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Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls ( Ambystoma mexicanum

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Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was
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  Abstract Immunoreactivity against vasoactive intestinalpolypeptide (VIP), neurotensin (NT), substance P (SP),calcitonin gene-related peptide (CGRP), gastrin/chole-cystokinin (GAS/CCK), somatostatin (SOM), serotonin(SER), and nitric oxide synthase (NOS) was investigatedin the gastrointestinal tract of the urodele  Ambystomamexicanum , the axolotl, by the use of immunohisto-chemical techniques. The study also compares the distri-bution patterns and frequencies of the neurohormones,and NOS in neotenic and thyroxine-treated (metamor-phosed) individuals. GAS/CCK, SP, NT, SOM, and SERimmunoreactivities occurred in endocrine mucosal cellsand VIP, SP, CGRP, NTSER, SER, and NOS immunore-activities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the uppersmall intestine. NT-IR and SP-IR endocrine cells werefound in the entire gastrointestinal tract and were mostprominent in the distal large intestine. The density of theSOM-IR cells decreased from the stomach toward thelarge intestine. SER-IR endocrine cells were foundthroughout the gastrointestinal tract, with particularlyhigh densities in the stomach and distal large intestine.The VIP-IR enteric nerve fibers were the most prominentones, present in all layers of the entire gastrointestinaltract, and supplied the smooth muscle and the vascula-ture. The SER-IR fibers exhibited similar distributionpatterns but were less numerous. Very few NT-IR butmany SP-IR fibers were found in the muscle and submu-cosal layers. The NT-IR fibers mainly supplied bloodvessels, while the SP-IR fibers were also in contact withthe smooth muscle. In the muscle and submucosal layers,CGRP-IR fibers were associated to the vasculature;CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact withsubmucosal arteries but were the least frequent . Aftermetamorphosis provoked by exogenous thyroxine, thenumber of SOM-IR endocrine cells in the stomach mu-cosa was increased as well as the density of VIP-IR,SER-IR, and SP-IR nerve fibers in the gastrointestinaltract. It is proposed that the observed increases may re-flect refinements of the neurohormonal system aftermetamorphosis. Key words Vasoactive intestinal polypeptide ·Neurotensin · Substance P · Calcitonin gene-relatedpeptide · Gastrin/Cholecystokinin · Somatostatin · Axolotl,  Ambystoma mexicanum (Urodela) Introduction The legend of Axolotl srcinates from the ancient cre-ation myths of the Aztecs in Mexico (Spence 1994). Thestory thematically highlightens the  puer eternus aspectof the old mesoamerican deity Xolotl, who refused to ex-emplify the principle of metamorphosis (Zavala 1977).This legend can be reconciled with the development of the urodele  Ambystoma mexicanum , the Mexican axolotl,whose transition from larva to juvenile does not includea visible metamorphosis. In contrast to most other am-phibians, the axolotl reaches sexual maturity in a mor-phologically larval form (referred to as neoteny or pedo-morphy). However,  A. mexicanum is a facultative neo-tenic species and under experimental conditions a com-plete metamorphosis can be induced.In amphibians, thyroid hormones are known to playan important role in controlling biochemical and mor-phological changes during metamorphosis (Kikuyama etal. 1993; Shi and Ishizuya-Oka 1996). Adult and devel- oping axolotls, treated with exogenous thyroid hormonesand thyroxine-stimulating hormone, respectively, under-go metamorphotic changes that resemble those of natu-rally metamorphosing amphibians (Rosenkilde and C. Maake · M. Szendefi · M. Reinecke ( ✉ )Division of Neuroendocrinology, Institute of Anatomy,University of Zürich, Winterthurerstr. 190,CH-8057 Zurich, Switzerlande-mail: reinecke@anatom.unizh.chTel: +41-1-635 53 70, Fax: +41-1-635 57 02W. KloasDepartment of Zoology II, University of Karlsruhe,Kaiserstr. 