Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Topography, architecture and structure of the plexus submucosus externus (Schabadasch) of the porcine small intestine in scanning electron microscopy

Whole-mount preparations of the porcine small intestine, consisting of the tela submucosa and the adjacent lamina muscularis mucosae, were used for scanning electron-microscopic investigation of the plexus submucosus externus (Schabadasch) after enzymatic digestion, fixation and HCI hydrolysis. The present results confirm previous light-microscopic data and provide irrefutable proof that within the submucosal plexus, considered by most authors as one ganglionated nerve plexus situated in the entirety of the tela submucosa, two distinct nerve meshworks can be distinguished, one lying close to the lamina muscularis mucosae, i.e., the plexus submucosus internus (Meissner), and the other, i.e., the plexus submucosus externus (Schabadasch), situated in the outer region of the tela submucosa against the circular smooth muscle layer. In addition to the distinct location of both plexuses, they are quite different with regard to the pattern and diameter of their nerve strands and the number and appearance of their ganglia.     

Calcitonin gene-related peptide-like immunoreactivity in the human small intestine.

Calcitonin-gene-related-peptide (CGRP)-like immunoreactivity was localized in nerve fibres, neuronal somata and in mucosal endocrine cells of the human small intestine. Immunoreactive enteric neurons were more numerous in the submucous plexuses than in the myenteric plexus. Morphologically, they predominantly had the appearance of type II neurons. The majority of the CGRP-like immunoreactive nerve fibres ran within the ganglionic nerve plexuses. Only a small proportion could be observed in the lamina propria, the lamina muscularis mucosae, or the circular and longitudinal outer smooth muscle layer. These findings suggest that within the wall of the human small intestine neuronal CGRP of either extrinsic or intrinsic origin exerts its effect chiefly on other enteric neurons, and might be indirectly involved in the regulatory functions of the human small intestine.     

Distinct distribution of CGRP-, enkephalin-, galanin-, neuromedin U-, neuropeptide Y-, somatostatin-, substance P-, VIP- and serotonin-containing neurons in the two submucosal ganglionic neural networks of the porcine small intestine

Summary In addition to differences between the two submucosal ganglionic neural networks, i.e., the plexus submucosus externus (Schabadasch) and the plexus submucosus internus (Meissner), with respect to the occurrence and distribution of serotonin as neurotransmitter, immunocytochemistry also revealed a distinct distribution for various neuropeptides in these two plexuses. Immunoreactivity for galanin, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, neuromedin U, enkephalin, somatostatin and neuropeptide Y was found in varicose and non-varicose nerve fibres of both submucosal ganglionic plexuses, albeit with a distinct distributional pattern. The difference in neurotransmitter and/or neuromodulator content between both neural networks became even more obvious when attention was focussed on the immunoreactivity of the nerve cell bodies for these substances. Indeed, neuropeptide Y, enkephalin-and somatostatin-immunoreactive neuronal perikarya as well as serotonergic neuronal cell bodies appear solely in the plexus submucosus externus. Neuromedin U-immunoreactive perikarya, mostly coexisting with substance P, are observed in large numbers in the plexus submucosus internus, whilst they are rare in the plexus submucosus externus. Double-labelling immunostaining for substance P with CGRP and galanin revealed a different coexistence pattern for the two submucosal ganglionic plexuses. The differing chemical content of the neuronal populations supports the hypothesis that the existence of the two submucosal ganglionic plexuses, present in most large mammals including man, not only reflects a morphological difference but also points to differentiated functions.     

Oxytocin is expressed by both intrinsic sensory and secretomotor neurons in the enteric nervous system of guinea pig

Single- and double-immunostaining techniques were used systematically to study the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. Oxytocin immunoreactivity was distributed widely in the guinea pig gastrointestinal tract; 3%, 13%, 17%, 15%, and 10% of ganglion neurons were immunoreactive for oxytocin in the myenteric plexuses of the gastric corpus, jejunum, ileum, proximal colon, and distal colon, respectively, and 36%, 40%, 52%, and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of the jejunum, ileum, proximal colon, and distal colon, respectively. In the myenteric plexus, oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbindin 28 K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secretomotor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer possibly arose from the myenteric oxytocin-ir neurons, and oxytocin-ir nerve fibers in the mucosa possibly arose from both the myenteric and submucosal oxytocin-ir neurons. Thus, oxytocin in the digestive tract might be involved in gastrointestinal tract motility mainly via the regulation of the inner circular muscle and the balance of the absorption and secretion of water and electrolytes.     

