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Behavioural Brain Research 225 (2011) 529– 537

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho mepage: www.elsev ier .com/ locate /bbr

esearch report

daptive changes in zebrafish brain in dominant–subordinate behavioral context

ichail Pavlidisa,b,∗, Maria Sundvika, Yu-Chia Chena, Pertti Panulaa

Neuroscience Center and Institute of Biomedicine/Anatomy, University of Helsinki, P.O. Box 63, 00014, Helsinki, FinlandDepartment of Biology, University of Crete, P.O. Box 2208, GR-71409, Heraklion, Crete, Greece

r t i c l e i n f o

rticle history:eceived 18 May 2011eceived in revised form 11 August 2011ccepted 13 August 2011vailable online 22 August 2011

eywords:ominantocial stressubordinateebrafish

a b s t r a c t

Male zebrafish were held in dyadic social stress situation for a period of 5 days, to characterize stresscoping styles and to investigate the role of the underlying neuroendocrine mechanisms in establish-ing dominant–subordinate relationships. A strong consistent dominant–subordinate relationship wasformed in ten out of the sixteen pairs of fish (62.5%). Both dominant (DOM) and subordinate (SUB) indi-viduals showed statistically significant higher trunk cortisol concentration than controls. Expression ofgenes encoding proteins involved in the functioning of the hypothalamus–hypophysis–interrenal axis(corticotropin releasing factor, CRF; glucocorticoid receptor, GR; mineralocorticoid receptor, MR); argi-nine vasotocin, AVT), in the biosynthesis and catabolism of catecholamines (tyrosine hydroxylase, TH1and TH2; DOPA decarboxylase, DDC), dopamine �-hydroxylase, DBH; catechol-O-methyl transferase,COMT), in the biosynthesis of histamine (histidine decarboxylase, HDC) and in the general stress response(galanin, GAL; hypocretin/orexin, Hcrt) was examined. The MR/GR ratio was higher in dominant and sub-

ordinate fish than in controls (P = 0.016). The mRNA levels of TH2 and HDC were up-regulated in DOM, ofAVT in SUB, while COMT mRNA levels were down-regulated in both DOM and SUB compared to controlfish. In addition, mRNA levels of hypocretin/orexin (Hcrt) were up-regulated in dominant compared tosubordinate and control males. There was a statistically significant correlation between mRNA expres-sion levels of TH2, HDC, Hcrt, GR, MR and CRF genes. The obtained results provide new evidences for the

mal m

use of zebrafish as an ani

. Introduction

Individuals of various animal species have developed differ-nt sets of behavioral and physiological adaptive responses ineaction to ordinary challenges and stress stimuli in the naturalnvironment or in captivity. Several studies in rodents, birds andammals, and to a less extent in fish, provide strong evidence for

he presence of two distinct coping styles, the proactive and reac-ive [8,13,14,26,41,76,78,81–83]. Individuals of the proactive stylere characterized by a fight-flight behavioral strategy, aggressivend bold emotional state, rigid and routine-like behavior and highnergy consumption [46,50]. The reactive coping style is charac-erized by a freeze-hide behavioral strategy, non-aggressive and

autious emotional state, flexible behavioral flexibility and energyonservation.

∗ Corresponding author at: Department of Biology, University of Crete, P.O. Box208, GR-71409, Heraklion, Crete, Greece. Tel.: +30 2810 394084;ax: +30 2810 394404.

E-mail addresses: pavlidis@biology.uoc.gr (M. Pavlidis),aria.sundvik@helsinki.fi (M. Sundvik), yu-chia.chen@helsinki.fi (Y.-C. Chen),

ertti.panula@helsinki.fi (P. Panula).

166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2011.08.022

odel to study social stress and allostasis in vertebrates.© 2011 Elsevier B.V. All rights reserved.

Behavioral differences in coping styles are associated withimportant differences in physiological and neuroendocrineresponses, as well as in neuronal structure [24,38,46,47,50].Proactive individuals show high sympathetic reactivity andhigh hypothalamus–hypophysis–gonadal axis activity, while reac-tive animals show high parasympathetic reactivity and highhypothalamus–hypophysis–adrenal axis activity [46]. Accordingto the concept of allostasis, the emotional brain plays a cru-cial role in the coordination of behavioral plasticity and severalneurotransmitters are considered important mediators of allosta-sis [47]. For example, increased serotonergic activity has beenreported in subordinate rats, mice and teleosts [11,42,43]. Argi-nine vasopressin (AVP) in mammals and arginine vasotocin (AVT)in fish, amphibians, reptiles and birds is also associated withdominant–subordinate relationships [42,51]. Chronic social stressaffects tyrosine hydroxylase mRNA and protein levels in rat locuscoeruleus [79], while recently it was proposed that endogenoushypocretin/orexin (Hcrt) may be an important factor in neuronalpathways that coordinate stress responses [66].

