Peptide-containing innervation of the human intestinal mucosa. An immunocytochemical study on whole-mount preparations

The three-dimensional distribution of the peptide-containing innervation in the human intestinal mucosa was studied by fluorescence immunohistochemistry on whole-mount mucosal preparations. An extensive VIP-immunoreactive nerve supply was demonstrated at all levels, but was markedly increased in density in the distal intestine, where it formed a particularly rich network in close contact with the luminal epithelium. In contrast, substance P-containing nerve fibres formed a looser and evenly distributed innervation at all levels. The muscularis mucosae was richly supplied by VIP- and substance P-containing fibres. Met-enkephalin immunoreactivity was confined to a few scattered nerve bundles running in the muscularis mucosae and around the bottom of epithelial crypts.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Substance P-containing nerve fibres in large peripheral blood vessels of the rat

Substance P-immunoreactive nerve fibres were localized by the indirect immunohistochemical method in the adventitia and the adventitial-medial border of large peripheral arteries and veins of the rat. Arteries showed a richer substance P-containing innervation than veins. The superior mesenteric artery was densely innervated, whereas no substance P-containing fibres were found around the carotid artery. Substance P produced a vasoconstriction of the veins, but was basically without effect on arteries, although with the carotid artery a dose-dependent relaxation was observed. The absence of a correlation between the degree of innervation of the blood vessels and their responsiveness to exogenous substance P suggests that there nerves do not subserve a vasomotor function. The depletion of substance P immunoreactivity from nerves in arteries and veins by capsaicin suggest that substance P-containing vascular nerves are primarily sensory in nature.     

The distribution and origin of calcitonin gene-related peptide-containing nerve fibres in feline dental pulp

Summary The origin and distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in feline dental pulp were studied using indirect immunofluorescence. Nerve fibres with varicosities exhibiting CGRP-like immunoreactivity were observed to enter the pulp with blood vessels. Many CGRP-containing nerve fibres were found to extend along blood vessels in the central pulp, and some of these fibres exhibited a network arrangement in the walls of dental pulp blood vessels. However, some of fibres were apparently not associated with blood vessels. Some thin, CGRP-containing nerve fibres formed a part of the nerve plexus in the subodontoblastic area and penetrated into the odontoblastic layer. In animals that had undergone transection of the inferior alveolar nerve, no CGRP-containing nerve fibres were observed. Application of a double-immunofluorescence staining technique also revealed that the distribution of CGRP-containing nerve fibres is very similar to that of substance P-containing nerve fibres.     

The distribution and origin of calcitonin gene-related peptide-containing nerve fibres in feline dental pulp. Relationship with substance P-containing nerve fibres

The origin and distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in feline dental pulp were studied using indirect immunofluorescence. Nerve fibres with varicosities exhibiting CGRP-like immunoreactivity were observed to enter the pulp with blood vessels. Many CGRP-containing nerve fibres were found to extend along blood vessels in the central pulp, and some of these fibres exhibited a network arrangement in the walls of dental pulp blood vessels. However, some of fibres were apparently not associated with blood vessels. Some thin, CGRP-containing nerve fibres formed a part of the nerve plexus in the subodontoblastic area and penetrated into the odontoblastic layer. In animals that had undergone transection of the inferior alveolar nerve, no CGRP-containing nerve fibres were observed. Application of a double-immunofluorescence staining technique also revealed that the distribution of CGRP-containing nerve fibres is very similar to that of substance P-containing nerve fibres.     

Synaptic connections of substance P-immunoreactive nerve terminals in the substantia nigra of the rat

Summary A monoclonal antibody that recognises the C-terminal part of substance P was used to study immunoreactive structures in the substantia nigra by the unlabeled antibody, peroxidase-antiperoxidase procedure. Immunoreactivity was present in nerve fibres in all parts of the substantia nigra, particularly in the pars reticulata and pars lateralis. Electron microscopically two types of bouton immunoreactive for substance P were found: Type 1 contained large electron-lucent vesicles, occasional large granulated vesicles and formed symmetrical synapses with dendrites. Type 2 boutons contained smaller, round electron-lucent vesicles, many large granular vesicles and formed asymmetrical synapses (having prominent postjunctional dense bodies) with dendrites and perikarya.Immunoreactive fibres with varicosities that had been identified light microscopically were studied in serial sections in the electron microscope. Each identified varicosity contained synaptic vesicles and formed a single synapse. An individual fibre formed boutons of only one kind (type 1 or type 2) and could form multiple synapses with the same neuron. Thus, an identified fibre in the pars compacta had eight varicosities, each of which was in synaptic contacts (type 2) with the dendrites or soma of the same neuron.The results are consistent with the concept that substance P is a synaptic transmitter in the substantia nigra and indicate that neurons in this region may receive a significant input from substance P-containing afferents, and that there are at least two types of such afferent fibres.     

