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A morphological and histochemical analysis of the neuroendocrine system of the gut in Acipenser transmontanus

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ABSTRACT Morphological, histochemical and immunohistochemical data are presented to demonstrate that the enteric nervous system of the sturgeon is in part composed and arranged differently from other fish. It is composed of neurons which distribute
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  J. zyxwvusrqponm ppl. Ichthyol. 15 1999), 81-86 ISSN zyxwvutsrqpon 175 8659 zyxwvutsrqpon   1999 Blackwell Wissenschafb-Verlag, Berlin A morphological and histochemical analysis of the neuroendocrine system of the gut in z cipenser transmontanus By C. Domeneghini, S. Arrighi, G. Radaelli ', G. Bosi, P. Berardinelli 2, F. Vaini and F. Mascarello Institute zyxwvutsr f Domestic Animals Anatomy, Histology and Embriology, Faculty of Veterinary Medicine, Milano, Italy. Qepartment of Animal Science, Faculty of Veterinary Medicine, Padova, Italy. 21nstitute zyxw   Veterinary Anatomy and Embriology, Faculty of Veterinary Medicine, Teramo. Italy. Veterinary Practitioner, Milano, Italy. Summary Morphological, histochemical and immunohistochemical data are presented to demonstrate that the enteric nervous system of the sturgeon is in part composed and arranged differently from other fish. It is composed of neurons which distribute both to the tunica propria-submucosa and tunica muscularis. Nerve cell bodies are small and nerve terminals run in bundles which are both unmyelinated and myelinated. The presence of myelinated nerve fibres in the enteric nervous system of vertebrates is infrequent. Ganglionated plexuses are only found in relation to the musculature. In contrast with the other tracts of the gut, the musculature of the oesophagus is arranged into two orthogonal layers, and the inner layer is composed of striated muscle fibres. Enzymes related to the classical neurotransmitters acetylcholine and adrenaline and some possible accessory neuromediators (CGRP-, somatostatin-, ANP-, substance P-, NPY-like peptides, and nitric oxide) were identified histochemically and immunohistochemically in components of the enteric nervous system, especially those which innervate the oesophagus. The diffuse endocrine system was limited to a gastric cell type, which synthesized a somatostatin-like peptide. Some of these special features of the enteric nervous system may possibly be related to functional properties peculiar to the sturgeon gut, which also shows aspects of morphological organization that are different to those of other fish. Introduction The function of vertebrate gut is based on the cooperation of the autonomic (ANS) and enteric nervous systems (ENS). The former nerve structures constitute the extrinsic component of the gut innervation, whereas the latter constitute the intrinsic one. Both cooperate with the epithelial cells of the diffuse endocrine system (DES), which in many instances synthesize and release the same regulatory substances as the nerve components. The interactions between the nervous components and the endocrine cells (neuroendocrine system) enable the gastroenteric apparatus to function independently from the central nervous system. Among vertebrates, the neuroendocrine system of mammals is the most studied. Nerve cell bodies and nerve terminals of the mammalian enteric nervous system are located in the submucosal and myenteric plexuses, together with nerve components of the ANS. Some neurons belong to the parasympathetic component of the ANS and have to be considered cholinergic. Both intrinsic and extrinsic neurons synthesize numerous neuromediators, which may be neuropeptides, as well zyxwvut s biogenic mines (serotonin) and gaseous mediators, such as nitric oxide (NO) (Sanders and Ward, 1992). All neuromediators may function as modulators of the cholinergic as well as noradrenergic innervation (H6kfelt et al., 1980). The neuropeptides belonging to the tachykinin family show an excitatory function. Other neuromodulators have an inhibitory role (VIP, NO, ATP). The DES contains numerous mucosal cell types (Solcia et al., 1981), which may be present all along the gut (serotonin- and somatostatin-immunoreactive cell types), or characterize with their presence a limited gut region (gastrin-immunoreactive endocrine cells of the pyloric glands). A limited knowledge exists as compared with mammals about the structure of the neuroendocrine system in fish gut and its possible functional correlates in regulating secretory and motor activities (Bjenning et