature of cercariae, pre-ovigerous, and ovigerous adults of Diplodiscus subclavatus was studied by means of TRITC-conjugated phalloidin staining of filamentous actin and confocal scanning laser microscopy. The body wall appears to include four muscle layers as follows: circular, outer longitudinal, diagonal, and inner longitudinal. Two layers of longitudinal muscle fibers are arranged in different modes due to the secondary transformed paramphistomid body construction. The organization of the acetabulum turned out to be more complex than ever described, with a radial layer, two layers of circular, two layers of meridional, an additional starry layer of muscle fibers, as well as a few separate muscle layers of the accessory sucker. Within the pharynx, I found a group of alar muscle fibers, never described before for any paramphistomids, and some morphological features which were not considered to be characteristic for D. subclavatus (namely—the middle semicircular layer and the transverse muscle fibers in the pre-sphincteric space). No significant reorganizations of the somatic musculature occur throughout the development from the cercaria to the ovigerous adult worm, so the metamorphosis goes in the manner of completion. The cercarial tail includes a layer of circular muscle fibers and a longitudinal muscle layer beneath. The latter consists of two medial longitudinal bundles of smooth muscle fibers and two lateral longitudinal bands of obliquely striated muscle fibers which are partially divided in halves.     

The organization of the lamina muscularis mucosae in the human esophagus

The structural organization of the lamina muscularis mucosae of the human esophagus was studied by light microscopy and scanning electron microscopy (SEM). The organization of the lamina muscularis mucosae varied considerably among the cervical, the thoracic, and the abdominal part of the esophagus. In the cervical part, the lamina muscularis mucosae was not well developed and only islets of the smooth muscle bundles were scattered within the connective tissue. In the thoracic part, the lamina muscularis mucosae consisted of several layers of smooth muscle bundles, individual muscle cells of which ran in a longitudinal direction. In the abdominal esophagus near the cardia, the muscular bundles in the lamina muscularis mucosae ran in various directions forming a reticular configuration. The differences in density and arrangement of the lamina muscularis mucosae are discussed in relation to the swallowing of food and submucosal invasion of esophageal cancer.     

Morphological analysis of the urethral muscle of the male pig with relevance to urinary continence and micturition

To investigate whether the pig could be considered a suitable model to study lower urinary tract function and dysfunction, the pelvic urethra of 24 slaughtered male pigs were collected, and the associated muscles were macroscopically, histologically and histochemically analyzed. In cross‐sections of the urethra, a muscular complex composed of an inner layer of smooth muscle and an outer layer of striated muscle that are not separated by fascial planes was observed. A tunica muscularis, composed of differently oriented smooth muscle bundles, is only evident in the proximal part of the pelvic urethra while, in the remaining part, it contributes to form the prostatic fibromuscular stroma. The striated urethral muscle surrounds the pelvic urethra in a horseshoe‐like configuration with a dorsal longitudinal raphe, extending from the bladder neck to the central tendon of perineum. Proximally to the bladder, it is constituted of slow‐twitch and fast‐twitch myofibers of very small diameter, and embedded in an abundant collagen and elastic fiber net. Moving caudally it is gradually encircled and then completely substituted by larger and compact myofibers, principally presenting circular orientation and fast‐twitch histochemical characteristics. So, like in humans, the cranial tract of the muscular system surrounding the pelvic urethra is principally composed of smooth musculature. The striated component cranially may have a role in blocking retrograde ejaculation, while the middle and caudal tracts may facilitate urine and semen flow, and seem especially concerned with the rapid and forceful urethral closure during active continence. Some differences in the morphology and structure between pigs and humans seem due to the different morphology of the ‘secondary’ sexual organs that develop from the urethral wall and to the different effect of gravity on the mechanics of the urinary system in quadruped and bipedal mammals.     