Zebrafish (Danio rerio) is an animal model in biomedical

research, developmental biology, genetics and neurobehavioralstudies [20,58,60,64]. Zebrafish in nature form shoals with maleshaving no preference for shoal size but to associate withfemale shoals, while females show a preference for larger shoals

5 Brain

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egardless of sex ratio [72]. Previous studies have shown thatebrafish, as many other social animals, when allowed to interactn pairs often establish dominant–subordinate relationships andisplay agonistic or anxiety-like behavior [51,72]. Aggressive inter-ctions involve chasing and biting and sex does not seem to be anmportant factor in determining fish’s dominance rank [35,72]. Inaboratory conditions, dominant–subordinate relationships appearo be relatively constant at least over the duration of 5-day socialnteraction experiments [71]. The fact that dominance hierarchiesre quickly established in males and are also present in femalesay be of great potential for partitioning the effects of social hier-

rchies and sex and thus for establishing zebrafish as a vertebrateodel for studying allostasis and phenotypic plasticity in behavior.The use of zebrafish as a model of stress has emerged

nly recently. The components of the hypothalamus–ypophysis–interrenal axis are expressed very early in devel-pment and cortisol is synthesized at hatch [3,4]. However,levated whole-body cortisol concentrations after exposure totressor are observed later on, at 97 h post fertilization [4]. Theain end product of the primary physiological response to stress

n zebrafish, like humans, is cortisol (rather than corticosterone, asn rodents). Whole-body cortisol concentrations in adult fish arelevated after exposure to acute net handling stress [63], chronicrowding stress [62] or social stress like direct and visual contactith a predator [7]. Recently it was also shown that whole-body

ortisol levels parallel behavioral indices of anxiety in zebrafishxposed to several anxiolytic and anxiogenic factors [30]. How-ver, there are few published reports on the neuroendocrine basisf stress and dominant–subordinate relationships in zebrafish30,51].

In this study we applied a dyadic social stress situation to charac-erize underlying neuroendocrinological changes in male zebrafish.

e investigated the presence and consistency of coping styles andhe role of the underlying neuroendocrine mediators of allostasisn establishing dominant–subordinate relationships in zebrafish.

. Materials and methods

.1. Animals and husbandry conditions

Zebrafish (D. rerio) were obtained from a breeding line maintained in theaboratory for more than 10 years [84]. In total, forty-two adult (body weight:6.4 ± 1.0 mg, Standard length: 35.9 ± 0.02 mm) males were used. Fish were heldnder a 14L:10D photoperiod, a water temperature of 27.5 ◦C, and were fed twoimes daily with fine flake food and once daily with Artemia spp. Fish were not fedn the day of sampling. An animal experiment permit was granted by the Office ofhe Regional Government of Southern Finland.

.2. Experimental design

Experiments were conducted in 3 l PVC tanks maintained in a recirculation sys-em. Each fish was anaesthetized in euglenol, weight and length measurements wereerformed, a tiny part of the upper or lower caudal fin lobe was cut for identification,nd then matched with a counterpart that did not differ in length by more than 1 mm.ehavioral interactions were checked in dyadic social interaction experiments. Fishere kept in pairs for a period of 5 days in 20 replicates.

Two to four hours from the beginning of the experiment identification of theominant individuals was carried out by two observers to avoid any subjective bias.ach tank was then observed twice daily to identify the dominant and subordinatendividual and recorded using a digital video camera for 5 min once daily for fiveonsecutive days. Recording was performed in the mean period between the twoeals. The order in which the tanks were recorded each day was selected using a

andom number generator.At the end of the experiment (Day 5) pairs of fish that showed a strong aggressive

dominant individuals) and freezing (subordinate individuals) behavior were nettedrom the tank and immediately killed by immersion in ice-cold water. In addition,

en males from the holding tanks were netted in less than 30 s, immediately killednd served as controls. Brains were dissected, frozen in 1.5-ml eppendorf tubes iniquid nitrogen, and placed at −80 ◦C for mRNA expression analysis. Then, fish headnd caudal fin were cut and trunks were weighed, frozen in 1.5-ml eppendorfs onry ice, and stored at −80 ◦C for cortisol determination.

