Available online at www.sciencedirect.com

3 (2008) 595–605www.elsevier.com/locate/phb

Physiology & Behavior 9

Sex differences in the effects of two stress paradigms ondopaminergic neurotransmission

C. Dalla 1, K. Antoniou 2, N. Kokras, G. Drossopoulou, G. Papathanasiou,S. Bekris, S. Daskas, Z. Papadopoulou-Daifoti ⁎

Department of Experimental Pharmacology, Medical School, University of Athens, M. Asias 75, Goudi, 115 27, Athens, Greece

Received 19 September 2007; accepted 24 October 2007

Abstract

Sex differences in behavioral and neurobiological responses to stress are considered to modulate the prevalence of some psychiatric disorders,including major depression. In the present study, we compared dopaminergic neurotransmission and behavior in response to two different stressparadigms, the Forced Swim Test (FST) and the Chronic Mild Stress (CMS). Male and female rats were subjected to one session of swim stress fortwo consecutive days (FST) or to a variety of mild stressors alternating for six weeks (CMS). Subsequently, the tissue levels of dopamine (DA)and its metabolites (HVA and DOPAC) in the hippocampus, the hypothalamus, the prefrontal cortex and the striatum were measured using high-performance liquid chromatography (HPLC). The ratios HVA/DA and DOPAC/DA were also calculated as indices of the dopaminergic activity.Results from the FST determined that males exhibited lower immobility, higher climbing duration and increased dopaminergic activity in theprefrontal cortex and the hippocampus compared to females. CMS induced alterations in sucrose intake in both sexes, while it only decreaseddopaminergic activity in the prefrontal cortex of females. These findings show that FST and CMS have different effects on the dopaminergicactivity of discrete brain regions depending on the sex of the animal. These data support the growing evidence that females display a differentialresponse and adaptation to stress than males.© 2007 Elsevier Inc. All rights reserved.

Keywords: Dopamine; Prefrontal cortex; Female; Rat; Forced swim test; Chronic mild stress; Male; Gender; Sucrose intake; Hippocampus; Hypothalamus; Striatum;Depression

1. Introduction

Stress exposure has been associated with the pathophysiol-ogy of several psychiatric disorders, such as major depression[1–3]. Therefore, exposure of rodents to various short-term orlong-term stressors has been used to study the behavioural andneurobiological response to stress [4]. Two stress paradigms

Abbreviations: 3,4 dihydroxyphenylacetate, DOPAC; Chronic mild stress,CMS; Dopamine, DA; Forced swim test, FST; High-performance liquidchromatography, HPLC; Homovanillic acid, HVA.⁎ Corresponding author. Tel.: +30 210 7462702; fax: +30 210 746 2554.E-mail address: zdaifoti@med.uoa.gr (Z. Papadopoulou-Daifoti).

1 Present address: Department of Psychology, Rutgers University, 152Frelinghuysen Road, Piscataway, 08854, New Jersey, USA.2 Present address: Department of Pharmacology, Medical School, University

of Ioannina, 45110, Ioannina, Greece.

0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.physbeh.2007.10.020

that have been validated and widely used are the Forced SwimTest (FST) and the Chronic Mild Stress (CMS). The FST is ashort-term paradigm, in which rats are forced to swim in acylinder for 15 min and the next day they are exposed again tothe swim stress for 5 min [5]. FST has been widely used forantidepressant screening, because most antidepressant com-pounds reverse FST induced manifestations, such as increasedimmobility and decreased swimming/climbing, during thesecond session of the paradigm [6,7]. These behaviouralresponses have been considered as indications/symptoms ofdespair and “depressive-like” behavior [8–10]. CMS is achronic paradigm that is considered to be a model of depressionwith high construct and face validity. CMS is based on theapplication of different mild stressors that alternate for a periodof 6 weeks [11–13]. In addition, CMS has very high predictivevalue; antidepressant administration reverses the CMS-induced

596 C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

alterations, including decreased sucrose intake, an index ofanhedonia and a core symptom of depressive symptomatology[14].

The role of monoamines, particularly serotonin and nor-adrenaline, in the stress response and the mechanism ofantidepressant action is well recognised and thoroughly studied[15–17]. In addition, evidence suggests that there are alterationsin dopaminergic neurotransmission in depression and in theresponse to antidepressant treatment [18–26]. It is known that anumber of acute or chronic stressors alter dopaminergic activityin a region-specific manner [27–34]. For example, dopaminer-gic activity has been reported to be increased in the striatum[35,36] and prefrontal cortex [37] of male rats exposed to FST.

Most preclinical and clinical studies examining the neuro-biological substrate of stress-related psychiatric disorders havebeen conducted on male subjects, although it is known thatwomen are more susceptible to some of them, such as majordepression, generalized anxiety disorder and post-traumaticstress disorder [38–45]. Numerous studies have determined thatsex differences exist in the response to stress and themanifestation of “depressive-like” behavior in various experi-mental procedures [46–56]. Notably, previous studies from ourlaboratory have shown a sex-differentiated response to FST andCMS, especially in regard with the serotonergic function andHPA axis activity [57,53]. In particular, serotonergic activitywas decreased in the hypothalamus and hippocampus offemales exposed to the FST, while it was increased in thehypothalamus of males [57]. In addition, CMS induced adecrease in hypothalamic and hippocampal serotonergicactivity in females that was not observed in males [53].

Based on the previous studies, we tested the hypothesis thatdopaminergic neurotransmission would differ between malesand females following a short-term (FST) or chronic stress(CMS) procedure. To determine whether there were differencesin dopaminergic function we examined tissue levels of DA andits metabolites in the hippocampus, the hypothalamus, theprefrontal cortex and the striatum; discrete brain regions,involved in the response to stress [16,3].

2. Experimental procedures

2.1. Animals

Adult male and female Wistar rats (∼3 months of age/300-350 and 250-300 g, respectively at the beginning of theexperiments) were used in the present study. For experiment 1,all rats were housed in groups of four in plastic, non-transparentcages (57×38×20 cm), while for experiment 2 all rats weresingly housed in plastic, non-transparent cages (40×25×15 cm), in order to conduct the behavioural testing of sucroseintake. All rats had free access to standard laboratory foodpellets (total protein content: 16.5%) and tap water throughoutthe experiments, unless it was indicated otherwise by theprotocol (experiment 2). Animal rooms were under controlledlight/dark cycle (12:12 h, lights on at 06:00 h) and temperature/humidity (22 °C, 30–40%) conditions. All animal experimentswere reviewed and approved by the local committee and all

studies have been carried out in accordance with the NationalInstitute of Health Guide for the Care and Use of LaboratoryAnimals (NIH Publications No. 80-23) revised 1996.

Females were housed in separate rooms than males and werecycling normally before the start of the experiment. However,vaginal smears were not collected during experimental pro-cedures, in order to assure that the amount of handling wasidentical in female and male rats. It is worth mentioning that thenormal distribution of female rats across the different phases ofthe estrous cycle ensures the average interaction of thehormonal milieu and points towards its median effect. Thus,the aim of the present study was to investigate the impact of thestress paradigms on male and female rats and not the differencesdue to the estrous cycle.

