The effects of cortisol administration on social status and brain monoaminergic activity in rainbow trout Oncorhynchus mykiss
1 Ottawa-Carleton Institute of Biology, Ottawa, ON, K1N 6N5
Canada
2 Department of Psychology, Carleton University, Ottawa, ON, K1S 5B6
Canada
Author for correspondence (e-mail:
Katie.Gilmour{at}science.uottawa.ca)
Accepted 17 May 2005
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Summary |
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Key words: rainbow trout, Oncorhynchus mykiss, cortisol, social status, monoamines, serotonin, dopamine, RU486
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Introduction |
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A number of social (e.g. prior residence, prior winning/losing experience)
and inherent (e.g. size, aggressiveness) factors are known to influence the
outcome of social interactions in salmonids
(Huntingford and Turner, 1987;
Fernandes and Volpato, 1993
;
Metcalfe, 1998
;
Rhodes and Quinn, 1998
).
Recent work has also raised the possibility that physiological factors, more
specifically the physiological condition of a fish, can impact on an
individual's initial success during competitive interactions and affect its
ultimate social status (e.g. Johnsson and
Björnsson, 1994
;
Björnsson, 1997
;
Sloman et al., 2001
). For
example, Sloman et al. (2001
)
reported that plasma cortisol concentrations were significantly higher prior
to pairing in size-matched rainbow trout that subsequently become subordinate,
suggesting that individuals with high plasma cortisol levels are predisposed
to become subordinate. In another study, experimental elevation of plasma
cortisol concentrations reduced appetite, growth rate and condition in rainbow
trout, and fin damage was greater in cortisol-treated trout held in mixed
groups with untreated controls (Gregory
and Wood, 1999
). These data suggest that elevated cortisol
concentrations might in fact be symptomatic of an individual fish's poor
condition, placing it at a physiological and/or competitive disadvantage
(Gregory and Wood, 1999
).
Thus, the main objective of the present study was to test the hypothesis that
circulating cortisol concentrations affect the outcome of social interactions
within pairs of rainbow trout. In particular, high plasma cortisol levels were
predicted to predispose a fish to low social status. In accordance with the
work of Gregory and Wood
(1999
), two possible
mechanisms through which cortisol might influence social status can be
envisaged. Elevated cortisol levels, by reducing physiological condition
(Barton et al., 1987
;
Barton and Iwama, 1991
;
Gregory and Wood, 1999
), could
impact on competitive ability directly. Alternatively, interactions between
cortisol and brain monoaminergic activity could affect competitive ability
indirectly by modulating behaviour.
Many of the behavioural consequences of social status are thought to be the
outcome of changes in brain monoaminergic activity that accompany victory or
loss in competitive interactions (reviewed by
Winberg and Nilsson, 1993).
For example, subordinate fish generally exhibit significantly higher turnover
of serotonin (5-HT), reflected by 5-hydroxyindolacetic acid (5-HIAA)
accumulation and elevated 5-HIAA/5-HT ratios within the telencephalon,
hypothalamus, and brain stem relative to dominant individuals (Winberg et al.,
1991
,
1992
,
1993
,
1997b
). These social
stress-induced increases of brain 5-HT activity are likely, at least in part,
to be responsible for the marked behavioural inhibition commonly observed in
subordinate fish; namely decreases in feeding, aggression, and spontaneous
locomotor activity (Winberg and Nilsson,
1993
; Winberg et al.,
1997a
; Øverli et al.,
1998
). In tetrapod vertebrates, experimentally elevated
serotonergic activity causes a reversal of dominance relationships in a number
of model systems (e.g. Sanchez and Hyttel,
1994
; Villalba et al.,
1997
; Larson and Summers,
2001
), suggesting that high brain 5-HT levels in these organisms
have the capacity to act as antecedents for subordinance. Winberg et al.
(1992
) reported that the
relationship between brain 5-HT turnover rate and social rank in fish
developed through social interactions and was not caused by intrinsic
differences in brain 5-HT activity. However, it is conceivable that high
circulating cortisol levels could influence the outcome of social interactions
by affecting central monoaminergic activity, specifically increasing
serotonergic activity and/or decreasing dopaminergic activity. These changes,
in turn, could alter behaviour (reducing aggression, locomotion, etc.) in such
a way as to reduce competitive ability, resulting in low social status. Thus,
in the present study the hypothesis that circulating cortisol levels influence
brain monoaminergic activity was also tested as one potential mechanism
underlying any observed relationship between cortisol treatment and the
outcome of social interactions.
