cortisol reduces cell proliferation in the telencephalon of rainbow trout (oncorhynchus mykiss)

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Cortisol reduces cell proliferation in the telencephalon of rainbow trout (Oncorhynchus mykiss) Christina Sørensen a, , Linda C. Bohlin a , Øyvind Øverli b , Göran E. Nilsson a a Department of Molecular Biosciences, University of Oslo, P.O. Box 1041, N-0316 Oslo, Norway b Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, 5003 Ås, Norway abstract article info Article history: Received 11 October 2010 Received in revised form 20 December 2010 Accepted 22 December 2010 Keywords: Cortisol Neurogenesis Brain Telencephalon PCNA Immunohistochemistry The sh brain grows throughout life, and new cells are added continuously in all major brain areas. As in mammals, the rate of adult brain cell proliferation in sh can be regulated by external factors including environmental complexity and interaction with conspecics. We have recently demonstrated that the stress experienced by subordinate rainbow trout in social hierarchies leads to a marked suppression of brain cell proliferation in the telencephalon, and that this is accompanied by an increase in plasma levels of cortisol. Corticosteroid hormones are known to suppress adult neurogenesis in mammals, and to investigate whether this is also the case in sh, rainbow trout were fed feed containing either a low or a high dose of cortisol for 6 days. Compared to control animals receiving regular feed, both cortisol treated groups had signicantly elevated cortisol levels 24 h after the last feeding, with the high group having levels comparable to those previously reported in socially stressed sh. To quantify cell proliferation, immunohistochemistry for proliferating cell nuclear antigen (PCNA) was performed to identify actively cycling cells. The density of PCNA-positive nuclei in the telencephalon was reduced by about 50% in both cortisol treated groups. The effect of cortisol on brain cell proliferation did not reect a general down regulation of growth, as only the high cortisol group had reduced growth rate, and there was no correlation between brain cell proliferation and growth rate in any group. These results indicate that the reduced proliferative activity seen in brains of socially stressed sh is mediated by cortisol, and that there is a similar suppressive effect of cortisol on brain cell proliferation in the teleost forebrain as in the mammalian hippocampus. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Teleost shes of the family Salmonidae, in particular species of the genera Oncorhynchus and Salmo, have been frequently studied for their tendency to form clear dominance based social hierarchies both in the wild and under experimental conditions [18]. Territoriality is expressed at several life stages in salmonids, and subordinate individuals often show a range of behavioural and physiological indicators of chronic stress, including anorexia, altered locomotor patterns and reduced aggression, as well as increased plasma cortisol levels, brain serotonergic activity, and standard metabolic rate [1,6,7,912]. We have recently demonstrated that socially subordi- nate rainbow trout (Oncorhynchus mykiss) have reduced brain cell proliferation in the forebrain compared to socially isolated non- stressed individuals [13]. This effect is similar to that seen in contemporary models of psychosocial stress in mammals, where social subordination leads to reduced neurogenesis in the dentate gyrus of the hippocampus [1416]. A reduced rate of neurogenesis is in turn believed to affect mood and cognition, and is believed to be involved in the pathophysiology of depressive disorders in man (for reviews see Refs. [1720]). In comparison to mammals, sh have a much higher prevalence [2125], and rate of adult brain cell proliferation [24,26], and for this reason sh have been promoted as a potentially important model to understand the evolution and function of adult neurogenesis [27]. The neuroanatomy of adult brain cell proliferation and its role in regeneration after injury is well-studied in sh (for review see Ref. [28]), but it has only recently become evident that the rate of cell proliferation in the sh brain can be regulated in response to external factors. Environmental enrichment [29], pheromone exposure [30] and social communication [31,32] have all been shown to affect the rate of brain cell proliferation in parts of the teleost brain. Yet, very little is known about the mechanisms involved in this regulation. In our previous study, socially subordinate individuals, which had reduced telencephalic brain cell proliferation also showed signi- cantly elevated plasma levels of cortisol [13]. Cortisol is the main corticosteroid hormone in sh and in addition to its homeostatic function in osmoregulation and energy metabolism (For reviews see Refs. [33,34]) it is typically elevated during acute and chronic stress [3436]. Cortisol is involved in mediating several of the behavioural Physiology & Behavior 102 (2011) 518523 Corresponding author. Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, N-0316 Oslo, Norway. Tel.: +47 99471471; fax: +47 22856041. E-mail address: [email protected] (C. Sørensen). 0031-9384/$ see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2010.12.023 Contents lists available at ScienceDirect Physiology & Behavior journal homepage: www.elsevier.com/locate/phb