2.2. Experiment 1

2.2.1. Forced swim testAll FST (Males: N=12, Females: N=11) and Control

(Males: N=16, Females: N=10) rats were gently handled inthe same way for two weeks before the start of the experiment.Rats exposed to the FST were individually placed in acylindrical tank measuring 60 cm height×38 cm width. Thetank was filled with water (24±1 °C) at a height of 40 cm andthe water was changed after each animal. The animals wereforced to swim for a 15-min period (session 1) and 24 h laterthey were subjected to a 5-min swimming session (session 2)[58,6,57]. The FST behavior was scored manually (on line) andthe total duration (in seconds) of immobility, swimming andclimbing was registered from the summation of the timerecorded with the use of a computerized program. Rats wereconsidered to show immobility when they floated withoutstruggling and only made movements necessary to keep theirheads above the water. Swimming was recorded when theyactively swam around in circles. Climbing was considered whenthe rats were climbing at the walls of the cylinder. Followingeach swimming session, the rats were removed from the tank,carefully dried in heated cages and then returned to their homecages. They were then sacrificed by decapitation 20 min afterthe 2nd swim session. On the same day control rats were takenfrom their home cages and they were also sacrificed bydecapitation. The time point of 20 min was chosen based onprevious studies [35,57]. Specifically, Connor et al. [35] showedthat changes in corticosterone levels and dopaminergicneurotransmission peak 30 min after the second session ofFST exposure, while they attenuate 120 min later.

2.3. Experiment 2

2.3.1. Chronic Mild Stress (CMS)All CMS (Males: N=7 and Females N=9) and Control

(Males: N=6 and Females N=8) rats were singly housed5 weeks before the start of the CMS application. Control andCMS rats were matched and divided based on their baselinesucrose intake that was obtained twice weekly by a 4-weekadaptation period: 8 sessions in total consisting of a one-hourpresentation of one bottle containing 1% sucrose solution in

Table 1Weekly CMS protocol

Monday 10:00 Cage cleaning followed by no stressMonday 20:00 Food and water deprivation for 14 hTuesday 10:00 Sucrose test, followed by food or water deprivation for 10 hTuesday 20:00 Paired housing for 14 hWednesday 10:00 Lights switched on and off every 2 h for 10 hWednesday 20:00 Soiled cage (250 ml of water was poured into the sawdust bedding) for 14 hThursday 10:00 Cage cleaning, followed by water deprivation for 10 hThursday 20:00 Paired housing for 14 hFriday 10:00 Stroboscopic illumination in darkness for 10 hFriday 20:00 Food deprivation for 14 hSaturday 10:00 Tilting of the cages backwards (45°) for 10 hSaturday 20:00 Cages were put back in straight position/ followed by no stressSunday 10:00 Stroboscopic illumination in darkness for 10 hSunday 20:00 Soiled cage (250 ml of water was poured into the sawdust bedding) for 14 h

Table shows the weekly schedule for the Chronic Mild Stress (CMS) paradigm. CMS lasted for 6 weeks and sucrose test was performed once a week. Male and femalerats were kept in separate animal rooms, in which lights were on at 06:00 h and off at 18:00 h (12:12 h light /dark cycle).

597C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

water following a period of 14 h of food and water deprivation,as it has been previously described in detail [53]. The CMSprotocol that has been previously described for male rats [59]and modified by Papp et al. lasted for 6 weeks and consisted ofcontinuous stressors alternating during the day (two differentstressors per day) [60,53]. The stressors were: food or waterdeprivation, stroboscopic illumination (120 flashes/min), inter-mittent illumination (lights switched on and off every 2 h),paired housing (two animals in one cage with randomassignment), cage tilting (45°), soiled cage bedding (250 mlof plain water into the sawdust bedding) followed by cagecleaning. Each stressor lasted 10–14 h (schedule in Table 1).Once a week, a sucrose test (presentation of one bottlecontaining 1% sucrose solution in water) was carried out for1 h between 10:00–11:00. All animals (Control and CMSgroups) were deprived of food and water for a period of 14 hbefore the sucrose intake test. In week 6, Control and CMS ratswere sacrificed by decapitation 24 h after the application of thelast stressor, in order to detect neurochemical changes inducedby the chronic procedure.

2.3.2. Neurochemical measurementsFollowing decapitation, the brains were rapidly removed and

discrete brain regions, specifically the hippocampus, thehypothalamus, the striatum and the prefrontal cortex weredissected for both experiments 1 and 2. The dissected tissueswere weighted, homogenized and deproteinized in 500 μl of0.2 N perchloric acid solution (Merck KgaA, Darmstadt,Germany) containing 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA(both by Riedel-de Haën AG, Seelze, Germany). The homo-genate was centrifuged at 14,000 rpm for 30 min in 4 °C and thesupernatant was stored at −80 °C, until analysis.

The analytical measurements were performed using aPharmacia-LKB 2248 high-performance liquid chromatography(HPLC) pump coupled with a BAS LC4B electrochemicaldetector (Bioanalytical Systems Inc., West Lafayette, IN, USA),as previously described by us and others [61,62] with someminormodifications [63,53]. All samples were analyzed within onemonth after homogenisation. Previous studies have shown that all

monoamines measured in the present study remain stable up toonemonth following homogenisation [64]. In all samples reverse-phase ion pair chromatography was used to assay dopamine (DA)and its metabolites 3,4 dihydroxyphenylacetate (DOPAC) andhomovanillic acid (HVA). The mobile phase consisted of a50 mM phosphate buffer regulated at pH 3.0, containing 5-octylsulfate sodium salt at a concentration of 300 mg/L as the ionpair reagent andNa2EDTA at a concentration of 20mg/L (both byRiedel-de Haën AG, Seelze, Germany). Further on, acetonitrile(Merck &Co., Darmstadt, Germany) was added at a 7–10%concentration. The reference standards were prepared in 0.2 Nperchloric acid (Merck KgaA, Darmstadt, Germany) solutioncontaining 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA (both byRiedel-de Haën AG, Seelze, Germany). The sensitivity of theassay was tested for each series of samples using externalstandards. The working electrode was glassy carbon and thereference one was Ag/AgCl; the columns were Thermo Hypersil-Keystone, 150× 2.1 mm 5 μ Hypersil, Elite C18 (ThermoElectron, Cheshire, UK). Samples were quantified by comparisonof the area under the curve (AUC) against reference standardsusing a PC compatible HPLC software package (Chromatogra-phy Station for Windows ver.17 Data Apex Ltd). The limit ofdetectionwas 1 pg/27μl (volume ofHPLC injection loop) and thesignal to noise ratio was more than 3:1. Additionally, the ratios ofDOPAC/DA and HVA/DA were calculated as an index of DAturnover rates [65,66,60,63], in order to have a better evaluationof the dopaminergic activity. The turnover ratios represent indicesof the activity of the cells that integrate the synthesis, release,reuptake, and/or metabolism of monoamines [67]. In some casesdue to low, undetectable levels of DA and/or HVA in thehippocampus and prefrontal cortex, samples of 2–3 animals pergroup were not successfully assayed.

2.4. Statistics

The statistical analysis of the immobility, swimming andclimbing duration during the second session of FST was per-formed using a one-way ANOVA with sex as a between-subjects factor (male versus female).

Fig. 2. DA, DOPAC, HVA levels (μg/g of tissue) were determined in thehippocampus of male (N=12) and female (N=11) rats, 20 min after the secondsession of Forced Swim Test (FST), as well as in male (N=16) and female(N=10) control rats. DOPAC/DA, HVA/DA turnover ratios were calculated asan index of dopaminergic activity. Graph depicts means±SE. The KruskalWallis non-parametric H test revealed that FST increased DOPAC levels,DOPAC/DA and HVA/DA and decreased DA levels in the hippocampus ofmales exposed to FST, in comparison to male controls (⁎pb0.05). Additionally,it revealed that DA and HVA levels were lower, while DOPAC/DA and HVA/DAwere higher in the hippocampus of female controls, in comparison to malecontrols (#pb0.05).

598 C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

The statistical analysis of the sucrose intake at the CMS wasperformed using a repeated analysis of variance (ANOVA), withtwo between-subjects factors: sex (male versus female) andexposure to stress (control versus CMS) and one within-subjectfactor of time (week 0–6). Separate repeated ANOVAs with 7different levels (weeks 0–6) were performed, in order to elucidatespecific differences between groups. Post-hoc comparisons onrepeatedmeasuresANOVAs, specificallyBonferroni's tests, wereused for adjustment and control of the type I error rate.Greenhouse–Geisser corrections were used when Mauchly'stest of sphericitywas significant (only in the case of sucrose intakeof male controls). Finally, one-way ANOVAs with stress as abetween-subjects factor (control versus CMS) were performed inorder to elucidate specific differences between groups.

