An immunohistochemical study of somatostatin-containing nerves in the aganglionic colon of human and rat

Summary The distribution of somatostatin-like immunoreactive (SOM-LI) nerves was elucidated immunohistochemically in the gut tissues from patients with Hirschsprung's disease and congenital aganglionosis rats. In the normoganglionic human colon, SOM-LI nerve cell bodies were found to a greater extent in the submucous plexus and to a lesser extent in the myenteric plexus. However, they were rarely observed in both the plexuses of the oligoganglionic segment. SOM-LI nerve fibres were widely distributed in the aganglionic bowel. The circular muscle layer of the distal aganglionic segment was densely innervated by SOM-LI nerve fibres which are probably derived from the extrinsic, hypertrophic nerve bundles. A decreased number of the intramuscular nerves fibres were seen in the proximal aganglionic segment. In the colon and rectum from adult and 21-day-old rats, SOM-LI cell bodies were numerous in both plexuses. On the other hand, enteric neurons were completely lacking from the colon and rectum of congenital aganglionosis rats of 21 days old. No neuronal elements staining for SOM were disclosed in these aganglionic segments of mutant rats. A possible origin and pathophysiological role of the extrinsic nerve fibres containing SOM in the diseased bowel are discussed. It is concluded that SOM-LI nerves in the human distal colon comprise both intrinsic and extrinsic elements, while SOM nerves in the rat colon and rectum are of only intrinsic origin.     

Distribution of neuropeptide Y-like immunoreactivity in the normoganglionic and aganglionic segments of human colon

Summary The localisation and distribution of neuropeptide Y (NPY)-like immunoreactivity were studied by use of immunohistochemical methods in gut tissues from 19 patients with Hirschsprung's disease, including 4 cases of long segment aganglionosis. In the normoganglionic segment, immunoreactive cell bodies and nonvaricose processes were seen within both myenteric and submucous plexuses. A scarce supply of varicose fibres was found in the lamina propria mucosae, muscularis mucosae and longitudinal muscle layer. NPY fibres were more frequently encountered in the circular muscle layer, although with a weakly immunostaining intensity. In addition, blood vessels in the submucosal connective tissue were surrounded by a typical plexus of varicose, NPY-positive fibres. Immunoreactive endocrine cells could be detected in the colonic epithelium. In the aganglionic segment, numerous nerve fasciculi comprising a small to moderate number of NPY fibres with varicosities were observed throughout the entire layer of the colonic wall. A few varicose, NPY-positive fibres were also contained in the relatively large, hypertrophic nerve fasciculi located in the intermuscular zone and submucosal connective tissue. NPY-immunoreactive fasciculi were more densely distributed in the distal aganglionic segment than in the proximal aganglionic one. On the other hand, the distribution of NPY-positive fibres in long segment aganglionosis was quite different from that in short segment type; in cases of long segment type, no immunoreactive nerve fibres were detected within the circular muscle layer of the proximal aganglionic segment near the oligoganglionic segment and only a few fibres were observed within the hypertrophic nerve bundle of the intermuscular zone. The present results suggest that NPY-like immunoreactive nerves in the human colon have a dual origin of intrinsic and extrinsic elements. The origin and nature of extrinsic NPY nerve fibres in the human colon are discussed.     

An immunohistochemical study of glial fibrillary acidic (GFA) protein and S-100 protein in the colon affected by Hirschsprung's disease

Summary The supportive cells of the enteric nervous system were examined in gut tissues from 15 patients with Hirschsprung's disease by means of immunohistochemistry, utilizing antisera to glial fibrillary acidic (GFA) protein and S-100 protein. In the normoganglionic segment, GFA protein immunoreactivity was predominantly found in association with the myenteric plexus and to a lesser extent in the submucous plexus. On the other hand, the extrinsic, hypertrophic nerve fasciculi were selectively immunostained with GFA protein antiserum throughout the entire length of the aganglionic intestinal walls from all children studied. The large fasciculi were numerous in the distal aganglionic segment and commonly appeared in the intermuscular zone and submucosal connective tissue. Both small-and mediumsized nerve fasciculi with GFA protein immunoreactivity were also encountered within the circular muscle layer of the proximal aganglionic segment. A subpopulation of supportive cells within the hypertrophic nerve fasciculi showed immunoreactivity for GFA protein, while all supportive elements of these fasciculi were stained for S-100 protein. The intrinsic nerve fibers within the circular muscle layer of normoganglionic segments were stained for S-100 protein, but not for GFA protein. The present study supports our previous findings that two types of supportive cells can be differentiated by immunohistochemistry in the enteric nervous system, utilizing antisera to GFA protein and S-100 protein. It is also concluded that the demonstration of GFA protein by immunohistochemical methods favors the diagnosis of aganglionic colons with Hirschsprung's disease, since GFA protein immunoreactivity is confined to the extrinsic, hypertrophic nerve fasciculi characteristic of aganglionic bowels.     