12, D-76128 Karlsruhe, GermanyCell Tissue Res (1999) 297:91–101©Springer-Verlag 1999 REGULAR ARTICLEC. Maake · W. Kloas · M. Szendefi · M. Reinecke Neurohormonal peptides, serotonin, and nitric oxide synthasein the enteric nervous system and endocrine cellsof the gastrointestinal tract of neotenicand thyroid hormone-treated axolotls (  Ambystoma mexicanum  ) Received: 20 January 1999 / Accepted: 8 March 1999  Phaff-Ussing 1996; Brown 1997). Furthermore, parts of the genes for axolotl thyroid hormone receptors havebeen cloned and sequenced (Safi et al. 1997).The terrestrial life of postmetamorphotic amphibia ischaracterized by a different choice of nutritional sources.To gain further insight into the complex processes of am-phibian metamorphosis, we focused on the neurohor-monal system of the gastrointestinal tract. Few studieshave dealt with the presence of neurohormonal peptidesin amphibia, particularly in the enteric nervous system(see Discussion), and only one with the gastrointestinaltract of   A. mexicanum (Gabriel et al. 1992). This investi-gation concentrates on the myenteric plexus of the axo-lotl stomach and reports the presence of immunoreactivi-ty for some neurohormonal peptides and serotonin. Nodata exist on the occurrence of gastroenteroendocrinecells and the innervation patterns of the gastrointestinaltract; furthermore, there is no information about the ef-fect of provoked metamorphosis on the gastrointestinalneurohormonal peptide system in the axolotl.Thus, the present study deals with the immunohisto-chemical localization of some neurohormonal peptides,i.e., vasoactive intestinal polypeptide (VIP), neurotensin(NT), substance P (SP), calcitonin gene-related peptide(CGRP), gastrin/cholecystokinin (GAS/CCK), and so-matostatin (SOM), and nitric oxide synthase (NOS) andserotonin (SER) in the gastrointestinal tract of   A. mexi-canum and considers both the mucosal endocrine cellsand the enteric nervous system. In addition, the distribu-tion patterns and frequencies of the neurohormones andNOS are compared between adult neotenic and thyroidhormone-treated (metamorphosed) individuals. Materials and methods Fixation and embeddingAdult axolotls ( n =6) were decapitated, then stomach, small intes-tine, and large intestine were rapidly dissected and fixed by im-mersion in Bouin’s solution without acetic acid for 3–5 h. In a sec-ond set of adult axolotls ( n =6), metamorphosis was induced by ex-ogenous thyroxine. Five milliliters of T 4 (Serva, Heidelberg, Ger-many) were added per liter of surrounding water. The effect of T 4 treatment was monitored by observation of changes in externalmorphological criteria (Prahlad and DeLanney 1985) such as re-sorption of ventral tail fins and gill filaments, skin change, and adecrease in body weight. The complete metamorphosis occurredwithin 35 days. Principles of laboratory animal care and specificnational laws were followed. Thereafter, the metamorphosed axo-lotls were dissected and fixed as described above. All specimenswere dehydrated in ascending series of ethanol and routinely em-bedded in Paraplast. Sections were cut at 4 µm.Immunohistochemical techniqueTo reduce unspecific binding, sections were treated with phos-phate-buffered saline (PBS) containing 2% bovine serum albuminfor 30 min at room temperature and processed for immunofluores-cence. Sections were incubated with one of the antisera for 12 h at4ºC and washed three times in PBS (pH 7.4). The following antise-ra raised in rabbit were used: anti-vasoactive intestinal polypeptide(1:8000, B34; Bioscience Products, Emmenbrücke, Switzerland),anti-neurotensin (1:800, B44; Bioscience Products), anti-CCK-26–33 (1:600, 20078; Incstar, Stillwater, Calif., USA; Beorlegui etal. 1992; Reinecke et al. 1997), anti-serotonin (1:1000, B56–1; Serva, Heidelberg, Germany; Reinecke et al. 1997), anti-calcitoningene related peptide (1:800, 61011; Peninsula, Merseyside, UK),and anti-nitric oxide-synthase (1:200, A 564; Dako, Copenhagen,Denmark; Spinas et al. 1998). Furthermore, antisera were usedwhich were raised in rat against: somatostatin-14 (1:2000, NT116; Eugene Tech, N.J., USA; Reinecke et al. 1997) and substanceP (1:300, 80509; Serva). The primary antisera were detected usingbiotinylated goat anti-rabbit IgG or biotinylated goat anti-rat IgG(1:100; Bioscience Products) for 30 min at room temperature.Thereafter, sections were washed in PBS and incubated with strep-tavidin-fluorescein-isothiocyanate (FITC) (1:100; Bioscience Prod-ucts) for 30 min at room temperature in the dark.For analysis of the coexistence of SP and CGRP, single sec-tions were incubated consecutively for double immunofluores-cence with the rabbit CGRP antiserum and with the rat SP antise-rum. After buffer wash, the rat SP antiserum was detected withbiotinylated sheep anti-rat