Time-course of the effect of dietary L-tryptophan on plasma cortisol levels in rainbow trout Oncorhynchus mykiss
1 Evolutionary Biology Centre, Department of Comparative Physiology, Uppsala
University, Norbyvägen 18A, SE-752 36, Sweden
2 NERC Centre for Ecology and Hydrology, Windermere Laboratory, The Ferry
House, Far Sawrey, Ambleside, Cumbria, UK
* Author for correspondence (e-mail: Svante.Winberg{at}ebc.uu.se)
Accepted 15 July 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: serotonin, brain, fish, rainbow trout, Oncorhynchus mykiss, feed, stress, Salmonidae, aquaculture
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brain 5-HT is involved in the regulation of the
hypothalamus-pituitary-adrenocortical (HPA) axis in mammals (Chaoulof, 1993;
Dinan, 1996) as well as in the
control of the hypothalamus-pituitary-interrenal (HPI) axis in fish
(Winberg et al., 1997
;
Winberg and Lepage, 1998
;
Øverli et al., 1999
;
Höglund et al., 2002
).
Stressors, like social subordination, handling and predator exposure, usually
produce a rapid activation of the brain serotonergic system, revealed by an
increase in brain levels of the major serotonin metabolite,
5-hydroxyindoleacetic acid (5-HIAA), and/or elevated brain [5-HIAA]/[5-HT]
ratios (an index of 5-HT activity)
(Winberg et al., 1992
;
Winberg and Nilsson, 1993
). In
several studies, brain [5-HIAA]/[5-HT] ratios have been found to correlate
with plasma [cortisol] (Winberg and
Lepage, 1998
; Øverli et
al., 1999
) and adrenocorticotropin (ACTH) levels
(Höglund et al., 2000
),
suggesting that brain 5-HT has a stimulatory action on the HPI axis. However,
the role of the brain 5-HT system in the control of the HPI axis is still not
clear. For instance, 8-OH-DPAT, a selective 5-HT1A receptor
agonist, may have either stimulatory or inhibitory effects on HPI axis
activity in rainbow trout, depending on the dose and context. In undisturbed
fish 8-OH-DPAT stimulates the HPI axis
(Winberg et al., 1997
;
Höglund et al., 2002
),
whereas if administrated at low doses to stressed fish, 8-OH-DPAT has the
opposite effect, suppressing the stress-induced elevation of plasma [ACTH] and
[cortisol] (Höglund et al.,
2002
).
In a previous study we similarly showed that feeding rainbow trout
TRP-supplemented feed for 7 days results in a slight but significant elevation
of basal plasma levels of cortisol, but at the same time causes a significant
reduction in the stress-induced elevation of plasma cortisol concentrations
(Lepage et al., 2002). These
effects were suggested to occur as a consequence of the elevated 5-HT activity
caused by dietary TRP supplementation, although it is not clear through what
mechanisms 5-HT modulates HPI axis activity. Winberg et al.
(2001
) reported that dietary
supplementation with TRP for 7 days also results in an inhibition of
aggressive behaviour in rainbow trout, whereas 3 days of TRP supplementation
have no effect on aggressive behaviour. An anti-aggressive effect of the brain
5-HT system has been reported in a number of vertebrates
(Raleigh et al.,
1991
; Blanchard et al.,
1991
,
1993
;
Deckel, 1996
;
Deckel and Jevitts, 1997
;
Edwards and Kravitz, 1997
;
Larson and Summers, 2001
)
including teleost fish (Adams et al.,
1996
; Winberg and Nilsson,
1993
), and the suppression of aggressive behaviour induced by
elevated dietary TRP is believed to be mediated by elevated brain 5-HT
activity.
Norepinephrine (NE) and dopamine (DA) are also important in the control of
neuroendocrine release factors at the level of the hypothalamus and pituitary.
For instance, in teleost fish, the central NE system has been suggested to
stimulate HPI axis activity (Øverli
et al., 1999; Höglund et
al., 2000
). DA, on the other hand, might have effects that are to
some extent opposite to those of 5-HT
(Winberg and Nilsson, 1992
),
and L-dopa treatment, which elevates brain DA activity, has been
reported to induce social dominance
(Winberg and Nilsson, 1992
)
and to counteract the stress-induced elevation of plasma [cortisol] and brain
5-HT activity in Arctic charr Salvelinus alpinus
(Höglund et al.,
2001
).
These results suggest that brain catecholaminergic systems are interacting
with the 5-HT system and that NE and DA may modulate the effect of 5-HT on the
HPI axis. Moreover, catecholamines are synthesised from L-tyrosine,
another essential large neutral amino acid (LNAA), competing with TRP for
uptake to the brain (Fernstrom,
1983). Thus, elevated dietary intake of TRP may also affect brain
NE and DA activity.
In the present study we report the effects of feeding rainbow trout TRP-supplemented feed for 3, 7 and 28 days on plasma levels of cortisol and ACTH as well as on brain NE, DA and 5-HT activity. The effects of elevated dietary intake of TRP were studied in both stressed and undisturbed fish.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental protocol
The experiment was performed in two consecutive rounds, each round
including 48 individuals kept in eight 250 litre glass aquaria continuously
supplied with aerated Uppsala tapwater (0.8l min-1, 8-10°C).
