1999 Overli Et Al Brain Behav Evol 54 263-275

8/10/2019 1999 Overli Et Al Brain Behav Evol 54 263-275

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2000 S. KargerAG,Basel00068977/99/05450263$17.50/0

Fax +41 61 306 12 34E-Mail [email protected] Accessible online at:www.karger.com www.karger.com/journals/bbe

Short-Term Effects of Fights for SocialDominance and the Establishment ofDominant-Subordinate Relationships on BrainMonoamines and Cortisol in Rainbow Trout

yvind verli Charmaine A. Harris Svante Winberg

Evolutionary Biology Centre, Department of Animal Development and Genetics, Uppsala University,

Uppsala, Sweden

Original Paper

Brain Behav Evol 1999;54:263275

yvind verliEvolutionary Biology Centre, Department of Animal Development and Genetics

Uppsala University, Norbyvgen 18D, SE752 36 Uppsala (Sweden)Tel. +46 18 4712615, Fax +46 18 4712683E-Mail: [email protected]

Key Words

Aggression Behavior Brain Cortisol Dopamine

Hierarchy Monoamines Norepinephrine

Serotonin Stress response

Abstract

We report changes in brain serotonergic, noradrenergic

and dopaminergic activity, along with plasma cortisol

concentrations, occurring during the initial 24-h period

following the establishment of dominant-subordinate

relationships in pairs of rainbow trout. Immediately

(within 5 min) after the termination of staged fights for

social dominance, a large increase in blood plasma corti-

sol was observed in both fight losers (future subordinate

fish) and winners (future dominant fish). In dominant

fish, cortisol decreased rapidly (within 3 h) to the level of

unstressed controls, while continuing to increase in sub-

ordinate fish. At 3 h following fights, the brain seroto-

nergic system was activated in both dominant fish and

subordinate fish, at least in some brain regions (telen-cephalon). This effect was reversed in dominant individ-

uals within 24 h of social interaction, whereas in subor-

dinate fish a substantial activation of the serotonergic

system was manifest in all brain regions by 24 h. Simi-

larly, a strong increase in brain catecholaminergic activa-

tion was indicated after 24 h of social interaction in sub-

ordinate fish, but not in dominant fish. Relationships

between plasma cortisol and brain serotonergic and nor-

adrenergic activity in the various experimental groups

suggest that these systems influence cortisol secretion

under normal conditions and during moderate or short-

term stress.

Copyright 2000 S.Karger AG, Basel

Introduction

Increased glucocorticoid secretion, sustained sympa-

thetic activation, and other physiological stress responses

have repeatedly been observed in socially defeated animals

[Golub et al., 1979; Ejike and Schreck, 1980; Sapolsky,

1990; Blanchard et al., 1995; McLeod et al., 1996; Koolhaas

et al., 1997; Shively et al., 1997a; Winberg and Lepage,

1998]. Subordinate animals are also often characterized by a

general behavioral inhibition, including suppressed aggres-

sive behavior, reduced feeding, and low spontaneous loco-

motor activity [Abbott et al., 1985; Nelissen and Andries,

1988; Winberg et al., 1993; Blanchard et al., 1993; Meerlo

et al., 1997; Shively et al., 1997b; verli et al., 1998].Many of the behavioral changes seen in subordinate fish

also have been reported to occur during predator challenge

[Blanchard and Blanchard, 1971], as well as in response to

other types of stress [McNaughton, 1993]. Thus, the behav-

ioral characteristics of socially subordinate animals prob-

ably reflect a general response to chronically stressful, un-

predictable, and/or potentially dangerous situations where

flight is not feasible. The brain monoamine neurotrans-


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mitters serotonin (5-hydroxytryptamine, 5-HT), dopamine

(DA) and norepinephrine (NE) are involved in the media-

tion of behavioral and neuroendocrine responses to social

stress [Yodyingyuad et al., 1985; Winberg and Nilsson,

1993; Blanchard et al., 1993; Stanford, 1993; Shively et al.,

1997b; Haller et al., 1997]. The overall effect of 5-HT stim-

ulation appears to be a general inhibition of active behav-ioral responses such as feeding, locomotion, and aggressive