Altered immunoreactivity of HPC-1/syntaxin 1A in proliferated nerve fibers in the human aganglionic colon of hirschsprung’s disease

To clarify the pathogenesis of excessive proliferation of extrinsic nerve fibers in the aganglionic colon of patients with Hirschsprung’s disease (HD), we immunohistochemically determined the role that exocytosis-related proteins play in the regulation of exocytosis using the antibody to HPC-1/syntaxin 1A, an exocytosis-related protein. Localization of exocytosis-related proteins (HPC-1/syntaxin 1A, N-ethylmalemide-sensitive fusion protein (NSF), soluble NSF attachment protein (SNAP), synaptotagmin, synaptobrevin, and synaptosome-associated protein 25 (SNAP-25)) was determined in surgical specimens obtained from normal proximal and aganglionic distal segments of the colon of 7 infant patients with HD. In the normal ganglionic colon, Auerbach’s plexus, Meisner’s plexus, nerve fibers in the muscle layer, and ganglion cells were immunopositive for all six kinds of antisera. In the aganglionic segments, numerous proliferated nerve fibers and hypertrophied nerve bundles were detected in the submucosal layer and myenteric layer by NSF, SNAP, synaptotagmin, synaptobrevin, and SNAP-25. However, HPC-1/syntaxin 1A was not recognized in the proliferated nerve fibers of the submucosal layer or the hypertrophied nerve bundles of the aganglionic segment. These findings show that immunoreactivity of HPC-1/syntaxin 1A was decreased in the affected bowel segments of patients with HD and may be related to the pathogenesis of extrinsic nerve-fiber proliferation in the aganglionic colon of HD.     

Coexistence of immunoreactive substance P and serotonin in neurones of the gut

Serotonin (5-HT) and substance P (SP) were visualized by indirect immunofluorescence using two fluorochromes applied sequentially on the same preparation of myenteric plexus from the guinea pig gut. Three groups of labelled neurones were found: (i) neurones with 5-HT-like immunoreactivity only; (ii) neurones with SP-like immunoreactivity only; (iii) neurones with both 5-HT- and SP-like immunoreactivity, which represented approximately one-third of the 5-HT-like immunoreactive neurones.     

Localization of serotonin immunoreactivity in cephalopod visual system

The distribution of 5-HT-like immunoreactivity in paraformaldehyde-fixed sections of retina, optic nerve and the optic lobe of Octopus vulgaris was studied by both immunofluorescence and avidin-biotin complex (ABC) immunohistochemical methods utilizing polyclonal antibodies to 5-HT. Some immunoreactive serotonin-containing cells were demonstrated in the retinal plexus, optic nerve, the ciliary body and the lens-generating tissue by both methods. An analysis of dissected retina and optic nerve of Octopus vulgaris by high pressure liquid chromatography (HPLC) with an electrochemical detector (ECD) also showed the presence of 5-HT. In the optic lobe, three 5-HT-immunoreactive bands in the plexiform layer of the cortex were clearly immunostained, and in the medulla both the cell islands and the neuropil contained some cells immunostained by both fluorescein isothiocyanate (FITC) and ABC methods. This is the first report on the systemic immunocytochemical visualization of 5-HT-containing cells and/or fibers in the cephalopod visual system.     