The differentiation of cytotoxic T lymphocytes in vitro

Summary Previous neurohistological studies have been extended to include the structures contained solely or mainly within the junctional esophageal segment which may play an important role in the sphincter mechanism. The main findings were: 1) a progressive cranio-caudal thickening of the muscularis mucosae; 2) a conspicuous thickening of the circular muscle layer; 3) abundant and close interconnections between the esophageal striated fibres and gastric smooth muscle cells; 4) presence of annulo-spiral elastic fibres coiled around bundles of striated musculature; 5) increase of the intramural nerve component, particularly Auerbach's plexus, which consisted of a continuous nervous layer containing twice as many neurocytes as found in the upper esophageal segments; 6) presence of numerous interconnected motor endplates often possessing ultraexpansional fibres and secondary endplates. The findings are discussed with emphasis on functional correlations in order to attain a unitary morpho-functional view.Abbreviations used LES lower esophageal sphincter - HPZ high pressure zone; mm: muscularis mucosae - CNS central nervous system - CCK-PZ cholecystokinin-pancreozymin Dedicated to Prof. Dr. Wolfgang Bargmann for his fundamental contributions to Comparative Morphology     

Projections of nitric oxide synthesizing neurons in the guinea-pig colon

The neuronal form of the enzyme nitric oxide synthase, which is an obligatory constituent of neurons that utilise nitric oxide as a transmitter, was revealed histochemically in this study by its ability to transfer a proton from reduced nicotinamide adenine dinucleotide phosphate to nitro-blue tetrazolium. In the guinea-pig colon, nitric oxide synthase was located in numerous irregularly-shaped myenteric neurons with single axons. In the submucosa, a small number of neurons had strong enzyme activity, whereas many were weakly stained. Nerve fibres were found in the longitudinal muscle, circular muscle, muscularis mucosae and ganglia of the two plexuses. No nerve fibres were found in the lamina propria of the mucosa. The same distribution of nerve cells and fibres was revealed using immunohistochemistry for nitric oxide synthase. Lesion studies showed that the axons of myenteric neurons all projected anally. Myenteric cells were the source of nerve fibres in the circular muscle and in more anally located myenteric ganglia. The sparse innervation of submucous ganglia was intrinsic to the submucous plexus. It is suggested that nitric oxide synthase is one of the transmitters of inhibitory neurons to the muscle and is also utilized by descending interneurons of the myenteric plexus.     

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.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.     

Cholinesterase histochemistry of the rabbit distal colon

Summary The distribution of cholinesterase in the rabbit distal colon has been studied using histochemical methods. Non-specific cholinesterase predominates in muscle tissue, the longitudinal layer of the muscularis externa staining slightly more strongly than the circular layer. The muscularis mucosae stains the most intensely but its non-specific cholinesterase is markedly inactivated by 20 hr fixation in formaldehyde. Specific cholinesterase is present in ganglion cells in the myenteric and submucous plexuses, and these cells vary in staining intensity. Nerve fibres which stain for specific cholinesterase are present in the nerve plexuses and in the muscle layers. It is concluded that there are cholinergic nervous elements in the myenteric and submucous plexuses and in the muscle layers of the rabbit distal colon.M. R. C. Junior Research Fellow.     

Projections of 5-hydroxytryptamine-immunoreactive neurons in guinea-pig distal colon

The presence of 5-hydroxytryptamine in enteric neurons of the guinea-pig distal colon was demonstrated by immunohistochemistry and the projections of the neurons were determined. 5-Hydroxytryptamine-containing nerve cells were observed in the myenteric plexus but no reactive nerve cells were found in submucous ganglia. Varicose reactive nerve fibres were numerous in the ganglia of both the myenteric and submucous plexuses, but were infrequent in the longitudinal muscle, circular muscle, muscularis mucosae and mucosa. Reactivity also occurred in enterochromaffin cells. Lesion studies showed that the axons of myenteric neurons projected anally to provide innervation to the circular muscle and submucosa and to other more anally located myenteric ganglia. The results suggest that a major population of 5-hydroxytryptamine neurons in the colon is descending interneurons, most of which extend for 10 to 15 mm in the myenteric plexus and innervate both 5-hydroxytryptamine and non-5-hydroxytryptamine neurons.     