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Physiology & Behavior 102 (2011) 518–523

Contents lists available at ScienceDirect

Physiology & Behavior

j ourna l homepage: www.e lsev ie r.com/ locate /phb

Cortisol reduces cell proliferation in the telencephalon of rainbow trout(Oncorhynchus mykiss)

Christina Sørensen a,⁎, Linda C. Bohlin a, Øyvind Øverli b, Göran E. Nilsson a

a Department of Molecular Biosciences, University of Oslo, P.O. Box 1041, N-0316 Oslo, Norwayb Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, 5003 Ås, Norway

⁎ Corresponding author. Department of Molecular BiosBox 1041 Blindern, N-0316 Oslo, Norway. Tel.: +47 9947

E-mail address: [email protected] (C.

0031-9384/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.physbeh.2010.12.023

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 October 2010Received in revised form 20 December 2010Accepted 22 December 2010

Keywords:CortisolNeurogenesisBrainTelencephalonPCNAImmunohistochemistry

The fish brain grows throughout life, and new cells are added continuously in all major brain areas. As inmammals, the rate of adult brain cell proliferation in fish can be regulated by external factors includingenvironmental complexity and interaction with conspecifics. We have recently demonstrated that the stressexperienced by subordinate rainbow trout in social hierarchies leads to a marked suppression of brain cellproliferation in the telencephalon, and that this is accompanied by an increase in plasma levels of cortisol.Corticosteroid hormones are known to suppress adult neurogenesis in mammals, and to investigate whetherthis is also the case in fish, rainbow trout were fed feed containing either a low or a high dose of cortisol for6 days. Compared to control animals receiving regular feed, both cortisol treated groups had significantlyelevated cortisol levels 24 h after the last feeding, with the high group having levels comparable to thosepreviously reported in socially stressed fish. To quantify cell proliferation, immunohistochemistry forproliferating cell nuclear antigen (PCNA) was performed to identify actively cycling cells. The density ofPCNA-positive nuclei in the telencephalon was reduced by about 50% in both cortisol treated groups. Theeffect of cortisol on brain cell proliferation did not reflect a general down regulation of growth, as only thehigh cortisol group had reduced growth rate, and there was no correlation between brain cell proliferationand growth rate in any group. These results indicate that the reduced proliferative activity seen in brains ofsocially stressed fish is mediated by cortisol, and that there is a similar suppressive effect of cortisol on braincell proliferation in the teleost forebrain as in the mammalian hippocampus.

ciences, University of Oslo, P.O.1471; fax: +47 22856041.Sørensen).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

Teleost fishes of the family Salmonidae, in particular species of thegenera Oncorhynchus and Salmo, have been frequently studied fortheir tendency to form clear dominance based social hierarchies bothin the wild and under experimental conditions [1–8]. Territoriality isexpressed at several life stages in salmonids, and subordinateindividuals often show a range of behavioural and physiologicalindicators of chronic stress, including anorexia, altered locomotorpatterns and reduced aggression, as well as increased plasma cortisollevels, brain serotonergic activity, and standard metabolic rate[1,6,7,9–12]. We have recently demonstrated that socially subordi-nate rainbow trout (Oncorhynchus mykiss) have reduced brain cellproliferation in the forebrain compared to socially isolated non-stressed individuals [13]. This effect is similar to that seen incontemporary models of psychosocial stress in mammals, wheresocial subordination leads to reduced neurogenesis in the dentategyrus of the hippocampus [14–16]. A reduced rate of neurogenesis is

in turn believed to affect mood and cognition, and is believed to beinvolved in the pathophysiology of depressive disorders in man (forreviews see Refs. [17–20]).

In comparison to mammals, fish have a much higher prevalence[21–25], and rate of adult brain cell proliferation [24,26], and for thisreason fish have been promoted as a potentially important model tounderstand the evolution and function of adult neurogenesis [27]. Theneuroanatomy of adult brain cell proliferation and its role inregeneration after injury is well-studied in fish (for review see Ref.[28]), but it has only recently become evident that the rate of cellproliferation in the fish brain can be regulated in response to externalfactors. Environmental enrichment [29], pheromone exposure [30]and social communication [31,32] have all been shown to affect therate of brain cell proliferation in parts of the teleost brain. Yet, verylittle is known about the mechanisms involved in this regulation.