behavior [Olivier et al., 1989; Winberg et al., 1993; Lei-

bowitz and Alexander, 1998; verli et al., 1998]. This is

supported by the observation that social subordination, like

other stressors, leads to increased brain serotonergic activity

in a range of species [Yodyingyuad et al., 1985; Winberg et

al., 1992; Blanchard et al., 1993; Fontenot et al., 1995; Sum-

mers and Greenberg, 1995; Matter et al., 1998]. Opinions

are divided about the role of brain catecholamines (NE and

DA) in behavioral responses to stress, but NE and DA might

to some extent have behavioral effects opposite those of

5-HT [Eichelman, 1987; Winberg and Nilsson, 1992;Arregui et al., 1993; Haller et al., 1997]. Brain catechol-

aminergic activity has been reported to be increased, de-

creased, or remain unaffected by social stress [Yodyingyuad

et al., 1985; Blanchard et al., 1991; Summers and Green-

berg, 1995; Tidey and Miczek, 1996; Matter et al., 1998].

This probably reflects the observation that stress commonly

leads to a general increase of 5-HT neurotransmission in

most brain regions, whereas stress can have a biphasic effect

on brain catecholaminergic activity [Stanford, 1993]. Alter-

ations of brain catecholaminergic activity are also more

regionally specific than those observed for 5-HT and depend

more heavily on factors such as stressor specificity, condi-

tioning, and prior exposure to stress [Nisenbaum et al., 1991;

Stanford, 1993].

Behavioral inhibition in subordinate animals is particu-

larly well documented in small groups of salmonid fishes

[Winberg et al., 1993; verli et al., 1998]. These animals

can be highly aggressive, especially at life stages during

which they are territorial in nature and form distinct social

hierarchies both in the wild and when reared in captivity

[Keenleyside and Yamamoto, 1962; Noakes and Leather-

land, 1977; Winberg et al., 1992; Nakano, 1994]. The distri-

bution and functions of monoamine neurotransmitters infishes and higher vertebrates exhibit extensive similarities,

suggesting that these systems have been conserved during

vertebrate evolution and are thus phylogenetically primitive

[Parent et al., 1984; Hornby and Piekut, 1990; Jacobs and

Azmitia, 1992; Winberg and Nilsson, 1993; Ma, 1994]. It

has previously been shown that socially subordinate salmo-

nids display chronically elevated brain serotonergic activity,whereas, brain dopaminergic activity appears to be decreased

in long-term subordinate fish [Winberg et al., 1991]. As inmammals, changes in brain monoaminergic activity coincide

with behavioral inhibition in subordinate individuals [Win-

berg et al., 1993; verli et al., 1998]. Short-term effects of

social interactions on brain monoamine utilization have not

been investigated in fish. Escalated fights for social domi-

nance are probably stressful both for the eventual winner(dominant individual) and loser (subordinate individual)

[e.g. Summers and Greenberg, 1994], but behavioral inhibi-

tion is not seen in the dominant individual after the forma-

tion of a stable social hierarchy. On the contrary, dominant

individuals display constantly high locomotor activity,

aggression, and feeding [verli et al., 1998]. Thus, either

dominant individuals are able to quickly suppress stress re-

sponses resulting from aggressive interactions, or other neu-

roethological aspects of the dominant social position imply

that perceived stressors are not as likely to result in behav-

ioral inhibition in these animals.

In the present study we describe changes in blood plasmacortisol concentrations and brain monoaminergic activity

occurring during the 24 h in which rainbow trout become

dominant or subordinate after staged fights for social domi-

nance. Specifically, it was hypothesized that both winners

(i.e. future dominant fish) and losers (i.e. future subordinate

fish) would show a substantial stress response immediately

following fights. Furthermore, it was expected that domi-

nant fish, but not subordinate fish, would be able to suppress

this response within a short period of time. In line with

evidence from mammals, changes in brain monoaminergic

activity and blood plasma cortisol are expected to be corre-

lated.

Materials and Methods

Experimental fish were juvenile (2-year-old) rainbow trout, weigh-ing 120 20 g (mean SD, n = 72). Prior to the experiment, the fishhad been kept indoors in a 1 m3 holding tank at a rearing density ofapproximately 0.02 kg/l for >3 weeks. The holding tank was continu-ously supplied with aerated Uppsala tap water at 811C and the light-dark regimen was continuously adjusted to conditions at 51 northlatitude. In the holding tank and throughout the experiment, fish werefed daily with commercial trout pellets (EWOS ST40, Astra-EWOS)

at 12% of their body weight (BW).The experiment was conducted in seven glass aquaria (100 50

