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Neuroscience xx (2005) xxx

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ARTICLE IN PRESS

HRONIC MILD STRESS IMPACT: ARE FEMALES MORE

ULNERABLE?

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. DALLA, K. ANTONIOU, G. DROSSOPOULOU,. XAGORARIS, N. KOKRAS, A. SFIKAKIS AND. PAPADOPOULOU-DAIFOTI*

epartment of Pharmacology, Medical School, University of Athens,. Asias 75, Goudi, 11527 Athens, Greece

bstract—Despite the knowledge that women are more sus-eptible than men to stress-related mental illness, such asajor depression, there is no comprehensive estimation of

he role of gender in the detrimental effects of chronic stresshat might cause depression. Sex differences regarding thessociation of behavioral parameters with serotonergic andypothalamic–pituitary–adrenal axis activities were investi-ated in the chronic mild stress model of depression. Addi-ionally, the impact of chronic mild stress exposure on andditional/novel short-term stressful procedure, such as theorced swim test was examined in male and female rats.emale rats were found to be more vulnerable to chronic mildtress and that was depicted with disruption of sucrose in-ake, decreases in open field activity, increased corticoste-one levels, alteration in estrous cycle and decreased sero-onergic activity in hippocampus and hypothalamus. On theontrary, in males the current chronic mild stress protocollicited only behavioral changes, such as disruption in su-rose intake and decreased open field activity. Interestingly,n response to forced swim test, females previously sub-ected to chronic mild stress, were found to cope better byxhibiting increased active behavior in the second forcedwim test session and higher hypothalamic serotonergic ac-ivity in comparison with corresponding males. On the otherand, males were more affected by previous chronic mildtress exposure and that was manifested by decreased activeehavior in the first forced swim test session and increasedorticosterone levels following second forced swim testession.

These data indicate that although females are more vul-erable in the application of chronic mild stress than males,

n response to an additional-novel stressor (forced swim test)hey show better response. Therefore, both sex/gender andombination of stressful procedures should be carefully con-idered in the study of the pathophysiology of stress-relatedental illnesses. © 2005 Published by Elsevier Ltd on behalff IBRO.

ey words: rats, sucrose intake, forced swim test (FST),erotonin (5-HT), corticosterone, open field (OF).

omen are more susceptible than men to stress-relatedental illness and twice as likely to experience depression

Corresponding author. Tel: �30-210-7462702 or �30-210-7462579;ax: �30-210-7462554.-mail address: zdaifoti@med.uoa.gr (Z. Papadopoulou-Daifoti).bbreviations: ANOVA, analysis of variance; CMS, chronic mildtress; FST, forced swim test; HPA, hypothalamic–pituitary–adrenal;

cPG, hypothalamic–pituitary– gonadal; OF, open field; 5-HIAA,-hydroxyindoleatic acid; 5-HT, serotonin.

306-4522/05$30.00�0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO.oi:10.1016/j.neuroscience.2005.06.068

1

Kornstein, 1997; Kendler, 1998; Kendler et al., 2000).hese differences are probably underlined by sexual di-orphisms observed in the hypothalamic–pituitary–adre-al (HPA) axis activity/response to stress (Patchev andlmeida, 1998; Young, 1998) and its interaction with theerotonergic system (Heninger, 1997; Joffe and Cohen,998). Experimental studies examining the behavior ofale and female rats in response to stress (Kennett et al.,986; Alonso et al., 1991; Palanza, 2001; Beck and Luine,002; Bowman et al., 2003; Campbell et al., 2003; Dros-opoulou et al., 2004; Leuner et al., 2004) aim to thenderstanding of sex differences in the pathophysiology ofepression. Subsequently, sex differences in behaviorave been linked with indices of HPA axis activity anderotonergic function that are differently modulated in malend female rats following either short-term (Karandrea etl., 2002; Drossopoulou et al., 2004) or long-term stressfulrocedures (Duncko et al., 2001b; Beck and Luine, 2002;owman et al., 2003; Konkle et al., 2003; Westenbroek etl., 2003). Despite the availability of several relevant re-orts there is no comprehensive estimation of the role ofender in the detrimental effects of chronic stress thatight cause depression.