Research 225 (2011) 529– 537

2.3. Behavioral analysis

Behavioral videos were analyzed by VideoLAN – VLC media player using slow-motion analysis when needed. The incidence of dominant behavior was quantifiedby measuring the duration (relative to session) for chasing, the number of attacksand the amount of time spent by the dominant fish in the lower one quarter of thetank (i.e. patrolling the subordinate fish). Subordinate behavior was quantified bymeasuring the duration, relative to session of freezing. Chasing was defined accord-ing to [59] as “direct/aggressive swim toward another fish in the aquarium causingit to increase its speed and possibly change direction”. Freezing was defined accord-ing to [10] as “a motionless state during which only the gills and, occasionally theeyes may move” which occurred mostly while the fish was in the bottom and in acorner, and in a few cases right below the water surface. Attack was defined as thedominant fish attacking the subordinate without or associated with biting trials.

2.4. Cortisol determination

Cortisol extraction was performed according to [27]. Briefly, samples were par-tially thawed on ice and homogenized in 5× (w/v) ice-cold phosphate-bufferedsaline (pH 7.4) with a rotor homogenizer. Cortisol was extracted from 2 × 250 �l ofhomogenate with 3 ml of diethyl ether. The water phase of the extract was allowedto freeze by placing tubes in −80 ◦C and the combined diethyl ether layer was trans-ferred into a new tube. The ether was evaporated by placement of tubes in a 45 ◦Cwater bath for 1 h and in room temperature for an additional 3 h. Samples were thenreconstituted in 250 �l of enzyme immunoassay buffer.

Cortisol was quantified by the use of a commercial enzyme immunoassay kit(Cayman Chemical, MI, USA). All samples were tested in duplicate. Displacementcurves (six different homogenates in serial dilutions 1:2–1:20) demonstrated lin-ear parallelism after logit–log transformation (standard curve r2 = 0.992 ± 0.001,slope = 1.076 ± 0.101). The recovery of cortisol (10 and 20 pg) added to zebrafishpool homogenates was 91.2 ± 4.3% (mean ± SEM, n = 2).

2.5. RNA isolation and cDNA sysnthesis

Total RNA was extracted using RNeasy mini Kit (Qiagen Inc., CA, USA). Twomicrograms of total RNA was reverse-transcripted using SuperScriptTM III reversetranscription (Invitrogen, CA, USA) according to the instructions of the manufac-turer.

2.6. Quantitative real-time PCR (qPCR)

The mRNA expression of genes encoding for corticotropin releasing factor (CRF),glucocorticoid receptor (GR), mineralcorticoid receptor (MR), tyrosine hydroxylase1 (TH1), tyrosine hydroxylase 2 (TH2), DOPA decarboxylase (DDC), dopamine �-hydroxylase (DBH), catechol-O-methyl transferase (COMT), histidine decarboxylase(HDC), galanin (GAL), hypocretin/orexin (Hcrt) and arginine vasotocin (AVT) wasexamined in brains of dominant and subordinate fish showing a strong aggressiveand freezing behavior, respectively. Quantitative real-time PCR was performed onthe Cepheid SmartCycler II (Sunnyvale, CA) using the SYBR®Premix Ex TaqTM (TakaraBio Inc., Japan). Primer sequences are listed in Table 2. Cycling parameters were asfollows: 95 ◦C for 30 s followed by 45 cycles at 95 ◦C for 10 s and at 60 ◦C for 45 s. Flu-orescence changes were monitored with SYBR Green after every cycle. Dissociationcurve analysis was performed (0.2 ◦C/s increase from 60 to 95 ◦C with continuousfluorescence readings) at the end of cycles to ensure that single amplifications wereobtained. All reactions were performed in duplicate. Results were evaluated withthe SmartCycler II software. The data were calculated by the comparative methodusing Ct values of �-actin as the reference control [54].

2.7. Statistical analysis

All data are presented as means ± standard error of the mean. Percentages oftime that individuals spent chasing, freezing, and in the lower quarter of the tankwere converted to arcsin values prior to statistical analysis. One way repeated mea-sures ANOVA was used to determine whether there were statistically significantchanges in aggression by dominant individuals toward their subordinates over thestudy period (days). One-way ANOVA or Kruskal–Wallis non-parametric tests wasused to determine significant differences in whole trunk cortisol concentrationand in the relative mRNA expression levels of candidate genes between dominant,subordinate and control fish. If significant (P < 0.05), Tukey’s or Dunn’s multiplecomparison test was applied to identify groups that were significantly different.To investigate potential correlations among the tested parameters, Pearson corre-lation coefficients were calculated, and the correlation matrices were subjected toa principal component analysis (PCA). Data analysis was performed by SigmaStatv3.1 software (Jandel Scientific Software, Germany).