50 cm) continuously supplied with aerated Uppsala tap water (0.8 l/min, 810C). Light (12 h:12 h/light: dark) was provided by 2 20 Wwarm white fluorescent tubes placed 100 mm above the water surface.Each aquarium was divided into four 50 l compartments by removablePVC walls. At the start of the experiment, rainbow trout were selectedfrom the holding tank and weighed to allow for the formation ofsize-matched pairs (


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side of the caudal fin for the recognition of individual pair membersand placed in isolation (one fish per 50 l compartment) for acclimationto the experimental aquaria. During acclimation, fish were hand fedpelleted feed 12% BW/day, and when both members of a pair con-sumed 90100% of the available ration (after 510 days in isolation),they were considered fully acclimated and the PVC wall separatingthem was gently removed. After removal of the wall, escalated fightsfor social dominance (further described in the results section) started

after 0.590 min, and lasted for another 0.5120 min. These fightsinevitably ended with one fish retiring from further aggression andtrying to escape. In this manner, one subordinate (loser) and onedominant (winner) fish were readily identifiable in all pairs. After thetermination of fights for dominance, pairs of fish were allowed to inter-act for another 5 min, 3 h, or 24 h, before sampling for blood plasmaand brain tissue was performed. In addition, a number of undisturbedisolated fish were sampled to obtain non-stressed controls. In all, 7experimental groups were formed: Controls, and 5 min, 3 h, and 24 hpost-fight dominant fish and subordinate fish.

Upon sampling, fish were rapidly netted and anesthetized in500 mg/l ethyl m-aminobenzoate methanesulfonate. Thereafter, bloodsamples were collected from the caudal vasculature using a heparin-ized syringe and kept on ice. Fish were then decapitated and the brainwas dissected into four different brain regions: Telencephalon (exclud-ing the olfactory bulb), hypothalamus, optic tectum and brain stem.The pituitary, olfactory bulbs, and cerebellum were excluded fromanalysis. Brain samples were wrapped in aluminum foil, frozen in liq-uid nitrogen, and kept at 80 C. The sampling procedure was com-pleted within 2 min of removal of the fish from the aquarium. Finally,following centrifugation at 1,500 g for 3 min, plasma aliquots werefrozen and kept at 80 C.

The frozen brain samples were homogenized in 4% (wt/vol) ice-

cold perchloric acid containing 40 ng/ml epinine (deoxyepinephrine,

used as an internal standard) using a Potter-Elvehjem homogenizer

(brain stem) or an MSE 100-W ultrasonic disintegrator (other brain

parts). Samples were centrifuged at 27,000 g for 15 min at 4C and

the supernatants were analyzed for the following monoamines

and monoamine metabolites using high performance liquid chro-

matography with electrochemical detection (HPLC-EC): Dopamine

(DA), 3,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine

(NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), serotonin (5-hy-

droxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA).

The HPLC apparatus consisted of a solvent delivery system (Costa-

Metric II, LDC, USA), an autoinjector (Midas, Spark, Holland), a

reverse phase column (4.6 100 mm, Hichrom, C18, 3.5 m) kept at

40 C, and an ESA 5200 Coulochem II EC-detector (ESA, Bedford,

Ma., USA) with two electrodes at oxidizing potentials of +320 and

+450 mV, respectively. A conditioning electrode with a potential of

+40 mV was employed before the analytical electrodes to oxidize

potential contaminants. The mobile phase was delivered at 1 ml/min

and consisted of 75 mMsodium phosphate, 0.7 mMoctane sulfonic

acid, in deionized (18.2 M) water containing 10% methanol and

brought to pH 3.1. Samples were analyzed for concentrations of

monoamines (5-HT, NE and DA) and monoamine metabolites

(5-HIAA, MHPG, and DOPAC, respectively) by comparison with

standard solutions of known concentrations, and corrected for recov-

ery of the internal standard using HPLC software (CSW, DataApex

Ltd., the Czech republic). Plasma samples were analyzed for the

concentration of cortisol using the radioimmunoassay previously

described by Olsen et al. [1992], as modified by Winberg and Lepage

[1998].