Fluorescence microscopic study of the architecture and structure of an adrenergic network in the plexus myentericus (Auerbach), plexus submucosus externus (Schabadasch) and plexus submucosus internus (Meissner) of the porcine small intestine

The distribution of adrenergic fibres in the ganglionated plexuses of the porcine small intestine has been made on air-dried stretch preparations using the glyoxylic acid fluorescence method. Adrenergic fluorescent fibres occur in the ganglia and internodal strands of the three fundamental ganglionated plexuses: the myenteric plexus (Auerbach) and the two superimposed meshworks of the plexus submucosus , i.e. the plexus submucosus externus ( Schabadasch ) and the plexus submucosus internus (Meissner). The plexus Auerbach consists of densely glyoxylic acid induced fluorescent (GIF) elongated ganglia with in general a longitudinal axis running parallel to the circular muscle layer and large dense interconnecting fibre tracts with primary, secondary and tertiary subdivisions. In the ganglia, the fibres are varicose, forming large fluorescent 'baskets' which might be related to the occurrence of well defined enteric neurones. The plexus Schabadasch can be distinguished from the plexus Meissner by its size, strongly fluorescent ganglia and broad densely fluorescent internodal strands. The pattern of fluorescing ring-like formations at the margin and out of the nodes, clearly present in the Auerbach and Schabadasch plexuses, completely lack in the plexus Meissner, the latter being narrow-meshed with smaller fluorescent 'baskets', indicating that the corresponding neurones are smaller in size. In the ganglionic nodes of all three plexuses the axons display comparatively more varicosities than in the fibre tracts. Each of the three main ganglionated enteric plexuses are quite different with regard to the pattern of the adrenergic network both in the ganglia and in the strands.     

Distribution of NADPH Diaphorase-Positive Neurons in the Enteric Nervous System of the Rabbit Intestine

Nitric oxide (NO) has been proposed as an inhibitory transmitter in gastrointestinal muscle relaxation. We analyzed the distribution of nitric-oxide producing neurons in the rabbit intestine through nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. By this reliable and convenient method, we visualized neuronal nitric-oxide-synthase, the enzyme responsible for nitric oxide generation, in the rabbit intestine. In the ileum and rectum, nitric-oxide-synthase-related diaphorase activity was present in the myenteric plexus ganglion cells, and in the nerve fibers in the internodal strand, secondary, and tertiary plexuses. These fibers were particularly abundant in the deep circular rather than in the outer longitudinal muscle layer. In the inner submucosal plexus, we found scarce labeled neurons. Labeled neural somata showed a range of sizes and shapes suggesting different functional roles. The present basic information is required to use the rabbit as an experimental animal in neurochemical NO enteric research.     

NO-Ergic Innervation of the Intestine of the Snow Sculpin Myoxocephalus brandti

Distribution, localization, and morphological peculiarities of NO-ergic nerve cells in the intestine of the snow sculpin Myoxocephalus brandti (Cottidae family) were studied using histochemical staining for NADPH-diaphorase ( NADPH-d). These cells were shown to be present in the pyloric appendages, middle and posterior parts of the intestine and in its rectal part. The NO-ergic cells are the most numerous in the myenteric plexus and circular muscle layer of all studied parts of the intestine. Single NO-ergic nerve cells are revealed in the submucosal plexus of pyloric appendages, middle and posterior parts of the intestine. No NO-ergic neural cells were found in subserosal and subepithelial plexuses, longitudinal layer of smooth muscle in all studied parts, and in the submucosal plexus of the rectal part of the intestine.     

Topography, architecture and structure of the plexus submucosus internus (Meissner) of the porcine small intestine in scanning electron microscopy

Scanning electron microscopy of whole-mount preparations of the tela submucosa in the porcine small intestine, examined after trypsin digestion, fixation and HCl hydrolysis, visualized a clear differentiation of the submucosal plexuses, i.e., the plexus submucosus internus (Meissner) and the plexus submucosus externus (Schabadasch). The distinctive features refer to the topography, number, size and shape of the ganglia and the number and diameter of the nerve strands. The plexus of Meissner is closely apposed to the external surface of the lamina muscularis mucosae by the enveloping connective tissue and by connecting strands penetrating the lamina muscularis mucosae. Three distinctive subdivisions of connecting strands can be identified. Since the glial cells covering the ganglia and connecting strands have been preserved, neither individual neuronal cells nor axons can be observed.     

