Appetite-suppressing effects of ammonia exposure in rainbow trout associated with regional and temporal activation of brain monoaminergic and CRF systems
1 Department of Integrative Biology, University of Guelph, Guelph, ON,
Canada, N1G 2W1
2 Biology and Neuroscience, University of South Dakota, Vermillion, SD
57069, USA
* Author for correspondence (e-mail: nbernier{at}uoguelph.ca)
Accepted 9 March 2005
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Summary |
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Key words: feeding behavior, anorexia, HPI axis, stress, corticotropin-releasing factor, urotensin I, cortisol, rainbow trout, Oncorhynchus mykiss
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Introduction |
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Among the many factors identified as potential regulators of food intake in
fish (Lin et al., 2000),
several indirect lines of evidence suggest that the monoamine-containing
neuronal systems of the brain may be involved in mediating the
appetite-suppressing effects of ammonia. In channel catfish Ictalurus
punctatus, the growth-suppressing effects of chronic environmental
ammonia are associated with exposure-dependent decreases in whole brain
dopamine (DA) and serotonin (5-HT) concentrations and in an increase in 5-HT
turnover (Atwood et al., 2000
).
Moreover, intracerebroventicular (icv) injections of 5-HT and DA inhibit
feeding behaviour in goldfish Carassius auratus (De Pedro et al.,
1998a
,b
)
and intraperitoneal administration of the 5-HT-releasing agent, fenfluramine,
induces a short-term inhibition of food intake in rainbow trout
(Ruibal et al., 2002
).
Finally, in several species, including rainbow trout, there is an inverse
relationship within dominance-based hierarchies between brain serotonergic
activity and food intake where low-feeding fish have higher serotonergic
activity (Winberg et al.,
1993
; Alanara et al.,
1998
). Given this evidence, we investigated in this study the
effects of high external ammonia exposure on food intake and on the regional
brain turnover of 5-HT and DA in rainbow trout.
As indicated by the release of cortisol from the interrenal cells of the
head kidney, most stressors result in an activation of the
hypothalamic-pituitary-interrenal axis (HPI) in teleosts (reviewed by
Wendelaar Bonga, 1997;
Lovejoy and Balment, 1999
;
Bernier and Peter, 2001a
).
Similarly, the increase in plasma cortisol associated with ammonia exposure in
a variety of different fish species suggests an activation of the HPI axis in
response to this environmental stressor
(Tomasso et al., 1981
;
Spotte and Anderson, 1989
;
Person-Le-Ruyet et al., 1998
;
Wicks and Randall, 2002b
).
Although the hypophysiotropic factors involved in stimulating the HPI axis in
ammonia-exposed fish are not known, corticotropin-releasing-factor (CRF) and
the related neuropeptide urotensin I (UI) are likely candidates
(Lederis et al., 1994
). In
addition to their role as regulators of the HPI axis, CRF-related peptides
have been identified as potent anorexigenic factors in fish
(De Pedro et al., 1993
;
Bernier and Peter, 2001b
).
Therefore, we also examined the effects of high external ammonia exposure on
the forebrain levels of CRF and UI mRNA in rainbow trout.
Thus, to get a better understanding of the central mechanisms controlling
food consumption during ammonia toxicity in fish, one of the objectives of
this study was to determine whether the appetite-suppressing effects of
ammonia exposure are associated with changes in brain monoaminergic activity
and/or CRF and UI mRNA levels. However, since the appetite-suppressing effects
of chronic ammonia exposure can be transient
(Beamish and Tandler, 1990;
Wicks and Randall, 2002a
) and
there are known regional differences in the changes of both monoaminergic
activity and CRF-related peptide gene expression in response to stressors
(Winberg and Nilsson, 1993
;
Summers et al., 1998
;
Bernier and Peter, 2001b
), a
second objective of this study was to determine the temporal and regional
changes in brain monoaminergic activity and/or CRF and UI mRNA levels
associated with ammonia toxicity.
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Materials and methods |
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Experimental design
Two separate, but similar experiments were performed. Experiment 1 was a
flow-through 24 h ammonia exposure at three levels. Experiment 2 was a
flow-through 96 h ammonia exposure at the same exposure levels. Experimental
protocol and sampling procedure were identical for both and will be described
together. In each experiment, groups of 10-12 fish were randomly assigned to
one of eight tanks as described above. The eight tanks were paired to account
for tank effects and to allow for four treatment conditions: control, 500, 750
and 1000 µmol l-1 ammonia. Throughout the experiments, fish were
fed daily to satiety at 10:00 h and on the first day of the exposures the
ammonia treatments began 2 h post-feeding. Using prepared stock solutions of
ammonium chloride (NH4Cl), ammonia levels in the tanks were
increased instantaneously to their respective conditions and maintained using
a peristaltic pump (Perista Pump SJ-1220, Rose Scientific, Mississauga, ON,
Canada). Water samples were collected prior to feeding, immediately after the
start of the experiment, and every 5 h thereafter to monitor pH and water
ammonia levels. At the end of an experiment, fish were quickly netted from a
tank, terminally anesthetized with an overdose of 2-phenoxyethanol (2.0 ml
l-1; Sigma, St Louis, MI, USA), weighed, and a blood sample
collected by caudal puncture using a K2-EDTA (0.5 mol
l-1, pH 8.0)-treated syringe. The brain was then dissected into
three distinct regions, the telencephalon (TEL), hypothalamus (HYP), and
posterior brain (PB; which includes the midbrain, optic tectum, cerebellum and
brainstem), and immediately frozen in liquid nitrogen for subsequent
measurement of both CRF and UI gene expression or
serotonergic and dopaminergic activity. The sampling of each fish was
completed in less than 2 min and an entire tank in less than 10 min. Blood
samples were immediately centrifuged at 15 000 g for 5 min and
the separated plasma frozen at -80°C for subsequent measurement of plasma
ammonia and plasma cortisol concentrations.
Assessment of food intake
Using X-radiography (Jobling et al.,
2001), food intake was assessed twice from each individual fish
over the course of this study: 7 days prior to the start of the ammonia
exposures (control food intake) and immediately after the ammonia exposures.