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Materials and methods |
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Experiment 1: The effects of cortisol treatment on the outcome of social interactions
Fish were lightly (i.e. to the point of losing equilibrium) anaesthetized
in a solution of benzocaine (0.05 g l1
ethyl-p-aminobenzoate) and initial masses and fork lengths were
measured (mass 76.8±2.4 g; fork length 186.1±2.1 mm,
N=98). Abbott and Dill
(1985) reported that an initial
length difference of as little as 5% was sufficient to ensure dominant status
to the larger individual within pairs of rainbow trout. Therefore, two
separate series of experiments were carried out. In the first, fish were
paired with a conspecific that was size-mismatched by 520% on the basis
of fork length. Each pair of fish was then randomly assigned to either a
control group (14 pairs) or a cortisol treatment group (11 pairs), with the
larger fish within the pair in each case receiving the treatment. In the
second experimental series, fish were size-matched by fork length (<1.5%
difference), and each pair of fish was randomly assigned to either a cortisol
treatment group (14 pairs), or a cortisol plus RU486 treatment group (10
pairs), with one fish within the pair receiving the treatment. Because the
objective of these experiments was to investigate the effect of elevated
cortisol concentrations on the outcome of social interactions, sham treatment
groups (treatment with coconut oil or cocoa butter alone) were not included in
the experimental design. Both scrutiny of the literature, and pilot trials
using sham-treated fish, revealed that fish respond unpredictably to sham
treatment, with cortisol concentrations being elevated in some fish but not
others. Such unpredictability would cloud comparisons between fish expected on
the basis of their treatment group to have low cortisol levels, and those
expected to have high cortisol levels, and therefore sham treatments were not
employed.
Cortisol-treated fish received a coconut oil pellet (0.005 ml coconut oil
g1 fish) containing dissolved cortisol (110 mg
hydrocortisone 21-hemisuccinate kg1 fish) in the
intraperitoneal cavity. The coconut oil was injected as a liquid but
solidified rapidly within the fish and acted as a solid implant for the
remainder of the experiment. Previous work has demonstrated that a coconut oil
vehicle allows the delivery of a prolonged, slow-release dose of cortisol
within each injected fish (reviewed by
Gamperl et al., 1994); the
cortisol dose used was selected on the basis of pilot trials that indicated
that this dose provided a physiologically relevant elevation of plasma
cortisol values over the desired experimental period. Cortisol plus RU486
treatment was achieved by implanting a cocoa butter pellet (0.01 ml cocoa
butter g1 fish) containing a combination of dissolved
cortisol (110 mg hydrocortisone 21-hemisuccinate kg1 fish)
and the glucocorticoid receptor antagonist RU486 (1100 mg mifepristone
kg1 fish; Sigma, Oakville, ON, Canada). The concentration of
RU486 was chosen on the basis of previous work indicating that treatment with
this compound is most effective at a dose tenfold greater than that of
cortisol (Vijayan et al.,
1994
). Control fish were untreated.
Following preparation, pairs of trout were placed in 40 l flow-through Plexiglas observation tanks. The fish were separated by an opaque perforated divider for a 48 h recovery and acclimation period, and the dividers were then removed to allow pairs of fish to interact; a small piece of PVC tubing was placed within each tank to provide shelter. Behavioural observations were carried out on all paired fish twice a day for 5 days, and the fish were then terminally sampled. During the experiment, fish were hand-fed to satiation with commercial trout food pellets once a day, after all observations had been carried out. Behaviour observations were first conducted 15 min after the opaque divider was removed, and then for 10 min each, once between 9:00 h and 11:30 h and once between 15:00 h and 17:30 h. The order of tank observation was randomized to account for any observational bias.
