Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss)

Comparative Biochemistry and Physiology, Part C 145 (2007) 245 – 255 www.elsevier.com/locate/cbpc

Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss) R.I. Caamaño-Tubío a , J. Pérez a , S. Ferreiro b , M. Aldegunde a,⁎ a

Animal Physiology Laboratory, Department of Physiology, Faculty of Biology, University of Santiago de Compostela, Spain b Department of Cell Biology and Ecology, Faculty of Biology, University of Santiago de Compostela, Spain Received 24 July 2006; received in revised form 14 December 2006; accepted 15 December 2006 Available online 16 January 2007

Abstract Serotonin (5-hydroxytryptamine, 5-HT) occurs in a wide range of tissues throughout the body of the rainbow trout. Results reported here indicate that the main peripheral sources of serotonin are the intestinal tract and the gill epithelium (levels above 1500 ng/g). The high intestinal serotonin concentration is mostly due to serotoninergic nerve fibres, which are present at high density in the intestinal wall. Only about 2% of serotonin is associated with mucosal enterochromaffin cells. In the remaining tissues studied serotonin concentration was below 160 ng/g: the highest concentrations were seen in the anterior and posterior kidneys, followed by the liver, heart, and spleen. 5-Hydroxyindolacetic acid (5-HIAA) levels, except in plasma, were generally lower than serotonin levels, and were below our detection limits in heart, spleen and posterior kidney. Acute d-fenfluramine treatment (5 or 15 mg/kg i.p.) significantly increased 5-HIAA/5-HT ratio in the anterior intestine, pyloric caeca and plasma. Serotonin released from intestinal serotoninergic fibres in response to d-fenfluramine treatment is metabolized locally, and only a small part reaches the blood, from where it can be taken up and metabolized by other peripheral tissues, such as the liver and gill epithelium. The non-metabolized serotonin pool in the blood appears to be located extracellularly, not intracellularly as in mammals. In view of these findings, we present an overview of peripheral serotonin dynamics in rainbow trout. © 2007 Elsevier Inc. All rights reserved. Keywords: Serotonin; 5-Hydroxyindolacetic acid; Peripheral tissues; Plasma; Blood; Enteroendocrine cells; Fenfluramine; Oncorhynchus mykiss; Rainbow trout

1. Introduction A considerable body of biochemical, physiological and histochemical evidence suggests a transmitter function for serotonin (5-hydroxytryptamine, 5-HT) in the animal kingdom, and in vertebrates this molecule is a well-established central neurotransmitter. Its levels in different brain regions have been widely studied in higher vertebrates, mainly mammals (Essman, 1978; Durán et al., 1985; Míguez et al., 1994, 1999), though also to a lesser extent in fish (Pouliot et al., 1988; Nilsson, 1989; Rozas et al., 1990). Central and peripheral 5-HT synthesis takes place from tryptophan in a two-step process that is strongly conserved in the animal kingdom (Aldegunde, 1998). In the first step, 5-hydroxytryptophan (5-HTP) is formed from trytophan by the action of tryptophan hydroxylase, and the 5-HTP is then converted to 5-HT ⁎ Corresponding author. Laboratorio de Fisiología Animal, Facultad de Biología, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain. Tel.: +34 981 563100x13335; fax: +34 981 596904. E-mail address: [email protected] (M. Aldegunde). 1532-0456/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.12.017

by the action of 5-HTP decarboxylase. 5-HT is converted to its main metabolite, 5-HIAA, by monoamine oxidase (Senatori et al., 2003), and 5-HIAA is excreted in urine. In mammals the distribution of 5-HT in tissues outside the central nervous system is well known (Fozard, 1989; Martín and Aldegunde, 1995). In other vertebrates, however, there have been fewer studies of its distribution and concentration in tissues other than brain. Studies in some fish species have determined 5-HT levels in various tissues (for a review see Essman, 1978), including intestine, gills and kidney of Conger conger and Scyliorhinus stellaris (Piomelli and Tota, 1983). All these studies have mainly used bioassays for 5-HT determination. Very few studies have used high performance liquid chromatography with electrochemical detection, which offers very high sensitivity and specificity. The few published studies of this type include studies of 5-HT and 5-HIAA levels in the blood of Anguilla anguilla (Caroff et al., 1986) and in the intestine of Platycephalus bassensis (Anderson et al., 1989). In mammals, peripheral 5-HTshows a wide range of biological activities. It is known to modulate the activity of neurons of the peripheral nervous system (the sympathetic, parasympathetic and