In brief, the commercial trout food was grounded and re-pelleted with 450
µm steel beads (Draiswerke, Mahwah, NJ, USA) at a ratio of 5% by mass of
dry powdered food. On days where control food intake was assessed, fish were
fed to satiety at 10:00 h, anaesthetized 2 h later with tricaine
methanesulfonate (0.1 g l-1; MS-222; Syndel, Vancouver, BC,
Canada), weighed, X-rayed using an Acu Ray HFJ portable X-ray machine (1.05
mAs at 50 kV; Stern Medical Equipment, Brampton, ON, Canada), and returned to
their original tanks for recovery. Post ammonia exposure, food intake was
assessed as above immediately after terminal sampling of the fish. The beads
located in the digestive tract of the fish were counted from the X-ray films
and the amount eaten in grams was determined from a standard curve.
Preliminary experiments showed that re-pelleting and diet labelling do not
affect palatability and control food intake is not affected by the above
procedures as long as the fish are given a 72 h recovery period between
X-raying. Finally, during a 2-week acclimation period prior to the experiment,
and between the control and post-exposure X-rays, the fish were fed the same
re-pelleted diet without the label.
Ammonia and cortisol determinations
Water ammonia concentration was measured using the method described by
Verdouw et al. (1978). Plasma
samples were assayed for ammonia using the glutamate dehydrogenase enzymatic
assay of Kun and Kearney
(1971
). Briefly, plasma
samples were de-proteinized with 2 volumes of trichloroacetic acid (10% w/v)
and centrifuged at 15 000 g for 10 min at 4°C. The
supernatant was neutralized with potassium hydrogen carbonate
(KHCO3; 2 mol l-1) and centrifuged at 7500
g for 30 s at 4°C. The enzymatic assay was adapted for
96-well microtitre plates. All samples were run in triplicate at 24°C with
shaking in a SpectraMax 190 plate reader (Molecular Devices, Menlo Park, Ca,
USA) and changes in absorbance were recorded until all reactions had gone to
completion. Ammonia standards were prepared fresh in distilled water and the
plasma ammonia concentrations determined from a linear regression of the
standards using SOFTmax software 4.6 (Molecular Devices). Throughout, all
ammonia concentrations reported refer to total ammonia-N.
Plasma cortisol concentration was measured using the enzyme immunoassay of
Carey and McCormick (1998)
with the following modifications. The primary antibody (rabbit anti-cortisol,
cat. #F3-314, Esoterix Endocrinology, Calabasas Hills, CA, USA) and the
cortisol-horseradish peroxidase conjugate (Clinical Endocrinology Laboratory,
University of California Davis, Davis, CA, USA) were used in microtitre plates
at a final dilution of 1:20 000 and 1:70 000, respectively. The plates were
incubated at 26°C with shaking and read in a SpectraMAX 190 plate reader.
All samples were run in triplicate and the cortisol concentrations determined
from a 3-parameter sigmoidal standard curve (Sigma Plot 7.0; SPSS, Chicago,
IL, USA). A serial dilution of rainbow trout plasma gave a displacement curve
that was parallel to the standard curve and the lower detection of this assay
was 0.6 ng ml-1. Using a pooled plasma sample, the intra- and
inter-assay variations were 5.7% (N=10) and 12.6% (N=5),
respectively.
Monoamine determinations
For the three brain regions dissected, (TEL, HYP, and PB)
dihydroxyphenylacetic acid (DOPAC), DA, 5-hydroxyindoleacetic acid (5HIAA) and
5HT were measured using high performance liquid chromatography with
electrochemical detection as described by Renner and Luine
(1984), with several
modifications. Briefly, frozen tissue samples were weighed and placed in a 1.5
ml centrifuge tube. Sodium acetate buffer (pH 5) containing
1x10-7 dihydroxybenzylamine (internal standard) was added to
each sample (10:1 v/w), the samples were sonicated on ice, and freeze-thawed.
Samples were either processed directly for analysis or stored at -80°C
until analysis. Prior to analysis, 10 µl of a 1 mg per 10 ml H2O
ascorbate oxidase solution (Sigma Co., St Louis, MO, USA) were added to each
sample and the samples were centrifuged (17 000 g for 4 min).
The supernatant was removed and 30 µl was injected into a Waters
chromatography system (Waters Associates, Milford, MA, USA) and analyzed
electrochemically using a LC-4B potentiostat and a glassy carbon electrode
(Bioanalytical Systems, Inc., West Lafayette, IN, USA). The electrode
potential was set at +0.65 V with respect to an Ag AgCl-1 reference
electrode. Separation was accomplished using a 4 µm C-18 radial compression
cartridge (Waters Associates). The mobile phase consisted of 11 g citric acid,
8.6 g sodium acetate, 110 mg octylsulfonic acid (Sigma Co.), 250 mg EDTA and
100 ml methanol in 1 l of water.
The concentrations of the amines and amine metabolites were calculated with respect to the mean peak height values obtained from standard runs set in the internal standard mode using CSW32 software (Data Apex LTD, Prague, Czech Republic). The resulting values were corrected for volume and expressed as pg amine mg-1 tissue wet mass.
Total RNA extraction and first strand cDNA synthesis
Total RNA was extracted from the TEL and HYP brain regions using Trizol
Reagent (Invitrogen, Carlsbad, CA, USA) based on the acid guanidinium
thiocyanate-phenol-chloroform extraction method. Total RNA concentrations were
determined by ultraviolet spectrophotometry at 260 nm and diluted with
RNAse-free water to a working dilution of 0.5 µg µl-1. To
eliminate potential genomic contamination, 1 µg of total RNA was treated
with DNase I according to the manufacturer's protocol (DNase I amplification
grade, Invitrogen). The DNase-treated total RNA samples were reverse
transcribed to cDNA using SuperScript RNase H- reverse
transcriptase (Invitrogen).
Real-time reverse transcriptase-polymerase chain reaction
To quantify the mRNA expression of CRF and UI, the above cDNA products were
amplified using the ABI Prism 7000 sequence detection system (Applied
Biosystems; Foster City, USA). Each reaction contained 10 µl of SYBR green
PCR master mix (part# 4309155; Applied Biosystems), 5 µl cDNA template, and
2.5 µl each of forward and reverse primers (0.4 µmol l-1).