al., 1990; Jensen and Conlon, 1992; Himick and Peter, 1994; Kiliaan et al., 1997; Reinecke et al., 1997). The aim of this work was to describe both the endocrine cell populations and the presence and localization of possible excitatory and inhibitory neuromodulators in the enteric nervous system of the white sturgeon, Acipenser transmontanus. Up to date, the gut of white sturgeon and other chondrostei has been described from limited morphological aspects and functional correlates (Weisel, 1973, 1979; Buddington and Doroshov, 1986 a), as well as during development (Buddington and Doroshov, 1986 b; Gawlicka et al., 1995). Taking into account that the anatomical and functional relationships between cholinergic and adrenergic innervations of the gut are in part different in fish as compared with higher vertebrates (Burnstock, 1969), a morphological description of the autonomic innervation will also be provided, utilizing histochemical and immunohistochemical techniques, largely applied in mammals. Previous studies by this group (Domeneghini et al., 1991, 1993; Radaelli et al., 1996) have revealed some structural aspects in the fish gut that are remarkably different in comparison with other vertebrates, namely mammals. This study will give a deeper insight into the morphological and functional aspects of the gut in the sturgeon, a fish extensively reared in Italy, and will be the basis for a future comparison of gut morphology at different stages of growth. Also nutritional studies may take advantage from this morphological examination, which underlines some unknown functional aspects. Materials and Methods Adult Acipenser transmontanus (n=6), obtained at slaughter from Agroittica Lombarda fish hatchery, Calvisano, Italy, were used for this study. Body weight ranged between 8 and 10 Kg. Several samples of oesophagus (proximal, medium, distal), stomach (in its different glandular zones: cardiac, gastric proper, pyloric) and intestine (pyloric caeca, proximal intestine, medium intestine with spiral valve, rectum) were collected. The small pieces, fixed in 4 paraformaldehyde in phosphate buffered saline, were either paraffin-embedded or snap-frozen in liquid nitrogen-cooled isopentane. Some pieces were frozen without fixation. Dewaxed sections (4-6 pn) were stained with haematoxylin- eosin, Azan and Mallory trichromic stains for morphology. In addition, a Nissl-thionin staining for neurons was performed. US. opyright Clearance Center Code Statement: 01 zyxwvu 5 86591994 504-05-0081 14.00/0  82 zyxwvusrqponm Cryostat sections (10 zyxwvuts rn) from both fixed and unfixed samples were stained for acetylcholinesterase (AChEase) according to Karnowsky and Roots (1964) and Felipe and Lake (1983). This enzyme may be considered a marker of cholinergic neurons and nerve terminals, specially in fish where, in contrast to other vertebrates, unspecific cholinesterases are not present (Pecot-Dechavassine, 1961). Other sections were used for C. Domeneghini et al. demonstration of NADPH-diaphorase, according to Scherer- Singler et al. (1983). In mammalian species, this enzyme is related to the synthesis of NO, both in central and peripheral nervous systems. The negative controls for these histochemical reactions included the omission of acetylthiocholine and NADPH, respectively. Figure 1 A Medium oesophagus. Mallory stain. The multilocular adipose cells (A) are accompanied by a myelinated nerve fibre bundle (M). Scale bar 70 pm B Medium oesophagus. Nissl-thionin. A nerve cell body (arrow) is present in a small myelinated nerve bundle. Scale bar zyxwvuts 2 zyxwvuts m C Spiral valve. Azan stain. Lymphatic tissue (ly) is abundant and solitary lymphatic follicles are indicated by arrows. Scale bar 330 pm. D Medium oesophagus. Nissl-thionin. A nerve ending (arrow) is seen contacting a striated muscle fibre. Scale bar 32 zyx m. E Rectum. NF-immunoreactivity in a small myenteric plexus (arrow). Asterisks show melanophores in a subserous localization. Scale bar z 5 pm. F Cardiac gland zone of the stomach. AChEase reactivity is present in a nerve fibre bundle (arrows), running in the smooth musculature. Scale bar 55 pm  A Morphological and Histochemical Analysis of the Neuroendocrine System 83 For immunohistochemistry, other cryostat sections were incubated with the following primary antisera, all raised in rabbits: anti-calcitonin gene-related peptide (CGRP), anti- substance P, anti-vasoactive intestinal peptide (VIP), anti- cholecystokinin (CCK) octapeptide, anti-enkephalin, anti- bombesin, anti-neuropeptide Y (NPY), anti-somatostatin, anti- atrial natriuretic peptide (ANP), anti-serotonin (5-HT), anti- neurofilament 200 (NF200), anti-glial fibrillary acidic protein (GFAP), zyxwvutsr nti tyrosine hydroxylase. hese primary antisera were purchased from Peninsula or Genosys (England). The antibody- antigen complexes were visualized using both an indirect immunoperoxidase method (with goat-anti-rabbit immunoglobulins, GAR) and a peroxidase anti peroxidase PAP) technique (Sternberger, 1979). In both cases, the amino-9-ethyl- carbazole chromogen was used to mark the sites of reaction in red. The controls for immunohistochemical reactions were performed by incubating sections with: 1) normal rabbit serum, instead of specific antisera, and 2) antiserum pre-absorbed with the respective antigen (10-100 pg/ml). The fire-absorption procedures were carried out overnight at zyxwvutsr °C Peptides were purchased from Sigma (Italy). The results of these controls were negative. As positive controls fish and mammalian gut samples were used. Resu zyxwvutsrqpon ts General macro- and microscopic anatomy of the gut Although a detailed description of the anatomy of the sturgeon gut is not the purpose of this study, some peculiar features in comparison with other fish are reported. Some of these special aspects were described by Weisel (1973, 1979) in the gut of chondrostei. Oesophagus: the inner surface was white and lucent and organized into several conical papillae arranged in parallel longitudinal rows. The mucosal epithelium contained a large number of caliciform cells and numerous taste buds, above all in its proximal end. The deep tunica propria-submucosa contained lobules of multilocular adipose tissue (Fig. 1 A), which were supplied by an extensive vasculature and bundles of myelinated nerve fibres (Figs.lA,B), a feature that indicates an active metabolism. It is difficult at this moment to understand if this special adipose tissue is related to heat production, as is well described for some mammalian species, or depends on the lipid composition of diet, or overfeeding. Unlike the stomach and intestine which contained smooth muscle cells, the oesophageal tunica muscularis was made up of a large sheath of striated muscle fibres, arranged orthogonally to a thin, subserous smooth muscle layer. Stomach: was siphon-shaped and with a ciliated epithelium in cardiac and fundic zones, where, in addition, tubular glands were present in the tunica propria. The superficial epithelium and gastric pits were lined by columnar cells, the glandular cells showed a granular appearance. This latter aspect, similarly to what is generally known for fish (Buddington and Doroshow, 1986 a; Smith, 1989; Buddington et al., 1997), may be related to the synthesis of both pepsinogen and acid. The presence of ciliated regions of the developing gut is often described in fish larvae (Gawlicka et al., 1995; Morrison et al., 1997). In adult teleosts epithelial ciliated cells are not common and their presence is limited to ancient fish (Ishida, 1935; Weisel, 1973). When present, cilia may sustain movements of fluid mucosal secretions or of small food particles. Intestine: the intestinal mucosa was organized into folds, not true villi, and was characterized by the presence of numerous calicifonn cells (Fig. 1 C). Appendices pyloricae constituted a glandular body. The spiral valve of the medium intestine was made up by folds of the intestinal mucosa which projected into the lumen, with an elicoidal arrangement. In this intestinal tract the tunica propria-submucosa was filled by lymphatic tissue and large lymphatic follicles (Fig. IC), in such a way resembling the mammalian Peyer’s patches of the small intestine and colonic lymphoepithelial complexes. Mammalian gut-associated lymphoid tissue (GALT) is a major component of the total immune system of the gut, and also contributes to immune events in mucosal structures of other organs (Heel et al., 1997). It is possible that the sturgeon spiral valve supports intestinal immune events like those described in mammals, in addition to slow down the passage of digesta and increase the absorptive surface. The distal intestine (rectum) was the only tract showing a muscularis mucosae and this aspect may be tentatively related to special movement requirements of the mucosa in this tract. Pancreas: pancreatic lobules were present in the subserous connective tissue all along the stomach and intestine. A “diffuse” arrangement of the pancreatic parenchyma is common in most fish. The enteric nervous system As shown by the Nissl-thionin staining for neurons as well as by the neurofilament-immunohistochemistry, the intramural innervation of the sturgeon gut was composed of small neurons