IgG (1:100; Amersham, UK) and visu-alized with streptavidin-Texas red (1:100; Amersham). For the de-tection and visualization of the rabbit antiserum against CGRP,FITC-conjugated goat anti-rabbit IgG (1:40; Bioscience Products)was used.The specificity of the reactions obtained was tested using thefollowing controls: (1) Replacement of the primary antiserum bynon-immune rabbit or rat serum; (2) preabsorption of the primaryantiserum with its antigen (40 µg, 400 µg peptide/ml diluted anti-serum). As positive controls, sections of rat gastrointestinal tractwere also processed in every incubation series.Photomicrographs were taken with a Zeiss Axiophot (Zeiss,Zürich, Switzerland). For photography, the fluorochromes were vi-sualized with fluorescence modules for FITC (BP 450–490 nm,FT 510, LP 515–565 nm) and for Texas red (BP 546, FT 580, LP590 nm).Determination of nerve fiber densityFor the quantitative evaluation of VIP-IR and SER-IR nerve fi-bers, five individuals of both untreated or treated axolotls were in-vestigated, screening at least five sections of stomach, small intes-tine, and large intestine, respectively, in each individual. Sectionsprocessed for immunohistochemistry were photographed andprinted at a final magnification of × 450. On these photographs, thenumbers of immunoreactive varicosities within a grid square(4 cm 2 ) were counted (25–30 randomly chosen squares per photo-graph). Statistical analysis of the data was performed with a Stat-View 4.5 program including an ANOVA t  -test (unpaired). Results SerotoninSER immunoreactivity was present in mucosal endocrinecells and nerve fibers, the SER-IR mucosal cells beingpresent mainly in the stomach (Fig. 1a). They occurredin lower numbers in the small intestine (Fig. 1b) andlarge intestine but were found in higher density in themost distal part of the large intestine (Fig. 1c). SER-IRnerve fibers were present in the myenteric plexusthroughout the gastrointestinal tract, reaching their high-est density in the stomach plexus (Fig. 1d,e). These fi-bers were found in moderate numbers in the muscle lay-ers of the stomach (Fig. 1d,e) but were scarce in theintestinal smooth muscle. Very few SER-IR fibers werefound in the submucosal layer and the lamina propria.The metamorphosed individuals exhibited a denserSER-IR innervation, which was most pronounced in themuscular layer of the stomach (Fig. 1d,e; Tables 1, 2, 3), 92  93 Fig.1a–e Immunohistochemical localization of serotonin ( SER )and vasoactive intestinal polypeptide ( VIP ) in the axolotl gastroin-testinal tract. a–c SER-immunoreactive (-IR) endocrine mucosalcells were found in the stomach ( a ), the small intestine ( b ), andthe most distal part of the large intestine ( c ). a, c  × 410; b  × 210. d,e Distribution and frequency of SER-IR nerve fibers in the muscu-lar layers of the stomach. Numerous SER-IR fibers were presentin the myenteric plexus of a neotenic ( d ) and a metamorphosed ( e )individual. The number of SER-IR fibers in the muscle layer of the metamorphosed axolotl ( e ) was greater than in the neotenic in-dividual ( d ). d, e  × 320. f–h Distribution patterns and frequency of VIP-IR fibers in the stomach. f  High numbers of VIP-IR fiberswere found in the muscle and the submucosal layer,where theysupply smooth muscle fibers and the vasculature. Additional VIP-IR fibers were present in the lamina muscularis mucosae and thelamina propria. x450. g, h VIP-IR fibers were found in the myen-teric plexus and the muscle layer of the axolotl stomach. The den-sity of these fibers was greatly enhanced in the metamorphosed in-dividual ( h ) when compared with the neotenic ( g ). g, h  × 420  although this difference was not significant. Here thedensity of SER-IR fibers was 3.94±0.6/0.9 mm 2 in neo-tenic and 5.78±1.8/0.9 mm 2 in metamorphosed individu-als.Vasoactive intestinal polypeptideVIP immunoreactivity was restricted to the enteric ner-vous system. Occasionally, VIP-IR perikarya were pres-ent in the myenteric plexus. VIP-IR fibers occurredthroughout the gastrointestinal tract, with their highestdensity in the large intestine. They were found in highnumbers in the myenteric plexus but also occurred in thesubmucosal plexus. The VIP-IR innervation was mostpronounced in the circular muscle layer where the fiberswere associated with the smooth muscle and the vascula-ture (Fig. 1f). A dense VIP-IR innervation was present inthe lamina muscularis mucosae (Fig. 1f). VIP-IR