Structure of the musculature of the chicken small intestine

Summary The small intestine of the chicken was studied by light and electron microscopy. The musculature, measuring about 180 m in thickness in the distended intestine, consists of four layers (outer longitudinal, outer circular, inner circular and inner longitudinal) which are directly apposed to one another. There is no layer of connective tissue equivalent to the submucosa of mammalian intestine, and the intestinal glands lie close to the inner longitudinal muscle. Mucosal folds are not formed during isotonic contraction of the intestine. The muscle cells of the chicken small intestine are characterized by large, numerous and sharply outlined dense bodies, by the presence of an extremely thin basal lamina, by prominent dense bands at the cell surface but relatively few intermediate junctions. There are many areas of direct apposition between cell membranes of adjacent cells and little collagen between the muscle cells. The four muscle layers have each distinctive structural features. Gap junctions between muscle cells occur only in the outer circular layer. The outer circular and outer longitudinal layers are closely apposed and numerous junctions of the adherens type link cells of the two layers. Intramuscular blood capillaries are rare and are found virtually only in the outer circular layer; their endothelial cells are joined by tight junctions. In the outer circular layer (but not in the other layers) there are two further cell types, fibroblasts and interstitial cells, which can be clearly distinguished from one another. The latter cells are intimately related to nerve bundles and are connected by gap junctions to some muscle cells.     

Distribution of vagal CGRP-immunoreactive fibers in the lower esophagus and the cardia of the stomach of the rat

We have examined whether the smooth muscle fibers in the lower esophagus and the cardia of the stomach of the rat are innervated by calcitonin gene-related peptide-immunoreactive (CGRP-ir) fibers coming from the nucleus ambiguus. Immunohistochemical observations revealed that there were many CGRP-ir fibers and free endings in all external muscular layers of the lower esophagus and the cardia. Occasionally, bundles of CGRP-ir fibers were found in the inner oblique muscle layer of the cardia. There were also many CGRP-ir fibers in the mucous membrane in the lower esophagus and the cardia. When Fluorogold was injected into the junction of the lower esophagus and the cardia, many retrogradely labeled neurons were found in the compact formation of the nucleus ambiguus and the dorsal motor nucleus of the vagus nerve. Double labeling with immunohistochemistry for CGRP and the retrograde tracer Fluorogold showed that almost all of neurons (more than 90%) in the nucleus ambiguus that project to the lower esophagus or the cardia contained CGRP, while no CGRP-ir neurons were found in the dorsal motor nucleus of the vagus nerve. These results indicate that the vagal motor neurons of the nucleus ambiguus that contain CGRP project not only to the striated muscle fibers of the esophagus but also to the smooth muscle fibers of the external muscle layers of the lower esophagus and the cardia.     

Morphological and functional characteristics of the urogenital diaphragm in women

Macroscopical study of muscle layer of urogenital diaphragm in women has revealed two muscles situated between its superior and inferior fasciae--m. transversus perinei profundus (MTPP) and sphincter urethrae (SU),--which are separated by connective tissue. In females, SU is not ring-shaped, but is an arched structure situated in front of urethral lumen intertwining into urethral outer circular muscular layer. MTPP consists of three groups of bundles: anterior, medium and posterior. Anterior bundles of MTPP are periurethral and directly interlace with urethral muscular wall. Medium and posterior bundles of this muscle do not reach the urethral wall and act on the muscular wall of vagina. The study of histotopographic sections of urethral muscular wall made from the level of neck of the urinary bladder to the urogenital diaphragm, showed that the urethral outer circular muscular layer contains both bundles of smooth muscle cells and striated muscle fibers which ascend from urogenital diaphragm attaining an oblique-circular course. Major part of urethral striated muscle fibers, as well as SU and MTPP fibers belong to "red", slow-twitch, tonic fiber type.     