Immunohistochemical correlation of human adrenal nerve fibres and thoracic dorsal root neurons with special reference to substance P

Applying a double-labelling immunofluorescence technique, six types of substance P-containing nerve fibres were distinguished in the human adrenal gland according to the immunohistochemical colocalization of (I) calcitonin gene-related peptide (CGRP), (II) cholecystokinin, (III) nitric oxide synthase, (IV) dynorphin, (V) somatostatin, and (VI) vasoactive intestinal polypeptide. Fibre populations I to IV in their mediator content resembled the respective subpopulations of primary sensory neurons in human thoracic dorsal root ganglia, while populations V and VI revealed no correspondence with dorsal root neurochemical coding. Nerve fibres with the combination substance P/nitric oxide synthase occurred only in the adrenal cortex, whereas all other fibre types were present in both cortex and medulla. As revealed by immuno-electron microscopy, substance P-immunolabelled axon varicosities (a) exhibited synaptic contacts with medullary chromaffin cells or with neuronal dendrites, (b) were directly apposed to cortical steroid cells and (c) were separated from fenestrated capillaries only by the interstitial space. These findings provide immunochemical support for an assumed sensory innervation of the human adrenal gland, and additionally suggest participation of substance P in efferent autonomic pathways. Furthermore, the results are indicative for a differentiated involvement of substance P in the direct and indirect regulation of neuroneuronal and neuroendocrine interactions.     

Diabetic state affects the innervation of gut in an animal model of human type 1 diabetes

Gastrointestinal symptoms in diabetic patients are commonplace, and are believed to be due, at least partly, to neuropathy of the gut. In the present study, therefore, some important neurotransmitters in the myenteric plexus were investigated in non-obese diabetic mice, an animal model of human type 1 diabetes. For this purpose, immunocytochemistry was applied on sections from antrum, duodenum and colon, subsequently quantified by computerized image analysis. Whereas the number of vasoactive intestinal peptide (VIP)-positive neurons was increased in antral myenteric ganglia of diabetic mice, there was a decreased density of nerve fibres in muscularis propria. No difference was seen in the VIP of duodenum and colon. Acetylcholine-containing nerve fibres showed an increased volume density in muscularis propria of antrum and duodenum, but a decreased density in colon of diabetic mice, as compared with controls. There was a decreased number of neurons containing nitric oxide synthase (NOS) in myenteric ganglia of antrum and duodenum. No difference was seen in density of NOS-containing nerve fibres in muscularis propria. There was no difference regarding neuropeptide Y (NPY) and galanin between diabetic and control mice; nor was there any difference between pre-diabetic NOD mice and controls regarding all bioactive substances investigated. It is concluded that the diabetic state affects the innervation of gut in this animal model. The present findings may be of some relevance to the gastrointestinal symptoms seen in patients with diabetes.     

Ultrastructure and innervation of the swimbladder of Tetractenos glaber (Tetraodontidae)

Summary The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions.The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter.Type-I fibres took up -methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.     

Immunoreactive substance P in the chicken gut: Distribution,development and possible functional significance

Summary The distribution and cellular localization of substance P in the chicken gut was studied by immunocytochemistry and immunochemistry. Substance P-containing nerve fibers are numerous in the gut wall. They occur in the smooth muscle layer as well as in the mucosa, where they are associated with blood vessels or surround the intestinal crypts. The fibers are particularly numerous in the myenteric and submucosal plexuses, where substance P-containing nerve-cell perikarya are also encountered. Substance P was found also in scattered endocrine cells of the small intestine, caeca and colon. Previously, bombesin-containing cells, which are numerous in the proventriculus, have been mistakenly identified as substance P cells due to crossreactivity of certain antisera against substance P. Immunochemistry revealed the highest concentration of substance P in the duodenum. The gel chromatographic behavior of chicken substance P differs slightly from that of synthetic bovine substance P, suggesting that chicken substance P differs structurally from mammalian substance P. Substance P-containing nerve fibers in the chicken gut develop slowly after hatching, apparently beginning in the duodenum; at approximately 20 weeks after hatching the distribution pattern is fully developed.A functional investigation was performed on the isolated chicken caecum to clarify the role of substance P in the contractile behavior of smooth muscle. Substance P contracted the caecum over a wide dose range; the contractile response was greater in 20 week-old chickens than in 4 and 10 week-old animals. Electrical field stimulation caused a relaxation of the caecum and a contraction upon cessation of stimulation. Neither of these responses, both of which are neurally mediated, were inhibited by adrenergic and cholinergic blockade. It is conceivable that the contractile response following electrical stimulation is caused by substance P released from nerve fibers in the smooth muscle.     