in the tunica propria and tunica muscularis, small unmyelinated nerve fibres, with the same localizations, and more conspicuous myelinated nerve fibre bundles. The myelinated nerve fibre bundles were seen in the oesophagus (Fig. IA,B), in the deep tunica propria-submucosa in close relationship with the lobules of multilocular adipose tissue. They were also observed in the vicinity of the tunica muscularis of both the oesophagus and the cardiac stomach. The presence of nerve cell bodies was frequently observed in these myelinated nerve fibre bundles (Fig. 1B). The oesophageal striated muscle fibres were sometimes seen in close relationship with nerve terminals (Fig. 1D). A submucosal plexus, as evidenced in mammals, was not present, whereas nerve structures similar to the mammalian myenteric plexus were present in the fundic stomach and in the intestinal tracts (Fig. I E). Histochemistry AChEase activity was evidenced both in nerve cell bodies and small nerve terminals of oesophagus, stomach and intestine. Reactive neurons were observed both in the tunica propria- submucosa and tunica muscularis, where nerve terminals distributed, too (Fig. IF). The nerve terminals were either myelinated or unmyelinated. The myelinated fibres were seen all along the oesophagus and in the cardiac stomach. In the oesophagus, they were observed in close relationship with the lobules of multilocular adipose tissue. In the cardiac stomach, the myelinated nerve fibres were present near the tunica muscularis. Single fine nerve terminals were sometimes observed in contact with striated muscle fibres of the oesophagus. In the intestinal mucosa, cholinergic nerve fibres were abundant. Intramural ganglionated plexuses were almost exclusively seen in relation with the tunica muscularis, where this was composed of smooth muscle cells. The arrangement of these intramural plexuses resembled the mammalian myenteric plexus, except for the paucity of nerve cell bodies. Unlike AChEase reactivity, which was observed all along the sturgeon gut, the NADPH-diaphorase reactivity was present in a small number of nerve cell bodies and nerve terminals limited to oesophagus (Fig. 2A) and cardiac and fundic zones of the stomach. The nerve terminals were either unmyelinated or myelinated, and both were specially present in the deep propria- submucosa and near the musculature.  84 C. Domeneghini et al. lmmunohistochemistry Both immunohistochemical procedures (indirect immunoperoxidase and PAP) were effective in showing immunoreactive structures, and in an equal manner. zyxwvu yrosine-hydroxylase-immunoreactivity, which is a possible marker of the adrenergic innervation, was present in nerve terminals limited to oesophagus (Fig. 2B) and cardiac (Fig. 2C) and fundic zones of the stomach. These immunoreactive nerve terminals were larger than AchEase-reactive ones, and were present in both unmyelinated and myelinated bundles, namely in relationship with the zyxwvutsrqp unica muscularis. In addition, nerve cell bodies were sometimes observed in the oesophageal tunica propria-submucosa. The immunohistochemistry for GFAP, which is considered a marker of mammalian glial cells, did not give reaction in any of the organs examined. As regards the immunohistochemistry for possible accessory neuromediators of both the cholinergic and adrenergic innervations, the majority of the peptide-like immunoreactivities was observed in some nerve terminals which were shown in contact with the oesophageal striated muscle fibres. In these nerve terminals, the presence of CGW-, somatostatin-, ANP-, substance P- (Fig. 2D), and NPY-like peptides was showed. The diffuse endocrine system Somatostatin-like-immunoreactive endocrine cells (Fig. zy E, F). Figure 2 A zyxwvutsrqpon   Medium oesophagus, NADPH-diaphorase reactivity in a subserous nerve fibre bundle. Arrows show two neurons. M triated musculature. Scale bar zyxwvuts 5 pm. B Medium oesophagus. TH-immunoreactive nerve fibres arrows) in a myelinated nerve fibre bundle in the deep propria-submucosa.GAR. Scale bar 50 pm. C Cardiac gland zone of the stomach. TH-immunoreactivity is present in nerve terminals located in the deep propria-submucosa near the smooth musculature (M). GAR. Scale bar 85 pm. were identified in the stomach These mucosal endocrine cells were small and scarcely granulated, and a lumenal connection was rarely seen. Discussion Results show noteworthy differences in the neuroendocrine system of the sturgeon if compared with teleost fishes (Foucherau-Peron et al., 1990; Karila et al., 1993; Kiliaan et al., 1993; Venugopalan et al., 1995; Jensen, 1997; Olsson and Holmgren, 