Histochemical localization of NADPH-diaphorase-positive elements in the enteric nervous system of bivalve molluscs

Using a nicotinamide adenine dinucleotide phosphate-diaphorase(NADPH-d) histochemistry, localization and morphology of putativenitric oxide synthase-containing elements were studied in theintestine of the following bivalves: Ruditapes philippinarum,Callithaca adamsi, Mercenaria stimpsoni (Veneridae), Corbiculajaponica (Corbiculidae), Nodularia vladivostokensis and Cristariatuberculata (Unionidae). NADPH-d-positive nerve cells and plexuseswere found in the intestine of all species studied. Labelleddiformazan bipolar nerve cells were present in the epitheliumof the intestinal groove and typhlosole of the anterior midgut,the midgut proper, and the hindgut. Their apical process extendedtowards the gut lumen, whereas the basal one was connected withthe basiepithelial NADPH-d-positive nerve plexus. In the typhlosoleof N. vladivostokensis, these cells constituted up to 2.68%of the total number of epithelial cells. The bivalve speciesstudied exhibit a similar distribution pattern of NADPH-d-positivecells, which lie separately or form small groups of two to threein the basal part of the epithelium. Basiepithelial NADPH-d-positiveplexus was connected by separate fibres with subepithelial NADPH-d-positivenerve plexus. Both the plexuses were moderately developed inall intestinal regions in the majority of the species examined. (Received 27 March 2007; accepted 4 September 2007)     

Nitroxidergic elements in the digestive system of Mactra chinensis and Spisula sachalinensis (Mollusca: Bivalvia: Mactridae)

Localization and morphology of NO-ergic elements in the digestive system of bivalve molluscs Mactra chinensis and Spisula sachalinensis were studied using histochemical technique [1] for detection of NADPH-diaphorase (EC 1.6.99.1) [1]. The NO-producing elements were revealed in all parts of the digestive system of the studied animals. NADPH-diaphorase was found in cells of several morphological types as well as in nerve plexuses. The most abundant in the digestive tract parts of the studied molluscs were intraepithelial nerve cells of the “open” type, whose thin apical process is directed towards the gut lumen. Subepithelial NO-ergic neurons were revealed in stomach and crystalline style sac of Mactra chinensis. Besides, diformazan granules are present in brush-border epitheliocytes of the major and secondary ducts of the digestive gland as well as in cells of digestive tubules of this gland. All studied parts were found to contain basiepithelial and subepithelial NO-ergic nerve fibers forming networks of various densities from, most commonly, loose to dense plexuses particularly developed in labia, esophagus, and gut of the studied molluscs.     

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.     

Regulatory peptides in gut endocrine cells and nerves in the starfish Marthasterias glacialis

The endocrine cells of the starfish digestive tract are spindle-shaped, contacting both the lumen and the basiepithelial plexus. Silver impregnation labels the basiepithelial and subcoelomic plexuses as well as these cells. Twenty antisera have been tested using the avidinbiotin method, in order to identify the regulatory substances involved in this system. Endocrine cells and nerves immunoreactive to GFNSALMFamide- (S1), FMRFamide-, peptide tyrosine-tyrosine-(PYY), pancreatic polypeptide- (PP), melanocyte stimulating hormone- (MSH) and peptidylglycine alpha-amidating monooxygenase- (PAM) specific antisera have been found in the epithelium. The antibodies against S1, a peptide isolated from the nervous system of a starfish, and MSH, stain both the basiepithelial plexus and the subcoelomic plexus, but the others react only with nerves in the basiepithelial plexus. Absorption controls show that antibodies for S1 and FMRFamide totally crossreact recognizing the same molecule, possibly S1. The other antibodies do not show cross-reactivity to any of the rest, and thus we conclude that these regulatory peptides are present in starfish. This is the first report of the presence of FMRFamide, PYY, MSH and PAM in the Echinodermata. Under the electron microscope the endocrine cells exhibit secretory granules, microtubules and mitochondria. Direct contact with the subcoelomic plexus can be observed.     

