an immunohistochemical study on the neuroendocrine system in the alimentary canal of the brown...

General and Comparative Endocrinology 138 (2004) 166–181

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An immunohistochemical study on the neuroendocrine system in the alimentary canal of the brown trout, Salmo trutta, L., 1758

Giampaolo Bosi, Alessia Di Giancamillo, Silvana Arrighi, and Cinzia Domeneghini¤

Department of Veterinary Sciences and Technologies for Food Safety, University of Milan, via Trentacoste n. 2, I-20134 Milan, Italy

Received 30 September 2003; revised 24 May 2004; accepted 1 June 2004Available online 20 July 2004

Abstract

Several neurohormonal peptides of the gastrointestinal system of Wsh have been revealed by immunohistochemical methods.Among salmonids, the rainbow trout, Oncorhynchus mykiss (Walbaum) is the most studied species, whereas the informations aboutother species of the taxonomic group are lacking. The regional distribution and relative densities of cells belonging to the neuroendo-crine system have been in this paper demonstrated in the gut of the brown trout, Salmo trutta Linnaeus. In the gastric mucosa, endo-crine cells were detected, which were immunoreactive to bombesin-, gastrin-, and secretin-antisera. Endocrine cells containinggastrin-, bombesin-, cholecystokinin-8-, glucagon-, and leptin-like immunoreactivities were present in the pyloric caeca and intestine.The pancreatic endocrine islets contained glucagon-, and, possibly, secretin-like-immunoreactive endocrine cells, as well as a contin-gent of galanin-like-immunoreactive nerve Wbres. The exocrine pancreatic parenchyma showed bombesin-like-immunoreactive nerveWbres. Within the tested regulatory peptides, bombesin and leptin were observed in both endocrine cells and nerve cell bodies andWbres. Leptin was in addition detected in epithelial cells of the gastric glands. In the brown trout we have never observed any immu-noreactivity to the VIP antiserum (either in the stomach or in the intestine). Some special structural patterns (in particular those onesrelated to galanin- and leptin-immunohistochemical data) have thus been detected for the Wrst time in the brown trout, and providefurther data for a better knowledge of gut morpho-functional aspects in this economically important Wsh. 2004 Elsevier Inc. All rights reserved.

Keywords: Alimentary canal; Brown trout; Neuroendocrine system; Leptin; Immunohistochemistry; Confocal laser microscopy

1. Introduction

The enteric nervous and the endocrine diVuse systemsplay in Wsh, as in mammals, important roles in coordi-nating various intestinal processes such as motility,blood Xow, and secretion/absorption (Chang et al., 1998;Holmgren and Jönsson, 1988; Olsson et al., 1999). Sev-eral regulatory peptides are produced by the cells ofthese systems. The presence of regulatory peptides in theneuroendocrine system of the alimentary canal in Wshhas been reported by many authors (al-Mahrouki andYouson, 1998; De Girolamo et al., 1999; Domeneghiniet al., 1999, 2000; Gòmez-Visus et al., 1998; Karila and

¤ Corresponding author. Fax: +39-2-50315746.E-mail address: [email protected] (C. Domeneghini).

0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2004.06.003

Holmgren, 1997; Reinecke et al., 1997; Visus et al., 1996).Immunohistochemical methods, which utilize antiseraagainst both Wsh and mammalian antigens, are exten-sively used to detect the presence of a number of puta-tive regulatory substances, because their amino acidsequences are largely conserved. In teleosts, severalworks show that the presence and distribution of diVer-ent immunoreactive substances are often species-speciWc(Elbal et al., 1988). These morphological studies form animportant basic background to the Wsh gut physiologicalresearch.

The most studied species in salmonids is the rainbowtrout, Oncorhynchus mykiss (Walbaum), and manyimmunohistochemical studies have been carried out onthe presence of several neurohormonal peptides in neu-rons and endocrine cells of the gut and pancreas of thisspecies (Anderson and Campbell, 1988; Barrenechea

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G. Bosi et al. / General and Comparative Endocrinology 138 (2004) 166–181 167

et al., 1994; Beorlegui et al., 1992; Bjenning and Holm-gren, 1988; Dubois et al., 1979; Holmgren et al., 1982;Wagner and McKeown, 1981).

In this study, the regional distributions and relativedensities of the cells belonging to the neuroendocrinesystem in the alimentary canal and pancreas of thebrown trout, Salmo trutta Linnaeus, were investigatedby immunohistochemical methods using antisera againstbombesin, cholecystokinin-8 (CCK-8), gastrin, gluca-gon, secretin, galanin, leptin, and vasoactive intestinalpeptide (VIP). These regulative molecules share func-tions, which, among others, peripherally inXuence thecentrally regulated feeding behaviour. Within them,bombesin and CCK-8 are reputed Wsh peripheral satietysignals (Jensen, 2001), whereas galanin is regarded as anorexigenic signalling molecule in Wsh (Lin et al., 2000)like in mammals (Crawley, 1995). The knowledge aboutWsh leptin is at present far from being full, but recentimmunohistochemical studies (Johnson et al., 2000;Muruzábal et al., 2002) evidence the presence of leptin inWsh tissues diVerent from adipose. Lin et al. (2000) sug-gest that this hormonal regulator acts in Wsh upon foodintake via a central mechanism, which needs peripheralmessages, like in mammalian species (Schwartz and See-ley, 1997). When possible, the results here obtained willbe compared with those obtained by previous studies onthe related species O. mykiss, thus providing furtherinformation for a better knowledge of the gut in diversespecies of trouts, all of them of high commercial interest.

2. Materials and methods

2.1. Animals

Adults of S. trutta were collected by seine net at Car-turo (River Brenta, north of Padua, Italy). Ten Wsh,ranging from 20 to 40 cm in total length, were used forthis study. Samples of stomach (fundic region, pyloricregion), pyloric caeca, and intestine (proximal, middle,and distal regions) were collected immediately after thesacriWce by a blow to the head. Pancreatic tissue mixedwith adipose tissue was adherent to the gut wall of the

pyloric caeca and proximal intestine. The small pieces,Wxed for 7 h at 4 °C in Bouin's Xuid, were then paraYn-embedded after the dehydration treatment.

2.2. Immunohistochemistry

Dewaxed and re-hydrated sections (6 �m-thick) weretreated with 0.6% H2O2 in absolute methanol for 15 min,and with Normal Goat Serum (DAKO, Italy) diluted at1:250 for 30 min to inhibit non-speciWc reactivity. Sec-tions were then incubated overnight at 4 °C in a humidchamber with the primary antisera (Table 1). The pri-mary antisera have been diluted with 0.05 M Tris–HClbuVer containing 0.1% Triton X-100 and 0.1% bovineserum albumin (BSA). After rinsing in Tris–HCl buVersaline (TBS; 0.05 M, pH 7.4, 0.55 M NaCl), the sectionswere then incubated for 30 min with the secondary anti-serum (Goat anti-Rabbit IgG 1:100, DAKO) and,Wnally, for 30 min with Rabbit PAP (peroxidase–anti-peroxidase) complex (1:200, DAKO). The peroxidasereaction was developed in a solution of 3,3�-diam-inobenzidine tetrahydrochloride (Sigma, Italy) (0.04% w/v in Tris–HCl 0.05 M, pH 7.4) and H2O2 (0.005%). Devel-oped sections were counterstained with Mayer's hema-toxylin solution. Slides were observed and photographedunder an Olympus BX50 photomicroscope. The relativedensities of endocrine cells and nerve cell bodies andWbres immunoreactive to each antiserum were evaluatedby subjective estimates of all the authors, and repre-sented with symbol scales as in similar studies on thedigestive neuroendocrine system of the rainbow trout(Barrenechea et al., 1994; Beorlegui et al., 1992).