Immunocytochemical localization of taurine in different muscle cell types of the dog and rat

The presence and distribution of the amino acid taurine in different muscle cell types of the dog and rat was examined by immunocytochemical methods. The light microscope study revealed that smooth muscle cells were similarly immunoreactive for taurine, whereas skeletal muscle fibres showed wide differences in taurine immunoreactivity among individual cells. Some skeletal fibres were strongly immunoreactive whereas others did not display immunolabelling. Mononucleated satellite cells, found adjacent to skeletal fibres in a quiescent stage, were also immunostained. Other myoid cells, such as testicular peritubular cells showed a cytoplasmic and a nuclear pool of taurine. By means of electron microscope immunolabelling, the subcellular localization of taurine was studied in vascular and visceral smooth muscle cells. Taurine was present in most subcellular compartments and frequently appeared randomly distributed. Taurine was localized on myofilaments, dense bodies, mitochondria, the plasma membrane and the cell nucleus. Moreover, the labelling density within individual smooth muscle cells was variable and depended on the state of contraction of each single fibre. Contracted cells showed a higher density of gold particles than relaxed cells. Unmyelinated nerve fibres, found adjacent to smooth muscle cells from the muscularis mucosae and the lamina propria of the stomach, were unstained or poorly stained.     

胃良性病变不同部位粘膜肌形态改变及其意义

观察胃良性病变不同部位粘膜肌形态学改变及其意义。用目镜测微尺对不同部位的胃粘膜肌厚度进行测量,部分病例做网状纤维的VG染色。结果显示根据粘膜肌的形态学变化,把增厚之粘肌分作三个类型：即炎性增厚、肌性增厚和纤维性增厚,粘膜肌的改变与相应区的胃粘膜病变有关,结果表明粘膜肌的改变有三种类型,胃窦区的病变较体区重,粘膜的形态改变可能影响胃粘膜腺体的分泌,并有利于粘膜内癌向粘膜下侵犯。     

Adrenergic innervation of the oesophagus in the cat (Fellis domestica) and rhesus monkey (Macacus rhesus)

Summary The distribution of monoamines in the pharynx and oesophagus of the rhesus monkey (Macacus rhesus) and the cat (Felis domestica) was investigated by means of fluorescence microscopical and chemical methods. Fluorimetric determinations reveal the presence of varying amounts of noradrenaline in the pharynx and oesophagus of the rhesus monkey. The lowest amount (0.05 (g/g) was found in the lower part of the oesophagus, the so-called sphincter-segment. The middle and upper part of the oesophagus contain medium amounts of noradrenaline (0.06–0.09 g) whereas the highest concentration was detected in the pharynx (0.14 (g/g). Neither dopamine nor adrenaline occurred in the tissue pieces analyzed. Fluorescence microscopically noradrenaline was found to be located in varicose intramural nerve fibre plexus which innervate mucous glands and blood vessels in the pharynx of both species. In the rhesus monkey, the lamina muscularis mucosae of all parts of the oesophagus is supplied by a well developed noradrenergic ground-plexus. Preterminal and terminal varicose nerve fibres are distributed in myenteric and submucous ganglia of the oesophagus; the number of such ganglia decreases towards the lower segment. The density of the adrenergic innervation is higher in myenteric when compared to submucous ganglia. The arrangement of the intraganglionic terminals suggests that both axosomatic and axodendritic contacts occur in Auerbach's ganglia whereas axodendritic contacts seem to predominate in Meissner's ganglia. Myenteric ganglia situated close to the submucosa as well as true submucous ganglia may be occasionally seen to be traversed by faintly fluorescent non-varicosed fibres which do not establish any synaptic contacts. The fluorescence intensity of intraganglionic varicosities varies considerably; accordingly the transmitter content of individual varicosities seems to be very variable. The adrenergic innervation of the lamina muscularis is restricted to single contorted fibres being sparsely distributed throughout the longitudinal smooth muscle layer. The circularly arranged smooth musculature of the sphincter-segment lacks an adrenergic nerve supply. The vagus nerve carries sympathetic adrenergic fibres to the lower oesophagus and the cardia. Species differences between the innervation pattern in rhesus monkeys and cats are outlined: No adrenergically innervated ganglia occur in the submucosa of the cat. However, part of the myenteric ganglia in cats exhibit an adrenergic innervation pattern similar to that seen in submucous ganglia of the rhesus monkey. They might therefore be regarded as morphologically equivalent to the plexus submucosus which is, however, present in the whole gut. The density of the noradrenergic ground-plexus in the muscularis mucosae of the cat's oesophagus is less than that of the corresponding plexus in rhesus monkeys.The influence of noradrenaline upon the smooth musculature and the neurons from myenteric as well as submucous ganglia is discussed. From the point of view of the adrenergic innervation there is no structure corresponding to the sphincterlike lower oesophageal segment.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius-Gesellschaft zur Förderung der Wissenschaften, Hamburg.