In our previous study, socially subordinate individuals, which hadreduced telencephalic brain cell proliferation also showed signifi-cantly elevated plasma levels of cortisol [13]. Cortisol is the maincorticosteroid hormone in fish and in addition to its homeostaticfunction in osmoregulation and energy metabolism (For reviews seeRefs. [33,34]) it is typically elevated during acute and chronic stress[34–36]. Cortisol is involved in mediating several of the behavioural

519C. Sørensen et al. / Physiology & Behavior 102 (2011) 518–523

and physiological effects typically seen in subordinate fish, andtreatment with cortisol has been shown to reduce appetite, growthand physical condition in group reared fish [37–39]. Cortisoltreatment also affects aggression and locomotor activity during socialinteraction [40,41], and chronic cortisol treatment leads to increasedserotonergic activity in the telencephalon [42]. Cortisol also affectscell addition in the fish brain [31], and is known to reduce adultneurogenesis in the rodent hippocampus [43,44]. Cortisol is thus alikely candidate for mediating the suppressive effect of social stress ontelencephalic brain cell proliferation in rainbow trout.

Consequently the aim of the current study was to investigate theeffect of cortisol treatment on brain cell proliferation in the rainbowtrout. This aim was approached by giving cortisol containing feed torainbow trout and assessing cell proliferation in the telencephalon bymeasuring the density of proliferating cell nuclear antigen (PCNA)positive nuclei. This approach allowed for investigation of the effectsof cortisol without influence of additional stress associated withinjecting the fish with cortisol and amarker of cell proliferation like 5-bromo-3′-deoxyuridine (BrdU).

2. Materials and methods

2.1. Experimental animals and procedure

Juvenile rainbow trout were obtained from a commercial breeder(Valdres Ørretoppdrett, Valdres, Norway) andwere transported to theUniversity of Oslo aquarium facility where a group of approximately250 individuals was maintained in a 750 l holding tank continuouslysupplied with dechlorinated Oslo tap water (100 lh−1) at 4–6 °C.Lighting followed a 12 h light/12 h darkness cycle, and the fish werefed daily 1% of their body weight with pelleted trout food (3.0 mm,Skretting, Stavanger, Norway) between 12.00 and 16.00. After6 weeks of group holding, 32 fish weighing from 91.9 g to 225.3 g(156.3 g±6.2 g) were isolated in 50 l compartments separated byopaque PVC walls in 250 l glass observation aquaria (c.f. [7,11]).Aquaria were continuously aerated and supplied with dechlorinatedOslo tap water (4.7–5.4 °C). The fish were allowed to acclimate to theexperimental set-up for 14 days, during which they were fed pelletedtrout feed (1% of body weight) once daily between 12.00 and 16.00 bydropping pellets one by one into the aquarium. After 5 min anyremaining food was removed. After acclimation, the fish wereanesthetised lightly in a bath of 0.06 mg l−1 eugenol (Sigma Aldrich)and weighed. Starting the following day the daily food ration wasreplaced by control (n=8), low (n=12) and high (n=12) cortisolcontaining feed. Cortisol was incorporated into the diet by immersingfeed pellets in 96% ethanol containing dissolved cortisol (hydrocor-tisone, Sigma) corresponding to 75 mg cortisol kg−1 pellets (lowcortisol feed) and 600 mg kg−1 pellets (high cortisol feed). Controlfeed was immersed in pure 96% ethanol. The ethanol was evaporatedover night in room temperature to render the cortisol incorporated inthe food [45]. The concentrations used were based on the results fromØverli et al. [40], where a concentration of 600 mg kg−1 pellets and adaily ration of 1% of the body weight caused plasma cortisol levelssimilar to those observed in highly stressed rainbow trout(N100 ng ml−1). To address possible dose dependent effects, a lowconcentration (75 mg kg−1 pellets), that was found in a pilot study toproduce a low, but significant increase in plasma cortisol levels, wasalso used. Experimental feed was administered as during acclimationonce daily between 12.00 and 16.00 for six days and the exact dailyfood intake for each fish was recorded by counting the number of feedpellets eaten by each fish.