Histo-topographic study of the longitudinal anal muscle

The longitudinal anal muscle (LAM) has been described as a vertical layer of muscular tissue interposed between the circular layers of the internal (IAS) and external (EAS) anal sphincters. There is, however, no general agreement in the literature on its composition and attachments. The aim of this study was to investigate the histological structure, attachments, and topography of the LAM in order to evaluate its role in continence and defecation, thus enhancing knowledge of the surgical anatomy of this region. After in situ formalin fixation, the pelvic viscera were removed from eight male and eight female cadavers (age range: 52-72 years). Serial macrosections of the bladder base, lower rectum and anal canal, cervix and pelvic floor complex, cut in the transverse (six specimens) and coronal (six specimens) planes, underwent histological and immunohistochemical studies. Four specimens were studied using the E12 sheet plastination technique. The LAM was identified in 10/12 specimens (83%). Transverse and coronal sections made clear that it is a longitudinal layer of muscular tissue, marking the boundary between the internal and external anal sphincters. From the anorectal junction it extends along the anal canal, receives fibers from the innermost part of the puborectalis and the puboanalis muscles, and terminates with seven to nine fibro-elastic septa, which traverse the subcutaneous part of the external anal sphincter, reaching the perianal dermis. In the transverse plane, the mean thickness of the LAM was 1.68 +/- 0.27 mm. Immunohistochemical staining showed that the LAM consists of predominantly outer striated muscle fibers and smaller numbers of inner smooth muscle fibers, respectively coming from the levator ani muscle and from the longitudinal muscular layer of the rectum. The oblique fibers suggest that the LAM may represent the intermediate longitudinal course of small bridging muscle bundles going reciprocally from the striated EAS to the smooth IAS and vice versa. The spatial result is the helical course of striated and smooth muscle fibers between the EAS and IAS, which contribute not only to the narrowing but also to some shortening of the anal canal during sphincter contraction. Thus, rather than being a boundary, the LAM gives anatomical evidence of a functional connection between two muscle systems with different structures and topography.     

The Distribution of Muscles Fibers and Their Types in the Female Rat Urethra: Cytoarchitecture and Three‐Dimensional Reconstruction

An attempt to explore urethral cytoarchitecture including the distribution of smooth muscles and fast and slow striated muscles of adult female Sprague Dawley rat—a popular model in studying lower urinary tract function. Histological and immunohistochemical stainings were carried out to investigate the distribution of urethral muscle fibers and motor end plates. The urethral sphincter was furthermore three‐dimensionally reconstructed from serial histological sections. The mucosa at the distal urethra was significantly thicker than that of other segments. A prominent inner longitudinal and outer circular layer of smooth muscles covered the proximal end of urethra. Thick circular smooth muscles of the bladder neck region (urethral portion) decreased significantly distalward and longitudinal smooth muscles became 2‐ to 3‐fold thicker in the rest of the urethra. An additional layer of striated muscles appeared externally after neck region (urethra) and in association with motor end plates ran throughout the remaining urethra as the striated sphincter layer. Most striated muscles were fast fibers while relatively fewer slow fibers often concentrated at the periphery. A pair of extraneous striated muscles, resembling the human urethrovaginal sphincter muscles, connected both sides of mainly the distal vagina to the dorsal striated muscles in the wall of the middle urethra. The tension provided by this pair of muscles, and in conjunction with the striated sphincter of the urethral wall, was likely to function to suspend the middle urethra and facilitates its closure. Comprehensive morphological data of urethral sphincter offers solid basis for researchers conducting studies on dysfunction of bladder outlet. Anat Rec, 296:1640–1649, 2013. © 2013 Wiley Periodicals, Inc.     

食管胃连接处的肌纤维排列形式

观察３０例成人尸体食管胃连接处的肌纤维排列形式，该处肌肉分为浅，深两层。浅层肌为纵简装排；虺支肌为环斜排列。在“食管前庭”上端的浅层，可见两条肌束，其肌纤维为管状食管纵肌的延续，斜行向下移行为胃纵肌。深层的环形肌纤维在管状食管与前庭连接处增厚，位于贲门切迹上方２７。０６±２。１１ｍｍ外。在“食管前庭”中段，其深层环形肌纤维移行为螺旋形和扣状排列形式。在“食管前庭”末端，两条半环形肌束分别位于胃底测     