3. Results

Aggressive behavior was observed in all pairs of fishsoon after the start of the experiment. However, a strong

M. Pavlidis et al. / Behavioural Brain Research 225 (2011) 529– 537 531

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eFig. 1. Quantification of zebrafish behavior throughout the course of the experiment. The incidence of dominant behavior was quantified by measuring the amount of time,relative to session (5 min), spent by the dominant fish on chasing or on patrolling the subordinate fish (lower one quarter of the tank), and the number of attacks per session.S essions

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for TH1, DOPA decarboxylase (DDC) and dopamine �-hydroxylase(DBH) (Fig. 4). The mRNA levels of TH2 were up-regulated in domi-nant fish compared to controls (P = 0.045), while COMT mRNA levels

CTRL DOM SUB0.0

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ubordinate–fear behavior was quantified by measuring the duration, relative to sampling day).

ominant–subordinate relationship was formed in ten out of theixteen pairs of fish (62.5%). This behavioral pattern was consistentnd robust until the end of the experiment. One day after the startf the experiment (Day 1) dominant males spent on average 36.7%range 1.9–85.5%) of their time on chasing, 30% (range 14.5–43.1%)f their time patrolling the lower quarter of the tank (i.e. swim-ing around on the lower quarter of the tank and confining the

ubordinate male to that level) and performed on average 140.7ttacks (range 2.4–338.7) per 5 min on the subordinate individualFig. 1). Toward the end of the experiment, there was a tendency for

decrease in the percent time spent on chasing (Day 5: 19.8 ± 6.4%,ange 0.7–37.5%) and the numbers of attacks per 5 min (Day 5:5.6 ± 23.9, range 1.0–130.7), and a tendency for an increase in theime spent patrolling the subordinate fish and (Day 5: 49.7 ± 12.1%,ange 19.6–94.1%), but these differences were not statistically sig-ificant (Fig. 1). The subordinate males displayed a strong freezingesponse soon after the start of the experiment (Day 1: 63.3 ± 14.1%,ange: 14.5–100%) and the rest of their time was spent on escap-ng from the attacks of the dominant fish. There was a tendencyor an increase in fear behavior toward the end of the experimentDay 5: 80.2 ± 6.4%, range: 62.5–99.3%), but this difference was nottatistically significant (Fig. 1).

Cortisol measurements were done to characterize one ofhe best markers of stress under the dyadic social stressondition. There was no statistically significant difference inhole-trunk cortisol concentrations between dominant and

ubordinate males (Fig. 2). However, both groups showed sta-istically significant higher trunk cortisol than controls (DOM:.98 ± 0.76 ng g−1 trunk weight (t.w.); SUB: 3.47 ± 1.31 ng g−1 t.w.;ON: 0.08 ± 0.02 ng g−1 ng g−1 t.w.; P = 0.008).

Expression of genes encoding proteins critical for the function-ng of the hypothalamus–hypophysis–interrenal axis showed that

here were no statistically significant differences in mRNA tran-cripts for CRFand GR (Fig. 3). The mRNA levels of mineralocorticoideceptor (MR) were up-regulated in dominant (approximately 4-old change) and subordinate (approximately 2.5-fold change) fish

of freezing. Bars indicate mean values of the observed time (%) ± SEM (n = 10 per

compared to controls, but this difference was statistically signifi-cant only among dominant and control zebrafish (P = 0.034, Fig. 3).The MR/GR ratio was higher in dominant and subordinate fish thanin controls (P = 0.016, Fig. 3).

Neurotransmitters play an important role in the regulation ofthe stress response. Catecholamines and histamine are among themost important mediators of adaptive changes in stressful con-ditions. Expression of genes encoding proteins involved in thebiosynthesis and catabolism of catecholamines showed that therewas no statistically significant differences in mRNA transcripts

Fig. 2. Differences (mean ± SEM, n = 10 per group) in whole-trunk (t.w.) cortisol(F) values between control (CTRL: 0.08 ± 0.01 ng g−1 t.w.), dominant (DOM) andsubordinate (SUB) adult zebrafish. Means with different letters indicate significantdifferences (P < 0.05) between the experimental groups.