The controls for the speciWcity of immunohistochemi-cal reactions were performed by the pre-absorption ofeach antiserum with the corresponding antigen (10–100 mg/ml) (Table 2). Treating sections with the pre-absorbed antisera overnight at 4 °C, no immunoreactivitywas detected. Mammalian (swine, rat) gut samples wereused as positive controls. Samples of the alimentarycanals of other teleosts (eel, Anguilla anguilla, and chub,Leuciscus cephalus) were tested for additional positivecontrols. These sections gave the expected immunoreac-tivities.

Table 1Primary antisera used in this study

a CCK-8, cholecystokinin-8; VIP, vasoactive intestinal peptide.

Antisera raised in rabbit against Code Source Dilution

Bombesin AB905 Chemicon, Temecula, CA (USA) 1:400CCK-8a AB1973 Chemicon, Temecula, CA (USA) 1:600Galanin IHC 7153 Peninsula Laboratory, Belmont, CA (USA) 1:500Gastrin AB930 Chemicon, Temecula, CA (USA) 1:200Glucagon IHC 7165 Peninsula Laboratory, Belmont, CA (USA) 1:500Leptin (Ob1) SC 842 SantaCruz Biotechnol., Santa Cruz, (USA) 1:50Secretin IHC 7313 Peninsula Laboratory, Belmont, CA (USA) 1:1000VIPa CA-08-340 Genosys Biotechnol., Cambridge (UK) 1:500

168 G. Bosi et al. / General and Comparative Endocrinology 138 (2004) 166–181

2.3. Double immunoXuorescence

Sections (8 �m-thick) of the pyloric caeca with pan-creatic tissue, and proximal intestine were dewaxed,re-hydrated, and rinsed in TBS with 0.1% Triton-X 100(TBS-T). A treatment with Normal Goat Serum, 1:20 inTBS for 30 min was used for the inhibition of non-spe-ciWc reactivity. Sections were then incubated with theWrst-step primary anti-sera, either rabbit polyclonal anti-gastrin (Chemicon, 1:200, 3 h at 37 °C) or rabbit poly-clonal anti-secretin (Peninsula, 1:500, overnight at roomtemperature, RT). The slides were then washed in TBS-T, and subsequently treated with the biotin–avidinblocking kit solutions (Vector Laboratory, USA). Thesections were rinsed again in TBS-T and incubated with10 �g/ml goat biotinylated anti-rabbit IgG (Vector Lab-oratory) in TBS-T for 1 h at RT. After rinsing twice inTBS-T, the sections were treated with 10�g/ml Fluores-cein Avidin D (Vector Laboratory) in NaHCO3 0.1 M,pH 8.5, with 0.15 M NaCl for 1 h at RT.

For the second step of the double immunoXuores-cence, the slides previously (Wrst step) incubated for thedemonstration of gastrin-like-immunoXuororeactivitywere treated with rabbit polyclonal anti-cholecystoki-nin-8 (CCK-8) serum (Peninsula, 1:500, 3 h at 37 °C),and the slides previously (Wrst step) treated for the dem-onstration of secretin-like-immunoXuororeactivity wereincubated with rabbit polyclonal anti-glucagon (Penin-sula, 1:400, 4 h at RT). A blocking step with the biotin–avidin kit solutions (Vector) was performed, and thensections were rinsed in TBS-T and incubated with goatbiotynilated anti-rabbit IgG in TBS-T for 1 h at RT. Thesections were again washed twice in TBS-T, then treatedwith 10�g/ml Rhodamine Avidin D (Vector Labora-tory) in NaHCO3 0.1 M, pH 8.5, with 0.15 M NaCl for1 h at RT. Finally, slides with tissue sections wereembedded into Vectashield Mounting Medium (VectorLaboratory) and observed under a Confocal laser scan-ning microscope (Fluoview FV300, Olympus Italy). Thesections were excited using Argon/Helio-Neon-Green

Table 2Peptides used for absorption tests

a CCK-8, cholecystokinin-8; VIP, vasoactive intestinal peptide.

Peptide Code Source

Bombesin B4272 Sigma Chemicals, St. Louis, MO (USA)Pancreozymin P4429 Sigma Chemicals, St. Louis, MO (USA)CCK-8a H2085 Bachem AG, Bubendorf, SwitzerlandGalanin G112 Sigma Chemicals, St. Louis, MO (USA)Gastrin G3131 Sigma Chemicals, St. Louis, MO (USA)Glucagon G7774 Sigma Chemicals, St. Louis, MO (USA)Glucagon H6790 Bachem AG, Bubendorf, SwitzerlandLeptin (Ob1) SC 842 P SantaCruz Biotechnol., Santa Cruz,

(USA)Secretin S7147 Sigma Chemicals, St. Louis, MO (USA)VIPa V3628 Sigma Chemicals, St. Louis, MO (USA)

lasers with excitation and barrier Wlters set for Xuores-cein and rhodamine. Images containing superimpositionof Xuorescence were obtained by acquiring the imageslice of each laser excitation or channel sequentially.With this image capturing method, the image of thedouble-dyed specimens (either gastrin/CCK-8 or secre-tin/glucagon) can be obtained by sequentially acquiringimage slice of each type of Xuorescence, reducing noisebackground. Evaluation of the percentages of immuno-Xuororeactive endocrine cells was performed counting ineach section the cells with a single versus double immu-noXuororeactivity.

3. Results

3.1. Immunohistochemistry

Both endocrine cells (ECs) and nerve cell bodies andWbres were observed in the whole gastro–entero–pancre-atic system of the brown trout. ECs immunohistochemi-cally detected with the tested antisera were eitherroundish to ovoidal or elongated in shape. The formerendocrine cell type was interpreted as close in type, thelatter one as open in type (Fujita, 1989), reaching thelumen with an apical cytoplasm process. Immunoreac-tive neurons were detected within the myenteric plexus,as well as in a sub-serosal localization. Immunoreactivenerve Wbres were observed running in both the tunicapropria-submucosa and tunica muscularis. The relativedensities of both endocrine cells and nerve cell bodiesand Wbres observed in diVerent organs are summarizedin Table 3.

We never detected any immunoreactivity to theVIP-antiserum either in endocrine cells or in intramu-ral neurons. A VIP-like immunoreactivity was on thecontrary detected in the samples of the gut of the otherWsh species (eel, chub) tested as positive controls.