Localization of NO-Ergic Structures in Oral Lobes, Labia, and Esophagus of the Bivalve Mollusc Crenomytilus grayanus

Localization of NO-ergic elements in oral lobes, labia, and esophagus in the bivalve mollusc, mussel Crenomytilus grayanus was studied using histochemical technique [1] for detection of NADPH-diaphorase (EC 1.6.99.1). The NO-producing elements were revealed in all studied parts of the digestive system. NADPH-diaphorase was found in nerve and secretory cells as well as in nerve plexuses. Numerous NO-ergic nerve cells were observed in the basal part of epithelium of labia and of the initial part of esophagus as well as in the subepithelial area of these organs. In the middle and posterior parts of esophagus, only subepithelially located NO-ergic nerve cell are present. Basiepithelial NO-producing secretory cells are found in all the parts, but most of these cells are observed in labia and the initial part of esophagus. Subepithelial secretory cells labeled with diformazan granules are spread from the folded surface of oral lobes to the initial part of esophagus; no such cells were found on the smooth surface of the lobes. The deposit labeled basi- and subepithelial nerve plexuses in all studied organs except for oral lobes. These plexuses are the most developed in labia and the initial part of esophagus of the studied mollusc.     

NADPH-diaphorase activity in the digestive system of gastropod molluscs <Emphasis Type="Italic">Achatina fulica</Emphasis> and <Emphasis Type="Italic">Littorina littorea</Emphasis>

Distribution and morphological peculiarities of nitroxidergic elements throughout the entire length of digestive tract was studied for the first time in gastropod molluscs Littorina littorea (Prosobranchia) and Achatina fulica (Pulmonata) using histochemical detection of NADPH-diaphorase (NADPHd). NO-ergic cells and fibers were revealed in all parts of the mollusc digestive system beginning from esophagus. Intensive NADPHd activity is found in a great number of intraepithelial cells of the open type and their processes in the intraand subepithelial nerve plexuses, subepithelial neurons, granular connective tissue cells, and multiple nervous fibers distributed among muscular elements of digestive tract as well as those in nerves innervating the tract. NADPHd was also revealed in receptor cells in the oral area and in the A. fulica CNS ganglia innervating the digestive tract. A. fulica has a more complicated organization of A. fulica nitroxidergic system of the digestive tract. A system of glomerular structures formed by thin NADPHd-positive neural fibers coming from epithelium is found directly beneath the epithelium in esophagus, stomach, and midgut of the mollusc. More superficially under the main groups of muscular elements there are revealed small clusters of NADPHd-positive neurons that can be classified as primitive, non-structured microganglia. The distribution pattern and a possible functional role of nitroxidergic elements in digestive tract of molluscs as compared with other invertebrate and vertebrate animals are discussed.     

The basiepithelial nerve plexus of the viscera and coelom of eleutherozoan echinodermata

Summary The organisation of the basiepithelial nerve plexus in the alimentary canal of a starfish and the water vascular system of a sea-urchin is described. The plexus contains varicose aminergic neurones which terminate adjacent to the ciliated epithelial cells. It is proposed that the basiepithelial plexus innervates these cells and controls ciliary beating. The distribution of the basiepithelial plexus in various tissues described by other workers is dicscussed particularly in relation to whether it is the coelomic epithelium or the luminal epithelium which is innervated. It is concluded that where there is both an endothelium and a coelomic epithelium only one is innervated. The muscles, where present, of the viscera are innervated by a separate nervous system. The muscles are always on the opposite side of the non-cellular connective tissue sheath to the basiepithelial plexus.     

Morphology of the digestive system in carnivorous freshwater dourado Salminus brasiliensis

The digestive system of teleost shows remarkable functional and morphological diversity. In this study, the digestive tract and accessory organs of dourado Salminus brasiliensis are characterized using anatomical, histological, histochemical and immunohistochemical analyses. The existence of taste buds bordered by microridges in the oesophagus of dourado was recorded for the first time, thus showing that the species drives food intake by either swallowing or rejecting the food item. The Y-shaped stomach of dourado consisted of cardiac, cecal and pyloric regions with tubular gastric glands registered solely in the cardiac and cecal segments. The intestine is a short N-shaped tube with two loops, an intestinal coefficient of 0.73. The structure of pyloric caeca is similar to that of the intestine wall, comprising tunica mucosa, tela submucosa, tunica muscularis and tunica serosa layers. Histochemical analyses revealed an increased incidence of goblet cells from the midgut to the hindgut segment. A well-developed enteric plexus of scattered nerve cell and fibres are found along the digestive tract, and the calcitonin gene-related peptide (CGRP) immunoreactive neurons and fibres were identified in the myenteric plexus from the oesophagus to the hindgut. The exocrine pancreas appears diffuse in the mesentery around the stomach, intestine and also reaches the liver, and the endocrine pancreas is organized as a few islets of Langerhans. The liver comprises three distinct, asymmetric lobes, and the portal triad arrangement was registered in this tissue.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