532 M. Pavlidis et al. / Behavioural Brain Research 225 (2011) 529– 537

CRF

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ig. 3. Expression levels of corticotrophin releasing factor (CRF), glucocorticoid

CTRL), dominant (DOM) and subordinate (SUB) adult zebrafish. Expression levels oepresented as the mean ± SEM (n = 8–10 fish per group). Means with different lette

ere down-regulated in both dominant and subordinate fish com-ared to control (P = 0.023, Fig. 4).

Neuropeptide galanin coexists frequently within aminergic neu-ons and is involved in cognition, mood and neuroendocrineegulation [85]. There was no statistically significant differencen mRNA levels of GAL between dominant, subordinate and con-rol fish (Fig. 5). The mRNA levels of HDC were up-regulated inominant fish (4-fold change, P = 0.043) compared to control indi-iduals. In addition, mRNA levels of hypocretin/orexin (Hcrt) werep-regulated in dominant (6-fold change) compared to subordi-ate and control males (P = 0.008), suggesting a stimulatory role ofrexin in the hypothalamus–hypophysis–interrenal axis. Finally,RNA transcripts for arginine vasotocin (AVT), a known mediator

f stress and social behavior, were significantly higher in subordi-ate fish compared to controls (P = 0.014) (Fig. 5).

There was a statistically significant correlation between mRNAxpression levels of TH2, HDC, Hcrt, GR, MR and CRF genes (Table 1).

able 1tatistically significant Pearson product-moment correlation coefficient (r) in mRNAranscripts of tested genes.

HDC ORX GR MR CRF

TH2 0.965 0.954 0.770 0.858 0.708P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001

HDC 0.904 0.773 0.869 0.799P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001

ORX 0.774 0.871 0.615P < 0.0001 P < 0.0001 P = 0.005

GR 0.734 0.646P < 0.0001 P = 0.002

MR 0.855P < 0.0001

or (GR) and mineralocorticoid receptor (MR) genes, in brain samples of controlidate transcripts were obtained from Ct values normalized to ˇ-actin. Values wereicate significant differences (P < 0.05) between the experimental groups.

4. Discussion

In several vertebrate species adult males have been the subjectsof social stress studies, mainly because male dominance hierar-chies are much more evident and tend to influence a wider rangeof behaviors than in females [11]. Laboratory studies involving twoindividuals in dyadic situations are suitable in evoking agonisticinteractions comprising of aggressive or offensive and defensivecomponents. Among fish species, aggressive–dominant relation-ships have been documented in stickleback, Gasterosteus aculeatus[41], in the cichlid Nannacara anomala [16] and in rainbow trout,Oncorhynchus mykiss [70,82,83]. Aggressive behavior and domi-nance hierarchies have also been reported in zebrafish [51,56,72].Our results confirmed that dominant–subordinate relationshipsare present in male zebrafish housed in pairs. Dominant individ-uals utilize the entire aquarium and spend their time in patrolling,chasing and/or biting the subordinate individual, resembling aproactive coping style. The subordinate males display a strongfreezing behavioral strategy, typical for a reactive coping style [50].Dominance relationships, once established, are stable over the timecourse of the experiment (5 days), which is in accordance withprevious published data in zebrafish [35,71] (Table 2).

Our data demonstrate that differences in behavior betweendominant (DOM) and subordinate (SUB) zebrafish are associatedwith differences in several primary neuroendocrine mediators ofallostasis. Whether the observed changes are the prime cause forthe expression of the dominant/subordinate behavior or they rep-resent the outcome of the expressed behavior to provide adaptivefitness to the individual, cannot be answered by the present study.

Baseline whole-body cortisol concentrations ranged from 0.06 to0.13 ng g−1 and were similar or lower to those reported in pre-vious studies in adult zebrafish (approximately 0.03–0.09 ng g−1

[30]; 0.4–1.0 ng g−1 [61], 2.1–4.7 ng g−1 [62]). This may reflect

M. Pavlidis et al. / Behavioural Brain Research 225 (2011) 529– 537 533

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Fig. 4. Expression levels of tyrosine hydroxylase (TH1 and TH2), DOPA decarboxylase (DDC), dopamine �-hydroxylase (DBH) and catechol-O-methyl transferase (COMT)genes, in brain samples of control (CTRL), dominant (DOM) and subordinate (SUB) adult zebrafish. Expression levels of candidate transcripts were obtained from Ct valuesnormalized to ˇ-actin. Values were represented as the mean ± SEM (n = 8–10 fish per group). Means with different letters indicate significant differences (P < 0.05) betweenthe experimental groups.