fiberswere observed frequently in contact with the submucosalvasculature and some were also found in the lamina pro-pria (Fig. 1f). In the metamorphosed individuals, thedensity of the VIP-IR fibers was greatly and, in part,statistically significantly enhanced (Fig. 1g,h; Tables 1,2, 3), e.g., 6.86±0.95 varicosities/0.9 mm 2 comparedwith 1.87±0.46 varicosities/0.9 mm 2 in the stomach mus-cles.NeurotensinNT immunoreactivity occurred in endocrine cells andenteric nerves. NT-IR cells were present in the mucosalepithelium throughout the gastrointestinal tract (Fig.2a–c). They were more numerous in the intestine than inthe stomach with a preference for the upper and middlesmall intestine. Additionally, in the distal large intestinea particular accumulation of NT-IR cells was present(Fig. 2c). Very few NT-IR nerve fibers were observedalong the gastrointestinal tract in the muscle and submu-cosal layers (Fig. 2d) and the lamina propria (Fig. 2b).The majority of the NT-IR fibers were in association toarteries and arterioles. The NT-IR endocrine cells andnerve fibers exhibited no obvious differences betweenneotenic and metamorphosed individuals (Tables 1, 2). 94 Table 1 Distribution and relative frequency of endocrine mucosal cells and enteric nerve fibers in the neotenic axolotlStomachSmall intestineLarge intestineVasoactive intestinalMucosaEndocrine cells–––polypeptideNerve fibers+++SubmucosaNerve fibers++++++MuscularisNerve fibers++++++++++++SerotoninMucosaEndocrine cells+/++++/++Nerve fibers±±±SubmucosaNerve fibers+±±MuscularisNerve fibers++±±NeurotensinMucosaEndocrine cells±++/++Nerve fibers±±±SubmucosaNerve fibers++±MuscularisNerve fibers±±±SomatostatinMucosaEndocrine cells+/+++±Nerve fibers–––SubmucosaNerve fibers–––MuscularisNerve fibers–––Substance PMucosaEndocrine cells±/+±++Nerve fibers±––SubmucosaNerve fibers++±MuscularisNerve fibers++±Calcitonin gene-related MucosaEndocrine cells–––peptideNerve fibers–––SubmucosaNerve fibers±±±MuscularisNerve fibers±±±Gastrin/CCKMucosaEndocrine cells–+/++–Nerve fibers–––SubmucosaNerve fibers–––MuscularisNerve fibers–––NO synthaseMucosaEndocrine cells–––Nerve fibers±±±SubmucosaNerve fibers+++MuscularisNerve fibers±±±  Table 3 ANOVA for analysis of densities of vasoactive intestinalpolypeptide ( VIP )- and serotonin ( SER )-immunoreactive varicosi-ties in the gastrointestinal tract of neotenic versus metamorphosedaxolotls. Densities are expressed as mean numbers of immunore-active varicosities per 0.9 mm 2 tissue (smooth muscle)95 Table 2 Distribution and relative frequency of endocrine mucosal cells and enteric nerve fibers in the metamorphosed axolotlStomachSmall intestineLarge intestineVasoactive intestinal MucosaEndocrine cells–––polypeptideNerve fibers+++SubmucosaNerve fibers+++++++++MuscularisNerve fibers++++++++++++++++SerotoninMucosaEndocrine cells+/++++/++Nerve fibers±±±SubmucosaNerve fibers+++±MuscularisNerve fibers++++±NeurotensinMucosaEndocrine cells±++/++Nerve fibers±±±SubmucosaNerve fibers++±MuscularisNerve fibers+±±SomatostatinMucosaEndocrine cells+/++++/++±Nerve fibers–––SubmucosaNerve fibers–––MuscularisNerve fibers–––Substance PMucosaEndocrine cells±/+±++Nerve fibers±––SubmucosaNerve fibers+/+++±MuscularisNerve fibers++±Calcitonin gene-related MucosaEndocrine cells–––peptideNerve fibers–––SubmucosaNerve fibers±±±MuscularisNerve fibers±±±Gastrin/CCKMucosaEndocrine cells–+/++–Nerve fibers–––SubmucosaNerve fibers–––MuscularisNerve fibers–NO synthaseMucosaEndocrine cells–––Nerve fibers±±±SubmucosaNerve fibers+++MuscularisNerve fibers±±±StomachSmall intestineLarge intestineNeotenicT 4 NeotenicT 4 NeotenicT 4 VIPMean1.876.8612.9025.9325.4022.22Standard deviation0.670.950.574.226.4510.98Standard error0.390.470.332.443.726.34 P -value  ≤ 0.0006  ≤ 0.0060.69SERMean3.945.78Standard deviation0.621.83Standard error0.441.06 P -value0.28 Substance PSP immunoreactivity was present in endocrine cells(Figs. 2e, 3a) and enteric nerve fibers (Figs. 2e, 3a,c,e).The SP-IR endocrine mucosal cells were found in mod-erate numbers in all parts of the gastrointestinal tract andshowed a particular density in the distal large intestine.Only a few SP-IR perikarya (Fig. 3c) and numerousSP-IR fibers were present in the myenteric (Fig. 3a,c)and submucosal plexus. SP-IR fibers were found in mod-erate numbers both in the muscle layers (Fig. 3a,c) andthe submucosa, where they were in close association
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