Development of the submucous plexus in the large intestine of the mouse

In the small intestine of both embryonic birds and mammals, neuron precursors aggregrate first at the site of the myenteric plexus, and the submucous plexus develops later. However, in the large intestine of birds, the submucosal region is colonised by neural-crest-derived cells before the myenteric region (Burns and Le Douarin, Development 125:4335-4347, 1998). Using antisera that recognize undifferentiated neural-crest-derived cells (p75NTR) and differentiated neurons (PGP9.5), we examined the colonisation of the murine large intestine by neural-crest-derived cells and the development of the myenteric and submucosal plexuses. At E12.5, when the neural crest cells were migrating through and colonising the hindgut, the hindgut mesenchyme was largely undifferentiated, and a circular muscle layer could not be discerned. Neural-crest-derived cells migrated through, and settled in, the outer half of the mesenchyme. By E14.5, neural-crest-derived cells had colonised the entire hindgut; at this stage the circular muscle layer had started to differentiate. From E14.5 to E16.5, p75NTR- and PGP9.5-positive cells were observed on the serosal side of the circular muscle, in the myenteric region, but not in the submucosal region. Scattered, single neurons were first observed in the submucosal region around E18.5, and groups of neurons forming ganglia were not observed until after birth. The development of the enteric plexuses in the murine large intestine therefore differs from that in the avian large intestine.     

Avian enteric nerve plexuses

Summary The enteric nerve plexuses of the domestic fowl (Gallus domesticus) were investigated in sections and stretch preparations by means of the cholinesterase and glyoxylic acid fluorescence histochemical techniques. Cholinesterase-positive and varicose and non-varicose fluorescent nerve fibres were distributed at all levels of the gut in myenteric, submucosal, muscle and mucosal plexuses, and in a perivascular plexus. The density of the innervation and the detailed distribution of the nerves varied in different parts of the intestinal tract. All nerve plexuses appeared to be best developed in the rectum. Whereas the circular muscle coat contained a substantial number of nerves at all levels of the gut, the longitudinal coat was well innervated only in the rectum. The major portion of the mucosal plexus appeared to be associated with the intestinal glands. The nerve cell bodies were restricted to the myenteric and submucosal plexuses and were mainly cholinesterase-positive. Fluorescent ganglion cells were not observed. Pretreatment of stretch preparations with NADH: Nitro BT to stain ganglion cells showed that the majority of the cells were surrounded by a meshwork of fluorescent varicose fibres, although none of the fibres appeared to be associated with individual cells. The perivascular plexus was mainly associated with the arteries. The functional significance of the innervation is discussed.We would like to thank the British Council for financial support for Mr. H.A. Ali     

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).     

Structural organization and neuropeptide distributions in the equine enteric nervous system: an immunohistochemical study using whole-mount preparations from the small intestine

The architecture and neurochemistry of the enteric nervous system was studied by use of whole-mount preparations obtained by microdissection of the horse jejunum. A myenteric plexus and two plexuses within the submucosa were identified. The external submucosal plexus lying in the outermost region of the submucosa had both neural and vascular connections with the inner submucosal plexus situated closer to the mucosa. Counts of neurones stained for NADH-diaphorase demonstrated the wide variation in size, shape and neurone content of individual ganglia in both the external and internal submucosal plexuses. The average number of cells/ganglion was similar in each plexus (about 25 cells). Immunoreactivities for galanin, vasoactive intestinal peptide and neuropeptide Y were observed in nerve cell bodies and fibres of each of the plexuses. Immunoreactivity for substance P was extensive and strong in nerve fibres of all plexuses but was weaker in cell bodies of the submucosal neurones and absent in the cell bodies of the myenteric plexus. Comparative quantitative analysis of immunoreactive cell populations with total cell numbers (enzyme staining) was indicative of neuropeptide colocalization in the external submucosal plexus.     

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

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

Two submucosal nerve plexus in human intestines

We examined the architecture of human submucosal nerve networks of gut segments derived from 12 individuals (each six from small and large intestines). Twelve undivided submucosal wholemounts were prepared and immunohistochemically stained for peripherin (nerve elements) and for α-smooth muscle actin (remnants of attached muscle bundles). We found two ganglionic nerve networks. The plexus submucosus externus was generally monolayered and located under the outermost surface of the submucosal wholemounts. Its nerve fibre strands frequently joined each other in acute or obtuse angles, the meshes of the network were relatively wide and frequently polyangular shaped. The plexus submucosus internus was generally multi-(mostly two- or three-)layered and occupied at least the inner half of the thickness of the wholemount, sometimes extending abluminally beyond the great submucosal vessels. Its meshes were irregular. The shapes of ganglia of the two plexus were generally different, those of the internal plexus were frequently grape-like whereas the neurons of external ganglia were mostly embedded in the contoures of the joining nerve fibres. Both plexus were intensely connected via coiled interconnecting strands, either with or without intercalated ganglia. For use of eponyms for two different submucosal plexus, the names of Meissner (inner) and Schabadasch (outer) are historically justified.     

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm² for myenteric neurons, 750 μm² for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons. This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.     