Default cycling conditions were used: 10 min at 95°C followed by 40 cycles
of 15 s at 95°C and 1 min at 60°C. This protocol was followed by a
melting curve analysis to verify the specificity of the PCR products. To
account for differences in amplification efficiency between the different
cDNAs, standard curves were constructed for each target gene using serial
dilutions of cDNA samples known to have high expression levels of the target
gene (Giulietti et al., 2001).
Using the threshold cycle of each unknown, the relative dilution of a given
sample was extrapolated using the linear regression of the target-specific
standard curve. Finally, to correct for cDNA loading and RNA reverse
transcriptase efficiency, each sample was normalized to the expression level
of the housekeeping gene acidic ribosomal phosphoprotein A0 (ARP). All samples
were assayed in triplicate and only one target was assayed per well.
Specific primers for either rainbow trout CRF (GenBank accession no. AY651777), UI (GenBank accession no. AY651778) and ARP (GenBank accession no. AY685220) were designed using the software program Primer Express (Applied Biosystems). To prevent co-amplification of genomic DNA, the sequence of each forward primer was chosen to flank an intron based on the position of known intron-exon junctions. To maximize amplification efficiency, the CRF (forward: 5'-ACA ACG ACT CAA CTG AAG ATC TCG-3'; reverse: 5'-AGG AAA TTG AGC TTC ATG TCA GG-3'), UI (forward: 5'-AGG AGA CAA AAT ACC GGG CA-3'; reverse: 5'-CTT CAT AGT GCT GGA CAG ACG G-3') and ARP (forward; 5'-TGA AAA TCA TCC AAT TGC TGG A-3'; reverse 5'-CGC CGA CAA TGA AAC ATT TG-3') primers were designed to amplify the shortest product possible (CRF, 54 bp; UI, 51 bp; ARP, 51 bp).
Statistical analysis
All data are presented as mean values plus one standard error of the mean
(S.E.M.). Differences among treatments were
assessed by one-way analysis of variance (ANOVA) followed by a pairwise Tukey
multiple comparison test. Within a given treatment, the effects of treatment
duration were assessed by unpaired Student's t test. Differences
between the pre- and post-ammonia exposure food intake values were assessed by
pairwise Student's t test. The software SigmaStat 3.0 was used for
statistical analyses (SPSS, Chicago, IL, USA), while SigmaPlot 7.0 (SPSS) was
used for correlation analyses. The significance level for all statistical
tests was P<0.05.
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Results |
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Prior to ammonia exposure, there was no difference in basal food intake between any of the 24 h (Fig. 2A) or 96 h (Fig. 2B) treatments. Food intake decreased both significantly and dose dependently after 24 h ammonia exposure, with complete appetite suppression in the highest dose (1000 µmol l-1) (Fig. 2A). All doses did show some recovery after 96 hexposure, with the lowest dose (500 µmol l-1) approaching control levels and the highest dose recovering by approximately 40% (Fig. 2B).
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The NH4Cl treatments were associated with changes in brain monoaminergic activity that were both brain region specific and dependent on exposure duration. In the HYP, serotonergic activity increased dose-dependently and peaked in the first 24 h (Fig. 3A). Although the 96 h activity level remained elevated relative to control, it was less pronounced than at 24 h, increasing significantly only in the highest dose (1000 µmol l-1). By contrast, while there was no change in serotonergic activity in the TEL and PB after 24 h of ammonia exposure, there were significant increases in the 750 and 1000 µmol l-1 doses after 96 h (Fig. 3B,C). Furthermore, in the TEL, there was a significant difference between sampling times in both the 500 and 750 µmol l-1 doses with the PB only displaying significant differences between exposure times in the 750 µmol l-1 dose. When plasma ammonia is regressed against 5-HT turnover after 24 h, although there is a significant linear relationship between these two variables in all three brain regions, the relationship is strongest in the HYP (r2=0.39, P<0.0001, Fig. 3D) and more variable in the TEL (r2=0.10, P=0.045) and PB (r2=0.11, P=0.040). These relationships, however, disappear after 96 h of ammonia exposure in all three brain regions. There are also significant relationships between 5-HT turnover and water ammonia. Again the strongest relationship is in the HYP (r2=0.40, P<0.0001), and more variable in the TEL (r2=0.16, P=0.0114) and PB (r2=0.12, P=0.0333). After 24 h of ammonia exposure significant linear relationships exist in the regression of plasma ammonia against 5-HT concentration in all brain regions; however, the relationship is strongest in the PB (r2=0.31, P<0.0001), intermediate in the TEL (r2=0.23, P<0.0001), and weakest in the HYP (r2=0.11, P<0.0001). Overall, while both decreases in 5-HT and increases in 5-HIAA contributed to the significant increases in serotonergic activity in the HYP after 24 h of ammonia exposure, decreases in 5-HT concentrations appear to be the primary determinant of the changes in 5-HIAA:5-HT ratios in the HYP after 96 h of exposure and in the two other brain regions (Table 1).
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Due to low DOPAC concentrations in the TEL and HYP, detection by high pressure liquid chromatography (HPLC) was not possible. DOPAC was, however, detectable in the PB, allowing dopaminergic activity to be measured in this region. Dopaminergic activity increased in the two highest ammonia dose samples after 24 h of exposure (Fig. 4A). Dopaminergic activity values remained elevated after 96 h and were not significantly different than after 24 h of ammonia exposure. A significant linear relationship exists in the regression of plasma ammonia against both dopaminergic activity (r2=0.45, P<0.0001, Fig. 4B) and DA concentrations (r2=0.31, P<0.0001) in the PB at 24 h. However, as previously observed with 5-HT turnover in the brain, the relationship between dopaminergic activity and plasma ammonia breaks down after 96 h of ammonia exposure. Overall, a decrease in DA concentration appears to be the primary determinant of the changes in DOPAC:DA ratios in the PB (Table 1).
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In the HYP, ammonia exposure was associated with an increase in CRF mRNA levels after 96 h (Fig. 5A). This increase peaked at the highest ammonia dose (1000 µmol l-1) and was significantly higher than the 24 h value. By contrast, only the lowest ammonia dose (500 µmol l-1) was associated with an increase in TEL CRF mRNA levels after 24 h exposure (Fig. 5B). UI expression followed similar patterns in both the HYP and TEL, with the highest mRNA levels observed after 24 h in fish exposed to the lowest ammonia dose (500 µmol l-1) and no apparent effect in the fish exposed to the highest ammonia dose (1000 µmol l-1; Fig. 5C,D). After 96 h ammonia exposure, HYP and TEL UI mRNA levels in the ammonia-exposed fish did not differ from controls.