Light (12 h:12 h light:dark) was provided by a 30 W Lumilux daylight
fluorescent tube placed 100 mm above the water surface of each aquarium. Each
aquarium was divided into four individual 65 litrecompartments by removable
PVC walls.
At the start of the experiment, fish were selected from the holding tank, weighed and transferred to individual compartments within the experimental aquaria. The first week after transfer to social isolation, fish were hand-fed with commercial feed (EWOS ST40) until satiation. Individual feed intake was quantified by counting the number of pellets consumed. For quantification of fed intake, individual fish were fed with one pellet at the time until the fish rejected three pellets in a row. Pellets not consumed were removed from aquarium. Following this week of acclimation, commercial feed was exchanged for an experimental feed, supplemented with TRP to a level (3.57 g total TRP kg-1 dry feed) corresponding to 8 times the TRP content of the commercial feed (0.44 g total TRP kg-1 dry feed), but otherwise identical to this feed. A similar number of fish were fed with a control feed, not supplemented with TRP. Fish were fed once a day to satiation, or at maximum until the fish consumed a number of pellets corresponding to 1.5% of the body mass, for 3, 7 or 28 days, and individual feed intake was quantified. At the end of the experimental feeding period, half of the fish fed TRP-supplemented feed and half of the fish that received control feed, were exposed to a standardised stressor for 2 h. The stressor consisted of lowering the water level in the aquaria until the dorsal fin of the fish was exposed above the water surface. The remaining fish were left undisturbed and served as non-stressed controls. Following stress, fish were killed, and blood and brain tissue samples collected.
Blood and brain tissue sampling
Upon sampling, fish were rapidly netted and anaesthetised in 500 mg
l-1 ethyl-m-aminobenzoate methanesulphonate. Blood (1 ml
approximately) was collected from the caudal vasculature with a heparinized
syringe and kept on ice. Fish were then decapitated, and the brain was rapidly
(within 2 min) removed and dissected into four different regions:
telencephalon (excluding the olfactory bulb), hypothalamus (excluding the
pituitary gland), brain stem (including the medulla and part of the spinal
cord but excluding the cerebellum), and the optic tectum. Each brain part was
wrapped in aluminium foil, frozen in liquid nitrogen and stored at -80°C.
Finally, following centrifugation at 27 000 g for 10 min,
plasma portions were frozen and kept at -80°C.
Assays
The frozen brain samples were homogenized in 400 µl of sodium acetate
buffer (0.1 mol l-1, pH 5), containing 0.1 mg ml-1
pargyline (a monoamineoxidase inhibitor; Sigma P-8013), using a
Potter-Elvenhjem homogeniser (brain stem and optic tectum) or an MSE 100 W
ultrasonic disintegrator (telencephalon and hypothalamus).
After centrifugation (27 000 g, 10 min), 8 µl of ascorbic acid oxidase (Sigma A-0157; 100 units/800 µl H2O) was added to the supernatant, which was then left for 10 min at room temperature. Thereafter, 200 µl of 4% (w/v) ice-cold perchloric acid (PCA) containing 0.2% EDTA and 40 ng ml-1 epinine (deoxyephinephrine, used as an internal standard) was added. Following centrifugation (27 000 g, 10 min), the samples were rapidly frozen and stored at -80°C.
Brain [5-HT], [5-HIAA], [DA], [DOPAC] (3,4-dihydroxyphenylacetic acid),
[NE] and [MHPG] (3-methoxy-4-hydroxyphenylglycol) were quantified using
high-performance liquid chromatography with electrochemical detection
(HPLC-EC), as described by Øverli et al.
(1999).
Plasma and brain [TRP] were analysed using the same HPLC system but with
the oxidizing potential set at 600 mV
(Lepage et al., 2002).
Cortisol analysis was performed directly on rainbow trout plasma without
extraction, using a validated radioimmunoassay (RIA) modified from Olsen et
al. (1992), as described by
Winberg and Lepage (1998
).
Plasma ACTH concentrations were determined using a validated heterologous
radioimmunoassay (Balm and Pottinger,
1993). In brief, standards (0-160 pg; NIBBS hACTH1-39, Herts, UK)
or unknowns (25 µl) were incubated together with antibody (IgG-ACTH-1;
Campro Scientific, Veenendaal, The Netherlands) for 72 h at 4°C.
Radio-labelled ACTH (3-[125I]iodotyrosyl2)ACTH1-39; Amersham IM
216, Buckinghamshire, UK; 74 TBq/mmol; 4000 c.p.m./tube) was added to each
tube and these were incubated for a further 24 h. Immunoprecipitation was
achieved by adding 100 µl of a sheep anti-rabbit antiserum (SAR-IgG; Sigma
R-9754) solution containing rabbit IgG (Sigma I-5006) to each tube, vortex
mixing and then incubating at room temperature for 20 min. A 1 ml portion of
ice-cold PEG solution (7.5% polyethylene glycol; PEG 6000) was added to each
tube and tubes were centrifuged. The supernatants were aspirated and the
activity remaining in the pellets counted in a liquid scintillation counter
(Packard Tri-Carb 1900TR; Illinois, USA) using gammavials (Zinsser Analytic,
Berkshire, UK). A standard curve (3-parameter hyperbolic decay) was fitted and
the unknowns interpolated.