Social status was determined by assigning points to each fish based on its
food acquisition, position, aggressive behaviour and fin damage; high scores
in each case were indicative of dominant behaviour or characteristics. This
method has been used previously for assigning social status among salmonids
(Johnsson et al., 1996; Sloman
et al.,
2000a
,b
,
2001
). In brief, to score fish
on food acquisition, one pellet of food was dropped into the tank at the
beginning of each observation period and the first fish to take the pellet was
given a score of one, while the other fish scored zero points. Fish that
maintained their position within the water column scored ten points, whereas
fish that rested on the bottom of the tank or hid within the PVC tubing scored
five points, and fish that attempted to swim at the surface (a behaviour
indicative of subordinance; Sloman et al.,
2000a
) scored zero points. Fish directing five or more aggressive
attacks towards the other individual within an observation period were given a
score of two, fish performing between one and four aggressive attacks were
given a score of one, and those individuals performing no aggressive attacks
received a score of zero. Finally, fish were scored according to the extent of
dorsal and caudal fin damage sustained during the 5 day interaction period.
The mean dorsal and caudal fin damage scores were calculated and then combined
into a total fin score. Previous work demonstrated that the severity of fin
damage is likely to reflect the social rank of the individual
(Abbott and Dill, 1985
;
Moutou et al., 1998
).
Therefore, fish having no fin damage were given a score of three, minor damage
(<30% of the fin missing) a score of two, severe damage (3070% of
the fin missing) a score of one, and very severe damage (>70% of the fin
missing) a score of zero. A single behaviour score was calculated from all
observations by means of a principal components analysis (PCA; SPSS 10.1)
(Sloman et al., 2000c
). The
fish with the higher overall behaviour score within each pair was assigned
dominant social status, whereas that with the lower score was classified as
subordinate.
Fish were rapidly killed by immersion in a lethal dose of anaesthetic
solution (ethyl-p-aminobenzoate, 0.5 g l1). Pairs
were removed simultaneously from their tanks and sampled within 1 min of each
other; the sampling order within each pair was randomized to control for any
sampling bias. Final masses and fork lengths were measured and a blood sample
(1 ml) was removed by caudal puncture. Following centrifugation (13 200
g for 3 min), plasma was removed, immediately frozen in liquid
nitrogen, and subsequently stored at 80°C until analysis. Plasma
cortisol concentrations were measured using a commercially available
radioimmunoassay kit (ICN pharmaceuticals). The condition factor (CF) of each
fish was calculated as CF=100Mb/Lx,
where Mb=mass of fish in grams, L=length of fish
in cm, and x=slope of regression line for all fish of
logMb vs logL (
3). The specific
growth rate (SGR) of each fish was calculated as
SGR=[ln(MbFinal)ln(MbInitial)]
x100/D, where D=number of days elapsed.
Experiment 2: The effect of cortisol administration on brain monoamine levels
Fish were lightly anaesthetized in a solution of benzocaine (0.05 g
l1 ethyl-p-aminobenzoate), initial masses were
measured (mass 92.5±2.3 g, N=34) and fish were randomly placed
within groups of twelve in 780 liter holding tanks. Following a 5 day
acclimation period, fish were again lightly anaesthetized and injected
intraperitoneally with a cocoa butter pellet (0.005 ml coconut oil
g1 fish) containing dissolved cortisol (50 mg hydrocortisone
21-hemisuccinate kg1 fish; N=10) or a combination
of dissolved cortisol (50 mg hydrocortisone 21-hemisuccinate
kg1 fish) and RU486 (500 mg mifepristone
kg1 fish; N=12); an additional group of untreated
fish served as controls (N=12). The group and tank sizes were chosen
to avoid the formation of dominance hierarchies, as hierarchy formation in
this experiment would have a confounding effect on brain monoaminergic
activities. In addition, fish were fed by scattering food on the water
surface. Examination of the control group suggested that hierarchies did not
form, in that the group exhibited positive growth and plasma cortisol
concentrations in all cases were typical of unstressed fish (<10 ng
ml1).
Fish were sampled 5 days after receiving treatment in groups of four, to
minimize disturbance of the fish remaining within each tank. Fish were killed
by immersion in a lethal dose of anaesthetic (ethyl-p-aminobenzoate,
0.5 g l1), mass was measured and the brain was rapidly
removed. Two discrete brain regions were dissected out (on ice) for analysis,
the telencephalon (excluding the olfactory bulbs), and the hypothalamus
(excluding the pituitary gland). These brain areas were selected on the basis
of earlier studies showing that monoamine activity within these regions was
particularly influenced by social stress (Winberg et al.,
1991,
1992
,
1997b
). Brain samples were
frozen in liquid nitrogen and stored at 80°C. Blood samples (
1
ml) were removed via caudal puncture, and separated plasma was frozen
in liquid nitrogen and stored at 80°C until analysis for plasma
cortisol concentration using a commercial RIA kit (ICN pharmaceuticals). All
blood and tissue samples were collected between 11:30 h and 14:30 h to control
for any diurnal variations in either plasma cortisol and/or brain monoamine
concentrations.