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Changes in plasma cortisol levels were similar to the changes exhibited in plasma ammonia, with dose-dependent increases after 24 h exposure and a recovery towards control values after 96 h (Fig. 6A). After 24 h exposure, plasma cortisol values were respectively 3-, 9- and 14-fold higher in the low, medium, and high ammonia treatments than in the control fish. In contrast, plasma cortisol levels had returned to control values after 96 h in the 750 µmol l-1 treatment and decreased by more that 50% in the 1000 µmol l-1 treatment. There is a significant linear relationship between plasma ammonia and plasma cortisol after 24 h of ammonia exposure (r2=0.41, P<0.001; Fig. 6B). Similarly, when 5-HT turnover is regressed against the 24 h plasma cortisol data, there are significant linear relationships between these variables in both the HYP and PB, however the relationship is stronger in the HYP (r2=0.25, P=0.002, Fig. 6C) than in the PB (r2=0.15, P=0.015).
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Discussion |
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The appetite-suppressing effects of high ammonia exposure observed in this
study are consistent with previous observations made in rainbow trout
(Wicks and Randall, 2002a) and
lake trout Salvelinus namaycush
(Beamish and Tandler, 1990
).
While low levels of exogenous ammonia (
225 µmol l-1) have no
effect on food intake (Wood,
2004
), above a certain species-specific threshold, chronic
increases in water ammonia concentrations elicit an initial dose-dependent
reduction in food intake followed by a gradual recovery. The acute
hyperammonemia-associated reduction in food intake will protect fish from the
significant postprandial increase in plasma ammonia levels
(Mommsen and Walsh, 1992
;
Wicks and Randall, 2002a
).
Therefore, the appetite-suppressing effects of ammonia may represent an
immediate behavioural strategy aimed at minimizing ammonia toxicity. On the
other hand, the gradual recovery in appetite associated with ongoing
hyperammonemia probably results from biochemical adjustments aimed at
converting ammonia into less toxic substances
(Randall and Tsui, 2002
).
The partial recovery in plasma ammonia levels after 96 h of chronic water
ammonia exposure observed in this study and the similar results reported by
Wicks and Randall (2002b),
suggest that rainbow trout can detoxify ammonia. In fact, since all the fish
in this study were sampled 2 h after being fed and all the ammonia-exposed
fish ate significantly more after 96 h of ammonia exposure than after 24 h,
the reductions in plasma ammonia levels after 96 h exposure are likely to be
an underestimation. Similarly, the post-prandial increase in plasma ammonia
levels likely contributed to the relatively high plasma ammonia concentrations
of the control fish. In rainbow trout
(Arillo et al., 1981
;
Vedel et al., 1998
;
Wicks and Randall, 2002b
), as
in several other species (Walsh and
Milligan, 1995
; Peng et al.,
1998
; Jow et al.,
1999
), available data suggest that an important mechanism of
ammonia detoxification involves the conversion of excess ammonia to glutamate
via glutamate dehydrogenase and then to neutral and storable
glutamine via glutamine synthetase (GSase). In the brain of trout,
there is a significant decrease in glutamate and increase in glutamine
concentrations after 24 h of chronic ammonia exposure and these changes are
maintained through 96 h (Vedel et al., 1988). The changes in brain glutamate
and glutamine concentrations are associated with increases in brain and liver
GSase and an increase in muscle glutamine levels, suggesting that the ammonia
detoxified by the brain and liver may be stored in the muscle
(Wicks and Randall,
2002b
).
Rainbow trout chronically exposed to high ammonia concentrations in this
study displayed dose-dependent increases in brain 5-HIAA:5-HT ratios in the
TEL, HYP, and PB. These results support the findings of Atwood et al.
(2000) in channel catfish,
where 9 weeks of exposure to environmental ammonia was associated with a
dose-dependent increase in whole brain serotonergic activity. Besides
environmental ammonia exposure, several other stressors have been shown to
affect the 5-HT system in fish. For example, social subordination, chronic
shallow water disturbances and predator exposure are all associated with an
increase in brain serotonergic activity
(Winberg and Nilsson, 1993
).
In mammals, the increased brain serotonergic activity associated with
hyperammonemia appears to be caused by an increased availability in brain
tryptophan (TRP). The high brain glutamine concentrations associated with
hyperammonemia are known to stimulate blood-brain barrier transport of large
neutral amino acids such as TRP, which in return can increase 5HT turnover
(for reviews, see Bachmann,
2002
; Felipo and Butterworth,
2002
). Whether a similar glutamine-stimulated increase in brain
TRP uptake or a different mechanism is responsible for the increase in the
5-HIAA:5-HT ratios observed in this study remains to be determined. However,
after 96 h of ammonia exposure, rather than an increase in 5-HT turnover, the
decreases in HYP and PB 5-HT concentrations without change in 5-HIAA levels
suggest that 5-HT production is decreasing.
After 24 h of environmental ammonia, we observed exposure-dependent
increases and decreases in hypothalamic serotonergic activity and food intake,
respectively. In contrast, the exposure-dependent increases in serotonergic
activity in the TEL and PB occurred after 96 h of chronic ammonia, at a time
when food intake had partially recovered and hypothalamic serotonergic
activity was almost back to control levels. Although correlative, together
this evidence does suggest that serotonergic activity in the HYP may play an
important role in the regulation of food intake during ammonia toxicity in
trout. In support of such a role for 5-HT, it is interesting to note that
anorexia is also a characteristic feature of hyperammonemia in mammals
(Bachmann, 2002) and that
reducing dietary TRP supply counteracts the appetite-suppressing effects of
hyperammonemia in humans (Hyman et al.,
1986
). Anatomically, although the largest density of serotonergic
cell bodies are found in the raphe nuclei of the PB in fish
(Parent, 1983
), 5-HT
immunoreactive cell bodies are also found in several hypothalamic nuclei in
rainbow trout (Frankenhuis-van den Heuvel
and Nieuwenhuys, 1984
). These 5-HT positive cell bodies contribute
fibers to the hypothalamic inferior lobe
(Frankenhuis-van den Heuvel and
Nieuwenhuys, 1984
), a hypothalamic region previously identified as
an important integration center for the regulation of appetite in fish
(Peter, 1979
;
Wullimann and Mueller, 2004
).