Neural crest regionalisation for enteric nervous system formation: Implications for Hirschsprung's disease and stem cell therapy

Midbrain, hindbrain and vagal neural crest (NC) produced abundant enteric nervous system (ENS) in co-grafted aneural hindgut and midgut, using chick-quail chorio-allantoic membrane grafts, forming complete myenteric and submucosal plexuses. This ability dropped suddenly in cervical and thoracic NC levels, furnishing an incomplete ENS in one or both plexuses. Typically, one plexus was favoured over the other. This deficiency was not caused by lower initial trunk NC number, yet overloading the initial number decreased the deficiency. No qualitative difference in neuronal and glial differentiation between cranial and trunk levels was observed. All levels formed HuC/D+ve, NOS+ve, ChAT+ve, and TH-ve enteric neurons with SoxE+ve, GFAP+ve, and BFABP+ve glial cells. We mathematically modelled a proliferative difference between NC populations, with a plexus preference hierarchy, in the context of intestinal growth. High proliferation achieved an outcome similar to cranial NC, while low proliferation described the trunk NC outcome of incomplete primary plexus and even more deficient secondary plexus. We conclude that cranial NC, relative to trunk NC, has a positionally-determined proliferation advantage favouring ENS formation. This has important implications for proposed NC stem cell therapy for Hirschsprung's disease, since such cells may need to be optimised for positional identity.     

Intestinal coelomic transplants: a novel method for studying enteric nervous system development

Normal development of the enteric nervous system (ENS) requires the coordinated activity of multiple proteins to regulate the migration, proliferation, and differentiation of enteric neural crest cells. Much of our current knowledge of the molecular regulation of ENS development has been gained from transgenic mouse models and cultured neural crest cells. We have developed a method for studying the molecular basis of ENS formation complementing these techniques. Aneural quail or mouse hindgut, isolated prior to the arrival of neural crest cells, was transplanted into the coelomic cavity of a host chick embryo. Neural crest cells from the chick host migrated to and colonized the grafted hindgut. Thorough characterization of the resulting intestinal chimeras was performed by using immunohistochemistry and vital dye labeling to determine the origin of the host-derived cells, their pattern of migration, and their capacity to differentiate. The formation of the ENS in the intestinal chimeras was found to recapitulate many aspects of normal ENS development. The host-derived cells arose from the vagal neural crest and populated the graft in a rostral-to-caudal wave of migration, with the submucosal plexus being colonized first. These crest-derived cells differentiated into neurons and glial cells, forming ganglionated plexuses grossly indistinguishable from normal ENS. The resulting plexuses were specific to the grafted hindgut, with quail grafts developing two ganglionated plexuses, but mouse grafts developing only a single myenteric plexus. We discuss the advantages of intestinal coelomic transplants for studying ENS development. This work was supported by NIH K08HD46655 (to A.M.G.).     

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.     

豚鼠小肠神经节丛的NADPH—黄递酶组织化学观察

目前已知,ＮＡＤＰＨ－－黄递酶组化法可选择性地显示－－氧化氮合成酶（ＮＯｓｙｎｔｈａｓｅ,ＮＯＳ）神经元。因此,我们以ＮＡＤＰＨ－黄递酶组化法,观察了豚鼠小肠肌间神经丛和粘膜下神经丛的神经网格以及ＮＯＳ神经元。结果表明,三段小肠肌间神经丛的神经网眼大小和形态有明显差异,与对应的粘膜下神经丛相比,差异更显著。在肌间神经丛中,ＮＡＤＰＨ－黄递酶阳性神经元胞体大小不等;其长突起伸入节间束,而短突起较多,并可见短突起彼此连接．构成节内偶见的局部神经元回路。从小肠上段到下段,ＮＯＳ神经元数量呈下降趋势。在粘膜下神经丛,我们也观察到少数ＮＯＳ神经元。     