3.1.1. StomachA bombesin-like material was found in ECs of the

gastric mucosa (Figs. 1A and B). In addition, numerousbombesin-like-immunoreactive (IR) nerve Wbres wereobserved: (i) below the stratum granulosum of the stom-ach (Fig. 1A); (ii) in the connective axis of the gastricfolds in the pyloric region (Fig. 1B); (iii) in the innercircular musculature (Fig. 1C); and (iv) surroundingthe vessels that run between the musculature and thetunica serosa of the gastric wall (Fig. 1D). On the otherhand, we never found bombesin-like (IR) neurons inthe myenteric plexus. Gastrin- and secretin-antiserarevealed IR ECs in the epithelium of the gastricmucosa (Figs. 2A and B, respectively). Gastrin-like-IRECs were more numerous, especially in the pyloricstomach, than the endocrine elements containing asecretin-like material. CCK-8- and glucagon-antisera


did not reveal any immunoreactivity. The galanin-anti-serum revealed roundish IR nerve cell bodies in themyenteric plexus of the stomach (Fig. 2C), as well as IRnerve Wbres running in the gastric tunica propria-sub-mucosa and inner circular musculature (Fig. 2D). TheseIR nerve Wbres were rather numerous in the inner mus-culature of the proximal stomach (Fig. 2D), but theirnumber evidently decreased in the pyloric stomach(Fig. 2E). A leptin-like material was detected in IRnerve Wbres, which run in the muscular sheet of theproximal stomach, specially where the striated oesoph-ageal musculature conWned with the smooth gastricmusculature (Fig. 2F). In addition, a leptin-like mate-rial was shown in epithelial cells of the gastric glands,especially in their deep half. This leptin-like-immunore-activity was present in form of small cytoplasmic gran-ules (Fig. 2G).

3.1.2. Pyloric caecaThe mucosal folds of the pyloric caeca contained

ECs, which showed a CCK-8-like-IR material (Fig. 3A).Other ECs were gastrin-, glucagon- (Fig. 3B), and bom-besin-like-IR (Fig. 3C). The additional presence of subtlebombesin-like-IR nerve Wbres in the connective axis ofthe intestinal folds was observed (Fig. 3C). On the otherhand, we never found bombesin-like-IR neurons in themyenteric plexus.

3.1.3. PancreasNumerous glucagon-like-IR ECs were observed in

the endocrine islets of the diVuse pancreas (Fig. 4A). Aweak immunopositive reaction for the secretin-antise-rum was noticed in pancreatic endocrine cells, too(Fig. 4B). Thin bombesin-like-IR nerve Wbres and nervebundles were detected among endocrine cells of the islets(Fig. 4C), in the exocrine pancreatic parenchyma(Fig. 4D), and often placed around the blood vessels. Agalanin-like-immunoreactivity was evidenced in rathernumerous nerve Wbres, which were seen running among

the endocrine cells of the pancreatic islets (Fig. 4E) andrunning around the ducts of the exocrine parenchyma(Fig. 4F).

3.1.4. IntestineGastrin-, glucagon-, and CCK-8-like-IR ECs

(Fig. 5A) were detected in the proximal intestine, andwere rather numerous. Similarly, in the middle intes-tine, numerous CCK-8-, and glucagon-like-IR ECswere always detected. The glucagon-like-IR ECs werealways localized in the upper half of the intestinal folds(Fig. 5B). A leptin-like-immunoreactivity was shown innot-numerous endocrine cells of an elongated shape(Fig. 5C) and in subtle nerve Wbres running in the innermusculature of both the proximal and middle intestine(Fig. 5D). Immunoreactivity against glucagon-antise-rum was detected in ECs of the distal intestine(Fig. 6A). Bombesin-like-IR nerve cell bodies andWbres were observed in the myenteric and sub-serosalplexuses of this intestinal region (Fig. 6B), where, inaddition, a galanin-like immunoreactivity was detected(Fig. 6C).

3.2. Double immunoXuorescence

In the pyloric caeca, the gastrin and CCK-8 antiserarevealed two diVerent cell populations. One of them,small in number (13%), only contained CCK-8-rhoda-mine material (Fig. 7A). The most diVuse (87%) celltype population was constituted by elements, whichshowed immunoXuorescence for both the Xuorophores,thus positive for gastrin-Xuorescein- and CCK-8-rho-damine-antisera (Fig. 7B).

In the proximal intestine, the relative density of cellimmunoXuororeactive to the CCK-8-rhodaminealone was higher (25%) than that in the pyloric caeca,but low if compared with the percentage (75%) ofendocrine cells reactive to both the antisera (Figs. 8Aand B).

Table 3Relative densities of both endocrine cells and nerve cell bodies and Wbres in the alimentary canal of the brown trout detected by immunohistochem-istry (peptides are listed in alphabetical order)

Relative densities of endocrine cells: +++, numerous; ++, moderately numerous; +, scarce; ¡, not detected.Relative densities of nerve cell bodies and Wbres: *, scarce; **, moderately numerous; ***, numerous; and no symbol, not detected.a CCK-8, cholecystokinin-8; VIP, vasoactive intestinal peptide.b gg, immunoreactive cells of the gastric glands.

Stomach Pyloriccaeca

Pancreas Intestine

Fundic region Pyloric region Proximal Middle Distal

Bombesin ++*** ++*** +** ¡** ¡ ¡ ¡**

CCK-8a ¡ ¡ ++ ¡ +++ ++ ¡Galanin *** ** ¡ *** ¡ ¡ **

Gastrin + ++ ++ ¡ + ¡ ¡Glucagon ¡ ¡ ++ +++ ++ + +Leptin (Ob1) ggb** ** ** ¡ +** +** ¡Secretin + + ¡ + ¡ ¡ ¡VIPa ¡ ¡ ¡ ¡ ¡ ¡ ¡


In the pancreas, endocrine cells, which were anti-secretin immunoXuororeactive were positive to the glu-cagon-antiserum, too (Fig. 9A). On the contrary, mostof the endocrine cells revealed by this latter antiserumwere not shown containing a secretin-like material(Fig. 9A). Intestinal glucagon-like-IR endocrine cellsdid not show secretin-like immunoreactivity (Fig. 9B).

4. Discussion

The basic histology of the digestive tract of the salmo-nids was extensively described in the last century(Greene, 1912; Weinreb and Bilstad, 1955). There are nomarked diVerences in the microscopical anatomy of thegut in the diverse species of this family (Bullock, 1963).

Fig. 1. Stomach. Bombesin-like immunoreactivity is detected in: (A and B) epithelial endocrine cells (arrows); (A) subtle nerve Wbres (asterisks)placed between the stratum granulosum (arrowheads) and the tunica muscularis (tm) (gg: gastric glands); (B) thin nerve Wbres (arrowheads) running inthe connective axis of the gastric folds; (C) nerve Wbres (arrows) in the circular muscle layer; and (D) nerve Wbres (arrows) in a sub-serosal localiza-tion near vascular structures (a: artery; v: vein; arrowheads: melano-macrophages). Scale bars D 50 �m.