Statistics
All values are means ± standard error of the mean
(S.E.M.). Since there was no difference between fish fed control
feed for 3, 7 and 21 days, either in stressed or undisturbed fish, in any of
the parameters analysed, data from these groups were pooled to create two
groups, one consisting of stressed fish fed control feed for 3, 7 and 21 days
following acclimation and the other of undisturbed fish fed control feed
during the same time periods.
The effects of feeding TRP-supplemented feed (controls fed TRP-supplemented feed for 0 days, and TRP-supplemented fish fed TRP-supplemented feed for 3, 7 or 21 days) and stress on [cortisol], [TRP], [5-HT], [5-HIAA], [5-HIAA]/[5-HT], [DA], [DOPAC], [DOPAC]/[DA], [NE], [MHPG] and [MHPG]/[NE] were analysed using a two-way analysis of variance (2-way ANOVA) followed by the least significance difference (LSD) post-hoc test. Correlations were tested using Spearman rank-correlation coefficients. All statistical analyses were performed using STATISTICA software.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasma [TRP], [ACTH] and [cortisol]
Exposing the fish to the standardised stressor had a significant effect on
plasma [cortisol] (F1,77=49.63, P<0.0001),
fish subjected to stress showing elevated plasma [cortisol] as compared to
non-stressed fish (Fig. 2A).
Feeding the fish TRP-supplemented feed had no significant effects on plasma
[cortisol] by itself. There was, however, a significant
(F3,77=2.75, P=0.0485) interaction between TRP
supplementation and stress. In fish fed TRP-supplemented feed for 7 days there
was no significant difference in plasma [cortisol] between stressed and
non-stressed fish (Fig. 2A).
Thus, in fish fed TRP-supplemented feed for 7 days, exposure to the stressor
did not result in any elevation of plasma [cortisol]. By contrast, in fish fed
TRP-supplemented feed for 3 or 28 days stress resulted in an elevation of
plasma [cortisol] no different from that seen in fish fed control feed
(Fig. 2A).
|
In non-stressed fish, feeding with TRP-supplemented feed for 3 days resulted in significantly higher plasma [cortisol] as compared to non-stressed fish fed control feed (P=0.0233). There was, however, no significant difference in plasma [cortisol] of non-stressed fish fed control feed and non-stressed fish fed TRP-supplemented feed for 7 or 28 days (Fig. 2A).
The effects observed for plasma [ACTH] mirrored those for plasma [cortisol] (Fig. 2B). Exposing the fish to the standardised stressor had a significant effect on plasma [ACTH] (F1,77=30.86, P<0.0001), fish subjected to stress showing elevated plasma [ACTH] as compared to non-stressed fish (Fig. 2B). There was also a significant effect (F3,77=4.77, P=0.0042) of feeding the fish TRP-supplemented feed on plasma [ACTH] but no significant interaction (F3,77=1.18, P=0.3244) between stress and TRP supplementation.
As expected, feeding the fish TRP-supplemented feed resulted in a significant effect on plasma [TRP] (F3,73=16.41, P<0.0001), with fish fed TRP-supplemented feed for 3, 7 and 28 days showing elevated plasma [TRP] (P=0.0015, P<0.0001, P<0.0001, respectively) (Fig. 2C). There was, however, no significant effect of stress on plasma [TRP]. Correlations were found between plasma [TRP] and [ACTH] (r=0.208; P=0.0396) and between [ACTH] and [cortisol] (r=0.348; P=0.0010).
Brain [TRP], [5-HT], [5-HIAA] and [5-HIAA]/[5-HT] ratios
Subjecting the fish to stress had a significant effect on [TRP] only in the
brain stem (F1,79=3.99, P=0.0492), where stress
resulted in elevated [TRP] (Fig.
3D). As expected feeding the fish TRP-supplemented feed had a
significant effect on [TRP] in the telencephalon
(F3,76=6.06, P=0.0009), hypothalamus
(F3,79=7.09, P=0.0003) and optic tectum
(F3,76=3.73, P=0.0146). Fish fed TRP-supplemented
feed for 3 (P=0.0014), 7 (P=0.0008) and 28 days
(P=0.0003) showed elevated hypothalamic [TRP] as compared to fish fed
control feed (Fig. 3B).
Telencephalic [TRP] was elevated in fish fed TRP-supplemented feed for 3
(P=0.0182) and 7 days (P=0.0001), whereas [TRP] in
telencephalon of fish fed TRP-supplemented feed for 28 days did not differ
from telencephalic [TRP] of fish fed control feed
(Fig. 3A). In the optic tectum
the increase in [TRP] of fish fed TRP-supplemented feed appeared most
pronounced at 3 days (Fig. 3C).