In addition, central injection of 5-HT inhibits food intake in goldfish
(De Pedro et al., 1998a
), and
intraperitoneal administration of the 5-HT-releasing agent, fenfluramine,
simultaneously increases serotonergic activity in the HYP and temporarily
stops food intake in rainbow trout (Ruibal
et al., 2002
).
Along with the changes in serotonergic activity, chronic ammonia exposure
elicited an increase in PB dopaminergic activity. Unlike the regional changes
in serotonergic activity that were either rapid (HYP) or delayed (TEL and PB),
the dose-dependent increase in dopaminergic activity was significant after 24
h of ammonia exposure and maintained through 96 h. As previously observed by
Atwood et al. (2000) in channel
catfish, increasing ammonia exposure in this study resulted in significant
decreases in whole brain DA and no changes in DOPAC concentrations, changes
that are indicative of a decrease in DA production. As with 5-HT, the central
DA system in fish appears to be sensitive to a variety of stressors
(Overli et al., 2001
;
De Pedro et al., 2003
). In
general, results from these experiments and others suggest that the effects of
stress on the central DA circuitry in fish is both stressor- and brain
region-specific and may involve both changes in DA synthesis and metabolism.
Although icv injections of DA agonists in goldfish have anorectic effects
(De Pedro et al., 1998b
), in
mammals there is an extensive body of evidence showing that DA can both
inhibit and stimulate food intake (for a review, see
Meguid et al., 2000
). In this
study, after 24 h of ammonia exposure, the dose-dependent increase in
dopaminergic activity in the PB concurred with the reduction in food intake.
In contrast, the sustained increase in dopaminergic activity after 96 h
exposure was associated with a partial recovery in food intake. Therefore,
while the 24 h data suggest an involvement of DA in the reduction of food
intake, the 96 h dopaminergic activity results suggest that DA is certainly
not the only contributor to the observed changes in food consumption.
Anatomically, several different dopaminergic nuclei have been located in the
region identified in this study as the PB (which includes the midbrain, optic
tectum, cerebellum and brainstem;
Wullimann and Mueller, 2004
).
While none of these dopaminergic nuclei has specifically been linked to the
regulation of appetite in fish, the posterior tuberculum in the midbrain is
the origin of the ascending dopaminergic system in teleosts
(Rink and Wullimann, 2001
), a
major activating/modulatory system in vertebrate brains known for its
involvement in the regulation of feeding, drinking, and locomotion.
In addition to the potential involvement of central monoamines in the
control of food intake during high ammonia exposure, our data implicate the
CRF family of peptides in this regulatory process. Exposure to ammonia
elicited an increase in CRF and UI mRNA levels in both the HYP and TEL. Since
CRF-related peptides are potent anorexigenic signals in fish
(De Pedro et al., 1993;
Bernier and Peter, 2001b
), our
results suggest that CRF and UI may be involved in mediating at least a
portion of the anorexigenic effects associated with ammonia toxicity. However,
changes in gene expression do not necessarily predict peptide secretion, and
besides its role in the regulation of food intake, the CRF system in fish is
involved in regulating several other aspects of the stress response
(Lovejoy and Balment, 1999
).
Overall, the changes in CRF and UI gene expression with
ammonia exposure were region-, dose- and time-dependent. Whereas the mRNA
increases in TEL and HYP UI and in TEL CRF were only observed after 24 h of
exposure and were inversely related to the ammonia concentration, the
increases in hypothalamic CRF mRNA levels were observed after 96 h of exposure
and were dose-dependent. Although the exact cause is not known, the mechanism
responsible for the inverse relationship between the increase in mRNA levels
and the dose of ammonia after 24 h of environmental ammonia may involve
circulating glucocorticoids. In goldfish, cortisol exerts a negative feedback
action on forebrain CRF and UI gene expression (Bernier et
al., 1999
,
2004
). After 24 h of ammonia
exposure in this study, plasma ammonia and cortisol levels were positively
correlated. Therefore, the high circulating cortisol levels in the fish
exposed to the highest ammonia concentration may have negated the stimulatory
effects of hyperammonemia on TEL and HYP UI and on TEL CRF
gene expression.
The stimulatory effects of environmental ammonia on plasma cortisol
observed in this study are consistent with previous results in a variety of
fish species (Swift, 1981;
Spotte and Anderson, 1989
;
Person-Le-Ruyet et al., 1998
;
Wicks and Randall, 2002b
). As
in channel catfish (Tomasso et al.,
1981
), chronic exposure of trout to high ammonia concentrations
was associated with an initial dose-dependent increase in plasma cortisol
followed by a gradual return to basal values. The return of plasma cortisol
concentrations to basal conditions in chronically exposed fish may be a
reflection of ammonia detoxifying mechanisms. In fact, there is evidence to
suggest that the ammonia exposure-elicited surge in plasma cortisol promotes
the detoxification of ammonia. In the gulf toadfish Opsanus beta,
dexamethasone injections increase hepatic GSase
(Mommsen et al., 1992
) and
cortisol-synthesis inhibition prevents the acute hepatic GSase activation
induced by confinement stress (Hopkins et
al., 1995
). Whether the ammonia exposure-elicited surge in plasma
cortisol stimulates brain and liver GSase and contributes to the
detoxification of ammonia to glutamine in rainbow trout needs to be
ascertained. The stimulatory effects of cortisol implants on urea synthesis in
rainbow trout do, however, suggest that cortisol can stimulate the enzymes of
the uricolytic pathway in this species
(McDonald and Wood, 2004
). On
the other hand, while there are still numerous questions as to the precise
role of cortisol in the regulation of food intake in fish, since only chronic
elevations in plasma cortisol appear to affect appetite, the ammonia-elicited
surge in plasma cortisol was probably not a significant contributor to the
control of food intake in this study
(Bernier and Peter, 2001a
;
Bernier et al., 2004
).