On the contrary, we have here shown that immunohisto-chemistry of regulatory peptides within the gut neuroen-docrine system reveals a structural pattern in S. trutta,

which only in part resembles that of the most studiedspecies in salmonids, O. mykiss (Jensen et al., 2001; Ols-son et al., 1999).

Fig. 2. Stomach. The surface epithelium of the gastric mucosa shows (A) endocrine cells immunoreactive to the gastrin-antiserum, and (B) an endo-crine cell containing a secretin-like substance. Scale bars D 10 �m. (C) A roundish galanin-like immunoreactive neuron is seen in the myenteric plexus(bv: blood vessel; e: erythrocytes). Scale bar D 20 �m. (D) Subtle galanin-like immunoreactive nerve Wbres are shown running in the tunica propria-submucosa (tp) (arrowheads) and in the inner musculature (tm) (arrows). Scale bar D 50 �m. (E) A thin solitary galanin-like immunoreactive nerveWbre (arrows) is seen running in the inner musculature of the pyloric stomach. Scale bar D 50 �m. (F) A leptin-like immunoreactivity is detected innerve Wbres (arrows) which appose to striated muscle Wbres (tm) of the transition zone between oesophagus and stomach. Scale bar D 50 �m. (G)Cytoplasm of some epithelial cells (arrows) of gastric glands shows an evidently granular immunoreactivity towards the leptin-antiserum (tp, tunicapropria-submucosa; gg, gastric gland). Scale bar D 20 �m.


Bombesin and gastrin releasing peptide (GRP) belongto the same peptide family (Jensen, 2001). Closely relatedforms of either peptide are found in all the major verte-brate groups (Holmgren and Jensen, 1994). The stomachof S. trutta contained a bombesin-like-immunoreactivityin nerve Wbres that run in the connective axis of thegastric folds, in the submucosal plexus, in the circularmuscle, and around blood vessels. We have in additionobserved gastric bombesin-like-immunoreactive endo-crine cells. The presence of bombesin-like-immunoreac-tive neurons in the stomach has been described in otherteleosts, too (Holmgren and Jönsson, 1988). Holmgrenet al. (1982) and Barrenechea et al. (1994) have reportedthe occurrence of bombesin-like-immunoreactive endo-crine cells in the stomach of the rainbow trout, S. trutta.Bombesin has been shown to display an excitatory eVecton isolated strip preparations of both longitudinal andcircular muscle from the rainbow trout stomach (Holm-gren, 1985), and to upgrade the eVect of acetylcholine atthe receptor sites in the cod and in O. mykiss (Thorn-

dyke and Holmgren, 1990). In addition, it may act uponintestinal blood Xow modulating the blood vessels pres-sure, and really we have found immunoreactive nerveWbres around small arteries and veins of the gastrointes-tinal wall. In addition to the stomach, in the pyloriccaeca also of S. trutta we have found both bombesin-like-immunoreactive endocrine cells and nerve Wbres. Onthe other hand, we never observed bombesin-like-immu-noreactive nerve cell bodies either in the stomach or inthe pyloric caeca. It is thus conceivable that the observedimmunoreactive nerve Wbres come from the autonomicnervous system, and in particular its parasympatheticcomponents. On the other hand, we observed bombesin-like-immunoreactive nerve cell bodies in the myentericplexus of the distal intestine. We can therefore hypothe-size that the bombesin-like-immunoreactive nerve Wbresobserved in this intestinal region come from theobserved intramural neurons. It is noticeable thatHolmgren et al. (1982) showed rare endocrine cellsimmunoreactive to this neuropeptide in the rectum of

Fig. 3. In the pyloric caeca, endocrine cells (arrows) are immunoreactive to the CCK-8 antiserum (A). Scale bar D 100 �m. (B) A strong glucagon-likeimmunoreactivity is detected in endocrine cells (arrows). Scale bar D 20 �m. (C) A bombesin-like immunoreactivity is detected in a small endocrinecell (arrow) and in a thin nerve Wbre bundle (arrowheads) running in the connective axis of an intestinal fold. Scale bar D 20 �m.


O. mykiss. Moreover, for the Wrst time in salmonids, wereport the presence of a bombesin-like material in subtlenerve Wbres placed in the connective septa of theexocrine pancreas of S. trutta, as well as around itsexcretory ducts. Fish pancreatic tissue is richly inner-vated by the autonomic nervous system, and nerve Wbrespenetrate the exocrine parenchyma, where they becomeincreasingly thinner (Putti et al., 2000). Among teleosts,

Jönsson (1991) observed occasional nerve Wbres immu-noreactive to bombesin- and VIP-antisera in the pan-creas of the cod, Gadus morhua.

In the stomach of the brown trout, S. trutta, we foundgastrin-like-immunoreactive endocrine cells locatedamong the other epithelial cells of the fundic and pyloricmucosal regions. In the rainbow trout, O. mykiss, Bar-renechea et al. (1994) showed a high number of gastrin-

Fig. 4. Pancreas (ad, adipose cells; ex, exocrine pancreas; is, endocrine islets; pd, pancreatic duct). Endocrine cells (arrows) contain a glucagon-likematerial (A), others (arrows) are secretin-like immunoreactive (B). Scale bars D 20 �m. (C) Thin nerve Wbres (arrows) running in an endocrine isletshow an immunoreactivity to the bombesin-antiserum. Scale bar D 50 �m. (D) Bombesin-like immunoreactive nerve Wbres (arrows) are seen in theexocrine parenchyma and around an excretory duct (square). Scale bar D 50 �m. (E) Galanin-like immunoreactive nerve Wbres (arrows) are shown inone endocrine islet. Scale bar D 50 �m. (F) Galanin-like immunoreactive nerve Wbres are detected around an excretory duct. Scale bar D 50 �m.


immunoreactive endocrine cells in the pyloric regiononly. Gastrin-like-immunoreactive endocrine cells were alsodetected in the pyloric caeca and proximal intestine of thebrown trout. As shown by double immunoXuorescence, themajor part of these latter gastrin-like-immunoreactiveendocrine cells is also CCK-8-like-immunoreactive. Inmammals, gastrin is a peptide hormone regulating the gas-tric acid secretion and growth of the gastrointestinal epi-thelium (Kinoshita and Ishihara, 2000; Larsson, 2000).In Wsh also, like in mammals, the speciWc target of gas-trin are the fundic exocrine cells, which produce HCl andpepsinogen, and are thus called oxyntopeptic cells (Bjen-ning and Holmgren, 1988).

In the pyloric caeca of the brown trout, S. trutta, wehave observed rather numerous endocrine cells, whichwere immunoreactive to the CCK-8 antiserum. In addi-tion, a CCK-8-like material has been found in endocrinecells of the proximal and middle intestine. In Wsh, chole-cystokinin inXuences stomach motility (Olsson et al.,1999) and acts in the control of food intake (Le Bail andBoeuf, 1997).