1997) and elasmobranchs (Bjenning et al., 1990; Chiba et al., 1995). The pattern especially of the intramural D Proximal oesophagus. Substance P-like immunoreactivity in a nerve terminal contacting a striated muscle fibre. Note the presence of melanophores (M). PAP. Scale bar 50 pm. E Gastric proper gland zone of the stomach. Somatostatin-like- immunoreactivity in an epithelial endocrine cell arrow). Note that the epithelium is ciliated (c).PAP. Scale bar 8 pm F Pyloric zone of the stomach. Somatostatin-like-immunoreactivity in a small epithelial endocrine cell arrow). PAP. Scale bar I5 pm innervation possibly evidences peculiar functional attitudes of the gut, above all of its proximal segment. In addition, the morphological aspects of the sturgeon gut itself may be associated with the expression of some special functions, which need physiological approaches for understanding. The oesophagus and the cardiac gland zone of the stomach show the presence of nerve fibre bundles in which the presence of both myelinated and unmyelinated nerves is unusual. The unmyelinated nerves are reduced in size and possibly cholinergic, whereas the myelinated ones are larger and tyrosine-hydroxylase-  A Momholoeical and Histochemical Analvsis of the Neuroendocrine System 85 immunoreactive. If it is true for fish, like for mammals, that tyrosine-hydroxylase is a marker of the adrenergic structures, it may be that the myelinated, large-sized nerves are adrenergic, antagonistic to the cholinergic ones. These composite nerve fibre bundles are seen in relation with the lobules of multilocular adipose cells which characterize the deep oesophageal zyxwvutsr ropriu- submucosa and with the smooth musculature of the cardiac stomach. The presence of myelinated nerve fibres in the enteric nervous system of vertebrates is exceptional (Gabella, 1979). Quite recently, myelinated peptidergic nerve fibres have been described in the gut of cetaceans (Domeneghini et al., 1997), where the authors related the presence of them to an exceptionally rapid response of the gastrointestinal structures to nerve inputs. It is conceivable that also in sturgeon some esophageal (multilocular adipose cells, striated muscle fibres of the tunica muscularis), as well zyxwvuts s gastric (smooth muscle cells) structures share rapid responses to nervous stimulations. One other noteworthy observation is that in these nerve fibre bundles some nerve cell bodies are present. To our knowledge, this is a very unusual finding, whose significance is at present unknown. The observation also that cholinergic nerve structures are present along the whole gut, whereas the putative adrenergic ones are limited to oesophagus and stomach, is at present difficult to understand. Its significance may be tentatively related to the peculiar pattern of the fish gut ANS, which lacks a true caudal parasympathetic system (Bumstock, 1969). The NADPH-diaphorase activity is present in neurons sharing the same localizations with tyrosine-hydroxylase- immunoreactivity. Likely, the former nervous structures synthesize and release NO and, like in mammals, birds and some fish (Karila and Holmgren, 1995; Olsson and Holmgren, 1997), are possibly involved in the regulation of muscle tone and peristaltic reflexes. Only few of the numerous peptides tested as accessory neuromediators were immunohistochemically shown, and these almost exclusively in nerve terminals apposing to single striated muscle fibres of the oesophageal inner musculature. The presence of CGRP-, somatostatin-, ANP-, substance P-, NPY-like peptides was shown in this localization, suggesting that these peptides play a role in the modulation of contraction in the oesophageal striated muscle fibres. In addition, a somatostatin-like-immunoreactive endocrine cell type was identified in the stomach. These mucosal endocrine cells were small and poorly granulated. When identified in the gut of vertebrates, somatostatin-like- immunoreactive endocrine cells are widely distributed (Grube, 1986; Domeneghini and Arrighi, 1994, 1997), and somatostatin peptides are recognized to be inhibitory towards other endocrine or exocrine secretions. zyxwvutsrqp Acknowledgements This work was supported by the Italian Minister0 Universita Ricerca Scientifica Tecnologica (MURST 60 ). zyxwvut uthors wish to thank Mr. Giovanni Caporale for his valid technical assistance. References Bjenning, C., Jonsson, A.C. and Holmgren, S.. 1990: Bombesin-like immunoreactive material in the gut, and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus acanthias. Regul. Peptides 28, 57-69. 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