Table 2PCR primer sequence.

gene Forward primer Reverse primer Accession no.

AVT CCCAGCCGGAGCCCATCAGA CCATGCAGACCTGCGCCTCC NM 131327.1COMT ACTCGACCACAGCGTCTGCT AGCCCATTCGCGGTGTCTGC NM 001083843CRH (CRF) ACGGTGGCTCTGCTCGTTGC GTCCGCGGCTGGCTGATTGA NM 001007379.1ddc CTGAGGAGGCCCCGGAGGAG GGGCTGTGCCAGTGGGTGAC NM 213342.1dbh TGCAACCAGTCCACAGCGCA GCTGTCCGCTCGCACCTCTG NM 001109694hcrt TCTACGAGATGCTGTGCCGAG CGTTTGCCAAGAGTGAGAATC BX005093hdc TTCATGCGTCCTCTCCTGC CCCCAGGCATGATGATGTTC EF150846.1nr3c1 (GR) ACAGCTTCTTCCAGCCTCAG CCGGTGTTCTCCTGTTTGAT NM 001020711gal GACCAACTGATACTCAGGATGCA ATCCCGAGTGTTTCTGTCAGAA XM 001920300nr3c2 (MR) CCCATTGAGGACCAAATCAC AGTAGAGCATTTGGGCGTTG NM 001100403.1th1 GACGGAAGATGATCGGAGACA CCGCCATGTTCCGATTTCT XM 682702.1th2 CTCCAGAAGAGAATGCCACATG ACGTTCACTCTCCAGCTGAGTG NM 001001829ˇ-actin CGAGCAGGAGATGGGAACC CAACGGAAACGCTCATTGC RTPrimerDB ID: 705

534 M. Pavlidis et al. / Behavioural Brain Research 225 (2011) 529– 537

AVT

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GALANIN

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F tidined ndidatr rs ind

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ig. 5. Expression levels of arginine vasotocin (AVT), Hypocretin/Orexin (Hcrt), hisominant (DOM) and subordinate (SUB) adult zebrafish. Expression levels of caepresented as the mean ± SEM (n = 8–10 fish per group). Means with different lette

ifferences among strains (genetic), degree of domestication orn the analytical procedure used for the determination of corti-ol. Both DOM and SUB fish showed statistically significant higherhole-body cortisol concentrations than controls. This difference

annot be related to differences in body side or to chronic crowdingtress, since our fish were sized-matched and held in a low densityapproximately 0.7 fish/l). Average whole-body cortisol concen-rations in DOM and SUB individuals were 1.98 and 3.47 ng g−1,ower than that reported for zebrafish exposed to 5 days ofrowding stress (5.2–14.3 ng g−1, [62] or mild transportation stress9.0 ng g−1, [61]). Thus, both DOM and SUB zebrafish displayedn increased hypothalamus–pituitary–interrenal (HPI) axis outputnd maintained a chronic mild increased secretion of cortisol due toocial stress. Most social stress studies conducted in rodents, guineaigs and non-human primates demonstrate that a reactive strategyi.e. a behavioral strategy typical of subordinate individuals) is asso-iated with higher hypothalamus–pituitary–adrenal (HPA) axis (i.e.he mammalian equivalent of the teleosts HPI axis) output anddrenal cortex sensitivity than dominant animals [21,24,48,50].owever, in many cases the dominants show HPA axis activation asell. Group housing of previously isolated mice increased plasma

orticosterone in both SUB and DOM but concentrations returno basal levels more rapidly in DOM than in SUB individuals [17].herefore, it seems that the relationship between rank and corti-ol (or corticosterone) is not identical among vertebrates, and thatocial instability is generally viewed as stressful in zebrafish.