Many authors (Barrenechea et al., 1994; GarcíaHernández et al., 1994; Jönsson et al., 1987; Kiliaanet al., 1992; Reinecke et al., 1997) have shown that theimmunoreactivities against gastrin and CCK-8 peptidesare located in the same endocrine cell population of thepyloric stomach and pyloric caeca of Wsh. Since gastrinand CCK-8 share a common C-terminal pentapeptideamide (Jönsson et al., 1987), the highly conservedsequence contained within both peptides displaysunavoidable cross-reactivity with all gastrin/CCK-likepeptides (Himick and Peter, 1994). As regards our data,if is true that in the gastric mucosa of the brown trout wehave observed gastrin-like-immunoreactive endocrinecells, but no endocrine cells immunoreactive to theCCK-8 antiserum, and that, on the contrary, the middleintestine contained CCK-8-like immunoreactive andnot gastrin-like-immunoreactive endocrine cells, it isundoubted that the gastrin- and CCK-8-like immunore-activities are possibly colocalized in a large number ofendocrine cells of the pyloric caeca and proximal intestineof S. trutta, as shown by the double immunoXuorescence.

Fig. 5. Intestine. (A) Numerous endocrine cells (arrows) are immunoreactive to the CCK-8-antiserum in the proximal intestine (arrowheads indicateun-reactive mucous cells). Scale bar D 50 �m. (B) Endocrine cells (arrows) containing a glucagon-like immunoreactivity are detected prevalently inthe upper half of the intestinal folds (middle intestine). Scale bar D 50 �m. (C) An elongated leptin-like immunoreactive endocrine cell (arrow) isshown in the mucosal epithelium of the middle intestine. Arrowheads show un-reactive mucous cells (sc, stratum compactum; sg, stratum granulosum).Scale bar D 20 �m. (D) Subtle immunoreactive nerve Wbres are seen running in the inner musculature of the middle intestine (arrows). Scalebar D 20 �m. (tp, tunica propria-submucosa; cm, circular muscular layer; and lm, longitudinal muscular layer.)


As a consequence, a minor percentage of endocrine cells,more conspicuous in the proximal intestine than in pylo-ric caeca, is CCK-8-like-immunoreactive only. We thinktherefore that in the brown trout gastrin- and CCK-8-synthesizing endocrine cells possibly constitute two sep-arate populations in both the stomach and middle intes-tine, but these peptides are in part synthesized by the

same endocrine cell populations in pyloric caeca andproximal intestine.

We have found in the stomach of the brown trout,S. trutta, secretin-like-immunoreactive endocrine cells,which were localized in the upper third of the gastricfolds in the fundic and pyloric gland regions. Secretin isa 27-amino acid peptide hormone belonging to the struc-

Fig. 6. Distal intestine. (A) Endocrine cells (arrows) are shown, which are immunoreactive to the glucagon-antiserum (sc, stratum compactum; sg,stratum granulosum). (B) A strong immunoreactivity is shown to the bombesin antiserum in nerve Wbres detected in a sub-serosal localization (arrow-heads) (tm, tunica muscularis; ts, tunica serosa). (C) A galanin-like immunoreactivity is shown in nerve Wbres running in a sub-serosal localization(arrowheads), near a blood vessel (bv). Scale bars D 20 �m.


turally related peptides of pituitary adenylate cyclase-activating polypeptide/glucagon superfamily (Sherwoodet al., 2000). In Sparus aurata, Linnaeus, the presence ofsecretin was reported in endocrine cells of the upper halfof the gastric folds (Abad et al., 1987). Until now, nodata on the occurrence of secretin-like endocrine cells inthe stomach of salmonids have been published. The pres-ence of secretin in a gastric localization may be related tothe inhibition of gastric emptying and a stimulation ofgastric acid secretion via an enhanced gastrin release, asshown in mammalian species (Jin et al., 1994; Raybouldand Holzer, 1993; Shiratori et al., 1993). We have inaddition detected a secretin-like-immunoreactivity in asmall number of pancreatic endocrine cells of the browntrout, and this Wnding too appears new in Wsh. If con-Wrmed, maybe that in the pancreatic localization, secre-tin-like-immunoreactive endocrine cells act in regulatingthe strictly nearby pancreatic exocrine cells (“paracrine”action).

In the pyloric caeca as well as in the entire intestine ofthe brown trout, S. trutta, we have observed endocrinecells that were immunoreactive to the glucagon-antise-rum. In addition, as expected, numerous glucagon-like-immunoreactive endocrine cells have been shown inpancreatic endocrine islets, where a minor percentage ofthem revealed an immunoreactivity also towards thesecretin-antiserum. This same possible colocalization is,however, negligible in intestinal endocrine cells, which

consequently may be hypothesized to perform theunique synthesis of glucagon. Glucagon, whose 29-amino acid sequence is well conserved across verte-brates, displays functions which are in part Wsh-speciWc,above all those related with its antagonism with insulin.It is, however, well established that glucagon is a hyper-glycemic and lipolytic substance in Wsh species so farexamined (Moon, 1998), and it may be a Wsh potentialanorexic factor (Le Bail and Boeuf, 1997; Navarro et al.,1993; Silverstein et al., 2001).

Galanin is an originally porcine peptide, which dis-plays a number of physiological actions upon mamma-lian gut, which are various and frequently depend uponspecies and diVerent parts of the alimentary canal(Rattan, 1991; Yagci et al., 1990). In the alimentary canalof Wsh its presence is sporadically demonstrated (Karilaand Holmgren, 1997; Karila et al., 1993; Kiliaan et al.,1993; Wang et al., 1999), and Anglade et al. (1994) havecharacterized trout (O. mykiss) galanin conWrming thatthe N-terminal part of the molecule is fully conservedacross vertebrates (Wang et al., 1999). We have shownthe presence of a galanin-like substance in intramuralneurons of the brown trout (S. trutta) alimentary canal,and, to our knowledge, this is the Wrst report about thispeptide in salmonids gut. Our immunohistochemicaldata are compatible with a possible signiWcance of it as aparasympathetic neuromodulator, as postuled by Karilaet al. (1993) in the Atlantic cod, in which galanin acts

Fig. 7. (A) In pyloric caeca, an endocrine cell, which is unreactive to the anti-gastrin serum (left) contains a CCK-8-like immunoXuororeactive mate-rial (right). Scale bars D 10 �m. (B) An endocrine epithelial cell shows immunoXuororeactivity to both gastrin (left) and CCK-8 (right) antisera. Scalebars D 10 �m.


upon the inner muscular layer. We have in additionshowed the presence of putative galaninergic nerve Wbresin the islets of the brown trout endocrine pancreas. Inthe endocrine pancreas of tompot blenny (Blenniusgattoruggine), too, Putti et al. (2000) have demonstratedgalaninergic nerve Wbres which are likely important forthe regulation of islets secretions.