2.2. Sampling and tissue preparation

After six days of cortisol treatment, 24 h after the last feeding, thefish were anesthetised in a bath of 0.1% Ethyl 3-aminobenzoate

methanesulfonate (Sigma), and blood samples were collected fromthe caudal vein whereupon the fish were killed by decapitation. Bloodwas centrifuged for 3 min at 4 °C before the plasma was frozen inliquid N2. Brains were dissected out and fixed in 4% paraformalalde-hyde in PBS (phosphate buffered saline, 0.1 mM, pH 7.4) for 24 h, andthen transferred to 15% sucrose for 24 h and finally 20% sucrose for24 h. The brainswere embedded in Tissue Tek O.C.T-medium (Sakura)and frozen in isopentane cooled to its freezing point (−160 °C) inliquid N2. The brains were stored at −80 °C until being sectioned at25 μm in a cryostat (MicromHM560) andmounted on SuperFrostPlus(Thermo Scientific) slides.

2.3. PCNA immunohistochemistry

The slides were thawed, washed in PBS (5×5 min), and post-fixedin paraformaldehyde (4%, 10 min). Epitope retrieval was performedusing citric acid buffer (10 mM, pH 6.0, 85 °C, 60 min). Slides werewashed in PBS and unspecific binding blocked with 6% skim milkpowder (Acumedic) and 0.03% Triton X-100 (Sigma) in PBS. Sectionswere treatedwith primary antibody for 24 h at 4 °C (1:50, Rabbit Anti-PCNA, Dako Cytomation in PBS with 0.6% skimmilk powder and 0.03%Triton X-100) and washed 3×5 min with PBS. Endogenous peroxi-dase activity was blocked with 3% H2O2 (Sigma, 15 min) and washed3×5 minwith PBS. Slides were incubatedwith secondary antibody for30 min (EnVision+® System Labelled Polymer-HRP, Anti-Rabbit,Dako) and washed 3×5 min with PBS. Finally peroxidase activitywas visualized with 3,3'-diaminobenzidine (DAB, Applichem,15 min), washed 2×5 min with dH2O and coverslipped usingpermanent mounting medium.

2.4. Quantification of PCNA-positive nuclei

Sections were analyzed and photographed using a Zeiss AxioplanImaging microscope with an Axiocam HR camera (2600×2060 pixelsresolution) and Axiovision 3.1 software. All images from each sectionwere stitched together in Adobe Photoshop CS3. Every fourth 25 μmsection throughout the telencephalon was analyzed, giving a mean of22 sections per fish. The number of stained nuclei per investigatedbrain volumewas calculated for each fish based on the total number ofstained nuclei in all sections and the investigated volume (calculatedfrom the area of each section as determined using Adobe PhotoshopCS3 and the thickness of 25 μm). Areas from the olfactory bulbs andthe preoptic areas were excluded from analysis when these werepresent in the photographed sections.

2.5. Radioimmunoassay quantification of plasma cortisol

Plasma cortisol levels were determined using a radioimmunoassaybased on the assay by Pottinger and Carrick [46]. Steroids wereextracted from plasma with ethyl acetate (Merck, 1:5 plasma:ethylacetate), and the extract was vortexed for 30 s followed bycentrifugation at 14.000 rpm for 2 min. Supernatants (20–200 μl,depending on the expected stress level of the fish, to ensure that thecortisol levels were within the range of the standard curve) weretransferred to 1.5 ml eppendorf tubes. A zero sample and anonspecific binding (blank) control were made with pure ethylacetate, and a dilution series standard curve of cortisol (hydrocorti-sone, Sigma) in ethyl acetate of concentrations from 8 pg/µl to0.125 pg/µl was produced. All samples, standards and controls wererun in duplicate. Identical aliquots (50 μl) of ethyl acetate withapproximately 15.000 cpm of [1,2,6,7-3H] cortisol (Amersham Phar-macia Biotech, 60 Ci mmol−1) were added to all tubes and the ethylacetate was evaporated in an exsiccator coupled to a water jet pump.Antibody (Donkey anti cortisol, AbD Serotec, 1:600) in assay buffer;phosphate buffered saline (PBS tablets, Sigma) containing bovineserum albumin (0.1%, Sigma), was added to each tube except the