Two-way ANOVA with two between-subjects factors: sex(male versus female) and stress FST or CMS (control versusstress) was used to analyze the neurochemical results in thehypothalamus, the striatum and the prefrontal cortex. Separateone-way ANOVAs were performed when there were statisticalsignificant interactions, in order to elucidate specific differencesbetween groups. In data derived from the hippocampus, thetwo-tailed non-parametric Kruskal Wallis H test was usedinstead of ANOVA, due to the smaller and uneven number ofsamples that were successfully assayed.

Mean values±SE from experimental data, are presented inall tables and figures.

3. Results

3.1. Experiment 1

3.1.1. Effect of Forced Swim Test

3.1.1.1. Sex differences in FST. During the second session ofFST, female rats exhibited a higher duration of immobility thanmales [F(1,21)=16.05, p=0.001], while swimming duration did notdiffer (Fig. 1). Male rats exhibited a higher duration of climbingbehavior, in comparison to females [F(1,25)=5.2; p=0.03] (Fig. 1).

Fig. 1. Male (N=12) and female (N=11) rats were exposed to one 15 min swimsession on day 1, followed by a 5 min swim session on day 2 (Forced SwimTest). Graph depicts means±SE of immobility, swimming and climbingduration during the second session of FST. One-way ANOVA revealed thatfemales exhibited higher levels of immobility and lower levels of climbingduration compared to males. (⁎pb0.05; ⁎⁎pb0.01).

3.1.1.2. FST increased dopaminergic activity in the hippocam-pus of male rats. FST increased DOPAC levels, DOPAC/DAand HVA/DA turnover ratios (x2 =4.735, 6.788, 5.199; df=1;p=0.03, p=0.01, p=0.02, respectively) and decreased DAlevels in male rats (x2 =5.745; df=1; p=0.02), while it had noeffect on hippocampal dopaminergic activity of female rats (forDA levels: x2 =0.535; df=1; p=0.465) (Fig. 2). In addition,basal levels of DA and HVA levels were lower in the hippo-campus of control females in comparison to control males

Table 2Effect of FST on dopaminergic neurotransmission in the hypothalamus

Male Female

Control FST Control FST

DA 0.380±0.022 0.414±0.019 0.261±0.020## 0.324±0.039#DOPAC 0.132±0.019 0.116±0.011 0.097±0.008 0.098±0.012HVA 0.033±0.005 0.029±0.002 0.032±0.005 0.031±0.002DOPAC/DA 0.360±0.052 0.287±0.030 0.399±0.048 0.329±0.049HVA/DA 0.078±0.009 0.071±0.005 0.146±0.025## 0.116±0.013##

DA, DOPAC, HVA levels (μg/g of tissue) were determined in the hypothalamusof male (N=12) and female (N=11) rats, 20 min after the second Forced SwimTest (FST) session, as well as in male (N=16) and female (N=10) control rats.DOPAC/DA, HVA/DA turnover ratios were calculated as an index ofdopaminergic activity. Table shows means±SE. One-way ANOVA revealedthat DA levels were lower and HVA/DA was higher in the hypothalamus offemale rats, in comparison to male rats (#pb0.05, ## pb0.01).

Fig. 3. DA, DOPAC, HVA levels (μg/g of tissue) were determined in theprefrontal cortex of male (N=12) and female (N=11) rats, 20 min after thesecond session of Forced Swim Test (FST), as well as in male (N=16) andfemale (N=10) control rats. DOPAC/DA, HVA/DA turnover ratios werecalculated as an index of dopaminergic activity. Graph depicts means±SE. One-way ANOVA revealed that FST increased HVA/DA in the prefrontal cortex ofmales exposed to FST, in comparison to male controls (⁎pb0.05). Additionally,one-way ANOVA indicated that DA levels were lower and DOPAC/DA andHVA/DA were higher in the prefrontal cortex of female rats, in comparison tomale rats (#pb0.05, ## pb0.01, ### pb0.001).

Fig. 4. Male (N=7) and female (N=9) rats were exposed to 6 weeks of ChronicMild Stress (CMS). All rats including control males (N=6) and females (N=8)were subjected to 14 h of food and water deprivation before the sucrose in-take test. Sucrose testing lasted for 1 h and was performed every Tuesday at10:00 a.m. by the presentation of one bottle containing 1% sucrose in water.Graph depicts means±SE of sucrose intake of control rats and rats exposed toCMS during week 0 (last sucrose test before the start of CMS: baseline) andweeks 1–6 (during CMS exposure). Two-way mixed factor ANOVA revealed amain effect of CMS (pb0.001), while separate repeated measures ANOVAsdetermined that sucrose intake was increased only in control males (time effect:⁎pb0.05) and to some extent in control females (⁎pb0.05, difference betweencontrol and CMS females only in weeks 1 and 4), while it remained stable inrats exposed to CMS. Baseline consumption was higher in females, in com-parison to males (sex effect: pb0.05).

599C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

(x2 =9.706, 5.357 df=1 p=0.002 p=0.021 respectively), whileDOPAC/DA and HVA/DAwere higher (x2 =6.615, 6.497 df=1p=0.010 p=0.011, respectively) (Fig. 2).

3.1.1.3. FST did not alter hypothalamic dopaminergic activity.DA levels were lower and subsequently the HVA/DA turnoverratio was higher in the hypothalamus of all females in com-parison to males [F(1,43)=15.693, pb0.001; F(1,34)=18.205,pb0.001, respectively] (Table 2).

3.1.1.4. FST increased dopaminergic activity in the prefrontalcortex of male rats. FST increased HVA/DA in the prefrontalcortex of male rats [F(1,24)=4.955, p=0.036], while it had noeffect on female rats (Fig. 3). All females had lower DA levels

Table 3Effect of FST on dopaminergic neurotransmission in the striatum

Male Female

Control FST Control FST

DA 7.96±0.44 8.42±0.617 12.388±2.35# 15.375±3.012#DOPAC 1.86±0.214 1.81±0.216 2.722±0.287# 2.534±0.374HVA 0.653±0.045 0.739±0.063 0.788±0.098 0.863±0.103DOPAC/DA 0.247±0.037 0.232±0.118 0.287±0.052 0.262±0.063HVA/DA 0.087±0.009 0.092±0.013 0.073±0.008 0.070±0.010

DA, DOPAC, HVA levels (μg/g of tissue) were determined in the striatum ofmale (N=12) and female (N=11) rats, 20 min after the second Forced Swim Test(FST) session, as well as in male (N=16) and female (N=10) control rats.DOPAC/DA, HVA/DA turnover ratios were calculated as an index ofdopaminergic activity. Table shows means±SE. One-way ANOVA revealedthat DA and DOPAC levels were higher in the striatum of female rats, incomparison to male rats (#pb0.05).

[F(1,43)=17.454, pb0.001] and subsequently higher HVA/DAand DOPAC/DA turnover ratios than males [F(1,31)=25.664,pb0.001; F(1,43)=67.309, pb0.001, respectively] (Fig. 3).

3.1.1.5. FST did not alter striatal dopaminergic activity.Females had higher DA levels in their striatum than males,irrespective of stress exposure [F(1,43) =9.276, p=0.004](Table 3). In addition, the female controls had higher striatalDOPAC levels than the male controls [F(1,23)=5.315, p=0.03](Table 3).