Innervation of the pancreas by neurons in the gut

Experiments were done in order to test the hypothesis that neurons in the bowel send axonal projections to the pancreas and can modify pancreatic activity. pancreatic injections of the retrograde tracer, Fluoro-Gold, labeled neurons in the myenteric plexus of the antrum of the stomach and in the first 6 cm of the duodenum. this labeling was not due to the diffusion of Fluoro-Gold from the pancreas, because the injections did not label longitudinal muscle cells overlying labeled ganglia in the bowel or neurons in the phrenic nerve nucleus or nucleus ambiguous; nor were enteric neurons labeled if insufficient time was allotted for retrograde transport. More Fluoro-Gold labeled neurons were found in the stomach (9.2 +/- 0.9/ganglion) than in the duodenum (3.8 +/- 0.3/ganglion; p less than 0.001). Neurons were found in myenteric ganglia of both duodenum and stomach that were doubly labeled by retrograde transport of Fluoro-Gold and anti-serotonin (5-HT) sera. In addition, thick bundles of 5-HT immunoreactive nerve trunks were found to run between the duodenum and the pancreas. Most 5-HT immunoreactive axons in the pancreas terminated in ganglia, although some fibers were also observed near acini, ducts, vessels, and islet cells. The B subunit of cholera toxin (B-CT) was microinjected into single myenteric ganglia in order to determine if axon terminals in the pancreas would become labeled by anterograde transport in the pancreas. B-CT labeled bundles of axons in the pancreatic stroma. Branches of these bundles entered the pancreatic parenchyma and varicose B-CT labeled terminal axons were found in pancreatic ganglia and in proximity to acinar and insulin immunoreactive cells. The intercalating fluorochrome 1, 1', dioctadecyl-3,3,3',3'-tetramethylcarbocyanine perchlorate (Dil), which moves by lateral diffusion to outline entire cells, was introduced by microinjection into individual myenteric ganglia of fixed preparations. Fluorescence was seen in sequential observations to move away from the injected ganglion along connectives of the myenteric plexus. After about a month, neurons in ganglia at some distance from the injection site displayed Dil fluorescence as did nerve bundles that exited from the myenteric plexus and pierced the longitudinal muscle in the direction of the pancreas. Varicose Dil fluorescent terminal varicosities were also observed int he pancreas. These observations indicate that there is an extensive entero-pancreatic innervation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spinal cord serotonin: A biochemical and immunohistochemical study following transection

The serotonin (5-HT) content of rat spinal cord was studied following complete cord transection, transverse hemisection and rhizotomy by high pressure liquid chromatography-electrochemical detection (HPLC-EC) chromatography and immunohistochemically with rabbit anti-5-HT antiserum. Spinal cord 5-HT decreased but did not disappear after complete cord transection when studied 5 or 10 days after lesioning. Indeed the indole content 5 or 10 days after section were similar. Below the transection 5-HT-like immunoreactive neuronal elements were present, appeared normal but were significantly reduced compared with control cord. Although neuronal fibers were present after transection, no immunoreactive neuronal cell bodies were observed. The neuronal elements remaining after transection were capable of synthesizing and metabolizing 5-HT as evidenced by elevated 5-HT and decreased 5-hydroxyindoleacetic acid (5-HIAA) after inhibition of monoamine oxidase. Complete cord transection resulted in a fall of 5-HT in the ventral roots suggesting that they contain efferent 5-HT elements that originate above the transection. Rhizotomy plus cord transection did not change cord indole content more than transection alone demonstrating that the indoles that remain in the cord after transection did not originate from peripheral afferent 5-HT neurons. Hemitransection resulted in partial loss of immunoreactive neuronal elements on the cut side, but 5-HT-like immunoreactive nerve fibers were observed crossing within the cord from the intact side by the spinal canal. Analysis of indole content in the hemitransected cord were consistent with crossing of 5-HT fibers within spinal segments. Our studies, taken together with reports by other laboratories, support the notion that significant 5-HT elements remain in the spinal cord after transection. These elements appear normal morphologically and biochemically.     