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enteral system), and thus mediates numerous physiological functions; however, its main peripheral function remains to be elucidated (Fozard, 1989; Doggreu, 2003; Côte et al., 2004). In fish, there is likewise evidence implicating 5-HT in the modulation of diverse physiological functions, such as nervous control of gastrointestinal function (Burka et al., 1989; Kiliaan et al., 1989; Mori and Ando, 1991; Venugopalan et al., 1995; Buddington and Krogdahl, 2004), the secretion of inter-renal catecholamines (Fritsche et al., 1993; Bernier and Perry, 1996; Tubío et al., 2002), branchial function (Sundin, 1995; Sundin et al., 1995; Forster et al., 1998; Sundin and Nilsson, 1998), cardiovascular function (Meghji and Burnstock, 1984; Janvier et al., 1996; Pellegrino et al., 2003) and lymphocyte proliferation (Ferriere et al., 1996). Given that in mammals it has been demonstrated that the presence of 5-HT in tissues and organs is related to functional activity, it seems reasonable to suppose that this same relationship between location and function will also be seen in lower vertebrates. In several fish, 5-HT levels have been determined in individual tissues; however, there have been no studies of serotonin metabolism in peripheral tissues, or of relationships between different peripheral 5-HT pools. We thus consider that it is of interest to determine levels of 5-HT and its main metabolite 5-HIAA in diverse peripheral tissues in the rainbow trout (Oncorhynchus mykiss), which will be additionally overviewed in the context of peripheral serotonin.

chilled saline; it was then dissected into three regions, the pyloric caeca (CP), the anterior intestine without pyloric caeca (AI), and the posterior intestine including the middle intestine (PI). Finally, samples of gill epithelium were obtained by scraping the gill arches, again on a chilled glass plate. All samples (organs, intestinal tract, gill epithelium, plasma) were frozen on dry ice and stored at −80 °C until analysis. In Experiment II we investigated the distribution (Experiment IIa) and origin (Experiment IIb) of 5-HT and 5-HIAA in the intestinal tract of the rainbow trout. In Experiment IIa we used 12 fish distributed in three 100-L tanks. After anaesthesia, the protocol was like that of Experiment I, but using a different dissection of the intestinal tract: the pyloric caeca (CP), the anterior intestine without pyloric caeca but with middle intestine (AIa), and the posterior intestine (PIa). Immediately, the intestinal mucosa was removed from each region by scraping, and the resulting mucosal cells and intestinal tissue without mucosa (denominated intestinal wall) were weighed and stored at − 80 °C. In Experiment IIb the protocol was similar to that followed for Experiment IIa, but in this experiment trout were anaesthetised with MS-222 (0.1% solution) before fixation of intestinal tissues, by transcardial perfusion with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The intestine was then carefully dissected out (anterior intestine, pyloric caeca and posterior intestine), cut

2. Materials and methods 2.1. Experimental animals Immature rainbow trout (O. mykiss) (86 ± 5 g) were obtained from a commercial trout farm (Soutorredondo, Noia) and acclimated for 3 weeks in running dechlorinated tap water (temperature 14 ± 0.5 °C, pH 6.3 ± 0.1) with continuous aeration, in 200-L tanks. Fish were maintained under a 12-h-light and 12-hdark photoperiod (lights on at 8:00 h), and during the acclimation period were fed once daily in the morning (at 12:00 h) with commercial dry pellets (ration equivalent to 1.5% of body weight per day), and were fasted 24 h prior to sampling or intraperitoneal injections (see below). All samples were obtained at the same time each morning to avoid possible effects of circadian variations. To minimize stress, fish were anaesthetised (50 mg L− 1 MS-222 buffered to pH 7.4 with sodium bicarbonate) before handling, injection or decapitation. Replicate or triplicate tanks were established for each experiment. 2.2. Experimental protocols In Experiment I we studied the levels of 5-HT and 5-HIAA in peripheral tissues of rainbow trout. Fish were distributed in 100-L tanks at 4 fish per tank. After anaesthesia, blood was obtained with ammonium-heparinized syringes from the caudal peduncle, then centrifuged to obtain plasma. Liver, heart, anterior and posterior kidney, and spleen were dissected out and weighed. The intestinal tract was removed and transferred to a chilled glass plate (2 °C), for removal of external fat followed by washing with

Fig. 1. 5-HT and 5-HIAA concentrations in (A) plasma and blood, (B) gill epithelium, (C) liver, heart and (D) spleen in rainbow trout. Each bar represents the mean ± SEM for n = 17 fish per group (plasma), n = 4 fish per group (blood) and n = 6–8 fish per group (liver, heart and spleen). 5-HIAA levels were below detection limits in heart and spleen.