Frozen brain samples were sonicated in a homogenizing solution comprising 0.1 mmol l1 Na2EDTA, 0.3 mol l1 ClCHCOOH, 10% methanol, and 12.5 pg µl1 DHBA (the internal standard). Brain monoamines were then quantified by high performance liquid chromatography (HPLC) using electrochemical detection. The HPLC consisted of a solvent-delivery system (Waters590/WaterPump, Mississuaga, ON, Canada), an autoinjector (Waters712WISP), a reverse-phase column (8 mm x100 mm, Waters, NovaPak, 4 µm) kept at 30°C and a 5100A Coulochem detector (ESA, Bedford, MA, USA) with two electrodes at oxidizing potentials of 330 mV and +350 mV. The mobile phase consisted of 1.3 g l1 heptanesulphonic acid sodium salt, 0.1 g l1 disodium ethylene tetracycline, and 7.3 ml triethyloamine adjusted to pH 2.45 with orthophosphoric acid. Sample monoamine levels were indexed to standard solutions of known concentration, corrected for recovery of the internal standard, and expressed relative to total tissue protein content.
The monoamines measured were 5-hydroxytryptamine (5-HT) and dopamine (DA),
as well as their major metabolites, 5-hydroxyindolacetic acid (5-HIAA) and
3,4-dihydroxyphenylacetic acid (DOPAC), respectively. The ratio of
[metabolite]/[parent monoamine] was used as an index of brain monoaminergic
activity. This index reduces variance related to tissue sampling and provides
a more direct measure of brain monoaminergic activity than do levels of
monoamine metabolites on their own
(Shannon et al., 1986).
Statistical analysis
All data are presented as means ± 1 standard error of the mean
(S.E.M.). 2 analysis was used
to evaluate the effects of treatment group on social status and behaviour
scores for all pairs of fish. Behaviour scores in Experiment 1 were compared
between treatment groups for fish in the same category (dominant treated,
subordinate treated, dominant untreated or subordinate untreated). Plasma
cortisol concentrations were analysed by two-way analysis of variance (ANOVA)
followed by Bonferroni-corrected t-tests, or by Student's
t-tests, as appropriate. A two-way ANOVA followed by
Bonferroni-corrected t-tests, as appropriate, was carried out on
specific growth rate and final condition factor in Experiment 1, using social
status and treatment group as factors. The statistical significance of
differences in mean brain monoamine concentrations in Experiment 2 were
assessed using one-way ANOVA on ranks followed by Dunn's post hoc
pairwise multiple comparisons test, as appropriate. Non-normally distributed
plasma cortisol concentrations were log transformed as needed. The level for
significance for all tests was set at P=0.05 and all statistical
analyses were performed using SigmaStat v3.0 (SPSS, Inc) or SPSS v10.1 (SPSS,
Inc) software.
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Results |
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An experimental protocol in which cortisol-treated or cortisol+RU486
treated individuals were paired with a conspecific that was <1.5% different
in fork length revealed significant differences in behaviour as a result of
treatment. 2 analysis indicated that cortisol-treated fish
became subordinate more often than expected by chance alone
(Table 1). This effect of
cortisol was eliminated by simultaneous treatment with RU486; untreated and
cortisol+RU486-treated fish were equally likely to be relegated to subordinate
social status (Table 1). In an
attempt to elucidate the importance of circulating cortisol concentrations
relative to a factor that is known to affect the outcome of social
interactions in rainbow trout (i.e. body size;
Abbott and Dill, 1985
), an
experiment was carried out in which untreated or cortisol-treated individuals
were paired with a conspecific that was at least 5% (range 4.617.4%)
smaller in fork length. The larger fish became dominant in 86% of
size-mismatched pairs of trout in which both fish were untreated
(N=14 pairs), a result that was confirmed to be significantly
different than that expected by chance alone via
2
analysis (Table 2). This size
effect was eliminated by cortisol treatment (N=10 pairs), in which
only 40% of larger (treated) fish became dominant.