Colonization of the bowel by the precursors of enteric glia: studies of normal and congenitally aganglionic mutant mice

The terminal portion of the ls/ls mouse is congenitally aganglionic because the precursors of enteric neurons fail to enter this region. This animal was studied in order to gain insight into the origin of enteric glia and into the process by which the precursors of these cells colonize the gut. In control (CD-1) mice, immunoreactivity of the glial marker, glial fibrillary acidic protein, appeared for the first time in the fetal bowel at day E16 and, in adults, was much more intense within intraenteric neural elements than in nerves outside the bowel. Glial fibrillary acidic protein developed in tissue cultures of fetal intestine explanted before the protein appeared in situ, and before the bowel became innervated by extrinsic nerves; thus, the precursors of cells able to elaborate glial fibrillary acidic protein must have been present, but unrecognizable, in the original explants. This explant assay demonstrated that these glial precursors were present in all regions of the bowel of control mice, but not in the presumptive aganglionic bowel of ls/ls mice. The nerves (of extrinsic origin) in the aganglionic tissue of ls/ls mice showed a high level of immunoreactive glial fibrillary acidic protein; nevertheless, their ultrastructure was typical of peripheral nerve, not enteric plexus, and they contained Schwann cells, not enteric glia. These observations support the view that enteric glia are derived from the single wave of neural crest colonists that populates the enteric nervous system before the gut receives its extrinsic innervation. These glial precursors, like neuronal precursors, tend to be excluded from the presumptive aganglionic ls/ls bowel. In contrast, Schwann cells grow into the abnormal ls/ls gut with the extrinsic innervation. The enteric microenvironment appears to promote the expression of glial fibrillary acidic protein in both enteric glia and Schwann cells; however, even within the bowel, Schwann cells retain their characteristic morphology. It is thus probable that the normal enteric nervous system contains supporting cells of separate lineages, enteric glia and Schwann cells.     

Immunohistochemical demonstration of GABAergic neurons in the enteric nervous system

Application of a highly specific antiserum against GABA to whole-mount preparations of the guinea pig and rat myenteric plexus resulted in discrete and unambiguous immunolabeling of a subpopulation of myenteric neuronal cell bodies and fibers. The anti-GABA antiserum, which was raised against GABA conjugated by glutaraldehyde to BSA, was applied to glutaraldehyde-fixed whole mounts and subsequently visualized using the peroxidase-antiperoxidase method. In the guinea pig ileum and colon, immunoreactive varicose nerve fibers and scattered nerve cell bodies were found within the myenteric plexus. Immunostained fibers were also seen in the tertiary plexus and in the circular muscle, running parallel to the muscle bundles. GABA immunoreactivity in these intramuscular nerves was most pronounced in the colon. In the rat, immunoreactive fibers were prominent throughout the myenteric plexus. They formed dense networks within the myenteric ganglia, which also contained immunopositive nerve cell bodies, and ran between them in the interconnecting nerves. Some immunoreactive nerve fibers were seen in the circular muscle. Control experiments using non-immune sera or adsorbed anti-GABA antiserum showed no staining. These results add a definitive support to our previous suggestion that GABA serves as an autonomic neurotransmitter in vertebrates. In addition to the present immunohistochemical evidence, this hypothesis is supported by biochemical, autoradiographic, transmitter release, electrophysiological, and pharmacological studies on the enteric nervous system of several species. It is now important to determine the functional role of GABAergic neurons within the complex neuronal circuitry that controls gut functions.     

Ultrastructural analysis of substance P-immunoreactive nerve fibers in myenteric ganglia of guinea pig small intestine