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blank tubes that received only assay buffer and all tubes wereincubated over night at 4 °C. The tubes were placed on ice and 100 μlof ice cold, stirred dextran-coated charcoal in PBS (1.0% activatedcharcoal, Sigma; 0.2% dextran, Sigma) was added to each tube. Tubeswere vortexed and incubated at 4 °C for 10 min, whereupon theywere centrifuged (8000 rpm at 4 °C for 10 min). A 200 μl aliquot fromthe supernatant of each sample was added to 4.0 ml scintillation fluid(Ultima Gold, PerkinElmer) in a scintillation vial, mixed by inversion,and counted on a Packard Tri-Carb 1900 under standard 3Hconditions. A 3-parameter hyperbolic function was fitted to the plotof the percentage of 3H cortisol bound against cortisol concentrationin the standard curve samples, and the equation was used to findcortisol concentrations in the unknown samples, and the equationwas used to find cortisol concentrations in the unknown samples.Plasma cortisol levels were calculated by correcting for the amount ofextract analyzed. Sensitivity (minimal detection limit) of the assaywas 0.1 ng/ml and the inter- and intra-assay variation were both lessthan 10% (n=6 and n=5 respectively).

2.6. Statistical analyses

Values are presented as means±S.E.M. Statistical analyses wereperformed using Statistica 8.0 (StatSoft, Inc., Tulsa, Oklahoma) andSigmaPlot 11.0 (Systat Software, Inc., Evanston, Illinois). Data onplasma cortisol levels and feed intake did not show variancehomogeneity (Levene's test), and non-parametric Kruskal–Wallistest was used, followed by one-tailed Mann–Whitney U test withBonferroni correction for groupwise comparisons in the case ofplasma cortisol data. All other data (weight, growth rates, and PCNAimmunostaining) were analyzed by parametric ANOVA followed byTukey HSD post-hoc tests where relevant. The relationship betweenestimated cortisol intake and plasma cortisol levels, as well as thedensity of PCNA-positive nuclei and plasma cortisol levels, weight,growth and feed intake were analyzed within groups by linearregression. Comparison of means between subsets analyzed for braincell proliferation with means for whole groups was done by T-test forindependent samples by group. For groupwise comparisons of subsetmeans, the same tests that were used for whole groups were applied.Significance level was set to p≤0.05.

3. Results

3.1. Treatment effects on plasma cortisol

There was a significant effect of treatment with cortisol feed onplasma cortisol levels (Kruskal–Wallis ANOVA; pb0.001, Fig. 1).Thus, intake of cortisol treated feed for six days gave both cortisolgroups significantly elevated plasma cortisol levels (Low cortisol: 4.4±

Fig. 1. Effect of 6 days intake of feedwith either high (600 mg kg−1) or low (75 mg kg−1)cortisol content, compared to animals receiving cortisol free control feed. Different lettersindicate a significant difference between groups.

1.1 ng ml−1; High cortisol: 67.2±14.6 ng ml−1) compared to thecontrol group (1.3±0.3 ng ml−1), (p=0.04 and pb0.001, respectively)and low and high cortisol groups were also significantly different(pb0.001). Estimated daily cortisol intake per kg body weight for allcortisol treated animals was calculated on the basis of cortisolconcentration in the feed and average daily feed intake for theexperimental period, and found to be 0.20±0.02 mg kg−1day−1 forthe low cortisol group, and 1.02±0.16 mg kg−1day−1 for the highcortisol group. Within the high cortisol group there was a significantcorrelation between estimated cortisol intake and plasma cortisol level(linear regression, r2=0.61, p=0.003), while no such correlationexisted in the low cortisol group (linear regression, r2=0.01, p=0.74).

3.2. Weight, feed intake and growth rates

There was no difference in weight between the groups either at thebeginning of the cortisol treatment period (ANOVA; F(2,29) =1.21,p=0.31), or at the time of sampling (ANOVA; F(2,29) =1.12, p=0.34).Cortisol treatment had no effect on feed intake (Kruskal–WallisANOVA; p=0.07), with the groups taking in an average of 0.19±0.07% bw day−1, 0.27±0.02% bw day−1 and 0.17±0.03% bw day−1

(Control, low cortisol and high cortisol groups respectively). There was,however, a significant effect of the treatment on growth rate (ANOVA,F(2,29) =6.43, p=0.005, Fig. 2), with the high cortisol group having asignificantly lower growth rate than both the control and low cortisolgroups (TukeyHSDpost-hoc; p=0.01 and p=0.02 respectively),whiletherewasnodifference between the two latter groups (TukeyHSDpost-hoc; p=0.86).