Corticosteroid hormones exert their action at the cellu-ar/molecular level by binding to two classes of receptors – the

ineralocorticoid receptors (MRs) which, at least in higher verte-

rates, have low affinity for corticosteroids and have an importantegulatory role under normal baseline glucocorticoid concentra-ions, and the glucocorticoid receptors (GRs) which are thoughto become activated in response to stress and to be important in

decarboxylase (HDC), and galanin (GAL) genes, in brain samples of control (CTRL),e transcripts were obtained from Ct values normalized to ˇ-actin. Values wereicate significant differences (P < 0.05) between the experimental groups.

terminating the stress response [18,22,50]. Most teleost speciesstudied today possess two GRs, however, zebrafish possess a singleGR gene [5,69,75]. The present study revealed no difference in brainGR mRNA expression levels, an up-regulation of MR mRNA tran-scripts in DOM and a significant increase in the MR/GR ratio in bothDOM and SUB compared to control individuals. Studies on miceshowed no differences in mRNA expression of MR and GR receptorsin the hippocampus of aggressive and non-aggressive individualsunder baseline conditions [50,77]. However, after forced swim-ming there was a significant increase in MR mRNA expressionlevels in hippocampal CA1 region of aggressive mice and in allhippocampal regions of non-aggressive mice but no difference inGR mRNA expression levels [50,77]. Other studies also suggestthat increased concentrations of corticosterone operating throughhippocampal MR induce freezing behavior in rats [49,50]. In wild-caught European starlings (Sturnus vulgaris), chronic stress resultedin significantly lower GR mRNA expression in the hypothalamicparaventricular nucleus and in hippocampal MR mRNA expression[29]. The present study demonstrated that social stress increasedMR/GR balance. It seems that the observed moderately high levelsof cortisol in both DOM and SUB fish lead to increased expressionof MR mRNA to guarantee a stable background of neuronal firing inorder to cope with the social stress [22,23]. From studies based onmammals and humans, imbalance in the ratio of GR to MR followingexposure to chronic stress increases the overall “wear and tear” inthe organism, and thus the allostatic load [55], and has been linkedto several health disorders, such as depression [25], post-traumaticstress [25] and anxiety [39].

The neuropeptides corticotropin-releasing factor (CFR) and argi-

nine vasopressin (AVP, the mammalian homologue of argininevasotocin—AVT) are known to be involved in the regulation ofHPA/HPI axis and in the mediation of stress related and socialbehaviors [18,2,11,12,6]. The present study did not find any effect

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M. Pavlidis et al. / Behavioural

f social stress on brain CRF mRNA transcripts but there was aignificant up-regulation of AVT mRNA levels in subordinate fishompared to controls. In a recent study CRF gene over-expressionas observed in the hypothalamus of dominant and in the telen-

ephalon of subordinate male zebrafish, held in pairs of two malesnd two females for one day, but there was no difference on Day 532]. Psychosocial stress in tree shrews (Tupaia belangeri) resultedn a down-regulation of CRF receptors in several brain regions ofubordinate animals [11,34]. Behavioral effects of AVP/AVT differetween species and the social system (colonial or territorial) ofhe particular species. Intracerebroventricular injection of AVT inuvenile rainbow trout induced suppression of aggressive behavioruggesting that AVT acts inhibitory on aggression in this species6]. Similarly, intraperitoneal injection of AVT in territorial pup-sh, Cyprinodon nevadensis amargosae, males resulted in decreasedggression [52]. On the contrary, intraperitoneal injection of AVTncreased aggression in non-territorial males of bluehead wrasse,halassoma bifasciatum [68]. Studies in hamsters and rats providelso evidences for a role of AVP in aggressive behavior. Recent stud-es showed that the effect of AVP is related to the level of aggression.n particular, more aggressive rats decreased septal release of AVP

hereas less aggressive rats increased septal release of AVP duringggressive interactions [9]. Zebrafish are a shoaling species, how-ver, both sexes can establish dominance hierarchies and males useo defend territories. Therefore, the observed AVT up-regulation inUB zebrafish indicates that AVT is needed to increase aggressionn non-territorial subordinate individuals. The conflicting resultseported in the literature fit with Larson et al.’s [51] observation ofarge-AVT expressing cells in dominants and a smaller, but moreumerous population of AVT cells (in a different hypothalamicegion) in subordinates pointing to a finer-grained organization ofhe neuronal basis of social behavior.

Acute and chronic stressors have been shown to stimulate theentral noradrenergic system on several animal models. Studiesn male mice have demonstrated that repeated aggression leadso the activation of brain dopaminergic systems [15]. Tyrosineydroxylase is the enzyme responsible for catalyzing the con-ersion of l-tyrosine to l-dihydroxyphenylalanine (DOPA), andhereby acts as a key modulator of dopaminergic and adrener-ic/noradrenergic neurotransmission, and it is commonly used as aarker to represent the catecholaminergic neuron [59]. Zebrafish

as two non-allelic th genes, th1 and th2 [20]. Enhanced expres-ion of th2 gene was associated in the present study with dominantsh. There was also a statistically significant correlation betweenRNA expression levels of TH2 and HDC, Hcrt, GR, MR and CRF