Late embryonic and postnatal development of interstitial cells of cajal in mouse esophagus: distribution, proliferation and kit dependence

This paper investigates alterations in interstitial cells of Cajal (ICC) in the esophagus of mice from embryonic day 13.5 (E13.5) to 36 days postpartum (P0-P36) using immunohistochemistry. At E13.5, Kit+ cells presented in clusters and differentiated into spindle-like cells with biopolar processes within the outer (longitudinal) and inner (circular) muscle layers at E17.5. These Kit+ ICC with long processes were also Ano1+ and prominent at birth. The density of ICC gradually decreased, and at P36 it became about one twentieth of that at birth. Kit ligand (stem cell factor) expression is lower in striated muscle cells than that in smooth muscle cells. The ICC number was higher in the distal (close to the cardia) than in the proximal esophagus (close to the pharynx). Some Kit+/Ki67+ and Kit+/bromodeoxyuridine+ cells were observed within the muscle layers, and proliferation persisted from birth through adulthood (P28) with a gradually decreasing cell number. At 24 h, Kit+ ICC were dramatically decreased and almost missing 48 h after administration of imatinib (a Kit inhibitor). Our results indicate that ICC proliferation is age dependent and persists throughout the postnatal period. There is a dramatic decrease in the ICC number from P0 to adult life. The Kit signal is essential for the postnatal development of ICC in the esophagus.     

Expression of alpha-smooth muscle actin as special and morphometric assessment in the small intestine during the postnatal development in hamster

Immunoreactivity of alpha-smooth muscle actin (ASMA), along, with morphometric values in the hamster small intestine, was examined during postnatal development. Twelve healthy hamsters were divided into three age groups of 2·5 months (group 1), 3 months (group 2), and 4 months (group 3). For morphometrical study, routine tissue processing was carried out by autotechnicon tissue processor, the processed tissues were embedded and sectioned serially into 5-μm thick sections. The sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed by primary antibodies against alpha-smooth muscle actin (ASMA). All samples showed ASMA expression in the muscularis mucosa and circular and longitudinal layers of muscularis propria. The small intestine samples in group 1 showed minimal ASMA expression in total muscular layers. Alpha-smooth muscle actin labeling was increased in groups 2 and 3 in the muscularis mucosa and both layers of inner and outer circular muscle as compared to group 1. A more intense ASMA expression was observed in the longitudinal layers in both groups 2 and 3. The morphometrical results showed that there was a smaller increase in thickness of the mucosa, submucosa, and muscular layers from group 1 to group 2 that was followed by a larger increase between groups 2 and 3. There were significant increases in thickness of all morphometrical parameters (mucosa, submucosa, and muscularis) in groups 2 and 3 in comparison with group 1, and also group 3 compared with group 2. In groups 2 and 3, thickness of the submucosa and muscular layers were significantly higher in the jejunum compared with the ileum.     

Neuronal substance P in the esophagus. Distribution and effects on motor activity

Substance P-immunoreactive nerve fibres were fairly numerous in the lower esophagus of the guinea-pig and cat but few in the pig. They were particularly numerous in the myenteric and submucosal plexuses but could be detected also in the circular and longitudinal smooth muscle and in the muscularis mucosae. Only in the cat were SP-immunoreactive cell bodies detected, albeit in low number, in the myenteric plexus. Radioimmunoassay showed that the lower part of the cat esophagus contained approximately 10 times more immunoreactive SP than the upper part and that the muscle layer contained more SP than the mucosa. Motor effects of synthetic SP were studied on segments from circular smooth muscle of cat esophagus. SP contracted the smooth muscle and enhanced the response to electrical stimulation. These effects of SP could be blocked by the specific SP antagonist (d -Pro,²d -Trp^7,8)-SP. The contractile response to electrical stimulation could be blocked by the cholinergic muscarinic blocker atropine and the opiate receptor agonist leu-enkephalin but not by the SP antagonist or by adrenergic blockers. Hence, the results suggest that cholinergic neurons innervate the circular smooth muscle, and that opiate receptor agonists suppress transmission in these neurons. Neuronal SP in the esophagus may serve to enhance the contractile responses of esophageal smooth muscle.     