There is considerable evidence implicating CRF-related peptides and 5-HT in
the control of the HPI axis in fish (e.g.
Lederis et al., 1994;
Winberg et al., 1997
;
Winberg and Lepage, 1998
;
Huising et al., 2004
). In this
study, relative to the changes in plasma cortisol, the timing of the increase
in the mRNA levels of TEL CRF, TEL UI, HYP UI and HYP serotonergic activity
suggest a potential involvement of both CRF-related peptides and 5-HT in the
control of the HPI axis during environmental ammonia exposure. However, based
solely on gene expression data that may be influenced by the negative feedback
effects of cortisol (see above), it is difficult to assess the extent of the
CRF and UI contribution to HPI axis regulation during ammonia exposure. In
contrast, the strong correlation between hypothalamic serotonergic activity
and plasma cortisol levels after 24 h of exposure, suggest an important role
of hypothalamic 5-HT in the regulation of the HPI axis in hyperammonemic fish.
Interestingly, however, there is also evidence suggesting that the 5-HT system
may be involved in suppressing the adrenocortical stress response in fish
(Lepage et al., 2002
).
Pharmacological and anatomical studies have identified complex interactions
between the dopaminergic, serotonergic and CRF systems in the brain of mammals
(Meguid et al., 2000;
Sullivan Hanley and Van de Kar,
2003
; Herman et al.,
2003
). Although our understanding of the interplay between these
various neuroendocrine systems in fish is cursory, there are a few examples of
such interactions. In goldfish, for example, CRF may partially mediate the
anorectic effects of 5-HT (De Pedro et
al., 1998a
) and the anorectic effects of CRF may involve the
dopaminergic system (De Pedro et al.,
1998b
). Anatomical evidence also suggests potential interactions
between the serotonergic and CRF systems in rainbow trout
(Terlou et al., 1978
;
Frankenhuis-van den Heuvel and
Nieuwenhuys, 1984
). Hypothalamic regions known to contain CRF and
UI cell bodies in fish, the preoptic area and the basal HYP
(Lederis et al., 1994
), are
richly innervated by 5-HT fibers. Whether such interactions are important to
the overall regulation of food intake and HPI axis during exposure to high
ammonia concentrations remains to be determined.
In summary, exposure to high external ammonia concentrations over a 96 h period are characterized by a transient reduction in appetite and activation of the HPI axis in rainbow trout. Here we show that these responses to ammonia exposure are associated with regional and time-dependent increases in the activity of neurotransmitters and expression of neuropeptides with known anorexigenic and hypophysiotropic properties, namely 5-HT, DA, CRF and UI. These results implicate the brain's monoaminergic and CRF systems in the neuroendocrine circuitry involved in sensing and coordinating the response to hyperammonemia stress. More specifically, our findings show that among the many excitatory and inhibitory inputs that control food intake and HPI responsiveness, hypothalamic 5-HT may be a key neurobiological substrate for the regulation of these processes during exposure to high external ammonia concentrations.
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References |
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Alanara, A., Winberg, S., Brannas, E., Kiessling, A., Hoglund, E. and Elofsson, U. (1998). Feeding behaviour, brain serotonergic activity levels, and energy reserves of Arctic char (Salvelinus alpinus) within a dominance hierarchy. Can. J. Zool. 76,212 -220.[CrossRef]
Alderson, R. (1979). The effect of ammonia on the growth of juvenile dover sole, Solea solea (L.) and turbot, Scophthalmus maximus (L.). Aquaculture 17,291 -309.[CrossRef]
Arillo, A., Margiocco, C., Melodia, F., Mensi, P. and Schenone, G. (1981). Ammonia toxicity mechanisms in fish: Studies on rainbow trout (Salmo gairdneri). Ecotoxicol. Environ. Safety 5,316 -325.[CrossRef][Medline]
Atwood, H. L., Tomasso, J. R., Ronan, P. J., Barton, B. A. and Renner, K. J. (2000). Brain monoamine concentrations as predictors of growth inhibition in channel catfish exposed to ammonia. J. Aq. Anim. Health 12,69 -73.[CrossRef]
Bachmann, C. (2002). Mechanisms of hyperammonemia. Clin. Chem. Lab. Med. 40,653 -662.[Medline]
Beamish, F. W. H. and Tandler, A. (1990). Ambient ammonia, diet and growth in lake trout. Aquat. Toxicol. 17,155 -166.[CrossRef]
Bernier, N. J. and Peter, R. E. (2001a). The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish. Comp. Biochem. Physiol. 129B,639 -644.
Bernier, N. J. and Peter, R. E. (2001b). Appetite-suppressing effects of urotensin I and corticotropin-releasing hormone in goldfish (Carassius auratus). Neuroendocrinol. 73,248 -260.[CrossRef][Medline]
Bernier, N. J., Lin, X. and Peter, R. E. (1999). Differential expression of corticotropin-releasing factor (CRF) and urotensin I precursor genes, and evidence of CRF gene expression regulated by cortisol in goldfish brain. Gen. Comp. Endocrinol. 116,461 -477.[CrossRef][Medline]
Bernier, N. J., Bedard, N. and Peter, R. E. (2004). Effects of cortisol on food intake, growth, and forebrain neuropeptide Y and corticotropin-releasing factor gene expression in goldfish. Gen. Comp. Endocrinol. 135,230 -240.[CrossRef][Medline]
Carey, J. B. and McCormick, S. D. (1998). Atlantic salmon smolts are more responsive to an acute handling and confinement stress than parr. Aquaculture 168,237 -253.[CrossRef]
De Pedro, N., Alonso-Gomez, A. L., Gancedo, B., Delgado, M. J. and Alonso-Bedate, M. (1993). Role of corticotropin-releasing factor (CRF) as a food intake regulator in goldfish. Physiol. Behav. 53,517 -520.[CrossRef][Medline]
De Pedro, N., Pinillos, M. L., Valenciano, A. I., Alonso-Bedate, M. and Delgado, M. J. (1998a). Inhibitory effect of serotonin on feeding behavior in goldfish: involvement of CRF. Peptides 19,505 -511.[CrossRef][Medline]
De Pedro, N., Delgado, M. J., Pinillos, M. L. and Alonso-Bedate,
M. (1998b). 1-Adrenergic and dopaminergic receptors
are involved in the anoretic effect of corticotropin-releasing factor in
goldfish. Life Sci. 62,1801
-1808.[CrossRef][Medline]
De Pedro, N., Delgado, M. J., Gancedo, B. and Alonso-Bedate, M. (2003). Changes in glucose, glycogen, thyroid activity and hypothalamic catecholamines in tench by starvation and refeeding. J. Comp. Physiol. B 173,475 -481.[Medline]
Felipo, V. and Butterworth, R. F. (2002). Neurobiology of ammonia. Prog. Neurobiol. 67,259 -279.[CrossRef][Medline]
Frankenhuis-van den Heuvel, T. H. M. and Nieuwenhuys, R. (1984). Distribution of serotonin-immunoreactivity in the diencephalon and mesencephalon of the trout, Salmo gairdneri. Anat. Embryol. 169,193 -204.[CrossRef][Medline]
Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R. and Mathieu, C. (2001). An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25,386 -401.[CrossRef][Medline]
Herman, J. P., Figueiredo, H., Mueller, N. K., Ulrich-Lai, Y., Ostrander, M. M., Choi, D. C. and Cullinan, W. E. (2003). Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Fr. Neuroendol. 24,151 -180.[CrossRef]
Hopkins, T. E., Wood, C. M. and Walsh, P. J. (1995). Interactions of cortisol and nitrogen metabolism in the ureogenic gulf toadfish Opsanus beta. J. Exp. Biol. 198,2229 -2235.[Medline]
Huising, M. O., Metz, J. R., vanSchooten, C., Taverne-Thiele, A.