Concentrations of blood plasma cortisol, brain monoamines,monoamine metabolites, and monoamine/metabolite ratios were com-pared among experimental groups by one-way analysis of variance(ANOVA), followed by the Tukey post hoc test for unequal n (Spjotvoll-Stoline test). When necessary to obtain homogeneity of variance prior tothe use of parametric ANOVA, data were log (concentrations) or arcsin(ratios) transformed. Homogeneity of variance was tested by the Levenetest. Correlations between brain monoaminergic activity and plasma

cortisol, or between brain monoamine metabolite concentrations and theduration of fights were tested by the Spearman rank test, because theexperimental groups were not large enough for multivariate analysis.

Results

Agonistic Behavior

After removal of the wall, fish first engaged in a series of

mutual displays and after a variable (0.590 min) latency

period began performing overtly aggressive behavior con-

sisting of violent attacks, biting, and circling. The durationof these aggressive encounters was also highly variable

(from 0.5120 min), but it inevitably ended with one fish

retiring from further aggression and trying to escape. Not

being able to flee from the arena after stopping the retalia-

tion of aggressive acts from the opponent, the subordinate

fish would usually seek a position out of the field of vision

of the dominant, e.g. close to the walls or in a corner of the

aquarium and remain inactive. Dominant fish, on the other

265Social Dominance and Brain Monoamines and

Cortisol in Trouts


Fig. 1. Effects of fights for social dominance and continued inter-action in an established dominant-subordinate relationship on bloodplasma cortisol in dominant and subordinate rainbow trout, as com-pared to non-stressed controls (bars indicate mean SEM). F, df andp values are the result of one-way ANOVA, followed by the Tukey posthoc test for unequal n (Spjotvoll-Stoline test). Post hoc significance

levels are indicated by asterisks, where * is used to indicate a differ-ence to controls and [*] indicates a difference between social ranks at agiven point in time. *p < 0.05, **p < 0.01, ***p < 0.001.


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hand, would tend to keep moving directly above the bottom

close to the center of the aquarium. Notably, even when

remaining passive, subordinate fish would frequently be

attacked, nipped, and chased by dominant fish. Thus, a

phase of bi-directional aggression (fight) and a phase of uni-

directional aggression (social dominance) could be distin-

guished in all cases.

Plasma Cortisol

Plasma cortisol concentrations in isolated controls and in

dominant and subordinate rainbow trout following fights for

dominance are shown in figure 1. Blood plasma cortisol was

drastically elevated directly (5 min) after fights in both dom-

inant (winners) and subordinate (losers) rainbow trout, as

compared to controls. On average, the increase was slightlyhigher (12 fold) in subordinate fish than in dominant fish

(10 fold), but the post hoc tests did not reveal a significant

difference between dominant and subordinate fish. How-

ever, 3 h following fights plasma cortisol had decreased in

dominant fish to approach the level of controls, whereas

subordinate fish now displayed significantly elevated corti-

sol values when compared to both dominant fish and to con-

trols. The pattern of plasma cortisol levels established in

dominant and subordinate fish at 3 h following fights was

still evident after 24 h.

Brain Monoaminergic Activity

At the sampling point 5 min after fights, the only effect

seen on brain monoaminergic activity was an elevation of

5-HIAA/5-HT ratios in the telencephalon of subordinate

fish (fig. 2). At 3 h both subordinate and dominant fish dis-

played further indications of activation of brain monoamin-

ergic systems (see fig. 2 for monoamine/metabolite ratios,

and table 1 for tissue concentrations). Telencephalic 5-HIAA/

5-HT ratios were elevated with respect to controls in sub-

ordinate fish as well as dominant fish, as was the level of

5-HIAA in the optic tectum. Furthermore, dominant fish

displayed an elevation of DOPAC concentrations in thebrain stem, which was not significant in subordinate fish. In

subordinate fish, on the other hand, hypothalamic DOPAC

levels, DOPAC/DA ratios, and MHPG levels were signifi-

cantly affected. At 3 h, subordinate fish also displayed a

large elevation in brain stem MHPG levels as well as

MHPG/NE ratios.