2 analysis
indicated that cortisol treatment (
2=0.8, d.f.=1,
P>0.25) decreased the probability, to the point where it was not
significantly different than that expected by chance alone, of larger fish
within each pair becoming dominant (Table
2).
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Condition factor and specific growth rate
Prior to the onset of social interactions, there were no significant
differences in initial condition factor (CFi) for either
size-matched or size-mismatched pairs of rainbow trout (size-matched pairs
CFi=1.08±0.014, N=48; size-mismatched pairs
CFi=1.11±0.021, N=48). Similarly, neither social
status (two-way ANOVA, P=0.141) nor treatment group
(P=0.937) had a significant effect on final condition factor
(CFf) within size-matched pairs of fish at the end of the 5-day
interaction period (Table 3).
By contrast, significant effects of social status (two-way ANOVA,
P=0.006) were present within pairs of size-mismatched fish for
CFf (Table 4), with
dominant fish exhibiting significantly higher CFf values than
subordinate fish (P=0.006).
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Determination of specific growth rates (SGR) revealed significant effects of both social status (two-way ANOVA, P<0.001) and treatment group (P=0.036), as well as significant interactions (P=0.003), within size-matched pairs of fish (Table 3). Dominant individuals demonstrated significantly higher growth rates than subordinate fish in the cortisol (P<0.001) but not the cortisol+RU486 treatment group (P=0.08). Furthermore, growth rates for dominant fish from the cortisol+RU486 treated group were significantly lower than those of dominants from the cortisol treatment group (P=0.01). Finally, SGR for cortisol-treated fish was significantly lower (P=0.005) than that of non-injected fish with which they were paired, while a similar analysis in the cortisol+RU486 group revealed no difference (P=0.72, data not shown), a finding that suggests that the observed differences in growth might be a cortisol-mediated effect. Within size-mismatched pairs of rainbow trout, both social status (two-way ANOVA, P<0.001) and treatment group (P=0.012) had a significant effect on SGR, although there were no significant interactions between these factors (P=0.154; Table 4). Growth rates were significantly higher for dominant over subordinate fish (P<0.001), and for control over cortisol-treated fish (P=0.012).
Experiment 2: The effect of cortisol administration on brain monoamine levels
To assess the impact of circulating cortisol concentrations on brain
monoaminergic activity, groups of trout were injected intraperitoneally with a
slow-release pellet of cortisol. Circulating plasma cortisol concentrations
increased significantly (one-way ANOVA on ranks, P<0.001) with
cortisol administration (Fig.
3). The levels attained in the plasma in both cortisol and
cortisol+RU486 injected fish were similar to those associated with moderately
stressed salmonids (Gamperl et al.,
1994; Wendelaar Bonga,
1997
), and marginally lower than those observed in previous
studies that adopted similar methods (e.g.
Vijayan et al., 2003
;
McDonald and Wood, 2004
).
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Surprisingly, the observed effects of cortisol treatment on serotonergic and dopaminergic activities in the hypothalamus were opposite to those in the telencephalon; hypothalamic serotonergic activity was significantly reduced by cortisol implants (one-way ANOVA on ranks, P<0.001; Fig. 5C), while dopaminergic activity was significantly increased (one-way ANOVA on ranks, P<0.001; Fig. 5F). The lower serotonergic activity occurred because of a significant reduction in 5-HIAA concentrations (one-way ANOVA on ranks, P<0.001; Fig. 5B) even though hypothalamic 5-HT was unchanged (one-way ANOVA on ranks, P=0.078; Fig. 5A). The higher dopaminergic activity reflected significant increases of hypothalamic DOPAC levels (one-way ANOVA on ranks, P<0.001; Fig. 5E) in combination with significantly lower dopamine concentrations (one-way ANOVA on ranks, P<0.001; Fig. 5D). However, in no case did simultaneous administration of RU486 abolish these effects.
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Discussion |
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Competitive ability is probably the key determinant of the winner of
agonistic contests within juvenile salmonid fish, although factors such as
prior residence can also play a role
(Bachman, 1984;
Rhodes and Quinn, 1998
;
Cutts et al., 1999
).