There was no significant effect of TRP-supplemented feed on brain stem [TRP]
(F3,79=2.61, P=0.0569), even though brain stem
[TRP] showed a tendency towards an increase after feeding the fish
TRP-supplemented feed for 7 and 28 days
(Fig. 3D). There was no
significant interaction effect between stress and TRP supplementation on [TRP]
in any part of the brain.
|
Stress had a significant effect on [5-HIAA] in the telencephalon (F1,74=22.34, P<0.0001), stressed fish showing elevated [5-HIAA] (Fig. 4A). Stress also tended to elevate hypothalamic [5-HIAA] in a similar way (Fig. 4B), even though this effect did not reach the level of statistical significance (F1,72=3.57, P=0.0688). Feeding the fish TRP-supplemented feed also had a significant effect on [5-HIAA], but only in the hypothalamus (F3,72=3.26, P=0.0264) and optic tectum (F3,70=2.84, P=0.0441) (Fig. 4B,C). Fish receiving TRP-supplemented feed for 7 days displayed a significant elevation of [5-HIAA] in the hypothalamus (P=0.0410) as compared to fish fed TRP-supplemented fed for 28 days (Fig. 4B). In the optic tectum [5-HIAA] was significantly higher in fish fed TRP-supplemented feed for 3 and 7 days (P=0.0045, P=0.0039, respectively) than in fish fed control feed, while fish fed TRP-supplemented feed for 28 days did not show any significant elevation of [TRP] in this brain part (Fig. 4C). In the brain stem neither stress nor TRP supplementation had any effect on [5-HIAA] (Fig. 4D), and there were no interaction effects between stress and TRP supplementation in any brain area.
|
Stress affected [5-HT] only in the brain stem (F1,73=4.90, P=0.0300), stressed fish showing slightly elevated [5-HT] as compared to non-stressed fish (Fig. 5D). In the optic tectum there was a significant (F3,70=3.26, P=0.0264) interaction effect between stress and TRP supplementation on [5-HT] (Fig. 5C), an effect not seen in other brain areas. In the optic tectum, stressed fish fed TRP feed for 3 days showed higher [5-HT] than stressed fish fed control feed (P=0.0108, Fig. 5C).
|
Stress also had a significant effect on telencephalic [5-HIAA]/[5-HT] ratios (F1,73=22.01, P<0.0001), stressed fish showing elevated telencephalic [5-HIAA]/[5-HT] ratios. A similar trend was observed in the hypothalamus (F1,72=2.66, P=0.0540) and brain stem (F1,66=2.69, P=0.0640), even though in these brain areas the effect did not reach the level of statistical significance (Fig. 6). Feeding the fish TRP-supplemented feed had no significant effect on [5-HIAA]/[5-HT] ratios in any brain area, even though [5-HIAA]/[5-HT] showed a tendency towards an increase in the optic tectum of fish fed TRP-supplemented feed (F3,70=2.67, P=0.0544) (Fig. 6C). There was no significant interaction effect between stress and TRP supplementation on [5-HIAA]/[5-HT] ratios in any brain area.
|
Brain [DOPAC], [DA] and [DOPAC]/[DA] ratios
Neither stress nor feeding TRP-supplemented feed had any significant
effects on either [DA] or [DOPAC] in any brain area (data not shown). There
was however an effect of feeding the fish TRP-supplemented feed on
hypothalamic [DOPAC]/[DA] (F3,70=3.42, P=0.0219),
fish fed TRP-supplemented feed for 28 days displaying a [DOPAC]/[DA] ratio of
0.064±0.025 as compared to a ratio of 0.108±0.030 in fish fed
control feed (P=0.0048).
Brain [NE], [MHPG] and [MHPG]/[NE] ratios
There was no effect of either stress or TRP-supplemented feed on [NE] in
any brain area (data not shown) but there was an effect of TRP supplementation
on [MHPG] in the optic tectum (F3,70=3.52,
P=0.0195) and an interaction effect between stress and TRP
supplementation on [MHPG] in the optic tectum (F3,70=3.27,
P=0.0262) and brain stem (F3,67=3.00,
P=0.0365) (Fig. 7).
Optic tectum [MHPG] was elevated in both non-stressed and stressed fish fed
TRP-supplemented feed for 7 days, as compared to levels in fish fed control
feed (P=0.0006) or TRP-supplemented feed for 3 and 28 days
(P=0.0010, P=0.0003, respectively)
(Fig. 7A). In the brain stem
[MHPG] was elevated in stressed fish fed TRP-supplemented feed for 3 days as
compared to stressed fish fed control feed (P=0.0389)
(Fig. 7B). Neither stress nor
feeding TRP-supplemented feed had any significant effects on [MHPG]/[NE] in
any brain area (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lepage et al. (2002)
similarly reported that feeding rainbow trout feed supplemented with three
different levels of TRP, corresponding to two, four and eight times the TRP
content of commercial trout feed, for 7 days resulted in a dose-dependent
suppression of the stress-induced elevation of plasma [cortisol], along with a
dose-dependent elevation of basal plasma [cortisol]. The elevation of basal
plasma [cortisol] could suggest that the suppression of the stress-induced
elevation of plasma [cortisol] observed in fish fed TRP-supplemented feed for
7 days is an effect of elevated negative feedback by cortisol. However, in the
present study, feeding the fish TRP-supplemented feed resulted in elevated
basal plasma [cortisol] only after 3 days, whereas the effect on post-stress
plasma [cortisol] were observed first after feeding the fish this feed for 7
days, at a time when basal plasma [cortisol] were not elevated. This
observation argues against the suggestion that reduced post-stress plasma
[cortisol] in fish fed TRP-supplemented feed is an effect of elevated negative
feedback of cortisol.