After 24 h of social interaction, brain monoaminergic

activity had increased even more in subordinate fish. At this

266 Brain Behav Evol 1999;54:263275 verli/Harris/Winberg

Fig. 2. Effects of fights for social domi-nance and continued interaction in an estab-lished dominant-subordinate relationship on

A 5-HIAA/5-HT; B DOPAC/DA; and CMHPG/NE ratios in different brain regions ofdominant and subordinate rainbow trout, ascompared to non-stressed controls (bars indi-cate mean SEM). F, d.f. and p values are theresult of one-way ANOVA, followed by theTukey post hoc test for unequal n (Spjotvoll-

Stoline test). Post hoc significance levels areindicated by asterisks, where * is used to indi-cate a difference to controls and [*] indicatesa difference between social ranks at a givenpoint in time. *p < 0.05, **p < 0.01, ***p 0.99 p = 0.49 p = 0.07 p = 0.22

DOPAC/DA n = 10 n = 13 n = 9 n = 7 n = 9 n = 9

r = 0.08 r = 0.37 r = 0.15 r = 0.57 r = 0.39 r = 0.30

p = 0.83 p = 0.23 p = 0.70 p = 0.18 p = 0.26 p = 0.43

Telencephalon

5-HIAA/5-HT n = 10 n = 17 n = 10 n = 9 n = 9 n = 9

r = 0.15 r = 0.37 r = 0.33 r = 0.43 r = 0.18 r = 0.17

p = 0.68 p = 0.14 p = 0.35 p = 0.34 p = 0.61 p = 0.97

MHPG/NE n = 10 n = 17 n = 10 n = 7 n = 9 n = 8

r = 0.28 r = 0.04 r = 0.61 r = 0.65 r = 0.24 r = 0.31

p = 0.43 p = 0.89 p = 0.06 p = 0.06 p = 0.49 p = 0.46

DOPAC/DA n = 10 n = 17 n = 10 n = 8 n = 9 n = 9

r = 0.16 r = 0.005 r = 0.02 r = 0.31 r = 0.55 r = 0.43

p = 0.65 p > 0.99 p = 0.96 p = 0.46 p = 0.10 p = 0.24

Optic tectum

5-HIAA/5-HT n = 10 n = 17 n = 10 n = 9 n = 9 n = 9

r = 0.82 r = 0.16 r = 0.33 r = 0.63 r = 0.19 r = 0.16

p = 0.004 p = 0.55 p = 0.35 p = 0.07 p = 0.59 p = 0.67

MHPG/NE not analysed not analysed not analysed not analysed not analysed not analysed

DOPAC/DA n = 10 n = 17 n = 10 n = 9 n = 9 n = 9

r = 0.13 r = 0.26 r = 0.24 r = 0.47 r = 0.09 r = 0.31

p = 0.73 p = 0.32 p = 0.51 p = 0.21 p = 0.81 p = 0.41

Spearman r and p values are given, and significant relationships are indicated by bold font. Relationships between plasma cortisol and sero-

tonergic and noradrenergic activity in the brain stem are illustrated in figure 3.


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fish did not differ from unstressed controls after 24 h of

social interaction.

Concerning possible relationships between plasma corti-

sol and brain monoaminergic activity as indexed by metabo-

lite/neurotransmitter ratios, it should be noted that in both

dominant and subordinate fish correlations appeared to

change markedly with time after termination of fights

(fig. 3). Data from 5 min fish were pooled, as dominant and

subordinate fish did not differ at this time point, but other-

wise, different experimental groups were treated separately.

Testing within groups, a number of significant correlations

with plasma cortisol were found in the brain stem (fig. 3).

Specifically, brain stem 5-HIAA/5-HT and MHPG/NE

ratios were found to correlate with plasma cortisol in con-


Cortisol in Trouts


Brain stem Hypothalamus Telencephalon Optic tectum

5-HIAA n = 18 n = 15 n = 20 n = 20

r = 0.60 r = 0.69 r = 0.52 r = 0.51

p = 0.012 p = 0.007 p = 0.02 p = 0.03

MHPG n = 18 n = 15 n = 20 not analysed

r = 0.76 r = 0.23 r = 0.26 not analysed

p < 0.001 p = 0.41 p = 0.28 not analysed

DOPAC n = 18 n = 14 n = 20 n = 20

r = 0.36 r = 0.12 r = 0.37 r = 0.50

p = 0.14 p = 0.68 p = 0.12 p = 0.03

Spearman r and p values are given, and significant relationships are indicated by bold font.

Relationships between fight duration and metabolite concentrations in the brain stem are illus-

trated in figure 4.

Table 3. Summary of relationships between

fight duration and brain monoamine metabo-

lite levels in rainbow trout sampled 5 min

following fights for dominance (data from

dominant and subordinate fish are pooled)

Fig. 4. The relationship between fight dura-tion and the concentrations of 5-HIAA;DOPAC, and MHPG in the brain stem in rain-bow trout sampled 5 min following the termi-nation of fights for social dominance. Notethat date from dominant and subordinate fishare pooled. Spearman r and p values aregiven.