Monoamine- and peptide-containing elements in the nemertine digestive tract

Detailed comparative-histological data on localization and morphological peculiarities of catecholamine-, serotonin-, neurotensin-and FMRFamide-containing elements in the nemertine digestive system are presented for the first time. Using fluorescent histochemistry and immunohistochemistry, pharynx, esophagus, and midgut were studied in five species from three genera of the White Sea nemertines. Described in various nemertine species are intra-and subepithelial cells of the open and closed type, containing biologically active substances. Their processes are distributed in basi-and subepithelial nerve plexuses and can make contact with lumen of the digestive tract. Species-specificity in localization of the cells containing certain biologically active substances is noticed both along the length of the tract and with respect to its epithelial layer. Peculiarities and common regularities in distribution are discussed, as well as a possible function of monoamine-and peptide-containing elements in the digestive tract of the studied nemertines and other invertebrates.     

Monoamine- and peptide-containing elements in the nemertine digestive tract

Detailed comparative-histological data on localization and morphological peculiarities of catecholamine-, serotonin-, neurotensin- and FMRFamide-containing elements in the nemertine digestive system are presented for the first time. Using fluorescent histochemistry and immunohistochemistry, pharynx, esophagus, and midgut were studied in five species from three genera of the White Sea nemertines. Described in various nemertine species are intra- and subepithelial cells of the open and closed type, containing biologically active substances. Their processes are distributed in basi- and subepithelial nerve plexuses and can make contact with lumen of the digestive tract. Species-specificity in localization of the cells containing certain biologically active substances is noticed both along the length of the tract and with respect to its epithelial layer. Peculiarities and common regularities in distribution are discussed, as well as a possible function of monoamine- and peptide-containing elements in the digestive tract of the studied nemertines and other invertebrates.     

Morphology and ultrastructure of the alimentary canal of the cicada Platypleura kaempferi (Hemiptera: Cicadidae)

The alimentary canal of cicada Platypleura kaempferi is described. It comprises the oesophagus, filter chamber, external midgut section and hindgut. The elongate oesophagus expands posteriorly, with its posterior end constricting to become a bulb. The filter chamber consists of two parts: a very thin sheath and a filter organ. The filter organ is composed of the anterior and posterior ends of the midgut (internal midgut section), and the internal proximal ends of the Malpighian tubules. The external midgut section differentiates into a collapsed sac and a midgut loop. The latter is divided into three distinct segments. The hindgut contains a dilated rectum and a long narrow ileum. The distal portions of the four Malpighian tubules are enclosed in a peritoneal sheath together with the distal ileum before reaching to the rectum. Ultrastructurally, the oesophagus and the hindgut are lined with a cuticle. The filter chamber sheath consists of cells with large irregular nuclei. Filamentous substances coat the microvilli of the cells of the internal midgut section. The posterior end of the midgut comprises two types of cells, with the first type of cells containing many vesicles and scattered elements of rough endoplasmic reticulum. The anterior and posterior segments of the midgut loop cells have ferritin‐like granules. The ileum cells have well‐developed apical leaflets associated with mitochondria. Accumulations of virus‐like particles enclosed in the membrane are observed in the esophagus, conical segment, mid‐ and posterior segments of the midgut loop.     

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement. Received: 14 July 1998 / Accepted: 2 September 1998     

Ultrastructure of the tentacle nerve plexus and putative neural pathways in sea anemones

Abstract. Neurons of sea anemone tentacles receive stimuli via sensory cells and process and transmit information via a plexus of nerve fibers. The nerve plexus is best revealed by scanning electron microscopy of epidermal peels of the tentacles. The nerve plexus lies above the epidermal muscular layer where it appears as numerous parallel longitudinal and short interconnected nerve fibers in Calliactis parasitica . Bipolar and multipolar neurons are present and neurites form interneuronal and neuromuscular synaptic contacts. Transmission electron microscopy of cross sections of tentacles of small animals, both C. parasitica and Aiptasia pallida , reveals bundles of 50–100 nerve fibers lying above groups of longitudinal muscle fibers separated by intrusions of mesoglea. Smaller groups of 10–50 slender nerve fibers are oriented at right angles to the circular muscle formed by the bases of the digestive cells. The unmyelinated nerve fibers lack any glial wrapping, although some bundles of epidermal fibers are partially enveloped by cytoplasmic extensions of the muscle cells; small gastrodermal nerve bundles lie between digestive epithelial cells above their basal myonemes. A hypothetical model for sensory input and motor output in the epidermal and gastrodermal nerve plexuses of sea anemones is proposed.