In the present study, plasma levels of ACTH correlated with plasma cortisol levels and the effects of stress and elevated dietary intake of TRP on plasma [ACTH] closely mirror the effects on plasma [cortisol]. This suggests that the effect of elevated dietary intake of TRP on plasma [cortisol] is mediated through effects on the HPI axis, at a level upstream of the interrenal tissue.
Treatments elevating plasma [TRP] and/or plasma [TRP]/[LNAA] ratios have
also been reported to counteract stress-induced elevations of plasma
[cortisol] in mammals, including humans
(Morméde and Dantzer,
1979; Markus et al.,
1998
,
1999
,
2000a
,b
),
an effect which is believed to be mediated by brain 5-HT. In the present
study, both plasma and brain [TRP] were elevated in fish fed TRP-supplemented
feed. Fish fed this feed also showed elevated [5-HIAA] in the hypothalamus and
optic tectum, but there were no significant effects of TRP on [5-HT] or
[5-HIAA]/[5-HT] ratios in any of the brain areas analysed. Lepage et al.
(2002
) reported that feeding
the fish TRP-supplemented feed for 7 days resulted elevated brain and plasma
[TRP] along with a suppression of post-stress plasma [cortisol], but only
small and not quite significant effects on brain [5-HIAA] and [5-HIAA]/[5-HT]
ratios. In the study by Winberg et al.
(2001
) the lowest level of
dietary supplementation of TRP (8.38 mg TRP g-1 dry feed, about
four times higher than the dose in the present study) also had modest effects
on brain [5-HIAA] and [5-HIAA]/[5-HT] ratios in fish fed this feed for 7 days,
but pronounced effects on aggressive behaviour. Still, it could not be
excluded that the effects of TRP-supplemented feed on HPI axis reactivity and
aggression in rainbow trout are mediated by the brain 5-HT system. However,
the time course of the effect of TRP on aggression and HPI axis reactivity
suggests that mechanisms other than a direct effect on 5-HT synthesis and
release are involved.
The effect of elevated dietary TRP intake on 5-HT synthesis and release
could be expected to be very rapid, but in the present study the effects of
TRP on post-stress plasma [cortisol] were manifested first after feeding the
fish TRP-supplemented feed for 7 days. Similarly, in the study by Winberg et
al. (2001), the effects of
elevated dietary TRP on aggression in rainbow trout were observed after
feeding the fish TRP-supplemented feed for 7 days, but not after feeding the
fish this feed for 3 days. Interestingly, the time course of the
anti-depressive effects of specific 5-HT re-uptake inhibitors (SSRI), such as
fluoxetine (Prozac), is strikingly similar, the anti-depressive effects of
these drugs occurring only after long-term treatment
(Mongeau et al., 1997
).
Moreover, the effects of SSRI and TRP on stress responses and aggression
appear to be similar. Larson and Summers
(2001
) showed that a 1-week
treatment with the SSRI, setralin, reduces aggressive behaviour and reverses
dominant social status in the lizard Anolis carolinensis. As seen
with elevated dietary intake of TRP, short-term treatment with SSRI activates
the HPA axis, whereas long-term treatment has the opposite effect,
desensitising the HPA axis in rats (Jensen
et al., 1999
). An effect on the densities and transduction
mechanisms of post- and/or pre-synaptic 5-HT receptors, resulting in a delayed
elevation of 5-HT post-synaptic effects in certain brain regions, has been
suggested as a mechanism involved in mediating the effects of long-term SSRI
treatment (Mongeau et al.,
1997
; Nutt et al.,
1999
).
Central 5-HT interacts with the brain NE system, and one possible mechanism
through which 5-HT could suppress HPI axis activity is by inhibiting central
NE activity (Aston-Jones et al.,
1991; Engberg,
1992
). However, fish fed TRP-supplemented feed for 7 days showed
significantly higher [MHPG] in the optic tectum than fish fed control feed or
TRP-supplemented feed for 3 or 28 days. If anything, this would argue against
the hypothesis that the effects of elevated dietary intake of TRP on stress
responsiveness are a result of a 5-HT-mediated inhibition of brain NE
activity.
Elevated dietary intake of TRP may also have a more direct effect on the
synthesis and release of DA and NE since the amino acid precursor of DA and NE
is tyrosine, a large neutral amino acid (LNAA), which enters the brain
via the same LNAA transport carrier as TRP
(Wurtman et al., 1974;
Fernstrom, 1983
;
Boadle-Biber, 1993
). Thus, a
rise in blood levels of TRP may competitively inhibit tyrosine uptake into the
brain (Wurtman et al., 1974
).
However, Lepage et al. (2002
)
showed an increase in brain [TRP] but no concomitant decrease in brain levels
of other LNAAs in rainbow trout fed TRP-supplemented feed for 7 days. In the
present study, feeding the TRP-supplemented feed had an effect on hypothalamic
[DOPAC]/[DA], but only after 28 days, when fish fed TRP-supplemented feed
showed lowered hypothalamic [DOPAC]/[DA] ratios.