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trols, 5 min fish, and 3 h subordinate fish. Monoaminergic

activity were found to correlate with plasma cortisol in other

brain regions than the brain stem only in one case, and that

was 5-HIAA/5-HT of the optic tectum which correlated sig-

nificantly with cortisol in controls (table 2).

The accumulation of monoamine metabolites in neural

tissue following the activation of monoaminergic systems isprobably time dependent, thus fish sampled after the termi-

nation of fights were investigated for possible relationships

between fight duration and concentrations of monoamine

metabolites. In 5 min fish, fight duration was significantly

correlated with 5-HIAA concentrations in all brain regions.

MHPG levels in the brain stem, and DOPAC levels in the

optic tectum were also correlated to the duration of fights

(fig. 4; table 3). Fighting time was not correlated to mono-

amine metabolite concentrations in any brain region at 3 h

or 24 h following fights (results not shown).

Discussion

Presupposing that plasma cortisol levels reflect stress-

induced activation of the hypothalamus-pituitary-interrenal

axis in fish [Sumpter, 1997], the results of the current study

suggest that fights for social dominance were highly stress-

ful to all contestants, winners as well as losers. Dominant

fish, but not subordinate fish, were able to rapidly reduce

their cortisol levels during continued interaction after domi-

nant-subordinate relationships had been established. These

data are in agreement with previous observations that gluco-

corticoid secretion is increased in socially subordinate ani-

mals [Golub et al., 1979; Ejike and Schreck, 1980; Sapolsky,

1990; McLeod et al., 1996; Winberg and Lepage, 1998]. It

should be noted that social stress is probably enhanced

under conditions of artificial rearing where opportunities for

social signaling and escape are limited. However, a majority

of the available studies concerning free-living as well as

captive animals consistently suggest hypersecretion of glu-

cocorticoids in subordinate animals. A general lack of con-trol and predictability, restricted access to food and other

resources, and a constant threat of aggressive actions from

dominant individuals are all possible factors that could pro-mote the stress response in subordinate animals.

When interpreting the results of brain monoaminergic

activity, it should be kept in mind that the analysis of tissue

concentrations of neurotransmitter metabolites does not

reflect instantaneous neural activity, as opposed to tech-

niques of in vivo voltammetry or microdialysis [Fillenz,

1993]. The metabolites 5-HIAA, DOPAC, and MHPG are

formed following re-uptake of the parent monoamines (5-

HT, DA, and NE, respectively) from the synaptic cleft, and

their accumulation in neural tissue is probably time depen-

dent. In addition, a fraction of the monoamine molecules is

always deaminated intraneurally prior to release, so metabo-

lite levels are even sensitive to changes in synthesis rate and

monoamine oxidase activity [Fillenz, 1993; Stanford, 1993].

Brain metabolite/monoamine ratios are less sensitive thanmetabolite concentrations to changes in other neural pro-

cesses than release rate, and are also less sensitive to vari-

ance related to tissue sampling and weight determination.

Statistically significant increases in metabolite concentrations

in response to stress are therefore more rarely observed than

increases in metabolite/monoamine ratios, but when they

are observed, increased metabolite level is still in most cases

considered an indicator of increased monoamine utilization

[for review see Fillenz, 1993; Stanford, 1993].

As in the current study, utilizing poikilothermic animals

living at 12 C, the effects of social interaction were re-

flected in increased monoaminergic activity only in the tel-encephalon (5-HIAA/5-HT ratios) of subordinate fish 5 min

following the termination of fights. However, the duration

of fights was highly variable (270 min in the case of 5 min

fish), and significant correlations were found between fight

duration and brain concentrations of 5-HIAA (in all brain

regions), MHPG (in the brain stem), and DOPAC (in the

optic tectum) at 5 min (fig. 4; table 3). Thus, it appears that

these monoaminergic systems were activated during fights,

leading to gradually increasing metabolite levels over time.

Due to the variation in fighting time, however, averaged

group values were in most cases not significantly affected at

5 min. A multivariate analysis with fight duration as a

covariate would be the preferred method to take this varia-

tion into account, but is not applicable in the current study

as fight duration would have less effect on later time points

than early in hierarchy formation. A better covariate to

explain variation in monoaminergic and endocrine activa-

tion would probably be the frequency of aggressive acts per-

formed by the contesting animals. This parameter was how-

ever not registered in the current experiment.