3.2. Experiment 2

3.2.1. Effect of Chronic Mild Stress

3.2.1.1. CMS affected sucrose intake in both sexes. Statisticalanalysis revealed an interaction of time with CMS [F(6,138)=2.361; pb0.05] on the sucrose intake. Sucrose intake wasincreased in control males [F(6,36)=3.723; p=0.05], while itremained stable in CMS males. Male CMS rats consumed lesssucrose than controls in all weekly tests (weeks 1–6) [F(1,12)=10.28; pb0.01, F(1,12)=4.65; p=0.05, F(1,12)=5.43; pb0.05,F(1,12)=5.101; pb0.05, F(1,12)=26.31; pb0.001, F(1,12)=5.441;pb0.05, for weeks 1–6, respectively] (Fig. 4).

In females, there was a tendency for sucrose intake to beincreased only in controls [F(6,36)=2.135; p=0.07], while it

Fig. 5. DA, DOPAC, HVA levels (μg/g of tissue) were determined in thehippocampus of male (N=7) and female (N=9) rats, 24 h after the end of theChronic Mild Stress (CMS) paradigm, as well as in male (N=6) and female(N=8) control rats. DOPAC/DA, HVA/DA turnover ratios were calculated as anindex of dopaminergic activity. Graph depicts means±SE. The Kruskal Wallisnon-parametric H test revealed that HVA levels were higher in the hippocampusof females exposed to CMS, in comparison to males exposed to CMS and thatHVA/DA was higher in the hippocampus of female controls in comparison tomale controls (#pb0.05).

Fig. 6. DA, DOPAC, HVA levels (μg/g of tissue) were determined in theprefrontal cortex of male (N=7) and female (N=9) rats, 24 h after the end of theChronic Mild Stress (CMS) paradigm, as well as in male (N=6) and female(N=8) control rats. DOPAC/DA, HVA/DA turnover ratios were calculated as anindex of dopaminergic activity. Graph depicts means±SE. One-way ANOVArevealed that CMS decreased HVA levels and HVA/DA in the prefrontal cortexof females exposed to CMS, in comparison to female controls (⁎pb0.05).Additionally, one-way ANOVA showed that HVA levels were higher in theprefrontal cortex of female control rats, in comparison to male controls(#pb0.05).

600 C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

remained stable in CMS females (Fig. 4). CMS femalesconsumed less sucrose than controls only during weeks 1 and4 [F(1,13)=15.102; pb0.01; F(1,13)=5.433; pb0.05, respec-tively]. Notably, the baseline sucrose intake (week 0, before thestart of stressful procedures) was higher in female, incomparison to male rats [F(1,25)=5.685; pb0.05] (Fig. 4).

3.2.1.2. CMS did not alter hippocampal dopaminergicactivity. HVA/DA turnover ratio was higher in the hippo-campus of female controls, in comparison to male controls[x2 =4.800; df=1; p=0.03] (Fig. 5). In addition, the HVA

Table 4Effect of CMS on dopaminergic neurotransmission in the hypothalamus

Male Female

Control CMS Control CMS

DA 0.44±0.055 0.49±0.03 0.35±0.02 0.32±0.03##DOPAC 0.04±0.004 0.05±0.006 0.04±0.007 0.04±0.01HVA 0.019±0.003 0.023±0.005 0.02±0.002 0.015±0.003DOPAC/DA 0.11±0.02 0.10±0.013 0.12±0.024 0.12±0.024HVA/DA 0.047±0.009 0.047±0.01 0.048±0.004 0.046±0.008

DA, DOPAC, HVA levels (μg/g of tissue) were determined in the hypothalamusof male (N=7) and female (N=9) rats, 24 h after the end of the Chronic MildStress (CMS) paradigm, as well as in male (N=6) and female (N=8) control rats.DOPAC/DA, HVA/DA turnover ratios were calculated as an index ofdopaminergic activity. Table shows means±SE. One-way ANOVA revealedthat DA levels were lower in the hypothalamus of female rats exposed to CMS,in comparison to male rats exposed to CMS (##pb0.01).

hippocampal levels were higher in females exposed to CMS, incomparison to respective males [x2 =4.135; df=1; p=0.04](Fig. 5). No other significant differences were detected.

3.2.1.3. CMS did not alter hypothalamic dopaminergicactivity. DA levels were higher in females exposed to CMS,in comparison to their male cohorts [F(1,14)=13.987; p=0.002](Table 4). No other significant differences were detected.

3.2.1.4. CMS decreased dopaminergic activity in the prefrontalcortex of female rats. There was an interaction of CMS with

Table 5Effect of CMS on dopaminergic neurotransmission in the striatum

Male Female

Control CMS Control CMS

DA 8.85±0.24 9.49±0.39 6.55±0.67# 6.6±0.34###DOPAC 0.96±0.16 1.03±0.12 0.72±0.15 0.82±0.14HVA 0.69±0.053 0.75±0.06 0.56±0.062 0.55±0.044##DOPAC/DA 0.11±0.019 0.11±0.016 0.11±0.025 0.13±0.03HVA/DA 0.08±0.006 0.08±0.006 0.08±0.008 0.08±0.01

DA, DOPAC, HVA levels (μg/g of tissue) were determined in the striatum ofmale (N=7) and female (N=9) rats, 24 h after the end of the Chronic Mild Stress(CMS) paradigm, as well as in male (N=6) and female (N=8) control rats.DOPAC/DA, HVA/DA turnover ratios were calculated as an index ofdopaminergic activity. Table shows means±SE. One-way ANOVA revealedthat DA and HVA levels were lower in the striatum of female rats, in comparisonto male rats (#pb0.05; ## pb0.01; ### pb0.001).

601C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

sex on HVA levels [F(1,20)=4.785; p=0.04] and a tendency foran interaction of CMS with sex on HVA/DA [F(1,20)=3.497;p=0.08] in the prefrontal cortex. CMS induced a decrease incortical HVA levels [F(1,11)=4.651; p=0.05], a tendency forenhanced DA levels [F(1,11)=2.584; p=0.1] in female rats,as well as a subsequent decrease of HVA/DA turnover ratio[F(1,11)=6.425; p=0.03] (Fig. 6). Also, the HVA levels werehigher in female controls compared to male controls [F(1,9)

=4.677; p=0.05] (Fig. 6).

3.2.1.5. CMS did not alter striatal dopaminergic activity.Females had lower DA levels in their striatum than males,irrespective of stress exposure [F(1,26) =30.489; pb0.001](Table 5). Additionally, females exposed to CMS had lowerHVA levels in their striatum thanmales exposed to CMS [F(1,14)=7.511; p=0.02] (Table 5).

4. Discussion

The present study investigated whether short-term (FST) orchronic (CMS) stress procedures altered dopaminergic neuro-transmission in specific brain regions of male and female rats.These data indicate that dopaminergic alterations in response tostress depend on the type/duration of stressors, the sex of theanimals and the brain region. FST increased the dopaminergicactivity in the prefrontal cortex and the hippocampus of males,while it had no effect on females. On the other hand, CMSdecreased dopaminergic activity in the prefrontal cortex offemales, while it had no effect on males. Furthermore, sexdifferences related to the dopaminergic neurotransmission werealso observed in the control groups of the FST and CMSparadigms.

4.1. Sex differences in response to FST

Exposure to the FST paradigm induced an increase inhippocampal DA turnover ratios (DOPAC/DA and HVA/DA)in males, indicating an enhanced dopaminergic activity thatwas not apparent in females. The increased hippocampaldopaminergic activity has been associated with the response tostress and possibly reflects the activation of both noradrenergicand dopaminergic neurons innervating the hippocampus [68].Given that the hippocampus is also critically involved inlearning and memory [69], it is possible that the hippocampalDA activation in males is associated with their enhancedperformance in certain learning tasks after acute stressexposure [70–74].