Since 5-HT is the precursor of melatonin, elevated dietary intake of TRP
may also increase plasma levels of melatonin, and elevated plasma [melatonin]
following TRP treatment have been reported in humans
(Hajak et al., 1991), rats
(Yaga et al., 1993
) and
chickens (Heuther et al., 1992). Melatonin is a hormone best known for its
role in synchronising circadian rhythms, but which has also been reported to
affect aggressive behaviour and post-stress plasma [cortisol]. For instance,
Munro (1986
) showed that
intracranial injections of melatonin suppressed aggressive responsiveness in
the cichlid Aequidens pulcher, and in mammals, melatonin has been
reported to exert a glucocorticoid antisecretagogue effect
(Xu et al., 1995
;
Rao et al., 2001
).
In the present study the effect of dietary TRP on the stress-induced
elevation of plasma [cortisol] was not observed after feeding the fish
TRP-supplemented fed for 28 days, suggesting that long-term dietary TRP may
activate compensatory mechanisms, normalizing brain [TRP] and cortisol
release. Notably, following 28 days of dietary supplementation of TRP, [TRP]
in the telencephalon and optic tectum no longer differed from that of fish fed
control feed. In mammals, the TRP catabolising enzyme
indoleamine-2,3-deoxygenase, which is present in the brain, is induced by TRP
(Gal, 1974). In the present
study, there was also a tendency towards decreased plasma [TRP] in undisturbed
fish fed TRP-supplemented feed for 28 days as compared to undisturbed fish fed
this feed for 3 and 7 days. A decline in plasma [TRP] following prolonged
dietary supplementation of TRP could be related to an activation of the enzyme
tryptophan pyrrolase in the liver
(Chaouloff, 1993
). Tryptophan
pyrrolase is another TRP catabolising enzyme, which in the rat liver is
regulated by the circulating concentration of its substrate
(Feigelson and Greegard,
1962
). However, Brown and Dodgen
(1968
) found that
administration of repeated doses of TRP into channel catfish failed to induce
the liver enzyme, and Walton et al.
(1984
) did not find any
relationship between plasma TRP levels and the activity of hepatic TRP
pyrrolase in rainbow trout.
In conclusion, the results from the present study confirm that supplemental
dietary L-tryptophan has an effect on stress responses in rainbow
trout. Fish fed TRP-supplemented feed for 7 days show reduced post-stress
plasma [cortisol], whereas feeding the fish this feed for 3 or 28 days has no
effect on post-stress plasma [cortisol]. Thus, the time courses of the effects
of elevated dietary TRP on aggressive behaviour
(Winberg et al., 2001) and
post-stress plasma [cortisol] are similar. Both these effects of dietary TRP
could be mediated by the brain 5-HT system, and, if so, probably through
effects on 5-HT receptor densities and receptor mechanisms resulting in a
delayed elevation of 5-HT post-synaptic effects. Elevated, dietary intake of
TRP does not appear to have any direct effects on the synthesis and release of
DA, and the results of the present study do not support the hypothesis that
the suppressive effect of dietary TRP on HPI axis reactivity is mediated by
5-HT inhibition of brain NE activity. However, possible effects of TRP on
circulating melatonin levels cannot be excluded as the mechanism of
action.
![]() |
List of abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, C. F., Liley, N. R. and Gorzalka, B. B. (1996). PCPA increases aggression in male firemouth cichlids. Pharmacol. 53,328 -330.[Medline]
Aldegunde, M., Garcia, J., Soengas, J. L. and Rozas, G. (1998). Uptake of tryptophan into brain of rainbow trout (Oncorhyncus mykiss). J. Exp. Zool. 282,285 -289.[CrossRef]
Aldegunde, M., Soengas, J. L. and Rozas, G. (2000). Acute effects of L-tryptophan on tryptophan hydroxylation rate in brain regions (hypothalamus and medulla) of rainbow trout (Oncorhynchus mykiss). J. Exp. Zool. 286,131 -135.[CrossRef][Medline]
Aston-Jones, G., Akaoka, H., Charletey, P. and Chouvet, G. (1991). Serotonin selectively attenuated glutamate-evoked activation of noradrenergic locus coerulus neurones. J. Neurosci. 11,760 -769.[Abstract]
Balm, P. H. M. and Pottinger, T. G. (1993). Acclimation of rainbow trout (Oncorhynchus mykiss) to low environmental pH does not involve an activation of the pituitary - interrenal axis but evokes adjustments in branchial ultrastructure. Can. J. Fish. Aquat. Sci. 50,2532 -2541.
Blanchard, D. C., Cholvanich, P., Blanchard, R. J., Clow, D. W., Mammer, R. P., Rowlet, J. K. and Bardo, M. T. (1991). Serotonin, but not dopamine, metabolites are increased in selected brain regions of subordinate male rats in a colony environment. Brain Res. 568,61 -66.[CrossRef][Medline]
Blanchard, D. C., Sakai, R. R., McEwen, B., Weiss, S. M. and Blanchard, R. J. (1993). Subordination stress: behavioral, brain and neuroendocrine correlates. Behav. Brain Res. 58,113 -121.[CrossRef][Medline]
Boadle-Biber, M. C. (1993). Regulation of serotonin synthesis. Prog. Biophys. Mol. Biol. 60, 1-15.[CrossRef][Medline]
Brown, J. N. and Dodgen, C. L. (1968). Fish liver tryptophan pyrrolase: The apparent absence of both hormonal regulation and substrate induction. Biochim. Biophys. Acta 165,463 -469.