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chromatographic analysis of 5-HT in blood. Plasma samples were obtained after blood centrifugation (5 min at 11,600×g, 4 °C). Plasma aliquots (250 μL) were deproteinized with 125 μL of a homogenization solution (HS) made up of 0.4 M perchloric acid, 0.4 mM sodium metabisulfite and 0.1 mM K2 EDTA; after stirring the homogenates were centrifuged (30 min at 40,000×g, 2 °C) and the supernatants were stored (− 80 °C) until use for HPLC-EC analysis of 5-HT and 5-HIAA in plasma. Heart, liver, intestinal tissue, spleen and kidney samples were homogenized with HS (1:5 w/v). The homogenates were centrifuged (30 min at 40,000×g, 2 °C), and the supernatants filtered through Nylon Acrodisc 13 (0.20-μm pore-size) filters and stored at − 80 °C until 5-HT and 5-HIAA analysis. 2.4. HPLC-EC analysis of 5-HT and 5-HIAA

Fig. 2. 5-HT and 5-HIAA concentrations in whole (A) anterior intestine (AI), pyloric caeca (CP) and posterior intestine (including middle intestine) (PI), and (B) anterior and posterior kidney, in the rainbow trout. Each bar represents the mean ± SEM for n = 7–8 fish per group (AI, CP and PI) and n = 4–6 fish per group (anterior and posterior kidney).

The analysis of 5-HT and 5-HIAA levels in plasma, blood and tissues was done using HPLC-EC as previously described (Martín and Aldegunde, 1989; Rozas et al., 1990). A Waters M510 solvent delivery system was coupled to an ESA Coulochem M5100A electrochemical detector with a 5010 dual analytical cell set at + 50 mV (first, screening cell) and +350 mV (second, analytical cell). A 5-μm C18 Spherisorb ODS2 (150 × 4 mm) analytical column was used. All analyses were performed at room temperature and with isocratic elution. The mobile phase was a mixture of acetic acid (0.3 M) and

into small pieces and stored at 4 °C in the same fixative for 12 h. After rinsing in PB, the pieces were immersed in cooled 30% sucrose in PB until they sank, and were then embedded in OCT Compound (Tissue Tek, Sakura, Torrence, CA), frozen with liquid-nitrogen-cooled isopentane, and serially sectioned in a transverse plane on a cryostat. Finally, the sections (18 μm thick) were mounted on Superfrost Plus slides. In a third set of experiments the effects of fenfluramine on levels of 5-HT and 5-HIAA in intestine (CP, AI and PI), anterior kidney and plasma of rainbow trout were evaluated (Experiment III). d-Fenfluramine (FF; Sigma Chemical Co., St. Louis, USA) (5 or 15 mg/kg), or its saline (0.6% NaCl) vehicle, was injected intraperitoneally (i.p., 125 μL/fish). For each of the two doses of FF 16 fish distributed in four 100-L tanks were used. In both cases the fish were killed 180 min after i.p. FF or saline administration. Plasma and tissues were obtained and processed as in Experiment I. 2.3. Tissue processing Whole blood samples (100 μL) were deproteinized with 100 μL of 0.8 M perchloric acid containing 0.1 M ascorbic acid and 0.01 M EDTA (Xiao et al., 1998). After stirring, the homogenate was centrifuged (10 min at 4500×g, 2 °C) and the resulting supernatant was again centrifuged (15 min at 30,000×g, 2 °C). The final supernatant was used for the

Fig. 3. 5-HT and 5-HIAA concentrations in (A) intestinal wall of the anterior intestine (including middle intestine) (AIa), pyloric caeca (CP) and posterior intestine (PIa), and (B) intestinal mucosa of the AIa, CP and PIa in the rainbow trout. Each bar represents the mean ± SEM for n = 10–12 fish per group. Means with the same letter do not differ significantly at the 5% level.

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Fig. 4. Photomicrographs showing occurrence and distribution of 5-HT-immunoreactive endocrine cells and nerve fibres in sections of different regions of the rainbow trout intestine. Proximal intestine (A): 5-HT-like-immunoreactive nerve fibres (thin arrows) in the submucosal plexus and inner musculature, with several fibres running in the connective axis of an intestinal fold. Pyloric caeca (B): 5-HT-like immunoreactivity (thin arrows) is seen in nerve fibres in the wall of the pyloric caeca and running in the connective axis of an intestinal fold. Distal intestine (C, D and E): 5-HT-like-immunoreactive nerve fibres (thin arrows) were detected at high density in the submucosal plexus and inner musculature and running in the connective axis of an intestinal fold (C). 5-HT-like-immunoreactive endocrine cells (thick arrows) can be seen in the mucosal epithelium of the distal intestine (D and E). A 5-HT neuronal soma (circle) is also seen in the fibres running into an intestinal fold (D). Scale bars = 100 μm (A, B), 50 μm (C, D) or 25 μm (E).