Competitive ability, in turn, reflects numerous factors including innate
aggressiveness (Adams et al.,
1998
; Cutts et al.,
1999
), prior experience of winning or losing social contests
(Abbott and Dill, 1985
;
Rhodes and Quinn, 1998
), body
size in some cases (Abbott and Dill,
1985
; Rhodes and Quinn,
1998
) and, presumably, physiological condition (e.g.
Guderley and Couture, 2005
).
Physiological parameters such as abundant energy reserves, good condition and
perhaps high metabolic capacity might be expected to correlate with
competitive strength. For example, among several salmonid species, fish with
higher metabolic rates prior to social interactions tended to achieve higher
social status (Metcalfe et al.,
1995
; Yamamoto et al.,
1998
; Cutts et al.,
1999
; McCarthy,
2001
). High metabolic rate was associated with greater levels of
aggression in juvenile Atlantic salmon
(Cutts et al., 1998
),
suggesting a mechanism through which high metabolic rate could translate into
competitive success, and emphasizing the complexity of factors that determine
competitive ability.
Previous work suggested that circulating cortisol levels might also be a
physiological factor that affects competitive ability
(Gregory and Wood, 1999;
Sloman et al., 2001
).
Specifically, Sloman et al.
(2001
) documented
significantly higher plasma cortisol levels prior to social interaction in
rainbow trout that were identified as subordinate following pairing with a
conspecific. Similarly, Gregory and Wood
(1999
) attributed the greater
fin damage sustained by cortisol-treated trout held together with untreated
trout to a cortisol-related competitive disadvantage. The results of the
present study confirmed and extended these observations by revealing a causal
relationship between high plasma cortisol concentrations prior to social
interactions, and subsequent subordinate social status within pairs of rainbow
trout. Experimental elevation of plasma cortisol levels was associated with a
statistically significant increase in the probability of relegation to
subordinate rank, an effect that was eliminated by blocking cortisol receptors
using the glucocorticoid receptor antagonist RU486
(Table 1). Notably, the few (4
of 14) cortisol-treated fish that did achieve dominant status failed to
exhibit elevated plasma cortisol concentrations
(Fig. 2). It is possible that
these fish simply did not receive an effective dose of cortisol via
the implant. As cortisol levels in the plasma reflect the balance between
cellular biosynthesis and secretion into the blood (i.e. production), as well
as clearance of the hormone from circulation
(Mommsen et al., 1999
), these
fish may alternatively have been able to combat cortisol treatment by
increasing cortisol clearance from the plasma, although this possibility
remains to be tested.
Cortisol treatment also countered the effect of large size in determining
dominance (Table 2). Larger
fish became dominant in 86% of pairs in which both fish were untreated, a
result that was in agreement with previous studies that reported a positive
correlation between body size and dominant social status in rainbow trout
(Bachman, 1984;
Abbott and Dill, 1985
). This
significant effect of size in assuring dominant status was lost when the
larger fish were treated with cortisol; only 40% of cortisol-treated large
fish became dominant. The effect of cortisol treatment on the outcome of
social interactions observed in the present study fits well with previous
reports in which social status was closely linked with the magnitude of the
cortisol response to an acute stressor
(Pottinger and Carrick, 2001
).
Pottinger and Carrick (2001
)
found that within genetically maintained lines of rainbow trout selected for
divergent cortisol responses to an acute confinement stress, high responding
(HR) trout (i.e. greater elevation of cortisol levels) preferentially became
subordinate when paired with size-matched, low-responsive (LR) trout in staged
social encounters. Although Pottinger and Carrick
(2001
) were unable to
determine whether the link between social status and cortisol responses to an
acute stress was causal or circumstantial, the results of the present study
point to a causal relationship.