Chaouloff, F. (1993). Physiopharmacological interactions between stress hormones and central serotonergic systems. Brain Res. Rev. 18,1 -32.[Medline]
Deckel, A. W. (1996). Behavioural changes in Anolis carolinensis following injection with fluoxetine. Behav. Brain Res. 78,175 -182.[CrossRef][Medline]
Deckel, A. W. and Jevitts, E. (1997). Left vs. right-hemisphere regulation of aggressive behaviours in Anolis carolinensis: Effects of eye-patching and fluoxetine administration. J. Exp. Zool. 278, 9-21.[CrossRef]
Dinan, T. G. (1996). Serotonin: Current understanding and the way forward. Int. Clin. Psychopharmacol. 11,19 -21.
Edwards, D. H. and Kravitz, E. A. (1997). Serotonin, social status and aggression. Curr. Opin. Neurobiol. 7,812 -819.[CrossRef][Medline]
Engberg, G. (1992). Citalopram and 8-OH-DPAT attenuate nicotine-induced excitation of central noradrenaline neurons. J. Neur. Transm. 89,149 -154.
Feigelson, P. and Greengard, O. (1962).
Immunochemical evidence for increased titers of liver tryptophan pyrrolase
during substrate and hormonal enzyme induction. J. Biol.
Chem. 237,3714
-3717.
Fernstrom, J. D. (1983). Role of precursor availability in control of monoamine biosynthesis in brain. Physiol. Rev. 60,484 -546.
Fernstom, J. D. and Wurtman, R. J. (1972). Brain serotonin content: regulation by plasma neutral amino acids. Science 178,414 -416.[Medline]
Gal, E. M. (1974). Cerebral tryptophan-2,3-deoxygenase (pyrrolase) and its induction in rat brain. J. Neurochem. 22,861 -863.[Medline]
Hajak, G., Huether, G., Blanke, J., Blomer, M., Freyer, C., Poeggeler, B., Reimer, A., Rodenbeck, A., Schulzvarszegi, M. and Ruther, E. (1991). The influence of intravenous L-tryptophan on plasma melatonin and sleep in men. Pharmacopsych. 24,17 -21.
Huether, G., Poeggeler, B., Reimer, A. and George, A. (1992). Effect of tryptophan administration on circulating melatonin levels in chicks and rats - evidence for stimulation of melatonin synthesis and release in the gastrointestinal tract. Life Sci. 51,945 -953.[CrossRef][Medline]
Höglund, E., Balm, P. H. M. and Winberg, S.
(2000). Skin darkening, a potential social signal in subordinate
Arctic charr (Salvelinus alpinus): the regulatory role of brain
monoamines and pro-opiomelanocortin derived peptides. J. Exp.
Biol. 203,1711
-1721.
Höglund, E., Balm, P. H. M. and Winberg, S. (2002). Stimulatory effects of 5-HT1A receptors on adrenocorticotrophic hormone and cortisol secretion in a teleost fish, the Artic charr (Salvelinus alpinus). Neurosci. Lett. 324,193 -196.[CrossRef][Medline]
Höglund, E., Kolm, N. and Winberg, S. (2001). Stress-induced effects on brain serotonergic activity, plasma cortisol and aggressive behaviour in Arctic charr (Salvelinus alpinus) is counteracted by L-dopa. Physiol. Behav. 74,381 -389.[CrossRef][Medline]
Jensen, J. B., Jessop, D. S., Harbuz, M. S., Mørk, A., Sanchez, C. and Mikkelsen, J. D. (1999). Acute and long-term treatments with the selective serotonin reuptake inhibitor citalopram modulate the HPA axis activity at different levels in male rats. J. Neuroendocrinol. 11,465 -471.[CrossRef][Medline]
Johnston, W. L., Atkinson, J. L., Hilton, J. W. and Were, K. E. (1990). Effect of dietary tryptophan on plasma and brain tryptophan, brain serotonin, and brain 5-hydroxyindoleacetic acid in rainbow trout. J. Nutr. Biochem. 1, 49-54.[CrossRef]
Larson, E. and Summers, C. H. (2001). Serotonin reverses dominant social status. Behav. Brain Res. 121,95 -102.[CrossRef][Medline]
Lepage, O., Tottmar, O. and Winberg, S. (2002).
Elevated dietary L-tryptophan counteracts the stress-induced
elevation of plasma cortisol in rainbow trout (Oncorhynchus mykiss).
J. Exp. Biol. 205,3679
-3687.
Markus, C. R., Olivier, B., Panhuysen, G., Van der Gugten, J.,
Alles, M. S., Tuiten, A., Westnberg, G. M., Fekkes, D., Koppeschaar, H. F. and
de Haan, E. E. H. F. (2000a). The bovine protein
-lactalbumin increases the plasma ratio of tryptophan to the other
large neutral amino acids, and in vulnerable subjects raises brain serotonin
activity, reduces cortisol concentration, and improves mood under stress.
Am. J. Clin. Nutr. 71,1536
-1544.