ammonium hydroxide (0.08 M) containing Na2 EDTA (0.1 mM) and 10% methanol, adjusted to pH 4.50. Flow rate was 0.9 mL/min. 2.5. Immunohistochemistry Intestinal tissue sections were processed for serotonin immunohistochemistry by the peroxidase–antiperoxidase (PAP) method. After pretreatment with 10% H2O2 in Trisbuffered saline (TBS) at pH 7.4 for 30 min at room temperature (RT) to eliminate endogenous peroxidase activity, the sections were incubated overnight in a humid chamber at RT with the polyclonal anti-serotonin antibody (Incstar, Stillwater, MN; dilution 1:5000). The sections were rinsed in TBS (3 rinses, 10 min each), incubated in goat anti-rabbit IgG (DAKO, Glostrup, Denmark, diluted 1:100) for 1 h, rinsed in TBS, then incubated in rabbit peroxidase–antiperoxidase (PAP) complex (DAKO, diluted 1:100) for 1 h. The antibodies and the PAP complex were diluted in TBS containing 0.2% Triton X-100 and 15% normal goat serum (Sigma). After two TBS rinses, the immune complex was developed with 0.5 mg/mL of 3-3′

diaminobenzidine tetrahydrochloride (DAB, Sigma) and 0.03% H2O2 in 0.05 M Tris–HCl buffer (pH 7.6) for 5– 15 min. Photomicrographs were taken with an Olympus DP-10 colour digital camera. The images were converted to a grey scale and adjusted for brightness and contrast using Corel Photopaint 9. Photomontage and lettering were also done using Adobe Photoshop (Adobe Systems, San José, CA). In a control group designed to confirm the specificity of immunostaining, the primary antiserum was replaced with nonimmune serum, and no immunoreactivity was observed except in a few lymphocyte-like cells in the lamina propia of the posterior intestine. 2.6. Statistical analysis All data are presented as mean values ± standard errors (SEM). Statistical significance was evaluated with Student's ttest for comparison of the two groups. In all other cases, oneway analysis of variance (ANOVA) followed by the Student– Newman–Keuls test was used. Unless otherwise stated, statistical significance is taken to be indicated by p b 0.05.

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Fig. 5. Effects of d-fenfluramine (FF) (180 min before sampling) on 5-HT and 5HIAA concentrations and 5-HIAA/5-HT ratio in anterior intestine. (A) FF dose 5 mg/kg i.p. and (B) FF dose 15 mg/kg i.p. Each bar represents the mean ± SEM for n = 7–8 fish per group. ⁎p b 0.05 vs. controls.

Fig. 7. Effects of d-fenfluramine (FF) (180 min before sampling) on 5-HT and 5HIAA concentrations and 5-HIAA/5-HT ratio in anterior kidney. (A) FF dose 5 mg/kg i.p. and (B) FF dose 15 mg/kg i.p. Each bar represents the mean ± SEM for n = 7–8 fish per group. ⁎p b 0.05 vs. controls.

Fig. 6. Effects of d-fenfluramine (FF) (180 min before sampling) on 5-HT and 5HIAA concentrations and 5-HIAA/5-HT ratio in the pyloric caeca. (A) FF dose 5 mg/kg i.p. and (B) FF dose 15 mg/kg i.p. Each bar represents the mean ± SEM for n = 6–8 fish per group. ⁎p b 0.05 vs. controls.

Fig. 8. Effects of d-fenfluramine (FF) (180 min before sampling) on 5-HT and 5HIAA concentrations and 5-HIAA/5-HT ratio in plasma. (A) FF dose 5 mg/kg i. p. and (B) FF dose 15 mg/kg i.p. Each bar represents the mean ± SEM for n = 7 fish per group (dose 5 mg/kg) and n = 5 fish per group (dose 15 mg/kg).⁎p b 0.05 vs. controls.

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Fig. 9. Schematic overview of peripheral serotonin dynamics in rainbow trout. (1) Serotonergic intestinal fibres synthesize 5-HT, release it, and deaminate it to produce 5-HIAA. (2) The enterochromaffin (EC) cells of the posterior intestine take up and metabolize 5-HT to produce 5-HIAA. (3) Both 5-HIAA and non-deaminated 5-HT are released to the circulation. (4) Plasma 5-HT is taken up by the gill epithelium and liver, contributing to the control of peripheral 5-HT levels. (5) In liver, 5-HT is deaminated to 5-HIAA, which then enters the blood. (6) The contribution of the heart, kidney and spleen to the peripheral homeostasis of 5-HT seems to be minor. (7) Blood is filtered through the kidney, and 5-HIAA is excreted into the urine.

All analyses were carried out with commercial software (SigmaStat v2.0; SPSS Scientific Inc.).

posterior kidney, although again these differences were not significant (Fig. 2B).