A causal relationship between high plasma cortisol levels and low social
status in rainbow trout could be the result of one or more underlying
physiological mechanisms. One possibility is that high circulating cortisol
levels affect competitive ability directly by depressing physiological
condition so that fish are not able to compete effectively. Prolonged
experimental administration of cortisol lowers growth rate and condition
factor, and increases mortality (Barton et
al., 1987; Pickering and
Pottinger, 1989
; Gregory and
Wood, 1999
), effects that have been attributed to appetite
suppression, the mobilisation of energy reserves, changes in digestive tract
morphology, reduced food conversion efficiency, increased metabolic rate, and
immune function suppression (Barton et al.,
1987
; Pickering and Pottinger,
1989
; Morgan and Iwama,
1996
; Gregory and Wood,
1999
; De Boeck et al.,
2001
). For example, the mean specific growth rate of
cortisol-treated fish in the present study was significantly lower over the 5
day interaction period than that of the untreated fish with which they were
paired, an effect that was cortisol-specific since it was eliminated by
co-administration of RU486. Similarly, the chronic elevation of plasma
cortisol attendant upon low social status probably accounted for, at least in
part (see Gilmour et al.,
2005
), the generally significantly lower growth rates of
subordinate fish relative to dominant fish (Tables
3,
4). These findings are in
agreement with previous reports in which lower growth rates were exhibited by
fish of low social status (Abbott and Dill,
1989
; Sloman et al.,
2000a
,b
).
However, the deleterious impact of cortisol on physiological condition
reflects prolonged elevation of the hormone, whereas cortisol levels were
raised in the present study only 48 h prior to the initiation of social
interactions. Thus, while cortisol-induced physiological depression may
diminish competitive ability, and may have contributed to the association
reported by Sloman et al.
(2001
) between higher plasma
cortisol prior to pairing and subsequent subordinate status, it is unlikely to
be the sole explanation for the results of the present study.
Alternatively, cortisol could affect competitive ability by modulating
behaviour in either a direct or indirect fashion. For example, time- and
context-dependent effects of cortisol administration were observed on
aggressive behaviour and locomotory activity in rainbow trout
(Øverli et al., 2002a).
The locomotory response to an intruder was enhanced after 1 h of cortisol
treatment, whereas both aggressive behaviour and activity in an intruder test
were inhibited following 48 h of cortisol treatment. Locomotory activity in
the absence of an intruder was unchanged by cortisol treatment, suggesting an
indirect role for cortisol in modifying behaviour, through interactions with
other signaling systems activated under particular circumstances
(Øverli et al., 2002a
).
Similarly, studies involving HR and LR rainbow trout found that HR trout
reacted to stress-induced increased plasma cortisol concentrations by marked
changes in locomotor activity, whereas LR trout did not (Overli et al.,
2001
,
2002b
). With respect to social
interactions in salmonid fish, brain monoamines, specifically serotonin and
dopamine, represent a signaling system of particular interest because the
behaviours characteristic of high or low social status are thought to result
in large part from the changes in brain monoaminergic activity that accompany
victory or loss in competitive encounters (reviewed by
Winberg and Nilsson, 1993
).
The results of the present study support, albeit not conclusively, a role for
cortisol in modifying brain monoaminergic activity, and hence suggest that the
causal link between high cortisol and low social status may reflect an
indirect modulatory action of cortisol on behaviour mediated through brain
monoaminergic systems.
Relative to control fish, serotonergic activity was markedly higher and
dopaminergic activity was lower in the telencephalon of cortisol-treated trout
(Fig. 4). This result is
consistent with work on other vertebrate groups in which corticosteroids have
been found to affect brain serotonergic activity (reviewed by Chaouloff,
1993,
2000
). For example,
intraperitoneal injection of corticosterone in male Anolis
carolinesis lizards significantly enhanced serotonergic activity within
20 min in two separate brain regions
(Summers et al., 2000
), and
intracorticol infusion evoked transient, dose-dependent increases in serotonin
overflow from neurons in the hippocampus
(Summers et al., 2003
). The
effects of cortisol administration on telencephalon serotonergic and
dopaminergic activity observed in the trout of the present study seemed to
mimic those produced by defeat in an agonistic contest (Winberg et al.,
1991
,
1992
). Experimental treatments
designed to increase brain 5-HT levels and/or serotonergic activity generally
have been reported to elicit behavioural inhibition in fish (but see
Stoddard et al., 2003
),
whereas high brain dopaminergic activity, on the other hand, seems to
facilitate aggressive behaviour (Winberg
and Nilsson, 1993
). For example, aggressive behaviour in rainbow
trout was suppressed by dietary administration of the 5-HT precursor
L-tryptophan, a treatment that also increased brain serotonergic
activity (Winberg et al.,
1991
). Similarly, territorial aggression in a coral reef fish was
depressed by intraperitoneal injection of the 5-HT selective reuptake
inhibitor fluoxetine (Perrault et al.,
2003
), while intracranial injection of either 5-HT or fluoxetine
inhibited aggressive `chirping' behaviour in a weakly electric fish
(Maler and Ellis, 1987
).