J., Hermsen, T., Verberg-vanKemenade, B. M. L. and Flik, G.
(2004). Structural characterization of a cyprinid (Cyprinus
carpio L.) CRH, CRH-BP and CRH-R1, and the role of these proteins in the
acute stress response. J. Mol. Endocrinol.
32,627
-648.
Hyman, S. L., Coyle, J. T., Parke, J. C., Porter, C., Thomas, G. H., Jankel, W. and Batshaw, M. L. (1986). Anorexia and altered serotonin metabolism in a patient with argininosuccinic aciduria. J. Pediatr. 108,705 -709.[Medline]
Ip, Y. K., Chew, S. F. and Randall, D. J. (2001). Ammonia toxicity, tolerance, and excretion. In Fish Physiology, vol. 20 (ed. P. A. Wright and P. M. Anderson), pp. 109-148. New York: Academic Press.
Jobling, M., Coves, D., Damsgard, B., Kristiansen, H. R., Koskela, J., Petursdottir, T. E., Kadri, S. and Gudmundsson, O. (2001). Techniques for measuring feed intake. In Food Intake in Fish (ed. D. Houlihan, T. Boujard and M. Jobling), pp.49 -87. Oxford: Blackwell Science.
Jow, L. Y., Chew, S. F., Lim, C. B., Anderson, P. M. and Ip, Y.
K. (1999). The marble goby Oxyeleotris marmoratus
activates hepatic glutamine synthetase and detoxifies ammonia to glutamine
during air exposure. J. Exp. Biol.
202,237
-245.
Kajimura, M., Croke, S. J., Glover, C. N. and Wood, C. M.
(2004). Dogmas and controversies in the handling of nitrogenous
wastes: the effects of feeding and fasting on the excretion of ammonia, urea
and other nitrogenous waste products in rainbow trout. J. Exp.
Biol. 207,1993
-2002.
Kun, E. and Kearney, E. B. (1971). Ammonia. In Methods of Enzymatic Analysis, vol.4 (ed. H.U. Bergmeyer), pp.1802 -1806. New York: Academic Press.
Lang, T., Peters, G., Hoffmann, R. and Meyer, E. (1987). Experimental investigations on the toxicity of ammonia: effects on ventilation frequency, growth, epidermal mucous cells, and gill structure of rainbow trout Salmo gairdneri. Dis. Aquat. Org. 3,159 -165.
Lederis, K., Fryer, J. N., Okawara, Y., Schonrock, C. and Richter, D. (1994). Corticotropin-releasing factors acting on the fish pituitary: experimental and molecular analysis. In Fish Physiology (ed. N. M. Sherwood and C. L. Hew), pp.67 -100. San Diego: Academic Press.
Lepage, O., Tottmar, O. and Winberg, S. (2002).
Elevated dietary intake of L-tryptophan counteracts the
stress-induced elevation of plasma cortisol in rainbow trout (Oncorhynchus
mykiss). J. Exp. Biol.
205,3679
-3687.
Lin, X., Volkoff, H., Narnaware, Y., Bernier, N. J., Peyon, P. and Peter, R. E. (2000). Brain regulation of feeding behavior and food intake in fish. Comp. Biochem. Physiol. 126A,415 -434.
Lovejoy, D. A. and Balment, R. J. (1999). Evolution and physiology of the corticotropin-releasing factor (CRF) family of neuropeptides in vertebrates. Gen. Comp. Endocrinol. 115, 1-22.[CrossRef][Medline]
McDonald, M. D. and Wood, C. M. (2004). The effect of chronic cortisol elevation on urea metabolism and excretion in the rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B 174,71 -81.[Medline]
Meguid, M. M., Fetissov, S. O., Varma, M., Sato, T., Zhang, L., Laviano, A. and Rossi-Fanelli, F. (2000). Hypothalamic dopamine and serotonin in the regulation of food intake. Nutrition 16,843 -857.[CrossRef][Medline]
Mommsen, T. P. and Walsh, P. J. (1992). Biochemical and environmental perspectives on nitrogen metabolism in fishes. Experientia 48,583 -593.
Mommsen, T. P., Danulat, E. and Walsh, P. J. (1992). Metabolic actions of glucagon and dexamethasone in the liver of the ureogenic teleost Opsanus beta. Gen. Comp. Endocrinol. 85,316 -326.[CrossRef][Medline]
Overli, O., Pottinger, T. G., Carrick, T. R., Overli, E. and Winberg, S. (2001). Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness. Brain Behav. Evol. 57,214 -224.[CrossRef][Medline]
Parent, A. (1983). The monoamine-containing neuronal systems in the teleostean brain. In Fish Neurobiology (ed. R. E. Davis and R. G. Northcutt), pp.285 -315. Ann Arbor: The University of Michigan Press.