3. Results

3.2. 5-HT tissue distribution in the intestinal tract

3.1. 5-HT and 5-HIAA levels in peripheral tissues, blood and serum

Fig. 3 shows the results of Experiment IIa, designed to evaluate whether the high concentrations of 5-HT found in intestinal regions (Experiment I) reflect enteroendocrine cells in the intestinal mucosa or serotoninergic nerve fibres associated with the intestinal wall (myenteric and submucosal plexus). 5-HT levels in the intestinal wall (expressed as ng/g of tissue) ranged from 4260 ± 448 ng/g in the anterior + middle intestine to 6070 ± 349 ng/g in the posterior intestine (Fig. 3A), while 5-HT levels in the intestinal mucosa ranged from 31.8 ± 4.9 ng/g in the anterior + middle intestine to 83 ± 15.2 ng/g in the posterior intestine (Fig. 3B). Mean 5-HT levels in the intestinal wall were about 73- to 133-fold higher (p b 0.05) than in intestinal mucosa. 5-HIAA levels were significantly lower than 5-HT levels in the wall of all three regions (anterior + middle intestine, posterior intestine, pyloric caeca) and in the mucosa of the posterior intestine, though not the mucosa of the anterior + middle intestine and the pyloric caeca (Fig. 3A and B). 5-HIAA level ranged from about 10% of 5-HT level in the wall of the posterior intestine to about 25% in the wall of the pyloric caeca and the mucosa of the posterior intestine (Fig. 3A and B). 5-HIAA levels were lowest and 5-HT levels highest in the wall and mucosa of the posterior intestine. Strongly 5-HT-immunoreactive nerve fibres were seen in the wall of the posterior intestine (Fig. 4C), with less marked immunoreactivity in the wall of the anterior intestine and pyloric caeca (Fig. 4A and B). Enterochromaffin cells showing

The results of Experiment I are shown in Figs. 1 and 2. The highest levels of 5-HT (expressed as ng/g of tissue) are seen in the anterior intestine (2624 ± 327), followed by the pyloric caeca (2272 ± 238), the middle + posterior intestine (1799 ± 566) (Fig. 2A), the gill epithelium (1569 ± 440) (Fig. 1B), the anterior kidney (160 ± 26), the posterior kidney (116 ± 38) (Fig. 2B), the liver (28.5 ± 8.5), the heart (23 ± 6) (Fig. 1C), and the spleen (0.22 ± 0.05) (Fig. 1D). 5-HT levels observed in blood (1173 ± 407 pg/mL) did not differ significantly from those observed in plasma (807 ± 99 pg/mL) (Fig. 1A). 5-HIAA levels were significantly lower (p b 0.05) than 5-HT levels in all tissues except plasma (1630 ± 194 pg/mL) (Fig. 1A). In liver, mean 5-HIAA concentration was about 58% of mean 5-HT concentration (Fig. 1C), while in the other tissues in which this metabolite was detected 5-HIAA levels were even lower with respect to 5-HT concentration, ranging from 1.3% in the gill epithelium to 18% in the pyloric caeca. Within the intestinal tract, 5-HT levels declined from anterior to posterior; this decline, though not statistically significant, was of considerable magnitude (0.32-fold). Similarly, a 0.32-fold reduction was also observed in 5HIAA levels (Fig. 2A), though again this decline was not statistically significant. In the renal regions, 5-HT and 5-HIAA concentrations were higher in the anterior kidney than the

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5-HT immunoreactivity were detected only in the mucosa of the posterior intestine (Fig. 4D and E). 3.3. Effects of acute fenfluramine on 5-HT release Figs. 5, 6, 7 and 8 show the effects of acute i.p. administration of d-fenfluramine (5 and 15 mg/kg) on 5-HT and 5-HIAA levels in those peripheral compartments in which highest 5-HT levels were observed in Experiment I (i.e. anterior intestine, pyloric caeca, and anterior kidney), as well as in plasma. In the anterior intestine, the administration of 5 mg/kg of fenfluramine induced significant increases in 5-HIAA level and in 5-HIAA/5-HT ratio (Fig. 5A); the higher fenfluramine dose likewise induced a significant increase in 5-HIAA/5-HT ratio (Fig. 5B). In the pyloric caeca, both fenfluramine doses induced a significant increase in 5-HIAA/5-HT ratio, and with the higher dose both the increase in 5-HIAA level and the reduction in 5-HT level were also statistically significant (Fig. 6A and B). In the anterior kidney fenfluramine had no significant effects on either 5-HT or 5-HIAA level (Fig. 7A and B). In plasma, both fenfluramine doses induced a significant increase in 5-HIAA/5-HT ratio, though a significant reduction in 5-HT level was observed only with the lower dose (Fig. 8A and B). d-Fenfluramine is a drug that acts specifically on serotoninergic terminals enhancing 5-HT release and suppressing its reuptake (Garattini, 1987). 4. Discussion In mammals, 5-HT is present at very high levels in platelets and enterochromaffin cells, and has also been detected at lower concentrations in other cells of peripheral tissues. In fish, there have likewise been some reports of 5-HT in peripheral tissues of different species, but these reports do not provide a firm basis for assessing the peripheral distribution of 5-HT in this group. The present study investigated the distribution of 5-HT and its principal metabolite 5-HIAA in various peripheral tissues of the rainbow trout, providing the basis for a model of serotonin dynamics in the peripheral tissues of teleosts. 4.1. Intestinal 5-HT metabolism In general, the peripheral tissues in which 5-HT levels were highest were the intestine, with the highest levels detected in the anterior intestine, followed by the pyloric caeca and the posterior intestine. Considering both intestinal tissues (wall and mucosa) together, the 5-HT concentrations seen are within the ranges observed in the small intestine of other species of fish, such as Amia calva (Bogdanski et al., 1963) and Cyprinus auratus (Bogdanski et al., 1963), and in the whole intestines of Scylliorhinus canicula, S. stellaris and Torpedo marmorata (Erspamer, 1954; Piccinelli, 1958; Piomelli and Tota, 1983). However, lower 5-HT levels than in the present study have been reported in the whole intestines of sturgeon (Acipenser sturio) and catfish (Ameiurus catus), and in the small intestine of the eel (Anguilla vulgaris) (Erspamer, 1954). A comparison of our results in the rainbow trout intestinal tract with results reported