In the present study increased dopaminergic activity in theprefrontal cortex, as reflected by the increased HVA/DA ratio,was only observed in males subjected to the FST. A number ofstudies have reported increased dopaminergic activity in theprefrontal cortex after exposure of male rats to a variety ofstressful procedures [27,75,29,30,37,31,76]. In agreement withthe present data, an in vivo microdialysis study by Petty et al.,has shown that dopaminergic activity in the prefrontal cortex ofmale rats is increased during the second swim session [37]. Theactivation of the mesocortical dopaminergic system has been

considered as an aspect of optimal cognitive function [77–79].It has also been proposed that the enhanced cortical DAfunction reflects an adaptive mechanism to stress, which in turnexerts an inhibitory control to the dopaminergic activity of thenucleus accumbens [28,30,76,80].

Interestingly, the “depressive-like” profile induced by FSTwas more pronounced in female than male rats, since durationof immobility was higher and climbing duration was lower infemales compared to males. The higher duration of climbing inmales might be related to the enhanced dopaminergic activity inresponse to FST, since climbing has been considered abehavioral measure of noradrenergic [81,10,82] and dopami-nergic neurotransmission [83,84]. Additionally, the lack ofcortical dopaminergic activation in females in response to FSTmay reflect their inability to cope with the stressful procedure,in contrast to males. This lack could be also related to the factthat female controls had higher basal dopaminergic activity, asreflected by increased DA turnover ratios (HVA/DA, DOPAC/DA), in most of the brain regions studied (hippocampus,hypothalamus and prefrontal cortex) than their male counter-parts. It is possible that the dopaminergic system cannot befurther activated in females exposed to FST, while this is not thecase in males. These sex differences in the basal dopaminergicneurotransmission, as well as in the response to stress could berelated to activational (during adulthood) and/or organizational(during development) effects of sex hormones [85,86]. Futurestudies will be needed to determine the exact role of sexhormones (i.e. estrogen, progesterone, and testosterone) onthese responses to stress.

4.2. Sex differences in response to CMS

CMS females exhibited a disrupted sucrose intake, but to alower extent than males. Interestingly, in male and to someextent in female (only a statistical significant tendency) controlrats, sucrose intake was increased, while it remained stable inrats exposed to CMS. Other studies have reported similar effectsin male and female rats [87,50,88]. Specifically for females,Bielajew and colleagues investigated the effects of CMS onsucrose preference/intake in different rat strains and they foundno effect of CMS on sucrose preference. However, theyreported an increase in the one-hour sucrose intake in controlsingly-housed rats and a decrease in a 24 h period sucrose intakein Sprague–Dawley female rats exposed to CMS [50,89].Notably, there are several other reports of decreased sucroseintake in female rats exposed to CMS [90,88]. Interestingly, inthe study of Grippo et al. (2005), the magnitude of the effect ishigher than in any other study [88]. The aforementioneddiscrepancies could be attributed to the different strains of rats,as well as the different duration and type of stressors used in thevarious studies. Additionally, the sex differences in sucroseintake in response to CMS may be attributed to the baseline sexdifferences in sucrose intake. In the present study, femalesstarted with a higher baseline of sucrose intake than malesbefore the application of any stressor, and consequently theyexhibited overall higher sucrose consumption and a more erraticincrease than males.

602 C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

Exposure to the CMS paradigm decreased dopaminergicactivity in the prefrontal cortex of females, as reflected bydecreased HVA levels and HVA/DA ratio, while it had no impacton males. These data, in combination with reduced serotonergicactivity in the hippocampus and the hypothalamus of femalesexposed to CMS [53], suggest that this stress paradigm decreasesmonoaminergic activity and may indicate a greater vulnerabilityof females in the CMS model of depression. Additionally,previous studies have shown that the application of CMS tofemales results in increased corticosterone levels [50,53] indicat-ing a sustained activation of the HPA axis. Based on our findingsand the aforementioned studies, it could be suggested that theapplication of the current CMS procedure in females results in amild decrease in sucrose intake and a reduced corticaldopaminergic activity, possibly related to a sustained activationof the HPA axis. These behavioral and physiological alterationsmight reflect the failure of female rats to adapt to theunpredictability of the CMS protocol.

There were no apparent alterations in the dopaminergicactivity in males following the application of the current CMSprotocol. Previous studies by Di Chiara et al. (1999), determinedthat there were no alterations in the basal cortical dopaminergicactivity of CMSmale rats, but there was an impact of CMS on theresponse of dopaminergic neurotransmission to aversive orhedonic stimuli [32]. However, we have previously found,using a more “severe” CMS protocol [59] that male Wistar ratsexhibit increased cortical and hypothalamic dopaminergicactivity, along with decreased striatal dopaminergic activity inresponse to CMS [34]. Additionally, a decrease in dopaminergicreceptor binding and mRNA expression has also been reported inmale rats exposed to the same more “severe” version of CMS[91–93], while D2/D3 dopamine receptor agonists exert anti-depressant effects by reversing the stress-induced anhedonia [94–96]. Based on our findings and the aforementioned studies inmalerats, it could be suggested that the current “mild” CMS protocol,as compared to a more “severe” version [59,34], induced adecrease in sucrose intake, reflecting a “depressive-like beha-vior”, but it resulted in a neurochemical adaptation, at least in thebrain regions studied here. This adaptation can also be reflectedby unaltered corticosterone levels in males subjected to the sameversion of CMS, suggesting a habituation of the HPA axis [53].More studies are needed in order to determine the potential sex-differentiated role of the mesolimbic dopamine system andparticularly the nucleus accumbens and the ventral tegmentalarea. These regions have been associated with depressionsymptoms, such as anhedonia and decreased motivation[97,98,25,26].

Sex differences in basal dopaminergic activity were not sopronounced in control groups of the CMS paradigm, incomparison to the control groups of the FST paradigm. Thisdifferentiation could be attributed to inadvertent stress in theprocedural testing of the control animals and might have animpact on the differential effects of the two stress paradigms.Control rats for the CMS paradigm were singly housed andsubjected to multiple sucrose intake tests and several periods offood/water deprivation. Previous studies have reported thatisolation is stressful for rats, especially for females [99,48,100],

while it influences the basal and the stress-induced mono-aminergic neurotransmission as well [101,102,76,103–105].

5. Conclusions

The present results indicate that the response of thedopaminergic system to stress is dependent on the interactionof the sex with the type/duration of the stressful procedure. Inparticular, FST, a well-known behavioral test that induced a“depressive-like” profile in both sexes, increased the dopami-nergic activity in the prefrontal cortex and hippocampus ofmales only. The CMS paradigm considered to be a model ofdepression induced behavioral alterations in both sexes anddecreased the dopaminergic activity in the prefrontal cortex offemales only. Future studies will determine whether theseeffects are mediated by effects of sex hormones duringdevelopment of the brain and/or during puberty and adulthood[106–108]. Alternatively, sex differences in response to stresscould be also determined by genetic factors [109,110].

Although, we should be careful when we extrapolatefindings from animal models to humans, the present findingsconcerning sex-differentiated effects of stress on dopaminergicneurotransmission seem to have some relevance to clinicalfindings. In humans, women have a differential response tostress than men, which may lead to higher incidence ofdepression [39,111–115,41,44]. It is possible that cortical andhippocampal dopaminergic activity contributes to the adapta-tion/ response to stress, which in turn might be involved in thesex-differentiated neurobiological substrate of depression.

Acknowledgments

Wewould like to thank Dr. G. Hodes for helpful comments onthe manuscript. This work was supported in part by the GeneralSecretariat of Research and Technology (GSRT) of Greece(PENED01, 01ED82). Dr. Christina Dalla is a Marie CurieInternational Fellow, funded from the European Commissionwithin the 6th European Community Framework Programme.

References

[1] Gold PW, Goodwin FK, Chrousos GP. Clinical and biochemicalmanifestations of depression. Relation to the neurobiology of stress (1).N Engl J Med 1988;319:348–53.