Aggressive behaviour in several salmonid species was increased following
treatment with the DA receptor agonist apomorphine
(Tiersch and Griffith, 1988
)
or DA itself (Nechayev and Musatov,
1992
), and oral administration of the DA precursor,
L-dopa, increased the probability of winning dominant social status
in Arctic charr (Winberg and Nilsson,
1992
). Thus, high circulating cortisol levels may be linked to low
social status through a pathway in which cortisol-induced increases in brain
serotonergic activity and/or decreases in dopaminergic activity result in the
inhibition of the aggressive behaviour critical for success in agonistic
encounters.
Within the hypothalamus, cortisol treatment resulted in a significant
decrease of serotonergic activity and an increase of dopaminergic activity
(Fig. 5), effects opposite to
those observed in the telencephalon of cortisol-treated fish
(Fig. 4), and opposite also to
the impact of social defeat on hypothalamic serotonergic activity
(Winberg and Nilsson, 1993).
The hypothalamus is a key component of the
hypothalamicpituitaryinterrenal (HPI) stress axis in fish
(Wendelaar Bonga, 1997
;
Mommsen et al., 1999
).
Hypothalamic corticotropin releasing factor acts on the pituitary to stimulate
the secretion of adrenocorticotropic hormone, which in turn elicits cortisol
synthesis and mobilisation from interrenal cells. Cortisol secretion
via this pathway may be modulated by the negative feedback actions of
cortisol at the levels of the hypothalamus and pituitary
(Mommsen et al., 1999
), and
several lines of evidence suggest that hypothalamic 5-HT also may be involved
in the regulation of the HPI axis (e.g.
Winberg et al., 1997a
; Lepage
et al., 2002
,
2003
;
Hoglund et al., 2002
).
Experimental elevation of plasma cortisol would be expected to downregulate
endogenous cortisol secretion via negative feedback. It is
conceivable that the lowering of hypothalamic serotonergic activity observed
in cortisol-treated fish reflected such a downregulation of cortisol secretion
pathways.
The changes of monoaminergic activity in the telencephalon and hypothalamus
of cortisol-treated rainbow trout were in general not abolished by
co-administration of the glucocorticoid receptor antagonist RU486 (Figs
4,
5), except in the case of
telencephalon serotonergic activity (Fig.
4C). While these findings raise concerns about whether the
responses were cortisol-specific, at least two plausible explanations exist.
First, the responses may be mediated via a mineralocorticoid receptor
(MR) rather than a glucocorticoid receptor (GR). In mammals, MRs and GRs
exhibit different expression patterns in the brain and play different roles in
mediating the effects of corticosteroids
(Chaouloff, 2000;
Korte, 2001
). GRs are widely
distributed in the forebrain of rainbow trout (Teitsma et al.,
1997
,
1998
) and the recently
identified fish MR (Colombe et al.,
2000
; Greenwood et al.,
2003
; Sturm et al.,
2005
) also appears to be present in the brain
(Greenwood et al., 2003
;
Sturm et al., 2005
). However,
the relative distributions and roles of the two corticosteroid receptors in
fish brains remain to be explored. Alternatively, cortisol may exert effects
in the brain via non-genomic mechanisms, a route of action that has
been well documented in mammals (reviewed by
Makara and Haller, 2001
). For
example, Mikics (2004) suggested that glucocorticoids rapidly increased
aggressive behaviour in rats via non-genomic mechanisms. The
non-genomic effects of corticosteroids are much more rapid than the genomic
responses, and resistant to both GR and MR blockade
(Makara and Haller, 2001
).
In conclusion, the results of the present study revealed a causal association between high plasma cortisol concentrations and low social status, and in general supported cortisol-induced changes in brain monoaminergic activity as a potential regulatory pathway for this effect. Clearly, however, additional work is required to validate the hypothesis that high circulating cortisol levels modify brain serotonergic activity and/or dopaminergic activity in trout, resulting in the suppression of aggressive behaviour and a consequent lowering of competitive ability that increases the likelihood of relegation to subordinate social status during agonistic contests.
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Acknowledgments |
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Footnotes |
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References |
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