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for mammals indicates (1) that concentrations of 5-HT are similar in the two groups (see Essman, 1978; Teff and Young, 1988; Martín et al., 1995; Côte et al., 2003), and (2) that both groups show declining 5-HT concentrations along the length of the intestinal tract (i.e. from anterior to posterior) (see Hansen and Skadhauge, 1997). In mammals it is known that intestinal 5-HT is located in two types of cell: in neurons of the myenteric nervous plexuses, and in enterochromaffin (EC) cells of the intestinal mucosa. About 95% of intestinal 5-HT is located in these latter cells (Gershon, 1982; Tyce, 1990); in mammals intestinal 5-HT level thus basically corresponds to the 5-HT level in EC cells. It is well known that 5-HT-containing EC cells are widely distributed in the intestinal tract of most vertebrates; however, they appear to be lacking from the intestine of cyclostomes and some teleosts (Erspamer, 1966; El-Salhy et al., 1985; Anderson and Campbell, 1988; Yui et al., 1988). This led to us question whether the high 5-HT levels detected by us in the whole intestine (Experiment I) corresponded to 5-HT in EC cells (i.e. endocrine cells in the mucosa) and/or to serotoninergic nerve fibres in the intestinal wall. The results of Experiment IIa clearly demonstrated that almost all of the intestinal 5-HT is located in the wall, and only about 2% in EC cells in the mucosa. This distribution is in striking contrast to that seen in mammals, and similar to that observed in the flathead (P. bassensis), a teleost without 5-HT-containing EC cells (Anderson, 1983; Anderson et al., 1989). In addition, 5-HT levels in the intestinal wall of rainbow trout (Fig. 3A) were within the range reported for the whole intestine without mucosa of flathead (Anderson et al., 1989). However, intestinal 5-HIAA levels and 5-HIAA/5-HT ratios were markedly higher in rainbow trout than flathead (Anderson et al., 1989). The different ratios may reflect a higher rate of metabolism and/or use of intestinal 5-HT in the intestine of rainbow trout. In general these differences between species may be a consequence of the structural and/or functional diversity of the digestive tract in relation to feeding habits (Chiba, 1998), which is in turn in line with observed species differences in intestinal monoamine oxidase activity (Hall and Urueña, 1982; Edwards et al., 1986; Senatori et al., 2003). Finally, we present for the first time in fishes data on variations in 5-HT metabolism along the length of the intestinal tract, both in the mucosa and in the intestinal wall. In both tissues 5-HT increased from anterior to posterior regions, while 5-HIAA levels showed the opposite trend. It is important to note that the apparent differences in levels and distribution of 5-HT between Experiment I and Experiment IIa simply reflect differences in the dissection procedure, and above all the separate analysis of intestinal wall and mucosa in Experiment IIa. Immunohistochemical study (Experiment IIb) revealed the same pattern of distribution of 5-HT between the intestinal wall and the mucosa, confirming the conclusions stated above. Throughout the intestinal tract most 5-HT is located in the intestinal wall, and very little in the mucosa: this is attributable (a) to the presence of a nerve plexus, containing 5-HT at very high concentrations, running along the length of the intestinal wall, and (b) to the practical absence of 5-HT-producing EC cells in the mucosa. These results are in general agreement with