Effects on brain monoaminergic activity were mani-

fested in several brain regions in both subordinate fish and

dominant fish at 3 h. In dominant fish, indicators of seroto-nergic activity were significantly increased in the telenceph-

alon (5-HIAA/5-HT ratios) and optic tectum (5-HIAA con-

centrations), and DOPAC concentrations were increased in

the brain stem. Subordinate fish displayed the same indica-

tors of serotonergic activation in the telencephalon and optic

tectum, and, in addition, noradrenergic activity was elevated

in the brain stem (MHPG concentrations) and hypothalamus

(MHPG concentrations and MHPG/NE ratios). Dopaminer-



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gic activity was significantly increased in the hypothalamus

of subordinate fish (DOPAC concentrations and DOPAC/

DA-ratios). These results indicate that some degree of

dopaminergic and serotonergic activation occurred during

the early stages of hierarchy formation in the dominant fish,

up to 3 h after the termination of fights, whereas activation

of noradrenergic systems was manifested only in subordi-nate fish. After 24 h of social interaction, brain monoamin-

ergic activity had increased additionally in subordinate fish,

with activation of serotonergic systems in all brain regions,

as well as catecholaminergic systems in the brain stem and

hypothalamus (table 1; fig. 2). In dominant fish, on the other

hand, effects of fights for social dominance on brain mono-

aminergic activity were apparently reversed and were not

evident after 24 h (as was the case for cortisol secretion).

These results are comparable to observations on the effects

of aggressive interactions on brain monoaminergic activity

in lizards (Sceloporus jarrovi and Anolis carolinensis)

[Summers and Greenberg, 1995; Matter et al., 1998; Sum-mers et al., 1998]. In these animals, it appears that dominant

individuals have a substantial, but quickly reversible, acti-

vation of the 5-HT systems during aggressive encounters,

whereas subordinate lizards exhibit chronically increased

serotonergic activity. Increased catecholaminergic activity

was only observed in dominant individuals in the above

studies, which could reflect differential activation of cate-

cholaminergic systems during social interactions in these

lizard species as compared to rainbow trout. Alternatively,

this discrepancy with the results of the current experiment

could reflect different time courses of the studies.

In many respects, brain catecholamines appear to oppose

the behavioral effects of 5-HT, at least with respect to

aggressive behavior and locomotor activity. In fish, as in

mammals, catecholaminergic systems are thought to stimu-

late aggressive behavior [Eichelman, 1987; Maler and Ellis,

1987; Winberg and Nilsson, 1992], whereas serotonin is

thought to inhibit aggressive behavior [reviewed by Win-

berg and Nilsson, 1993]. Thus, it is reasonable to suggest

that the widespread increase in brain serotonergic activity

seen in subordinate fish leads to behavioral inhibition in

these animals [Yodyingyuad et al., 1985; Winberg et al.,

1992; Blanchard et al., 1993; Fontenot et al., 1995; Sum-mers and Greenberg, 1995; Matter et al., 1998]. Further-

more, in mammals 5-HT has been reported to stimulate the

release of CRF and adenocorticotropin (ACTH) from the

hypothalamus and pituitary, respectively [Chaouloff, 1993;

Dinan, 1996]. In rainbow trout, pharmacological stimulation

of putative 5-HTIA receptors elevates plasma cortisol con-

centrations in a dose dependent manner [Winberg et al.,

1997]. Brain catecholamines are also thought to be involved

in behavioral as well as neuroendocrine stress responses

[Plotsky et al., 1989; Stanford, 1993; Winberg and Nilsson,

1993; Shively et al., 1997b], suggesting that socially

induced alterations in brain monoaminergic activity serves

to integrate behavioral and endocrine correlates of changing

social positions.