Electron microscopic immunocytochemistry has been used to study the distribution and synaptic relationships of substance P (SP)-immunoreactive nerve fibers in ultrathin sections from whole-mount preparations of myenteric ganglia from guinea pig small intestine. At the light microscopic level, myenteric ganglia stained for ultrastructural study contained dense arrays of SP-immunoreactive nerve fibers around and between the neuronal and glial cell bodies. At the electron microscopic level, SP-containing nerve fiber profiles occurred throughout the neuropil of the myenteric ganglia. Both vesicle-containing and nonvesiculated nerve fiber profiles were immunoreactive. The positive vesiculated profiles contained variable proportions of small clear and large granular vesicles. Two-thirds of the vesicle-containing nerve fiber profiles in myenteric ganglia were immunoreactive for SP. Many vesiculated SP-immunoreactive nerve fiber profiles were directly apposed to each myenteric neuron. However, only about 0.6% of these vesiculated profiles formed synapses that showed the ultrastructural specializations of vesicle clustering presynaptically and fuzzy electron-dense material on the postsynaptic membrane. On the other hand, morphologically identifiable synapses with SP-immunoreactivity comprised about half the total number of synapses in the ganglia. SP-immunoreactive synapses were observed on nonvesiculated nerve processes in the periphery of the ganglia and on nerve cell bodies. Most of the axosomatic SP synapses occurred on small neurons that lay either on the surfaces of the ganglia or near the fibers of the internodal strands where they traveled through the ganglia. Both SP-positive and SP-negative nerve cell bodies received SP synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galanin-immunoreactive neuronal system and colocalization with serotonin in the optic lobe and peduncle complex of the octopus (Octopus vulgaris)

Immunohistochemical techniques were used to investigate the distribution of galanin-like immunoreactivity and colocalization with serotonin (5-HT) in the optic lobe and peduncle complex of the octopus, Octopus vulgaris. Galanin immunoreactive (Gal-IR) fibers, but not cells, were seen in the plexiform layer of the optic lobe cortex. Gal-IR cells were scattered in the cell-islands of the optic lobe medulla and Gal-IR varicose fibers were observed to be abundant in the neuropil surrounding the islands. All Gal-IR cells were immunoreactive for 5-HT, and a few cells showed only 5-HT-like immunoreactivity. In the peduncle lobe, no Gal-IR cells were seen in the basal zone or spine, but in the basal zone, many Gal-IR fibers were seen. In the anterior olfactory lobule, only a few pyramidal Gal-IR cells were observed in the cell layer, and their apical processes were traced to the central neuropil. In the median olfactory lobule, ovoid Gal-IR cells were scattered in the peripheral cell layer. All Gal-IR cells in the anterior and median olfactory lobules showed 5-HT-like immunoreactivity. In the posterior olfactory lobule, ovoid and triangular Gal-IR cells were scattered in the cell layer. Some of them showed 5-HT-like immunoreactivity. Western blot analysis indicated an Gal-IR band at approximately 15.4 kDa. These results suggest the association of galanin-like substance and 5-HT with the visual system of octopus and that the main form of the octopus galanin might have a different molecular weight from vertebrate galanins.     

Histopathological and immunohistochemical study of the enteric innervations among various types of aganglionoses including isolated and syndromic Hirschsprung disease

We investigated enteric innervations in 15 isolated and five syndromic cases of Hirschsprung disease (HSCR) with immunohistochemistry for the S100 protein (S100), class III α‐tubulin (TUJ1), peripherin, neuronal nitric oxide synthase (nNOS) and CD34. The number of neurites per smooth muscle unit of the circular muscle layer (CML) was counted in the longitudinal sections. TUJ1 was the best marker to detect whole neuritic networks of the enteric nervous system. There were differences in the innervation patterns between isolated rectosigmoid aganglionosis (RS) and long segment aganglionosis (LS) including total colonic aganglionosis and extensive aganglionosis. In the aganglionic bowel (AGB) of LS, no nerve fibers innervated smooth muscle units in the CML in the area from the small bowel to the terminal descending colon. In the rectosigmoid region of every type of isolated HSCR, we observed transmural nerve fibers forming meshworks in the CML with TUJ1 and S100 antibodies. In RS, the neurites running parallel with smooth muscle cells gradually decreased in number in the distal portion. However, in the rectosigmoid AGB in LS, those neurites were absent and most neurites perpendicularly crossed the CML. Hypertrophic nerve trunks (HNT) in the submucous and myenteric plexuses were observed more frequently in the rectosigmoid region than in the rostral portion. Based on these data, it is suggested that the neuritic meshworks in the CML of the rectosigmoid AGB might derive from not only the sacral plexus, via HNT, but also intrinsic neurons in the oligoganglionic bowel. All of the syndromic HSCR were RS. In the AGB of RS with Down syndrome, the distribution of neurite meshworks in the CML is markedly reduced. In the AGB of RS with mental retardation suspected of having Mowat–Wilson syndrome, the density of intramuscular innervation was comparatively higher. In the rostral portion to the AGB of syndromic HSCR, myenteric ganglia were clearly small in size, and more numerous per smooth muscle unit with scarce internodal strands. These dysplastic features fall under neither hyperganglionosis nor hypoganglionosis classifications. We considered that syndromic HSCR might occur on the basis of a dysplastic enteric nervous system caused by genetic alteration.     