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those reported previously in rainbow trout (Anderson and Campbell, 1988; Beorlegui et al., 1992). The results of Experiment IIb also indicate that 5-HT levels in both wall and mucosa are higher in the posterior intestine, due to (a) the higher density of 5-HT fibres in this region, not to higher 5-HT content in fibres (cf. Anderson, 1983; Holmgren et al., 1985), and (b) the presence of EC cells in this region. We can thus conclude that the high concentrations of 5-HT and 5HIAA in the rainbow trout intestine mostly correspond to serotoninergic nerve fibres located in the muscle layers of the intestinal wall, which in turn are concentrated with higher density in the posterior intestine. 4.2. Blood and plasma 5-HT In mammals, the blood is the final destination of nonmetabolized 5-HT (mainly derived from intestinal EC cells) and of its metabolite 5-HIAA. In mammals, 5-HT from EC cells is secreted into the blood and taken up into platelets, where more than 99% of blood 5-HT is located; only a very small proportion is located free in the plasma, in what has been denominated the extracellular pool (Fozard, 1989; Celada et al., 1994; Martín et al., 1995). To date it has been unclear whether fishes have a cellular 5-HT storage pool equivalent to the platelet pool in mammals (for example thrombocytes, nucleated cells with similar functions to platelets in fish blood coagulation). The results obtained in the present study rule out this possibility, since 5-HT concentrations did not differ significantly between total blood and plasma. This finding is in full agreement with the fact that dogfish thrombocytes are incapable of accumulating 5-HT (Fänge, 1992). Thus in rainbow trout non-metabolized 5-HT reaching the blood from tissues appears to be stored in the extracellular pool only. Plasma levels of 5-HT were much lower in the present study than in most previous studies of other fish species (Essman, 1978). Very probably, however, this simply reflects the use of less selective and less sensitive analytical techniques in most previous studies, since the few studies that have used similarly sensitive techniques have obtained results similar to those obtained by us. In Caroff et al.'s (1986) study of the eel, for example, plasma 5-HT levels were below the limits of detection of the analytical technique used. Likewise, Handy's (2003) study of rainbow trout reported that serum levels were below 10 ng/mL. Plasma 5-HT levels measured in the present study were within the range previously reported for the extracellular pool (i.e. in platelet-free plasma) in mammals (Celada et al., 1994; Martín et al., 1995; Hirowatari et al., 2004). However, plasma concentrations of 5-HIAA were much lower (about 90% lower) than those seen in platelet-free rat plasma (Martín et al., 1995). We are not aware of any previously published data on 5HIAA levels in fish. The similar 5-HT levels seen in rainbow trout and in mammals are of interest, since in the latter uptake of 5-HT by platelets acts as a regulatory mechanism to maintain low levels of extracellular 5-HT. In the rainbow trout our results indicate that there is no cellular 5-HT pool in the blood, suggesting that some other type/s of regulatory mechanism must be acting, thus

avoiding high levels that may interfere with normal physiology of peripheral organs. Possible mechanisms include (a) efficient deamination of 5-HT in peripheral tissues, (b) efficient renal excretion, and (c) uptake/removal from blood. 4.3. 5-HT metabolism in liver, kidney, spleen, heart and gill epithelium In fishes, blood coming from the intestine and other organs eventually perfuses to the liver by means of the hepatic portal system (Olson, 2000). It thus seems likely that, as in mammals, 5HT from the intestine and other organs “overflows” to the liver, where it can be broken down. In mammals the liver is the most important peripheral tissue for 5-HT deamination (Verbeuren, 1989). Hepatic levels of 5-HT and 5-HIAA observed by us in rainbow trout are much lower than previously reported from rat (Martín et al., 1995). In contrast, 5-HIAA/5-HT ratio was much higher, suggesting that the liver of rainbow trout has somewhat higher capacity for metabolizing 5-HT. In line with this view, it has been observed that in rainbow trout hepatic cells are, after intestinal cells, those showing highest MAO activity (Edwards et al., 1986). These data seem to indicate that the liver of rainbow trout can efficiently take up and deaminate 5-HT from peripheral tissues that has not been metabolized in situ. Our data on renal levels of 5-HT and 5-HIAA are as far as we know the first published data on 5-HIAA levels in a fish, and thus the first to confirm in vivo renal breakdown of 5-HT to 5-HIAA. This is in line with the detection of MAO activity in the kidney of the rainbow trout (Edwards et al., 1986) and the goldfish (Hall and Urueña, 1982). Renal 5-HIAA levels were very low, possibly indicating highly efficient renal excretion of this metabolite. This hypothesis is supported by the high levels of 5-HIAA (about 320 ng/mL) reported in trout urine (Some and Helander, 2002), in contrast with the very low plasma levels (about 1.6 ng/mL) detected in the present study. Low plasma levels of 5-HIAA are indicative of efficient renal extraction of 5-HIAA, as reported in humans (Hannedouche et al., 1989). In rainbow trout spleen and heart we observed very low 5HT levels, and in both cases 5-HIAA levels were below the detection limits of the analytical technique used in the present study. These results clearly indicate that these tissues do not play an important role in peripheral 5-HT homeostasis. Finally, we studied levels of 5-HT and 5-HIAA in the gill epithelium. In fish, the peculiarities of the branchial circulation suggest an important role in the elimination of molecules from the plasma (Olson, 1998). In the present study we observed that 5-HT concentrations in the gill epithelium were very high, this being the peripheral tissue with the highest 5-HT content other than the intestine; these results are in line with data published on the gills in other fish species (Piomelli and Tota, 1983). The high 5-HT levels observed may have two possible explanations: (i) the existence of endocrine cells in the gill epithelium with the capacity for the synthesis of 5-HT-like substances (Goniakowska-Witalinska et al., 1995; Sundin, 1995; Franchini et al., 1999), and (ii) the capacity of the gills for uptake, storage and breakdown of serotonin, mechanisms