3.3. Cell proliferation

Immunohistochemistry for PCNA was performed on brains from17 individuals (control: n=6, low cortisol: n=6, and high cortisol:n=5) and staining resulted in distinctly labelled nuclei (Fig. 3).Nuclei positive for PCNAwere mainly found in the ventricular areas ofthe ventral and dorsal parts of the telencephalon. For quantification ofthe effect of cortisol on the prevalence of PCNA-positive cells, datafrom all telencephalic areas were pooled and divided by the totalinvestigated volume as determined from the area of each section andthe section thickness. There was no difference in analyzed volumebetween the groups (ANOVA; F(2,14) =2.21, p=0.15). Both cortisoltreated groups had about 50% fewer PCNA-positive nucleiper analyzed volume of the telencephalon than the control group(165±16; 89±10 and 110±24 PCNA-positive nuclei per mm3

analyzed telencephalon volume in control, low cortisol and highcortisol groups respectively. ANOVA; F(2,14) =12.83, pb0.001; Tukey

Fig. 2. Growth rate offish receiving feed containinghigh (600mg kg−1) or low(75mg kg−1)levels of cortisol compared to animals receiving cortisol free feed. Different letters indicate asignificant difference between groups.

Fig. 3. Photomicrograph of representative PCNA-immunoreactive cells localised in theventral area of the rainbow trout telencephalon. Scale bar=20 μm.

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HSD post-hoc: p=0.002 for both low and high cortisol vs. controlgroup, Fig. 4), while there was no significant difference between thecortisol treated groups (Tukey HSD post-hoc: p=1.00). There was nocorrelation (linear regression) between the density of PCNA-positivenuclei and weight, growth rate, plasma cortisol levels, estimatedcortisol intake or food intake (data not shown). To verify that thesubsets analyzed for brain cell proliferation correctly reflected theresults for the full groups, means of full groups and subsets werecompared for all measured parameters, and in no case were thesubsets significantly different from the full groups (data not shown).All groupwise comparisons were also done for the subsets. For data onweight, feed intake, estimated cortisol intake and growth during theacclimation period, the results did not deviate from those of the fullgroups (data not shown). As opposed to the whole groups, there wasno significant effect on growth during cortisol treatment in the subset(Kruskal–Wallis ANOVA; p=0.24). For plasma cortisol levels in thesubset, there was a significant effect of cortisol treatment (Kruskal–

Fig. 4. Density of PCNA-positive nuclei in the telencephalon of fish receiving feedcontaining high (600 mg kg−1) or low (75 mg kg−1) levels of cortisol compared toanimals receiving cortisol free feed. Different letters indicate a significant differencebetween the groups.

Wallis ANOVA; p=0.004), with the control group significantlydifferent from both low (p=0.05) and high (p=0.006) cortisoltreated groups. The two cortisol groups were not significantlydifferent (p=0.07). The lack of some significant differences in thesubsets compared to the whole data sets were likely related to loss ofstatistical power caused by the lower number of animals used in thesubset involving PCNA immunohistochemistry.

4. Discussion

The main finding of the present study was that 6 days of oraladministration of cortisol at two different doses (0.2 mg g−1day−1

and 1.0 mg g−1 day−1) causes a significant reduction of brain cellproliferation in the rainbow trout telencephalon as measured bydensity of PCNA-positive nuclei.