In the current study, significant correlations betweenblood plasma cortisol and 5-HIAA/5-HT ratios, notably in

the brain stem (fig. 3; table 2), were observed in several

experimental groups (controls, 5 min fish, and 3 h subordi-

nates). These results suggest that brain serotonergic systems

influence cortisol secretion under normal conditions and

during moderate or short-term stress. Under severe stress (as

in 24 h subordinate fish) and during recovery from stress (as

in 3 h and 24 h dominant fish), brain monoamines might

exert similar effects, but the apparent lack of correlations

suggest that plasma cortisol levels depend more heavily on

other factors such as the rate of clearance of cortisol from

circulation [Pottinger and Moran, 1993].Brain catecholaminergic responses to stress, although

extensively documented in mammals, have not been studied

in fish. Increased mesocortical dopamine release during

stress has been suggested as an essential coping mechanism,

because comparatively stress-resistant Wistar Roman High-

Avoidance rats react to stress with increased DOPAC accu-

mulation in this brain region during various forms of stress,

whereas stress-sensitive Low-Avoidance rats do not [Ber-

tolucci-DAngio et al., 1990]. Furthermore, a wide variety

of evidence from mammalian studies suggest that brain

catecholaminergic systems are involved in the activation of

the hypothalamus-pituitary-adrenal (HPA) axis [Plotsky et

al., 1989]. For instance, intraventricular administration of

0.55.0 nmol NE evoked CRF-41 secretion into the hypo-

physial portal circulation of rats in a dose dependent manner

[Plotsky, 1987]. Electrophysiological studies on the activity

of neurosecretory cells of the paraventricular nucleus (PVN)

suggest that medullary catecholaminergic projections exert

a strong facilitatory drive to parvicellular neurons in the

PVN [Day et al., 1985]. Results from catecholamine agonist

or antagonist challenge studies might initially seem contra-

dictory with respect to the direction or receptor mechanisms

of catecholamine action on HPA-axis function [Plotsky etal., 1989], but much of the discrepancy could be due to the

possible presence of inhibitory 2-receptors on catechol-

aminergic neurons.

The correlations between brain noradrenergic activity, as

indicated by brain stem MHPG/NE ratios, and plasma corti-

sol suggest an influence of brain stem noradrenergic neurons

on the hypothalamus-pituitary-interrenal axis (HPI-axis, the

teleost homologue of the mammalian HPA-axis) in fish.


Cortisol in Trouts



12/13

Further studies are needed, however, to establish the link

between brain noradrenergic activity and HPI-axis function.

Obviously, the simultaneous occurrence of elevated plasma

cortisol and increased neural activity need not imply that a

causal relationship exists. It should also be kept in mind that

glucocorticoids influence both the synthesis and release of

monoamine neurotransmitters, as well as receptor densities[Chaouloff, 1993; Stanford, 1993; Crayton et al., 1996;

Piazza et al., 1996], so the direction of causality is not

immediately obvious.

In summary, the results of the current study suggest that

dominant as well as subordinate fish show an extensive

stress response to fights for social dominance, but that this

response is quickly attenuated in dominant fish. Following

the settlement of fights, cortisol rapidly decreased in domi-

nant fish (by 3 h), while continuing to increase in sub-

ordinate fish, indicating that dominant fish experience a

generally non-stressful situation, whereas subordinate pair

members are subject to severe and sustained socially in-duced stress. In an early phase of hierarchy formation the

brain serotogenic system appears to be activated in both

dominant fish and subordinate fish, at least in some brain

regions (telencephalon). This effect is reversed in dominant

fish within 24 h of social interaction, whereas in subordi-

nate fish a substantial activation of the serotonergic system

is manifest in all brain regions by 24 h. Similarly, brain

catecholaminergic (DA and NE) systems are activated after

24 h of social stress in subordinate fish, whereas in domi-

nant fish effects on brain catecholaminergic systems are

abolished by 24 h of social interaction. Furthermore, rela-

tionships between plasma cortisol and brain stem 5-HIAA/5-HT and MHPG/NE ratios suggest that brain monoamin-

ergic systems influence cortisol secretion under normal

conditions and during moderate or short-term stress, but

not under severe stress or during recovery from stress. To

our knowledge, this is the first study to demonstrate a

relationship between brain catecholaminergic (NE) activity

and glucocorticoid secretion in a nonmammalian verte-

brate.

Acknowledgements

The methodology of this study was approved by Uppsala AnimalResearch Ethical Committee. Financing was granted by the Norwegian(NFR grant No. 119129/410 to ) and Swedish (SJFR grant No.41.0677/97 to SW) research councils, the Axelsson-Johnsson Founda-tion, the Lars Hiertas foundation, and the Hierta-Retzius Foundation(to ), and the Magnus Bergvall foundation (to SW). We thank E.T.Larson for valuable comments on the manuscript.


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1999 Overli Et Al Brain Behav Evol 54 263-275