Morphology of the interstitial cells of Cajal of the human ileum from foetal to neonatal life

The so-called interstitial cells of Cajal myenteric plexus (ICC-MP), interstitial cells of Cajal intramuscular (ICC-IM) and interstitial cells of Cajal deep muscular plexus (ICC-DMP) are the three types of ICC endowed within the intestinal muscle coat where they play different roles in gut motility. Studies on ICC ontogenesis showed ICC-MP in the human ileum by 7-9 weeks while information on ICC-IM and ICC-DMP in foetuses and newborns are not exhaustive. Functional recordings in the fasting state of prematurely born babies aged 28-37 weeks showed immature ileal motility. To gain more information on the time of appearance of the three ICC types in the human ileum and on the steps of the acquisition of mature features, we studied by c-kit immuno-histochemistry foetuses aged 17-27 weeks and newborns aged 36-41 weeks. In parallel, the maturative steps of enteric plexuses and muscle layers were immunohistochemically examined by using anti-neuron specific enolase (NSE), anti-S-100 and anti-alpha smooth muscle actin (alphaSMA) antibodies. The appearance and differentiation of all the ICC types were seen to occur in concomitance with those of the related nerve plexuses and muscle layers. ICC-MP appeared first, ICC-IM and ICC-DMP later and their differentiation was incomplete at birth. In conclusion, the ICC-MP, the intestinal pacemaker cells, in spite of absence of food intake, are already present during the foetal life and the ICC-IM appear by pre-term life, thus ensuring neurotransmission. The ICC-DMP and their related nerve plexus and smooth muscle cells, i.e. the intestinal stretch receptor, begin to differentiate at birth. These findings might help in predicting neonatal ileal motor behaviour and in interpreting the role of ICC abnormalities in the pathophysiology of intestinal motile disorders of neonates and young children.     

Immunohistochemical identification of serotonin and noradrenalin containing structures within the nerve plexuses of rat ileum

The distribution of 5-hydroxytryptamine (= serotonin = 5-HT) and noradrenalin (NA) in the enteric plexuses of the rat ileum was studied using immunocytochemical techniques. 5-HT-like immunoreactive fibers were observed only in the myenteric plexus, surrounding the ganglionic cells, which are all unreactive. NA-like immunoreactive fibers were present in all layers of the ileum: in the myenteric plexus, they were localized in the nodes, forming a network all round the neuronal perikarya; in the Meissner plexus, positive axons were arranged in a delicate network; submucosal blood vessels were often provided by NA-immunopositive nerve plexus. In the inner circular muscle layer the immunoreactive NA-positive fibers run within nerve bundles mainly parallel with the smooth muscle cells. The 5-HT immunoreactive material was depleted by treatment with reserpine; depletion of NA by 6-hydroxy-dopamine was also observed; on the contrary, no depletion of 5-HT by 5,7-dihydroxytryptamine was obtained. To confirm the validity of these results, specific antibodies to tyrosine hydroxylase (TH) and aromatic 1-aminoacid-decarboxylase (AADC), two enzymes involved in the synthesis of catecholamines, were used. In conclusion these experiments indicate that 5-HT is present, probably as a transmitter, in certain fibres of the rat myenteric plexus, distributed in a way similar to that of NA-containing fibers. However, at variance with NA fibers, 5-HT fibers are not present in other regions of the intestine wall.     

Somatostatin, substance P and calcitonin gene-related peptide-positive intramural nerve structures of the human large intestine affected by carcinoma

The aim of this study was to investigate the arrangement and chemical coding of enteric nerve structures in the human large intestine affected by cancer. Tissue samples comprising all layers of the intestinal wall were collected during surgery form both morphologically unchanged and pathologically altered segments of the intestine (n=15), and fixed by immersion in buffered paraformaldehyde solution. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against somatostatin (SOM), substance P (SP) and calcitonin gene-related peptide (CGRP). The microscopic observations revealed distinct morphological differences in the enteric nerve system structure between the region adjacent to the cancer invaded area and the intact part of the intestine. In general, infiltration of the cancer tissue resulted in the gradual (depending on the grade of invasion) first decomposition and reduction to final partial or complete destruction and absence of the neuronal elements. A comparative analysis of immunohistochemically labeled sections (from the unchanged and pathologically altered areas) revealed a statistically significant decrease in the number of CGRP-positive neurons and nerve fibres in both submucous and myenteric plexuses in the transitional zone between morphologically unchanged and cancer-invaded areas. In this zone, a decrease was also observed in the density of SP-positive nerve fibres in all intramural plexuses. Conversely, the investigations demonstrated statistically insignificant differences in number of SP- and SOM-positive neurons and a similar density of SOM-positive nerve fibres in the plexuses of the intact and pathologically changed areas. The differentiation between the potential adaptive changes in ENS or destruction of its elements by cancer invasion should be a subject of further investigations.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.