Development of serotonin, substance P and thyrotrophin-releasing hormone in mouse medullary raphe grown in organotypic tissue culture: developmental regulation by serotonin

Substance P (SP) and thyrotrophin-releasing hormone (TRH) are co-localized with serotonin (5-HT) in cells of the medullary raphe nuclei. In order to examine the factors that control development of multiple neurotransmitters within individual brain nuclei, we have grown presumptive raphe nuclei in organotypic tissue culture, an environment in which mammalian embryonic brain is easily accessible and manipulable. Tissue was obtained from E13 mice. A discrete midline segment of the rhombencephalon was dissected intact or was separated into 'rostral' (RR) and 'medullary' (MR) fragments. Tissue was explanted onto collagen coverslips and grown for up to two weeks in Maximow depression chambers. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, was barely detectable at explantation. During the first week in culture, however, TPH activity increased 7-fold. After two weeks, TPH activity increased almost 2.5-fold above the one-week level. Immunocytochemical analysis of the cultures confirmed a widespread distribution of 5-HT-positive cells and fibers throughout the explant. SP, monitored by radioimmunoassay, was detected after two days in culture, and attained a level of 111.7 +/- 9.8 pg/culture after two weeks. TRH activity was similarly elevated after two weeks in vitro. Therefore, developmental increases in TPH, SP, and TRH occurred in culture, mimicking the condition in vivo. RR and MR fragments, when grown apart on separate coverslips, developed 1.57-2.26 times the TPH activity that developed in the undivided piece. Inclusion of 1 microM pargyline in the fragments restored TPH to control levels. The effect of pargyline was blocked by methiothepin, suggesting autoreceptor-mediated regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical demonstration of GABA containing neurons in the guinea pig ileum using purified GABA antiserum

Antisera against GABA were prepared by immunizing rabbits with GABA conjugated to bovine serum albumin by glutaraldehyde and the antisera were then purified using a GABA immobilized epoxy-activated affinity column. Affinity chromatography and GABA-immobilized epoxy-activated agarose gels were made use of for the reduction of the cross-reactivities of GABA antiserum against endogenous amino acids. The purified GABA antiserum showed remarkably less cross-reactivity. Using this purified GABA antiserum, we noted numerous GABA-like immunoreactive (GABA-positive) nerve fibers in the myenteric meshworks and a few GABA-positive fibers exiting from the myenteric plexus of guinea pig ileum. In the myenteric ganglia, there were GABA-positive nerve cells and GABA-positive varicose fibers surrounding or running along the non-immunoreactive nerve cells. The direct visualization of enteric GABA neurons provides further support for the proposal that GABA is a neurotransmitter in the guinea pig small intestine.     

Sacral neural crest-derived cells enter the aganglionic colon of Ednrb-/- mice along extrinsic nerve fibers

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest-derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest-derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest-derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut.     

Galanin-immunoreactive neuronal system and colocalization with serotonin in the optic lobe and peduncle complex of the octopus (Octopus vulgaris)

Immunohistochemical techniques were used to investigate the distribution of galanin-like immunoreactivity and colocalization with serotonin (5-HT) in the optic lobe and peduncle complex of the octopus, Octopus vulgaris. Galanin immunoreactive (Gal-IR) fibers, but not cells, were seen in the plexiform layer of the optic lobe cortex. Gal-IR cells were scattered in the cell-islands of the optic lobe medulla and Gal-IR varicose fibers were observed to be abundant in the neuropil surrounding the islands. All Gal-IR cells were immunoreactive for 5-HT, and a few cells showed only 5-HT-like immunoreactivity. In the peduncle lobe, no Gal-IR cells were seen in the basal zone or spine, but in the basal zone, many Gal-IR fibers were seen. In the anterior olfactory lobule, only a few pyramidal Gal-IR cells were observed in the cell layer, and their apical processes were traced to the central neuropil. In the median olfactory lobule, ovoid Gal-IR cells were scattered in the peripheral cell layer. All Gal-IR cells in the anterior and median olfactory lobules showed 5-HT-like immunoreactivity. In the posterior olfactory lobule, ovoid and triangular Gal-IR cells were scattered in the cell layer. Some of them showed 5-HT-like immunoreactivity. Western blot analysis indicated an Gal-IR band at approximately 15.4 kDa. These results suggest the association of galanin-like substance and 5-HT with the visual system of octopus and that the main form of the octopus galanin might have a different molecular weight from vertebrate galanins.     