Leptin is a recently (Zhang et al., 1994) discoveredpeptide hormone which in mammals and other verte-brates (Lin et al., 2000; Muruzábal et al., 2002; Taouiset al., 1998) regulates food intake and energy balance.We have found immunoreactive nerve Wbres running in

the inner musculature of the brown trout (S. trutta)stomach and intestine (except for the distal part of it),and immunoreactive endocrine cells in the middle intes-tine. We did not Wnd, on the contrary, leptin-like-immu-noreactivity in the adipose tissue associated to thealimentary canal. This absence is noticeable, because onemight expect its presence considering the leptin sourcefrom mammalian adipocytes (Zhang et al., 1994). Theseobservations may be accordingly related to those ofMuruzábal et al. (2002) who have observed a leptin-immunoreactivity in the gut myenteric plexus and in gas-tric endocrine cells of the rainbow trout (O. mykiss), as

Fig. 8. (A) In the proximal intestine, an endocrine cell shows the CCK-8-like immunoXuororeactivity (right) only, and is not reactive to the gastrinantiserum (left). Scale bars D 20 �m. (B) One endocrine cell is shown, which is both gastrin-like (left) and CCK-8-like (right) immunoXuororeactive,with a weaker signal for the anti-gastrin serum (left) compared with CCK-8 antiserum. Scale bars D 20 �m.


well as the absence of leptin in the adipocytes. We have inaddition found a leptin-like-immunoreactivity in epithelialcells of the gastric glands. This latter observation is insharp contrast with the study of Muruzábal et al. (2002),but is in agreement with immunohistochemical studiesupon mammals, in which other authors have recentlyidentiWed leptin in parietal cells of human stomach (Mixet al., 2000; Sobhani et al., 2000). The further observationof ours that a leptin-like-immunoreactivity is detected innerve Wbres apposing striated muscle Wbres of the oesoph-ageal–gastric muscular layer is at present not reported inother Wsh species, but may be tentatively related to quiterecent studies which show a connection between the lep-tin-innervation of mammalian striated muscle Wbres andthe regulation of fatty acid metabolism (Bjorbaek andKahn, 2004; Steinberg et al., 2004). Our observations uponimmunohistochemical detection of leptin in the browntrout gut have surely a general value because describe the

presence of this peptide in a Wsh species, in some instancesfor the Wrst time, but in addition conWrm that also nearbyWsh species, like the brown trout (our study) and the rain-bow trout (other studies), may diVer in important aspects.

As a further conWrmation of this, in the brown trout wenever observed any immunoreactivity to the used VIPantiserum (either in the stomach or in the intestine). In asharp contrast, VIP-immunoreactive nerve cell bodies andWbres are reported in the intestine of the rainbow trout,O. mykiss (Beorlegui et al., 1992; Holmgren et al., 1982).The possible explanation for the evidenced absence ofVIP-like molecules in either intramural neurons or endo-crine cells of the brown trout, when these same moleculesare largely represented in several Wsh species (with theexception, however of the grass carp, according to Panand Fang, 1993) needs obviously further studies. In thisrespect, it is really to underline that the alimentary canalof the same species, S. trutta, naturally infected with

Fig. 9. (A) In a pancreatic islet, secretin-like immunoXuororeactive (left) endocrine cells are also glucagon-like immunoreactive (right). The secretin-like immunoreactive endocrine cells are in a small number, and especially placed in the central region of the islet. Scale bars D 20 �m. (B) In the mid-dle intestine, one glucagon-like immunoXuororeactive endocrine cell (right) does not show immunoXuororeactivity with the secretin antiserum (left).Scale bars D 20 �m.


parasite worms shows the presence of VIP-ergic neuronswithin the wall of the proximal intestine (Dezfuli et al.,2000, 2002). Considering that the concentration of VIP-ergic nerve Wbres is specially high in the reactive connec-tive capsule which encircles acantocephala parasites, wecan hypothesize that in the brown trout the presence ofparasite worms is able to elicit the occurrence of gut intra-mural VIP-ergic neurons, and this in the view of complexand articulate host–parasite interactions.

Taking into account the above mentioned data uponalimentary canal of the brown trout, S. trutta, and com-paring them to the Wndings reported by Holmgren et al.(1982), Beorlegui et al. (1992), and Barrenechea et al.(1994) on the rainbow trout, O. mykiss, we can underlinenoticeable diVerences about the presence and relative den-sities of neuroendocrine cell populations. In particular, abombesin-like-immunoreactivity is detected in neurons ofthe distal intestine, and in nerve Wbres of exocrinepancreas; a gastrin-like-immunoreactivity is detected inendocrine cells of both fundic and pyloric regions; a secre-tin-like-immunoreactivity is observed in endocrine cells ofboth fundic and pyloric regions, as well as possibly inendocrine cells of pancreatic endocrine islets; a galanin-like peptide is detected in intramural nerves of the stom-ach and pancreas; a leptin-like-immunoreactivity isdetected in intramural nerves of both the stomach andintestine, in intestinal endocrine cells, in the striatedoesophageal–gastric musculature; a VIP-like molecule hasnever been observed.

All these immunohistochemical observations are,even if upon a merely morpho-functional point of view,in agreement with a possible functional signiWcance ofthe immunohistochemically detected peptides towards aperipheral regulation of food intake, which in turn,through the presence of food in the lumen of the alimen-tary canal, may be potentially linked to the Wsh centralregulative mechanisms.

Acknowledgments

Thanks to Dr. Bahram S. Dezfuli of the University ofFerrara (Italy) for the supply of the specimens of S. tru-tta used in this work, and to Mr. Giacomo Grassi for itsexcellent technical assistance. This investigation wassupported by grants from the University of Milan(FIRST, 2001).

References

Abad, M.E., Peeze-Binkhorst, F.M., Elbal, M.T., Rombout, J.H.W.M.,1987. A comparative immunocytochemical study of the gastro–entero–pancreatic (GEP) endocrine system in a stomachless and astomach-containing teleost. Gen. Comp. Endocrinol. 66, 123–136.

al-Mahrouki, A.A., Youson, J.H., 1998. Immunohistochemical studiesof the endocrine cells within the gastro–entero–pancreatic system

of Osteoglossomorpha, an ancient teleostean group. Gen. Comp.Endocrinol. 110, 125–139.

Anderson, C., Campbell, G., 1988. Immunohistochemical study of 5-HT containing neurons in the teleost intestine: relationship tothe presence of enterochromaYn cells. Cell Tissue Res. 254, 553–559.

Anglade, I., Wang, Y., Jense, J., Tramu, G., Kah, O., Conlon, J.M.,1994. Characterization of trout galanin and its distribution in troutbrain and pituitary. J. Comp. Neurol. 350, 63–74.

Barrenechea, M.A., Lòpez, J., Martìnez, A., 1994. Regulatory peptidesin gastric endocrine cells of the rainbow trout, Oncorhynchusmykiss: general distribution and co-localizations. Tissue Cell 26,309–321.

Beorlegui, C., Martìnez, A., Sesma, P., 1992. Endocrine cells and nervesin the pyloric caeca and the intestine of Oncorhynchus mykiss (Tele-ostei): an immunocytochemical study. Gen. Comp. Endocrinol. 86,483–495.

Bjenning, C., Holmgren, S., 1988. Neuropeptides in the Wsh gut. Animmunohistochemical study of evolutionary patterns. Histochemis-try 88, 155–163.

Bjorbaek, C., Kahn, B.B., 2004. Leptin signalling in the central nervoussystem and the periphery. Rec. Progr. Horm. Res. 59, 305–331.