Peng, K. W., Chew, S. F., Lim, C. B., Kuah, S. S. L., Kok, W. K. and Ip, Y. K. (1998). The mudskippers Periophthmodon schlosseri and Boleophthalmus boddaerti can tolerate environmental NH3 concentrations of 446 and 36 µmol l-1, respectively. Fish Physiol. Biochem. 19, 59-69.[CrossRef]
Person-Le-Ruyet, J., Boeuf, G., Zambonino Infante, J., Helgason, S. and Le Roux, A. (1998). Short-term physiological changes in turbot and seabream juveniles exposed to exogenous ammonia. Comp. Biochem. Physiol. 119A,511 -518.
Peter, R. E. (1979). The brain and feeding behaviour. In Fish Physiology (ed. W. S. Hoar and D. J. Randall), pp. 121-159. London: Academic Press.
Randall, D. J. and Tsui, T. K. N. (2002). Ammonia toxicity in fish. Mar. Poll. Bull. 45, 17-23.[CrossRef]
Renner, K. J. and Luine, V. N. (1984). Determination of monoamines in brain nuclei by high performance liquid chromatography with electrochemical detection: young vs. middle aged rats. Life Sci. 28,2193 -2199.
Rink, E. and Wullimann, M. F. (2001). The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum). Brain Res. 889,316 -330.[CrossRef][Medline]
Ruibal, C., Soengas, J. L. and Aldegunde, M. (2002). Brain serotonin and the control of food intake in rainbow trout (Oncorhynchus mykiss): Effects of changes in plasma glucose levels. J. Comp. Physiol. A 188,479 -484.
Spotte, S. and Anderson, G. (1989). Plasma cortisol changes in seawater-adapted Mummichogs (Fundulus heteroclitus) exposed to ammonia. Can. J. Fish. Aquat. Sci. 46,2065 -2069.
Sullivan Hanley, N. R. and Van de Kar, L. D. (2003). Serotonin and the neuroendocrine regulation of the hypothalamic-pituitary-adrenal axis in health and disease. Vitamins Hormones 66,189 -255.[Medline]
Summers, C. H., Larson, E. T., Summers, T. R., Renner, K. J. and Greenberg, N. (1998). Regional and temporal separation of serotonergic activity mediating social stress. Neurosci. 87,489 -496.[CrossRef][Medline]
Swift, D. J. (1981). Changes in selected blood component concentrations of rainbow trout, Salmo gairdneri Richardson, exposed to hypoxia or sublethal concentrations of phenol or ammonia. J. Fish Biol. 19, 45-61.
Terlou, M., Ekengren, B. and Hiemstra, K. (1978). Localization of monoamines in the forebrain of two salmonid species, with special reference to the hypothalamo-hypophysial system. Cell Tissue Res. 190,417 -434.[Medline]
Tomasso, J. R. (1994). Toxicity of nitrogenous wastes to aquaculture animals. Rev. Fish. Sci. 2, 291-314.
Tomasso, J. R., Davis, K. B. and Simco, B. A. (1981). Plasma corticosteroid dynamics in channel catfish (Ictalurus punctatus) exposed to ammonia and nitrite. Can. J. Fish. Aquat. Sci. 38,1106 -1112.
Vedel, N. E., Korsgaard, B. and Jensen, F. B. (1998). Isolated and combined exposure to ammonia and nitrite in rainbow trout (Oncorhynchus mykiss): effect on electrolyte status, blood respiratory properties and brain glutamine/glutamate concentrations. Aquat. Toxicol. 41,325 -342.[CrossRef]
Verdouw, H., Van Echted, C. J. A. and Dekkers, E. M. J. (1978). Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12,399 -402.[CrossRef]
Walsh, P. J. and Milligan, C. L. (1995). Effects of feeding and confinement on nitrogen metabolism and excretion in the gulf toadfish Opsanus beta. J. Exp. Biol. 198,1559 -1566.[Medline]
Wendelaar Bonga, S. E. (1997). The stress
response in fish. Physiol. Rev.
77,591
-625.
Wicks, B. J. and Randall, D. J. (2002a). The effect of feeding and fasting on ammonia toxicity in juvenile rainbow trout, Oncorhynchus mykiss. Aquat. Toxicol. 59, 71-82.[CrossRef][Medline]
Wicks, B. J. and Randall, D. J. (2002b). The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout: the role of glutamine in regulation of ammonia. Comp. Biochem. Physiol. 132A,275 -285.
Wilkie, M. P. (2002). Ammonia excretion and urea handling by fish gills: present understanding and future research challenges. J. Exp. Zool. 293,284 -301.[CrossRef][Medline]
Winberg, S. and Nilsson, G. E. (1993). Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to stress. Comp. Biochem. Physiol. 106C,597 -614.
Winberg, S. and Lepage, O. (1998). Elevation of brain 5-HT activity, POMC expression, and plasma cortisol in socially subordinate rainbow trout. Am. J. Physiol. 274,R645 -R654.[Medline]
Winberg, S., Carter, C. G., McCarthy, I. D., He, Z.-Y., Nilsson,
G. E. and Houlihan, D. F. (1993). Feeding rank and brain
serotonergic acitivity in rainbow trout Oncorhynchus mykiss.
J. Exp. Biol. 179,197
-211.
Winberg, S., Nilsson, A., Hylland, P., Soderstom, V. and Nilsson, G. E. (1997). Serotonin as a regulator of hypothalamic-pituitary-interrenal activity in teleost fish. Neurosci. L. 230,113 -116.[CrossRef]
Wood, C. M. (2004). Dogmas and controversies in
the handling of nitrogenous wastes: Is exogenous ammonia a growth stimulant in
fish? J. Exp. Biol. 207,2043
-2054.
Wright, P. A. (1995). Nitrogen excretion: three end products, many physiological roles. J. Exp. Biol. 198,273 -281.[Medline]
Wullimann, M. F. and Mueller, T. (2004). Teleostean and mammalian forebrains contrasted: evidence from genes to behavior. J. Comp. Neurol. 475,143 -162.[CrossRef][Medline]