Administration of cortisol in the feed caused elevation of plasmacortisol levels in both the low and high cortisol groups 24 h after thelast feeding. Cortisol levels in the high cortisol groupwere in the rangeof those seen in subordinate rainbow trout after 1–7 days of socialinteraction [7,11,47], and also similar to the average cortisol level ofsubordinate fish in our previous studywhere social subordinationwasdemonstrated to reduce cell proliferation in the telencephalon [13].The plasma cortisol levels seen in the low cortisol treatment groupwere considerably lower, but still significantly increased compared tothe control group. Oral administration of cortisol is known to causeelevated plasma cortisol levels for 24–48 h after each meal, with peakvalues appearing between 1 and 24 h after food intake, depending onthe dosage [37,45,48], for review see Ref. [49]. Based on temporalprofiles of plasma cortisol levels after oral administration of similardoses of cortisol [48] it can be assumed that the cortisol levels in thehigh cortisol group have reached a daily peak value within 12 h offeeding, while the plasma cortisol levels of the low cortisol group havebeen relatively stable during the duration of the experiment. Plasmacortisol levels correlated with estimated cortisol intake in the high,but not in the low cortisol group. The lack of correlation in the lowcortisol group could indicate that the cortisol intake was low enoughto be influenced by individual differences in cortisol clearance. Ratesof cortisol clearance are known to vary with a multitude of factors,including stress, maturity state and nutritional state (for review seeRef. [33]).

The high cortisol group had a reduced growth rate compared to boththe control and low cortisol groups. This effect was only a tendencywhen comparing the subsets that were used for analysis of brain cellproliferation, though that is most likely a result of the low n in thesegroups. Corticosteroids are known to decrease growth rates in feedingfish [37,38]. Davis et al. [38] demonstrated that daily cortisol intakes inthe feed of 0.3, 1.5 and 3.0 μg g−1 caused treated fish to havesignificantly lower body weight than size matched control fish overthe course of 4–6 weeks, with higher doses leading to an earliersignificant effect. The same study showed that a daily cortisol intake of0.03 μg g−1 hadnoeffect onbodyweight. This indicates that the effect ofcortisol on growth is dose dependent, and supports the finding in thecurrent study where an average daily cortisol intake of 0.2 μg g−1 didnot affect growth rate over the experimental period, while 1.0 μg g−1

per daywas sufficient to significantly reduce growth. There are at least 3likely explanations for how cortisol suppresses growth. Firstly cortisoltreatments (injections or implants) have been found to cause anincrease in basal metabolic rate (measured as oxygen consumption) infish [50,51], which is likely to reduce the amount of energy that can beput into growth. Secondly, cortisol directly reduces cellular growth, as[3H]-thymidine incorporation intoDNAof cell cultures derived fromfishis reduced after cortisol treatment [52–55], and this effect is likelymediated through binding to glucocorticoid receptors [53–55]. Finally,Barton et al. [37] suggested that oral cortisol treatment may suppressgrowth by altering gut morphology, reducing nutrient uptake from thefood and thus restricting energy available for growth. In fish, these gut

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effects are seen not only after oral treatment [37], but alsowhen cortisolis elevated by cortisol implants [56,57] or chronic stress [58,59].

Injection of [3H]-thymidine has been used for identifying prolif-erating cells in intact fish [21,60], but a more commonly used methodfor investigating cell proliferation in the fish brain is injection of BrdU.BrdU functions as an S-phasemarker, since it is incorporated into DNAduring replication and can subsequently be visualized immunohisto-chemically [21–25]. We considered the BrdU method inappropriatefor the current study as the handling involved in injecting the fishwith BrdU appears to be stressful [13], and similar handling andinjection is known to elicit cortisol responses [35,61] that mightconfound effects of the cortisol treatment on brain cell proliferation.For this reason immunohistochemistry for PCNA was applied toquantify cell proliferation. PCNA is an auxiliary factor to DNApolymerase δ that is expressed in all cell cycle stages except G0, andis commonly used as a marker of actively cycling cells [62]. PCNA haspreviously been used as a marker of proliferation in the fish brain[22,29,63]. PCNA has a long half-life and may be present in detectablelevels in the cell for some time after cell cycle exit [64], and can also beinvolved in recombination and DNA repair in addition to replication[62]. This might lead to an overestimation of the number of activelycycling cells in a single staining for PCNA. A change in the number ofPCNA-positive cells will still most likely reflect a change in brain cellproliferation activity, but cannot be used to predict the exact numberof newborn cells (For further discussion regarding the use of PCNA asa non-invasive cell proliferation marker in fish, see [29,63]). In ourstudy both cortisol treated groups had a significant reduction ofPCNA-positive nuclei in the telencephalon compared to the controlgroup, indicating that cortisol treatment at both doses suppressedbrain cell proliferation in the rainbow trout telencephalon. Thetelencephalic cell proliferation was, however, not correlated withgrowth rate in any of the groups. When comparing groups, there is asignificant reduction in both cell proliferation and body growth in thehigh cortisol group, though in the low cortisol group the effect onbrain cell proliferation was not accompanied by a significant effect ongrowth. Other studies on telencephalic brain cell proliferation in fishhave also failed to find a relationship between telencephalic cellproliferation and body growth [13,29]. As multiple factors convene todetermine body growth in fish, it is not possible to make any definiteconclusions about the regulation of brain cell proliferation relative toregulation of cell proliferation in other tissues. The reduction indensity of PCNA-positive cells was approximately 50% in both groups,which is comparable to the 40% reduction in telencephalic cellproliferation found in socially subordinate rainbow trout [13]. As theplasma cortisol levels are also in the same range (55±13 ng ml−1 insubordinate individuals, 67±15 ng ml−1 in the high cortisol group),one can make the assumption that cortisol is involved in thesuppressive effect on brain cell proliferation seen during socialsubordination.