Bullock, W.L., 1963. Intestinal histology of some salmonid Wshes withparticular reference to the histopathology of acanthocephalaninfections. J. Morphol. 112, 23–35.

Chang, C.H., Chey, W.Y., Erway, B., Coy, D.H., Chang, T.-M., 1998.Modulation of secretin release by neuropeptides in secretin-pro-ducing cells. Am. J. Physiol. 275, G192–G202.

Crawley, J.N., 1995. Biological actions of galanin. Reg. Pept. 59, 1–16.De Girolamo, P., Lucini, C., Vega, J.A., Andreozzi, G., Coppola, L.,

Castaldo, L., 1999. Co-localization of Trk neurotrophin receptorsand regulatory peptides in the endocrine cells of the teleosteanstomach. Anat. Rec. 256, 219–226.

Dezfuli, B.S., Arrighi, S., Domeneghini, C., Bosi, G., 2000. Immunohis-tochemical detection of neuromodulators in the intestine of Salmotrutta L. naturally infected with Cyathocephalus truncatus Pallas(Cestoda). J. Fish Dis. 23, 265–273.

Dezfuli, B.S., Pironi, F., Giari, L., Domeneghini, C., Bosi, G., 2002.EVect of Pomphorhynchus laevis (Acantocephala) on putative neu-romodulators in the intestine of naturally infected Salmo trutta.Dis. Aquat. Org. 51, 27–35.

Domeneghini, C., Arrighi, S., Radaelli, G., Bosi, G., Berardinelli, P.,Vaini, F., Mascarello, F., 1999. Morphological and histochemicalanalysis of the neuroendocrine system of the gut in Acipenser trans-montanus. J. Appl. Ichthiol. 15, 81–86.

Domeneghini, C., Radaelli, G., Arrighi, S., Mascarello, F., Veggetti, A.,2000. Neurotransmitters and putative neuromodulators in the gutof Anguilla anguilla (L.). Localizations in the enteric nervous andendocrine systems. Eur. J. Histochem. 44, 295–306.

Dubois, M.P., Billard, R., Breton, B., Peter, R.E., 1979. Comparativedistribution of somatostatin, LH-RH, neurophysin and �-endor-phin in the rainbow trout: an immunocytological study. Gen.Comp. Endocrinol. 37, 220–232.

Elbal, M.T., Lozano, M.T., Agulleiro, B., 1988. The endocrine cells inthe gut of Mugil saliens Risso, 1810 (Teleostei): an immunocyto-chemical and ultrastructural study. Gen. Comp. Endocrinol. 70,231–246.

Fujita, T., 1989. Present status of paraneuron concept. Arch. Histol.Cytol. 52 (suppl.), 1–8.

García Hernández, M.P., Lozano, M.T., Agulleiro, B., 1994. Ontogenyof some endocrine cells of the digestive tract in sea bass (Dicentrar-chus labrax): an immunocytochemical study. Cell Tissue Res. 277,373–383.

Gòmez-Visus, I., Garcia-Hernàndez, M.P., Lozano, M.T., Agulleiro, B.,1998. Glucagon- and NPY-related peptide-immunoreactive cells inthe gut of sea bass (Dicentrarchus labrax L.): A light and electronmicroscope study. Gen. Comp. Endocrinol. 112, 26–37.


Greene, C.W., 1912. Anatomy and histology of the alimentary tract ofthe king salmon. Bull. Bureau Fisheries 32, 75–101.

Himick, B.A., Peter, R.E., 1994. CCK/gastrin-like immunoreactivity inbrain and gut, and CCK suppression of feeding in goldWsh. Am. J.Physiol. 267, R841–R851.

Holmgren, S., 1985. Neuropeptide functions in the Wsh gut. Peptides 6(Suppl. 3), 363–368.

Holmgren, S., Jensen, J., 1994. Comparative aspects on the biochemicalidentity of neurotransmitters of the autonomic neurons. In: Burn-stock, G. (Ed.), Comparative Physiology and Evolution of theAutonomic Nervous System. Harwood Academic Publishers, War-saw, pp. 69–95.

Holmgren, S., Jönsson, A.-C., 1988. Occurrence and eVects on motil-ity of bombesin related peptides in the gastrointestinal tract ofthe Atlantic cod, Gadus morhua. Comp. Biochem. Phys. C 89, 249–256.

Holmgren, S., Vaillant, C., Dimaline, R., 1982. VIP-, substance P-, gas-trin/CCK-, bombesin-, somatostatin- and glucagon-like immunore-activities in the gut of the rainbow trout, Salmo gairdneri. CellTissue Res. 223, 141–153.

Jensen, H., Rourke, I.J., Moller, M., Jonson, L., Johnsen, A.H., 2001.IdentiWcation and distribution of CCK-related peptides andmRNAs in the rainbow trout, Oncorhynchus mykiss. BBA-Mol.Cell Res. 1517, 190–201.

Jensen, J., 2001. Regulatory peptides and control of food intake in non-mammalian vertebrates. Comp. Biochem. Physiol. A 128, 471–479.

Jin, H.O., Lee, K.Y., Chang, T.-M., Chey, W.Y., Dubois, A., 1994.Secretin: a physiological regulator of gastric emptying and acidoutput in dogs. Am. J. Physiol. 267, G702–G708.

Johnson, R.M., Johnson, T.M., Londraville, R.L., 2000. Evidence forleptin expression in Wshes. J. Exp. Zool. 286, 718–724.

Jönsson, A.-C., 1991. Regulatory peptides in the pancreas of two spe-cies of elasmobranchs and in the Brockmann bodies of four teleostspecies. Cell Tissue Res. 266, 163–172.

Jönsson, A.-C., Holmgren, S., Holstein, B., 1987. Gastrin/CCK-likeimmunoreactivity in endocrine cells and nerves in the gastrointesti-nal tract of the cod, Gadus morhua, and the eVect of peptides of thegastrin/CCK family on cod gastrointestinal smooth muscle. Gen.Comp. Endocrinol. 66, 190–202.

Karila, P., Holmgren, S., 1997. Anally projecting neurones exhibitingimmunoreactivity to galanin, nitric oxide synthase and vasoactiveintestinal peptide, detected by confocal laser scanning microscopy,in the intestine of the Atlantic cod, Gadus morhua. Cell Tissue Res.287, 525–533.

Karila, P., Jönsson, A.-C., Jensen, J., Holmgren, S., 1993. Galanin-likeimmunoreactivity in extrinsic and intrinsic nerves to the gut of theAtlantic cod, Gadus morhua, and the eVect of galanin on thesmooth muscle of the gut. Cell Tissue Res. 271, 537–544.

Kiliaan, A.J., Holmgren, S., Jönsson, A.-C., Dekker, K., Groot, J.A.,1992. Neurotensin, Substance P, gastrin/cholecystokinin, and bom-besin in the intestine of the tilapia (Oreochromis mossambicus) andthe goldWsh (Carassius auratus): immunochemical detection andeVects on the electrophysiological characteristics. Gen. Comp.Endocrinol. 88, 351–363.