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by which about 80% of circulating 5-HT can be inactivated (Olson, 1998). Both possibilities would explain the raised levels of 5-HT in the gill epithelium. This author (Olson, 1998) postulates that the gills of fish play a role equivalent to that of the pulmonary epithelium in mammals, which is capable of extracting more than 90% of 5-HT administered as an intravenous infusion (Verbeuren, 1989), and in which raised levels of 5-HT are also seen (Martín et al., 1995) (similar to those found by us in the gill epithelium of the rainbow trout). However, 5-HIAA levels reported previously from the mammalian pulmonary epithelium are much higher than found by us in the present study of rainbow trout. That is, if the gills have the capacity not just to take up 5-HT but also to deaminate it (Olson, 1998), the low levels of 5-HIAA in the gill epithelium and in the plasma can only be explained, as we have suggested previously, by a highly efficient mechanism for elimination from the plasma.

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analytical method was insufficiently sensitive to resolve very small variations in 5-HT levels. 4.5. Conclusion: an overview of peripheral 5-HT in rainbow trout A proposed schematic overview of peripheral 5-HT dynamics in rainbow trout is shown in Fig. 9. Most peripheral 5-HT is synthesized in the serotoninergic fibres of the intestinal wall. 5-HT released and not broken down locally reaches the portal circulation, and is subsequently taken up from the blood for breakdown in the gills and liver. The supply of 5-HT from other tissues such as the spleen and heart appears to be negligible. Likewise, these organs do not appear to play an important role in maintaining the very low 5-HT levels seen in blood. Finally, our results indicate that 5-HIAA formed in the different tissues is eliminated from the blood by the kidney in a highly efficient manner.

4.4. Fenfluramine effects on tissue and plasma 5-HT Acknowledgements Bearing in mind that the principal peripheral 5-HT pool is in the intestine, our primary aim was to investigate how alterations in 5-HT levels within this peripheral pool affect plasma 5-HT. Previous studies in superfused isolated intestine in fish have demonstrated that 5-HT present in enteric neurons is sensitive to the administration of drugs that interfere withsynapse dynamics (Anderson et al., 1991). In the two intestinal regions studied by us, administration of fenfluramine (5 or 15 mg/kg) tended to reduce 5-HT concentrations and to induce a dose-dependent increase in 5-HIAA/5-HT ratio. Increases in 5-HIAA/5-HT ratio are usually interpreted as indicating enhanced functional release of the neurotransmitter (Øverli et al., 2001), so that the results obtained allow us to deduce that fenfluramine stimulates 5-HT release from neurons of the intestinal wall, as previously observed for neurons of the central nervous system of rainbow trout (Ruibal et al., 2002). However, we should note that in almost all cases fenfluramine induced an increase (in some cases statistically significant) in 5-HIAA levels, indicating that it does not even partially inhibit neuronal 5-HT uptake. It is evident that 5-HT released from these neurons is taken up again and metabolized in situ. This capacity to metabolize 5-HT in situ is clearly in line with the fact that in rainbow trout the tissue with highest MAO activity is the intestine (Edwards et al., 1986; Senatori et al., 2003). The capacity for in situ metabolization is considerable, given that – as can be seen in Fig. 8A and B – the administration of fenfluramine dose not increase plasma levels of 5-HT, but does increase plasma levels of 5-HIAA (though not significantly) and of 5-HIAA/5-HT ratio (significantly). In conclusion, these results indicate that in rainbow trout the 5-HT released from neurons in the intestinal wall (the most important peripheral 5-HT pool in this species) is metabolized in situ, so that probably only a small part will reach the plasma, from where it may be taken up and metabolized by other peripheral tissues (gills and liver). Although the effect of FF on serotonin dynamics in the anterior kidney is similar to that observed in the intestine, the absence of significant effects suggests either that renal intracellular 5-HT stores are less susceptible to this drug than intestinal 5-HT stores, or that our

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