125I-labeled galanin binding sites in congenital innervation defects of the distal colon

Neuropeptides have turned out to be promising new parameters, in addition to the routinely performed histochemical diagnosis, of Hirschsprung's disease (HD). Studies of the peptidergic innervation of the affected intestinal segment of patients with HD have demonstrated a marked reduction in the density of several neuropeptide-containing nerve fibers. The frequency of nerve fibers storing the neuropeptide galanin (GAL) was found to be unchanged or slightly reduced in HD, but nothing is known about the occurrence of GAL receptors. In this study, in vitro receptor autoradiography using (125)I-labeled GAL and GAL immunofluorescence have been performed on frozen tissue sections from colon biopsies of 10 patients diagnosed with HD, 8 patients with intestinal neuronal dysplasia (IND B) and 20 patients with chronic obstruction but normal innervation. Binding sites were mainly detected in the mucosal and muscular layer, in acetylcholinesterase-positive nerve fiber bundles and ganglia within the submucosal layer and in close association to blood vessels. An increased population of GAL receptor positive, parasympathetic nerve fibers was seen in the aganglionic segment of HD as compared to controls and IND B. In contrast, GAL immunostaining which was unchanged in HD revealed a significant lack of GAL-positive structures in IND B colon biopsies. Colocalization of GAL and GAL binding sites was only observed in thick nerve fibers in the submucosa. The presence of GAL binding sites in different cellular structures suggests an involvement of GAL in various physiological functions of the gastrointestinal tract.     

Three-dimensional distribution of vasoactive intestinal polypeptide-containing structures in the rat stomach and their origins using whole mount tissue

Summary The three-dimensional distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactive (VIPLI) structures in rat stomach and their origins were investigated using the indirect immunofluorescence method in whole mounted tissue.The present study demonstrates a very dense VIPLI fiber meshwork in the circular muscle layer, longitudinal muscle layer and myenteric plexus. In the muscle layers VIPLI immunoreactive fibers run parallel to the muscle. VIPLI fibers are distributed evenly throughout the entire stomach. We have also shown by experimental manipulations that the fibers in the stomach originate from VIPLI neurons in the myenteric plexus.     

Changes in pituitary adenylate cyclase activating peptide and vasoactive intestinal peptide innervation of rat oxyntic mucosa during ulcer healing

Pituitary adenylate cyclase activating peptide and vasoactive intestinal peptide belong to the same neuropeptide family. Both peptides are present in nerve fibers in the gastric wall and are thought to be involved in the regulation of inflammatory processes. Experimental ulcers were induced in the rat gastric mucosa by local application of acetic acid. During the healing process we examined the PACAP and VIP innervation by means of immunohistochemistry and in situ hybridization. The ulcer area was examined from day 1 to day 15 after ulcer induction. There was a marked depletion of PACAP in nerve fibers at the ulcer margin from day 1 and onwards. On day 10 and day 15, PACAP-immunoreactive nerve fibers could again be visualized at the ulcer margin. In contrast, VIP immunoreactive nerve fibers were present at the ulcer margin at all time points studied. From day 10 following ulcer induction PACAP- and VIP- immunoreactive nerve fibers were increased in frequency in the smooth muscle beneath the ulcer. An upregulation of VIP and PACAP mRNA was also demonstrated in the myenteric ganglia adjacent to ulcer. The present results indicate that neuronal PACAP and VIP react differently to the inflammation at the ulcer margin but similarly in the smooth muscle during the ulcer healing.