Kiliaan, A.J., Holmgren, S., Jönsson, A.-C., Dekker, K., Groot, J.A.,1993. Neuropetides in the intestine of two teleost species (Ore-ochromis mossambicus, Carassius auratus): localization and elec-trophysiological eVects on the epithelium. Cell Tissue Res. 271,123–134.

Kinoshita, Y., Ishihara, S., 2000. Mechanism of gastric mucosal prolif-eration induced by gastrin. J. Gastroenterol. Hepatol. 15 (suppl.),D7–D11.

Larsson, L.I., 2000. Developmental biology of gastrin and somato-statin cells in the antropyloric mucosa of the stomach. Microsc.Res. Tech. 48, 272–281.

Le Bail, P.Y., Boeuf, G., 1997. What hormones may regulate foodintake in Wsh. Aquat. Living Resour. 10, 371–379.

Lin, X., VolkoV, H., Narnaware, Y., Bernier, N.J., Peyon, P., Peter, R.E.,2000. Review. Brain regulation of feeding behavior and food intakein Wsh. Comp. Biochem. Physiol. A 126, 415–434.

Mix, H., Widjaja, A., Jandl, O., Cornberg, M., Kaul, A., Göke, M., Beil,W., Kuske, M., Brabant, G., Manns, M.P., Wagner, S., 2000.Expression of leptin and leptin receptor isoforms in the humanstomach. Gut 47, 481–486.

Moon, T.W., 1998. Glucagon: from hepatic binding to metabolism inteleost Wsh. Comp. Biochem. Physiol. B 121, 27–34.

Muruzábal, F.J., Fruhbeck, G., Gomez-Ambrosi, J., Archanco, M., Bur-rell, M.A., 2002. Immunocytochemical detection of leptin in non-mam-malian vertebrate stomach. Gen. Comp. Endocrinol. 128, 149–152.

Olsson, C., Aldman, G., Larsson, A., Holmgren, S., 1999. Cholecystoki-nin aVects gastric emptying and stomach motility in the rainbowtrout Oncorhynchus mykiss. J. Exp. Biol. 202, 161–170.

Navarro, I., Carneiro, M.N., Parrizas, M., Maestro, J.L., Planas, J.,Gutierrez, J., 1993. Post-feeding levels of insulin and glucagon introut (Salmo trutta fario). Comp. Biochem. Physiol. A 104, 389–393.

Pan, Q.S., Fang, Z.P., 1993. An immunohistochemical study of endo-crine cells in the gut of a stomachless teleost Wsh, grass carp,Cyprinidae. Cell Transplant. 2, 419–427.

Putti, R., Maglio, M., Odierna, G., 2000. An immunocytochemicalstudy of intrapancreatic ganglia, nerve Wbres and neuroglandularjunctions in Brockmann bodies of the tompot blenny (Blenniusgattoruggine), a marine teleost. Histochem. J. 32, 607–616.

Rattan, S., 1991. Role of galanin in the gut. Gastroenterology 100,1762–1768.

Raybould, H.E., Holzer, H., 1993. Secretin inhibits gastric emptying inrat via a capsaicin-sensitive vagal aVerent pathway. Eur. J. Pharma-col. 250, 165–167.

Reinecke, M., Müller, C., Segner, H., 1997. An immunohistochemicalanalysis of the ontogeny, distribution and coexistence of 12 regula-tory peptides and serotonin in endocrine cells and nerve Wbers ofthe digestive tract of the turbot, Scophthalmus maximus (Teleostei).Anat. Embryol. 195, 87–102.

Sherwood, N.M., Krueckl, S.L., McRory, J.E., 2000. The origin andfunction of the pituitary adenylate cyclase-activating polypeptide(PACAP)/glucagon superfamily. Endocr. Rev. 21, 619–670.

Schwartz, M.W., Seeley, R.J., 1997. Neuroendocrine responses to star-vation and weight loss. N. Engl. J. Med. 336, 1802–1811.

Shiratori, K., Watanabe, S., Takeuchi, T., 1993. Role of endogenousprostaglandins in secretin- and plaunotol-induced inhibition ofgastric acid secretion in rats. Am. J. Gastroenterol. 88, 84–88.

Silverstein, J.T., Bondareva, V.M., Leonard, J.B.K., Plisetskaya, E.M.,2001. Neuropetide regulation of feeding in catWsh, Ictalurus puncta-tus: a role for glucagon-like peptide-I (GLP-I). Comp. Biochem.Physiol. B 129, 623–631.

Sobhani, L., Bado, A., Vissuzaine, C., Buyse, M., Kermorgant, S., Laig-neau, J.-P., Attoub, S., Lehy, T., Henin, D., Mignon, M., Lewin,M.J., 2000. Leptin secretion and leptin receptor in the human stom-ach. Gut 47, 178–183.

Steinberg, G.R., Smith, A.C., Wormald, S., Malenfant, P., Collier, C.,Dick, D.J., 2004. Endurance training partially reverses dietary-induced leptin resistance in rodent skeletal muscle. Am. J. Physiol.Endocrinol. Metab. 286, E57–E63.

Taouis, M., Chen, J.-W., Daviaud, C., Dupont, J., Derouet, M., Simon,J., 1998. Cloning the chicken leptin gene. Gene 208, 239–242.

Thorndyke, M., Holmgren, S., 1990. Bombesin potentiates the eVect ofacetylcholine on isolated strips of Wsh stomach. Regul. Peptides 30,125–135.

Visus, I.G., Abad, M.E., Garcia-Hernandez, M.P., Agulleiro, B., 1996.Occurrence of somatostatin and insulin immunoreactivities in thestomach of the sea bass (Dicentrarchus labrax L.): light and elec-tron microscopic studies. Gen. Comp. Endocrinol. 102, 16–27.

Wagner, G.F., McKeown, B.A., 1981. Immunocytochemical localiza-tion of hormone-producing cells within pancreatic islets of the rain-bow trout (Salmo gairdneri). Cell Tissue Res. 221, 181–192.


Wang, W., Barton, B.A., Thim, L., Nielsen, P.F., Conlon, J.M., 1999. Puri-Wcation and charcaterization of galanin and schylyorhinin-I from thehybrid sturgeon, Scaphirhynchus platorynchus £ Scaphirhynchusalbus (Acipenseriformes). Gen. Comp. Endocrinol. 113, 38–45.

Weinreb, E.L., Bilstad, N.M., 1955. Histology of the digestive tract andadjacent structures of the rainbow trout, Salmo gairdneri irideus.Copeia 3, 194–204.

Yagci, R.V., Alpetkein, N., Rossowski, W.J., Brown, A., Coy, D.H.,Ertane, A., 1990. Inhibitory eVect of galanin on basal and pentagas-tri-stimulated gastric acid secretion in rats. Scand. J. Gastroenterol.25, 853–858.

Zhang, Y., Proenca, R., MaVei, M., Barone, M., Leopold, L., Friedman,J.M., 1994. Positional clonino of the mouse obese gene and itshuman homologue. Nature 372, 425–432.

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