[2] Gold PW,WongML, Chrousos GP, Licinio J. Stress system abnormalitiesin melancholic and atypical depression: molecular, pathophysiological,and therapeutic implications. Mol Psychiatry 1996;1:257–64.

[3] Tafet GE, Bernardini R. Psychoneuroendocrinological links betweenchronic stress and depression. Prog Neuropsychopharmacol BiolPsychiatry 2003;27:893–903.

[4] Anisman H, Matheson K. Stress, depression, and anhedonia: caveatsconcerning animal models. Neurosci Biobehav Rev 2005;29:525–46.

[5] Porsolt RD, Bertin A, Jalfre M. “Behavioural despair” in rats and mice: straindifferences and the effects of imipramine. Eur J Pharmacol 1978;51:291–4.

[6] Lucki I. The forced swimming test as a model for core and componentbehavioral effects of antidepressant drugs. Behav Pharmacol 1997;8:523–32.

[7] Cryan JF, Valentino RJ, Lucki I. Assessing substrates underlying thebehavioral effects of antidepressants using the modified rat forcedswimming test. Neurosci Biobehav Rev 2005;29:547–69.

603C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

[8] Porsolt RD, Anton G, Blavet N, Jalfre M. Behavioural despair in rats: anew model sensitive to antidepressant treatments. Eur J Pharmacol1978;47:379–91.

[9] Cryan JF, Markou A, Lucki I. Assessing antidepressant activity inrodents: recent developments and future needs. Trends Pharmacol Sci2002;23:238–45.

[10] Kelliher P, Kelly JP, Leonard BE, Sanchez C. Effects of acute and chronicadministration of selective monoamine re-uptake inhibitors in the ratforced swim test. Psychoneuroendocrinology 2003;28:332–47.

[11] Papp M, Willner P, Muscat R. An animal model of anhedonia:attenuation of sucrose consumption and place preference conditioningby chronic unpredictable mild stress. Psychopharmacology (Berl)1991;104:255–9.

[12] Willner P, Muscat R, Papp M. Chronic mild stress-induced anhedonia: arealistic animal model of depression. Neurosci Biobehav Rev1992;16:525–34.

[13] Willner P. Chronic mild stress (CMS) revisited: consistency andbehavioural–neurobiological concordance in the effects of CMS.Neuropsychobiology 2005;52:90–110.

[14] Willner P. Validity, reliability and utility of the chronic mild stress modelof depression: a 10-year review and evaluation. Psychopharmacology(Berl) 1997;134:319–29.

[15] Ressler KJ, Nemeroff CB. Role of serotonergic and noradrenergicsystems in the pathophysiology of depression and anxiety disorders.Depress Anxiety 2000;12(Suppl 1):2–19.

[16] Nestler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Monteggia LM.Neurobiology of depression. Neuron 2002;34:13–25.

[17] Wong ML, Licinio J. From monoamines to genomic targets: a paradigmshift for drug discovery in depression. Nat Rev Drug Discov2004;3:136–51.

[18] Spyraki C, Fibiger HC. Behavioural evidence for supersensitivity ofpostsynaptic dopamine receptors in the mesolimbic system after chronicadministration of desipramine. Eur J Pharmacol 1981;74:195–206.

[19] Borsini F, Nowakowska E, Pulvirenti L, Samanin R. Repeated treatmentwith amitriptyline reduces immobility in the behavioural ‘despair’ test inrats by activating dopaminergic and beta-adrenergic mechanisms. JPharm Pharmacol 1985;37:137–8.

[20] Borsini F, Pulvirenti L, Samanin R. Evidence of dopamine involvementin the effect of repeated treatment with various antidepressants in thebehavioural ‘despair’ test in rats. Eur J Pharmacol 1985;110:253–6.

[21] Klimek V, Nielsen M. Chronic treatment with antidepressants decreasesthe number of [3H]SCH 23390 binding sites in the rat striatum and limbicsystem. Eur J Pharmacol 1987;139:163–9.

[22] Tanda G, Carboni E, Frau R, Di Chiara G. Increase of extracellulardopamine in the prefrontal cortex: a trait of drugs with antidepressantpotential? Psychopharmacology (Berl) 1994;115:285–8.

[23] Gambarana C, Ghiglieri O, Graziella de Montis M. Desensitization of theD1 dopamine receptors in rats reproduces a model of escape deficitreverted by imipramine, fluoxetine and clomipramine. Prog Neuropsy-chopharmacol Biol Psychiatry 1995;19:741–55.

[24] D'Aquila PS, Collu M, Gessa GL, Serra G. The role of dopamine in themechanism of action of antidepressant drugs. Eur J Pharmacol2000;405:365–73.

[25] Willner P, Hale AS, Argyropoulos S. Dopaminergic mechanism ofantidepressant action in depressed patients. J Affect Disord2005;86:37–45.

[26] Dunlop BW, Nemeroff CB. The role of dopamine in the pathophysiologyof depression. Arch Gen Psychiatry 2007;64:327–37.

[27] Dunn AJ. Stress-related activation of cerebral dopaminergic systems.Ann N YAcad Sci 1988;537:188–205.

[28] Cabib S, Puglisi-Allegra S. Opposite responses of mesolimbic dopaminesystem to controllable and uncontrollable aversive experiences.J Neurosci 1994;14:3333–40.

[29] Finlay JM, Zigmond MJ, Abercrombie ED. Increased dopamine andnorepinephrine release in medial prefrontal cortex induced by acute andchronic stress: effects of diazepam. Neuroscience 1995;64:619–28.

[30] Horger BA, Roth RH. The role of mesoprefrontal dopamine neurons instress. Crit Rev Neurobiol 1996;10:395–418.

[31] Cuadra G, Zurita A, Lacerra C, Molina V. Chronic stress sensitizes frontalcortex dopamine release in response to a subsequent novel stressor:reversal by naloxone. Brain Res Bull 1999;48:303–8.

[32] Di Chiara G, Loddo P, Tanda G. Reciprocal changes in prefrontal andlimbic dopamine responsiveness to aversive and rewarding stimuli afterchronic mild stress: implications for the psychobiology of depression.Biol Psychiatry 1999;46:1624–33.

[33] Moore H, Rose HJ, Grace AA. Chronic cold stress reduces thespontaneous activity of ventral tegmental dopamine neurons. Neuropsy-chopharmacology 2001;24:410–9.

[34] Bekris S, Antoniou K, Daskas S, Papadopoulou-Daifoti Z. Behaviouraland neurochemical effects induced by chronic mild stress applied to twodifferent rat strains. Behav Brain Res 2005;161:45–59.

[35] Connor TJ, Kelly JP, Leonard BE. Forced swim test-induced neuro-chemical endocrine, and immune changes in the rat. Pharmacol BiochemBehav 1997;58:961–7.

[36] Connor TJ, Kelliher P, Harkin A, Kelly JP, Leonard BE. Reboxetineattenuates forced swim test-induced behavioural and neurochemicalalterations in the rat. Eur J Pharmacol 1999;379:125–33.

[37] Petty F, Jordan S, Kramer GL, Zukas PK, Wu J. Benzodiazepineprevention of swim stress-induced sensitization of cortical biogenicamines: an in vivo microdialysis study. Neurochem Res 1997;22:1101–4.

[38] Kessler RC, Sonnega A, Bromet E, Hughes M, Nelson CB. Posttraumaticstress disorder in the National Comorbidity Survey. Arch Gen Psychiatry1995;52:1048–60.

[39] Kornstein SG. Gender differences in depression: implications fortreatment. J Clin Psychiatry 1997;58(Suppl 15):12–8.

[40] Stein MB, Jang KL, Taylor S, Vernon PA, Livesley WJ. Genetic andenvironmental influences on trauma exposure and posttraumatic stressdisorder symptoms: a twin study. Am J Psychiatry 2002;159:1675–81.