Markus, C. R., Panhuysen, G., Jonkman, L. M. and Bachman, M. (1999). Carbohydrate intake improves cognitive performance of stress-prone individuals under laboratory stress. Br. J. Nutr. 82,457 -467.[Medline]
Markus, C. R., Pannhuysen, G., Tuiten, A. and Koppeschaar, H. (2000b). Effets of food on cortisol and mood in vulnerable subjects under controllable and uncontrollable stress. Physiol. Behav. 70,333 -342.[CrossRef][Medline]
Markus, C. R., Pannhuysen, G., Tuiten, A., Koppeschaar, H., Fekkes, D. and Peters, M. (1998). Does carbohydrate-rich, protein-poor food prevent a deterioration of mood and cognitive performance of stress-prone subjects when subjected to a stressful task? Appetite 31,49 -65.[CrossRef][Medline]
Mongeau, R., Blier, P. and de Montigny, C. (1997). The serotonergic and noradrenergic systems of the hippocampus: their interactions and the effects of antidepressant treatments. Brain Res. Rev. 23,145 -195.[Medline]
Morméde, P. and Dantzer, R. (1979). Effects of lithium on aggressive behavior in domestic pigs. J. Vet. Pharmacol. Ther. 2,299 -304.
Munro, A. D. (1986). Effects of melatonin, serotonin, and naloxene on aggression in isolated cichlid fish (Aequidens pulcher). J. Pineal Res. 3, 257-262.[Medline]
Nutt, D., Forshall, S., Bell, C., Rich, A., Sandford, J., Nash, J. and Argyopoulos, S. (1999). Mechanisms of action of selective serotonin reuptake inhibitors in the treatment of psychiatric disorders. Eur. Neuropsychopharmacol. 3, S81-S86.[CrossRef]
Olsen, Y. A., Falk, K. and Reite, O. B. (1992). Cortisol and lactate levels of Atlantic salmon Salmo salar developing infectious anemia (ISA). Dis. Aquat. Org. 14, 99-104.
Øverli, Ø., Harris, C. A. and Winberg, S. (1999). Short term effects of fights for social dominance and the establishment of dominant-subordinate relationships on brain monoamines and cortisol in rainbow trout. Brain. Behav. Evol. 54,263 -275.[CrossRef][Medline]
Raleigh, M. J., McGuire, M. T., Brammer, G. L., Pollack, D. B. and Yuwiler, A. (1991). Serotonergic mechanisms promote dominance acquisition in adult male vervet monkeys. Brain Res. 559,181 -190.[CrossRef][Medline]
Rao, N. V. A., Raza, B., Prasad, J. K., Razi, S. S., Gottardo, L., Ahmad, M. F. and Nussdorfer, G. G. (2001). Melatonin decreases glucocorticoid blood concentration in the rat and palm squirrel, acting directly on the adrenal gland. Biomed. Res. (Tokyo) 22,115 -117.
Walton, M. J., Colosso, R. M., Cowey, C. B., Adron, J. W. and Knox, D. (1984). The effects of dietary tryptophan levels on growth and metabolism of rainbow trout (Salmo gairdneri). Br. J. Nutr. 51,279 -287.[Medline]
Weyts, F. A. A., Cohen, N., Flik, G. and Verburg-Van Kemenade, B. M. L. (1999). Interactions between the immune system and the hypothalamo-pituitary-interrenal axis in fish. Fish Shellfish Immun. 9,1 -20.[CrossRef]
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. 43,645 -654.
Winberg, S. and Nilsson, G. E. (1992). Induction of social dominace by L-dopa treatment in Artic charr. Neurorep. 3,243 -246.
Winberg, S. and Nilsson, G. E. (1993). Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish. Comp. Biochem. Physiol. 106,597 -614.
Winberg, S., Nilsson, A., Hylland, P., Söderstöm, V. and Nilsson, G. E. (1997). Serotonin as a regulator of hypothalamic-pituitary-interrenal activity in teleost fish. Neurosci. Lett. 230,113 -116.[CrossRef][Medline]
Winberg, S., Nilsson, G. E. and Olsén, K. H. (1992). Social ranks and brain levels of monoamines and monoamine metabolites in Artic charr, Salnenius alpinus. J. Comp. Physiol. 168,241 -246.
Winberg, S., Øverli, Ø. and Lepage, O. (2001). Suppression of aggression in rainbow trout (Oncorhyncus mykiss) by dietary L-tryptophan. J. Exp. Biol. 204,3867 -3886.[Medline]
Wurtman, R. J., Larin, F., Mostafapour, S. and Fernstrom, J. D. (1974). Brain catechol synthesis: control by brain tyrosine concentration. Science 185,183 -184.[Medline]
Xu, F., Li, J. C., Ma, K. C. and Wang, M. (1995). Effects of melatonin on hypothalamic gamma-aminobutyric acid, aspartic acid, glutamic acid, beta-endorphin and serotonin levels in mice. Biol. Signals 4,225 -231.[Medline]
Yaga, K., Reiter, R. J. and Richardson, B. A. (1993). Tryptophan loading increases daytime serum melatonin levels in intact and pinealectomized rats. Life Sci. 52,1231 -1238.[CrossRef][Medline]