enes. This result is in line with the enhanced expression of tyro-ine hydroxylase and dopamine transporter genes responsible foropamine biosynthesis and re-uptake, respectively, in aggressiveebrafish [32]. It is also in accordance with enhanced expressionf the th gene in male mice following repeated positive fighting15], and of the stimulatory role of dopamine in dominant mam-

als [15,33]. In juvenile arctic char, Salvelinus alpinus, L-Dopa havelso been reported to stimulate aggressive behavior [42]. Catechol--methyltransferase (COMT) is an intracellular enzyme located in

he postsynaptic neuron and it is involved in the inactivation of theeurotransmitters dopamine, epinephrine, and norepinephrine.ebrafish has two putative COMT genes [59]. We observed down-egulation of COMT mRNA levels in both dominant and subordinatesh compared to control. This is a novel finding for fish and whether

t is related with changes in sex steroids (not analyzed in the presenttudy) [65] or with the regulation of pain perception, as in rodents28,57,74], remains to be tested.

The histaminergic system has been involved in hormone regu-ation, feeding and drinking, sleep–wake cycle, consciousness and

emory [37]. The zebrafish histaminergic system consists of pos-erior hypothalamic neurons with projections to other brain areas,

Research 225 (2011) 529– 537 535

such as dorsal telencephalon, optic rectum and brain stem [31,44].HDC is the enzyme involved in the biosynthesis of histamine fromL-histidine. In the present study, mRNA levels of HDC were up-regulated in dominant fish compared to control males. This is inaccordance with the overexpression of th gene in the hypothalamusof aggressive male zebrafish one day following exposure to socialstress [32] and support the evidences for a role of histamine in stressand arousal [37,73,80]. This is further supported by the observedstatistically significant correlation between mRNA expression lev-els of HDC, Hcrt, GR, MR and CRF genes. An indirect role of histaminethrough regulation of the release of prolactin, growth hormone,adrenocorticotropic hormone and thyrotropin-releasing hormonecannot be also excluded [37].

Galanin is a neuropeptide that can be found in approximately80% of the tyrosine hydroxylase containing neurons in the rat locuscoeruleus [11]. Galanin gene expression is regulated by steroids,thyroid hormones, nerve growth factor, and nerve injury. Chronicsubordination stress resulted in increased levels of preprogalaninmRNa in the locus coeruleus of subordinate rats and the levels werepositively correlated with the number of woods per animal [40].However, in our study there was no effect of social stress on galaninmRNA expression levels.

Orexin/hypocretin (Hcrt) is a neuropeptide that promotes wake-fulness and energy expenditure. Lack of functional hcrt or its Gprotein-coupled receptor is associated with narcolepsy in humansand experimental animals [19,53,59]. In zebrafish brain, hcrtmRNA-containing neurons are found bilaterally in the rostralhypothalamus, on the medial side of the medial forebrain bundleand caudally on the top of the lateral recess [45,59]. In the presentstudy, there was a clear up-regulation of hypocretin/orexin (Hcrt)mRNA levels in dominant compared to subordinate and controlzebrafish. Furthermore there was a statistically significant corre-lation mRNA expression levels between of Hcrt, GR, MR and CRFgenes. These novel findings extend the data obtained from studiesconducted in rodents and provide more evidences for a stimulatoryrole of orexin in the HPA/HPI axis [66]. Pharmacological adminis-tration of synthetic orexin in rats resulted in increased release ofadrenocorticotropin hormone (ACTH) and prolactin (PRL) throughthe enhancement of corticotropin-releasing hormone (CRH) releaseand in physical responses that mirrored stress/anxiety-relatedbehaviors [36,1,67,66].

In conclusion, the obtained results support the utility ofzebrafish as an animal model for studying social stress and allostasisin vertebrates. By focusing on strong dominant and freezing behav-ior we identified candidate genes that appear to play an importantrole in the neuroendocrine regulation of social stress in fish. Theproposed methodology may assist to a better understanding ofthe underlying mechanisms of chronic stress and anxiety in non-mammalian species.

Acknowledgments

The authors would like to thank Mr. Henri Koivula for fish hus-bandry and sampling and Mrs. Anna Lehtonen, Noora Kanerva andSusanna Norrbacka for their technical assistance. This study waspart of a research sabbatical leave granted to Dr. M. Pavlidis by theUniversity of Crete, Ministry of Education, Lifelong Learning andReligious Affairs.

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