[41] Kessler RC. Epidemiology of women and depression. J Affect Disord2003;74:5–13.

[42] Holden C. Sex and the suffering brain. Science 2005;308:1574.[43] Steiner M, Allgulander C, Ravindran A, Kosar H, Burt T, Austin C.

Gender differences in clinical presentation and response to sertralinetreatment of generalized anxiety disorder. Hum Psychopharmacol2005;20:3–13.

[44] Nemeroff CB, Bremner JD, Foa EB, Mayberg HS, North CS, Stein MB.Posttraumatic stress disorder: a state-of-the-science review. J PsychiatrRes 2006;40:1–21.

[45] Somers JM, Goldner EM, Waraich P, Hsu L. Prevalence and incidencestudies of anxiety disorders: a systematic review of the literature. Can JPsychiatry 2006;51:100–13.

[46] Young EA. Sex differences and the HPA axis: implications for psychiatricdisease. J Gend Specif Med 1998;1:21–7.

[47] Rivier C.Gender, sex steroids, corticotropin-releasing factor, nitric oxide, andthe HPA response to stress. Pharmacol Biochem Behav 1999;64:739–51.

[48] Palanza P. Animal models of anxiety and depression: how are femalesdifferent? Neurosci Biobehav Rev 2001;25:219–33.

[49] Bielajew C, Konkle AT, Kentner AC, Baker SL, Stewart A, Hutchins AA,et al. Strain and gender specific effects in the forced swim test: effects ofprevious stress exposure. Stress 2003;6:269–80.

[50] Konkle AT, Baker SL, Kentner AC, Barbagallo LS, Merali Z, Bielajew C.Evaluation of the effects of chronic mild stressors on hedonic andphysiological responses: sex and strain compared. Brain Res2003;992:227–38.

[51] Kitraki E, Kremmyda O, Youlatos D, Alexis MN, Kittas C. Gender-dependent alterations in corticosteroid receptor status and spatialperformance following 21 days of restraint stress. Neuroscience2004;125:47–55.

[52] Leuner B, Mendolia-Loffredo S, Shors TJ. Males and females responddifferently to controllability and antidepressant treatment. Biol Psychiatry2004;56:964–70.

[53] Dalla C, Antoniou K, Drossopoulou G, Xagoraris M, Kokras N, SfikakisA, et al. Chronic mild stress impact: are females more vulnerable?Neuroscience 2005;135:703–14.

[54] Hodes GE, Shors TJ. Distinctive stress effects on learning during puberty.Horm Behav 2005;48:163–71.

604 C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

[55] C.Dalla, C. Edgecomb, A.S.Whetstone, T.J. Shors. Females do not ExpressLearned Helplessness like Males do. Neuropsychopharmacology, advanceonline publication 22 August 2007; doi:10.1038/sj.npp.1301533.

[56] Shors TJ, Mathew J, Sisti HM, Edgecomb C, Beckoff S, Dalla C.Neurogenesis and helplessness are mediated by controllability in malesbut not in females. Biol Psychiatry 2007;62:487–95.

[57] Drossopoulou G, Antoniou K, Kitraki E, Papathanasiou G, Papalexi E,Dalla C, et al. Sex differences in behavioral, neurochemical andneuroendocrine effects induced by the Forced Swim Test in rats.Neuroscience 2004;126:849–57.

[58] Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal modelsensitive to antidepressant treatments. Nature 1977;266:730–2.

[59] Willner P, Towell A, Sampson D, Sophokleous S, Muscat R. Reductionof sucrose preference by chronic unpredictable mild stress, and itsrestoration by a tricyclic antidepressant. Psychopharmacology (Berl)1987;93:358–64.

[60] Papp M, Nalepa I, Antkiewicz-Michaluk L, Sanchez C. Behavioural andbiochemical studies of citalopram and WAY 100635 in rat chronic mildstress model. Pharmacol Biochem Behav 2002;72:465–74.

[61] Sharp T, Zetterstrom T, Series HG, Carlsson A, Grahame-Smith DG,Ungerstedt U. HPLC-EC analysis of catechols and indoles in rat braindialysates. Life Sci 1987;41:869–72.

[62] Papadopoulou-Daifotis Z, Antoniou K, Vamvakidis A, Kalliteraki I,Varonos DD. Neurochemical changes in dopamine and serotoninturnover rate in discrete regions of rat brain after the administration ofglycinergic compounds. Acta Ther 1995;21:5–18.

[63] Dalla C, Antoniou K, Papadopoulou-Daifoti Z, Balthazart J, Bakker J.Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit‘depressive-like’ symptomatology. Eur J Neurosci 2004;20:217–28.

[64] Kilpatrick IC, Jones MW, Phillipson OT. A semiautomated analysismethod for catecholamines, indoleamines, and some prominent metabo-lites in microdissected regions of the nervous system: an isocratic HPLCtechnique employing coulometric detection and minimal samplepreparation. J Neurochem 1986;46:1865–76.

[65] Karstaedt PJ, Kerasidis H, Pincus JH, Meloni R, Graham J, Gale K.Unilateral destruction of dopamine pathways increases ipsilateral striatalserotonin turnover in rats. Exp Neurol 1994;126:25–30.

[66] Cransac H, Cottet-Emard JM, Pequignot JM, Peyrin L. Monoamines(norepinephrine, dopamine, serotonin) in the rat medial vestibularnucleus: endogenous levels and turnover. J Neural Transm1996;103:391–401.

[67] Elhwuegi AS. Central monoamines and their role in major depression.Prog Neuropsychopharmacol Biol Psychiatry 2004;28:435–51.

[68] Zhang X, Kindel GH, Wulfert E, Hanin I. Effects of immobilization stresson hippocampal monoamine release: modification by mivazerol, a newalpha 2-adrenoceptor agonist. Neuropharmacology 1995;34:1661–72.

[69] Kim JJ, Diamond DM. The stressed hippocampus, synaptic plasticity andlost memories. Nat Rev Neurosci 2002;3:453–62.

[70] Shors TJ, Weiss C, Thompson RF. Stress-induced facilitation of classicalconditioning. Science 1992;257:537–9.

[71] Servatius RJ, Shors TJ. Exposure to inescapable stress persistentlyfacilitates associative and nonassociative learning in rats. Behav Neurosci1994;108:1101–6.

[72] Wood GE, Shors TJ. Stress facilitates classical conditioning in males, butimpairs classical conditioning in females through activational effects ofovarian hormones. Proc Natl Acad Sci U S A 1998;95:4066–71.

[73] Shors TJ. Acute stress rapidly and persistently enhances memoryformation in the male rat. Neurobiol Learn Mem 2001;75:10–29.

[74] D.A. Bangasser, and T. Shors. The hippocampus is necessary forenhancements and impairments of learning following stressful experi-ence. Nat Neurosci Nov 2007;10(11):1401–3.

[75] Gresch PJ, Sved AF, Zigmond MJ, Finlay JM. Stress-induced sensitiza-tion of dopamine and norepinephrine efflux in medial prefrontal cortex ofthe rat. J Neurochem 1994;63:575–83.

[76] Cabib S, Ventura R, Puglisi-Allegra S. Opposite imbalances betweenmesocortical and mesoaccumbens dopamine responses to stress by thesame genotype depending on living conditions. Behav Brain Res2002;129:179–85.

[77] Jentsch JD, Taylor JR, Elsworth JD, Redmond Jr DE, Roth RH. Alteredfrontal cortical dopaminergic transmission in monkeys after subchronicphencyclidine exposure: involvement in frontostriatal cognitive deficits.Neuroscience 1999;90:823–32.

[78] Chung YC, Li Z, Dai J, Meltzer HY, Ichikawa J. Clozapine increases bothacetylcholine and dopamine release in rat ventral hippocampus: role of 5-HT1A receptor agonism. Brain Res 2004;1023:54–63.