The animals used in the current study were juvenile rainbow trout.Salmonid fish commonly remain juvenile for most of their lifespan,and the major part of research on salmonids is done using animals injuvenile stages. Though no study has directly compared brain cellproliferation between juvenile and sexually mature salmonid fish,some results exist for other species. The brain of the weakly electricfish Apteronotus leptorhynchus grows throughout life, and the size ofand number of cells in the brain correlate linearly with body sizethroughout life with juvenile individuals falling along the sameregression line as adult individuals of both sexes [24]. It is thus likelythat in postembryonic stages, age or sexual maturation does not havea major impact on the rate of brain cell proliferation in fish, thoughsome caution should still be retained in comparing quantitativeresults on brain cell proliferation between studies done on juvenileand adult individuals.

A previous study has investigated the effect of cortisol onproliferation of brain cells in Apteronotus leptorhynchus. Dunlap et

al. [9] demonstrated that 7 days of treatment with cortisol usingperitoneal implants leading to plasma cortisol levels in the upperphysiological range caused a specific increase in brain cell prolifer-ation in the areas adjacent to the diencephalic prepacemaker nucleuswith no effect on surrounding areas. This area is implicated in theelectrocommunication behaviour known as chirping [65], and cortisoltreatment also leads to increased chirping [66]. This stimulatory effecton brain cell proliferation is the opposite direction of what we see inour study, despite the fact that the cortisol levels in the implanted fishare comparable to those of the high cortisol group in the currentstudy. Dunlap et al. [9] did, however, investigate a small area that isalso situated in a part of the brain we did not investigate, socomparison between the studies is difficult. The results still indicatethat cortisol can have a region specific effect on cell proliferation inthe fish brain.

Our results are in line with the effect of corticosteroids onneurogenesis in the rat dentate gyrus. Removal of circulating adrenalsteroids by adrenalectomy leads to an increase in the number ofnewborn cells of both glial and neuronal identity [43,44]. Corticoste-rone replacement abolishes this effect for newborn cells of glial butnot neuronal type [43,44]. An additional increase in plasmacorticosterone levels leads to a further reduction in the number ofnewborn neurons [43], and survival and differentiation of newbornneurons are also suppressed by exogenous corticosterone [67,68].Such a dose dependency of corticosterone on neurogenesis inmammals would lead to an expectation of a dose dependent responsein the current study, though this was not seen. The dose dependencyin rodents is, however, seen when corticosteroids are removed fromthe circulation, and to our knowledge it has not been determinedwhether there is a dose–response effect of additional corticosteronetreatment on cell proliferation or neurogenesis. It cannot be ruled outthat a dose–response relationship exists between cortisol and braincell proliferation in rainbow trout at other cortisol levels or differenttreatment periods than those used in the current study.

In conclusion, our results suggest that 6 days of oral cortisoltreatment leads to a suppression of cell proliferation in thetelencephalon of rainbow trout. This effect appears to be decoupledfrom the general growth depressive effect of cortisol, as low and highcortisol doses had different effects on growth rate, but identical effectson telencephalic cell proliferation. Also, no correlation existedbetween somatic growth and brain cell proliferation in any of thegroups. The results indicate that there is a similar suppressive effect ofcorticosteroids on brain cell proliferation in the fish forebrain as in themammalian hippocampus.

Acknowledgment

Funding for this study was provided by the Research Council ofNorway.

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