Mechanical activity of frog esophagus muscle in response to electrical stimulation of intramural nerves.

Microscopic observation of intramural nerves in the frog esophagus, fixed and stained with OsO(4) and ZnI(2), revealed that nerve cell bodies and bundles connecting the nerve cell bodies formed loose and irregular networks. The nerve cell bodies were mostly lying singly in the nerve bundles, with occasional observations of two closely linked nerve cell bodies. Isolated circular and longitudinal segments of esophageal muscle were spontaneously rhythmically contractile, with a frequency of 2.2-3.0 per min. This was not altered by tetrodotoxin (TTX). In longitudinal muscle segments, transmurally applied electrical stimulation produced contractile responses which were not inhibited by atropine or guanethidine, but were reduced in amplitude by TTX, suggesting a nonadrenergic-noncholinergic (NANC) excitatory innervation in the esophagus muscle. In circular muscle segments, transmural application of brief electrical stimulation evoked two types of mechanical response: a biphasic response consisting of an initial relaxation and a following contraction (type I) and a contraction alone (type II). These mechanical responses were not modulated by either atropine or guanethidine. In the type I response, TTX abolished the relaxation component, suggesting that this was produced by non-adrenergic non-cholinergic (NANC) inhibitory nerve excitation. In about half of the type II responses, the amplitude of the contraction was significantly reduced by TTX, suggesting that a part of the contraction was produced by activation of NANC excitatory nerves. Thus, the esophageal smooth muscle of the frog demonstrates myogenic activity, and is innervated by both excitatory and inhibitory NANC nerves.     

Projections of substance P, vasoactive intestinal peptide and tyrosine hydroxylase immunoreactive nerve fibres in the canine intestine, with special reference to the innervation of the circular muscle.

Antisera raised against neuron specific enolase (NSE), substance P, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) were used to reveal nerve fibres in the wall of the canine small and large intestine. The circular muscle of the colon was innervated by nerve fibre bundles that ran parallel to the muscle throughout its thickness. A plexus of fibre bundles was found against the inner (submucosal) surface of the circular muscle. Fibres with substance P, VIP and TH immunoreactivity all contributed to this innervation. The circular muscle of the small intestine was distinctly separated into outer and inner layers by a dense plexus of nerve fibres, the deep muscular plexus. The outer and inner circular muscle were innervated by substance P, VIP and TH fibres. Extrinsic denervation through the severing of nerve fibres in the mesentery caused TH fibres in the intestine to degenerate, but had no detectable effect on the fibres with substance P or VIP immunoreactivity. Myectomy (the removal of the myenteric plexus from the full circumference of the intestine over a distance of 2-3 cm), performed 7-13 days before tissue was taken, resulted in an almost complete loss of substance P fibres from the circular muscle of the colon and the outer circular muscle of the small intestine. However, many fibres persisted in the deep muscular plexus of the small intestine, and most fibres remained in its inner circular muscle. The changes in distribution of VIP fibres were almost identical, except that a small proportion of reactive fibres remained in the circular muscle of the colon and the outer circular muscle of the small intestine. It is concluded that the circular muscle layers of the small intestine and colon have dual sources of intrinsic nerve supply: the myenteric ganglia supply fibres primarily to the outer part of the muscle and the submucous ganglia supply fibres to the inner muscle. The present study further demonstrated that VIP fibres ran anally in the myenteric plexus of both the small and large intestine, whereas substance P fibres ran orally in the large intestine and both orally and anally in the small intestine. The innervation of the muscularis mucosae and mucosa by substance P and VIP fibres was not affected by myectomy or extrinsic denervation, and these structures are therefore likely to be innervated by nerve cells in the submucous ganglia.