[79] Li Z, Ichikawa J, Dai J, Meltzer HY. Aripiprazole, a novel antipsychoticdrug, preferentially increases dopamine release in the prefrontal cortexand hippocampus in rat brain. Eur J Pharmacol 2004;493:75–83.

[80] Ventura R, Cabib S, Puglisi-Allegra S. Genetic susceptibility ofmesocortical dopamine to stress determines liability to inhibition ofmesoaccumbens dopamine and to behavioral ‘despair’ in a mouse modelof depression. Neuroscience 2002;115:999–1007.

[81] Detke MJ, Rickels M, Lucki I. Active behaviors in the rat forcedswimming test differentially produced by serotonergic and noradrenergicantidepressants. Psychopharmacology (Berl) 1995;121:66–72.

[82] PageME, Brown K, Lucki I. Simultaneous analyses of the neurochemicaland behavioral effects of the norepinephrine reuptake inhibitor reboxetinein a rat model of antidepressant action. Psychopharmacology (Berl)2003;165:194–201.

[83] Reneric JP, Lucki I. Antidepressant behavioral effects by dual inhibitionof monoamine reuptake in the rat forced swimming test. Psychopharma-cology (Berl) 1998;136:190–7.

[84] Basso AM, Gallagher KB, Bratcher NA, Brioni JD, Moreland RB, HsiehGC, et al. Antidepressant-like effect of D(2/3) receptor-, but not D(4)receptor-activation in the rat forced swim test. Neuropsychopharmacol-ogy 2005;30:1257–68.

[85] Thompson TL, Moss RL. Modulation of mesolimbic dopaminergicactivity over the rat estrous cycle. Neurosci Lett 1997;229:145–8.

[86] Sfikakis A, Papadopoulou-Daifotis Z, Sfikaki M, Messari J. Mono-aminergic dysregulation on diestrus-2 and estrus through high emotionalreactivity. Pharmacol Biochem Behav 1998;60:285–91.

[87] Kioukia N, Bekris S, Antoniou K, Papadopoulou-Daifoti Z, ChristofidisI. Effects of chronic mild stress (CMS) on thyroid hormone function intwo rat strains. Psychoneuroendocrinology 2000;25:247–57.

[88] Grippo AJ, Sullivan NR, Damjanoska KJ, Crane JW, Carrasco GA, Shi J,et al. Chronic mild stress induces behavioral and physiological changes,and may alter serotonin 1A receptor function, in male and cycling femalerats. Psychopharmacology (Berl) 2005;179:769–80.

[89] Baker SL, Kentner AC, Konkle AT, Santa-Maria Barbagallo L, BielajewC. Behavioral and physiological effects of chronic mild stress in femalerats. Physiol Behav 2006;87:314–22.

[90] Duncko R, Kiss A, Skultetyova I, Rusnak M, Jezova D. Corticotropin-releasing hormone mRNA levels in response to chronic mild stress rise inmale but not in female rats while tyrosine hydroxylase mRNA levelsdecrease in both sexes. Psychoneuroendocrinology 2001;26:77–89.

[91] Muscat R, Towell A, Willner P. Changes in dopamine autoreceptorsensitivity in an animal model of depression. Psychopharmacology (Berl)1988;94:545–50.

[92] Papp M, Klimek V, Willner P. Parallel changes in dopamine D2 receptorbinding in limbic forebrain associated with chronic mild stress-inducedanhedonia and its reversal by imipramine. Psychopharmacology (Berl)1994;115:441–6.

[93] Dziedzicka-Wasylewska M, Willner P, Papp M. Changes in dopaminereceptor mRNA expression following chronic mild stress and chronicantidepressant treatment. Behav Pharmacol 1997;8:607–18.

[94] Muscat R, Papp M, Willner P. Antidepressant-like effects ofdopamine agonists in an animal model of depression. Biol Psychiatry1992;31:937–46.

[95] Papp M, Willner P, Muscat R. Behavioural sensitization to a dopamineagonist is associated with reversal of stress-induced anhedonia.Psychopharmacology (Berl) 1993;110:159–64.

[96] Willner P, Lappas S, Cheeta S, Muscat R. Reversal of stress-inducedanhedonia by the dopamine receptor agonist, pramipexole. Psychophar-macology (Berl) 1994;115:454–62.

[97] Schultz W. Dopamine neurons and their role in reward mechanisms. CurrOpin Neurobiol 1997;7:191–7.

605C. Dalla et al. / Physiology & Behavior 93 (2008) 595–605

[98] Naranjo CA, Tremblay LK, Busto UE. The role of the brain rewardsystem in depression. Prog Neuropsychopharmacol Biol Psychiatry2001;25:781–823.

[99] Brown KJ, Grunberg NE. Effects of housing on male and femalerats: crowding stresses male but calm females. Physiol Behav1995;58:1085–9.

[100] Baker S, Bielajew C. Influence of housing on the consequences ofchronic mild stress in female rats. Stress 2007;10:283–93.

[101] Harro J, Haidkind R, Harro M, Modiri AR, Gillberg PG, Pahkla R, et al.Chronic mild unpredictable stress after noradrenergic denervation:attenuation of behavioural and biochemical effects of DSP-4 treatment.Eur Neuropsychopharmacol 1999;10:5–16.

[102] Beck KD, Luine VN. Sex differences in behavioral and neurochemicalprofiles after chronic stress: role of housing conditions. Physiol Behav2002;75:661–73.

[103] Westenbroek C, Den Boer JA, Ter Horst GJ. Gender-specific effects ofsocial housing on chronic stress-induced limbic Fos expression.Neuroscience 2003;121:189–99.

[104] Westenbroek C, Ter Horst GJ, Roos MH, Kuipers SD, Trentani A, denBoer JA. Gender-specific effects of social housing in rats after chronicmild stress exposure. Prog Neuropsychopharmacol Biol Psychiatry2003;27:21–30.

[105] Westenbroek C, Den Boer JA, Veenhuis M, Ter Horst GJ. Chronic stressand social housing differentially affect neurogenesis in male and femalerats. Brain Res Bull 2004;64:303–8.

[106] Arnold AP, Breedlove SM. Organizational and activational effects of sexsteroids on brain and behavior: a reanalysis. Horm Behav 1985;19:469–98.

[107] McCarthy MM, Konkle AT. When is a sex difference not a sexdifference? Front Neuroendocrinol 2005;26:85–102.

[108] Sisk CL, Zehr JL. Pubertal hormones organize the adolescent brain andbehavior. Front Neuroendocrinol 2005;26:163–74.

[109] Arnold AP. Sex chromosomes and brain gender. Nat Rev Neurosci2004;5:701–8.

[110] DaviesW,Wilkinson LS. It is not all hormones: alternative explanations forsexual differentiation of the brain. Brain Res Dec 18 2006;1126(1):36–45.

[111] Sherrill JT, Anderson B, Frank E, Reynolds III CF, Tu XM, Patterson D,et al. Is life stress more likely to provoke depressive episodes in womenthan in men? Depress Anxiety 1997;6:95–105.

[112] Kessler RC, Walters EE. Epidemiology of DSM-III-R major depressionand minor depression among adolescents and young adults in theNational Comorbidity Survey. Depress Anxiety 1998;7:3–14.

[113] Kendler KS, Thornton LM, Prescott CA. Gender differences in the ratesof exposure to stressful life events and sensitivity to their depressogeniceffects. Am J Psychiatry 2001;158:587–93.

[114] Maciejewski PK, Prigerson HG, Mazure CM. Sex differences in event-related risk for major depression. Psychol Med 2001;31:593–604.

[115] Klein LC, Corwin EJ. Seeing the unexpected: how sex differences instress responses may provide a new perspective on the manifestation ofpsychiatric disorders. Curr Psychiatry Rep 2002;4:441–8.