Chronic mild stress (CMS) (Willner, 1997), despite itsimitations, is a widely accepted animal model of depres-ion and has been previously reported to induce a num-er of behavioral, endocrinological and neurobiologicalhanges in male rats (Willner et al., 1992; Moreau et al.,993; Di Chiara et al., 1999; Kioukia-Fougia et al., 2002;ekris et al., 2005). CMS consists of a variety of low gradetressors applied to rats for a prolonged period of time andauses reduction of sucrose solution consumption, which

s a behavior that reflects anhedonia, a core symptom ofepressive disorder (Willner et al., 1987, 1992). Although,few studies have examined the impact of CMS on some

eurobiological variables of males and females (Benelli etl., 1999; Duncko et al., 2001a,b; Konkle et al., 2003),here is little research (Grippo et al., 2005) focused on thessociation between sex differences in behavior and HPAxis activity along with serotonergic function. Therefore, inhe present study sex differences in the CMS impact werenvestigated regarding sucrose intake and open field (OF)ehavioral parameters, as well as serotonergic activity andorticosterone serum levels.

Additionally, the impact of CMS exposure on an addi-ional/novel short-term stressful procedure, such as theorced swim test (FST) (Porsolt et al., 1978) was examinedn both sexes. FST, apart from a useful screening test forntidepressants, has been also considered as a stressful

ondition inducing behavioral changes, which are indices

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f “depressive-like” symptomatology (Connor et al., 1997;ryan et al., 2002). We have recently found that FST

nduces a number of sex specific changes in behavior andn serotonergic and HPA axis indices (Drossopoulou et al.,004). Therefore, we hypothesized that the application ofST on male and female rats already disturbed by expo-ure to CMS, would give us additional information on sex-ifferentiated behavioral responses to stress and subse-uent biochemical changes.

EXPERIMENTAL PROCEDURES

nimals

dult male and female Wistar rats (approximately 3 months of ageeighing 300–350 and 250–300 g, respectively at the beginningf the experiment) were used throughout this study. All male andemale rats in both experiments were singly housed in plastic,on-transparent cages (40�25�15 cm), in separate rooms underontrolled 12-h light/dark cycle (lights on at 06:00 h) and temper-ture (22 °C) conditions. All rats had free access to food and waterhroughout the experiments, unless it was differently indicated byhe experimental procedure. All animal experiments were re-iewed and approved by the local committee and all studies haveeen carried out in accordance with the National Institutes ofealth Guide for the Care and Use of Laboratory Animals (NIHublications No. 80–23) revised 1996. Efforts were made in order

o minimize the numbers of animals used and to reduce theiruffering.

Female rats were cycling normally (4–5 days of estrous cycle)efore the start of the experiment and were characterized by anqual distribution of different stages of estrous cycle. The estrousycle was again detected during the last week of CMS with the usef vaginal smears, as it has been previously described (Sfikakis etl., 1996).

xperiment 1

CMS. A CMS protocol, that has been previously describedor male rats (Willner et al., 1987) and modified by Papp et al.,002, was employed to male and female rats. Specifically, maleN�13) and female (N�14) rats spent 4 weeks of adaptation toonsumption of 1% sucrose solution in water. The adaptationeriod took place before the beginning of any stressful proceduresnd consisted of eight one-bottled-sucrose test sessions. Eachession was performed twice weekly (with an interval of 3 days)ollowing a period of 14 h of food and water deprivation and lasted

h (between 10:00 and 11:00 h). Sucrose consumption wasalculated by weighting the bottles before and after the one hourest. The mean measurements of the three last tests were used ashe baseline sucrose consumption (week 0) and then rats wereatched and divided into four groups: Control Males, Controlemales, CMS Males and CMS Females groups. The differencesetween the baseline sucrose consumption of the two groups ofales and the two groups of females were not statistically

ignificant.The CMS protocol started one day after the last sucrose test

f the adaptation period and lasted for 6 weekly cycles thatonsisted of continuous stressors alternating during the day (twoifferent stressors per day, see Table 1, Papp et al., 2002). Thetressors were: food or water deprivation, stroboscopic illumina-ion (120 flashes/min), intermittent illumination (lights switched onnd off every 2 h), paired housing (two animals randomly assigned

nto one cage), cage titling (at 45 degrees), soiled cage (250 ml ofap water into the sawdust bedding) followed by cage cleaning.ach stressor lasted 10–14 h (schedule in Table 1). The bodyeight of each animal was measured once per week every Mon-

ay morning. a

Behavioral testing. Sucrose intake: Once a week duringMS procedure, a one-bottled-sucrose test was carried out forne-hour between 10:00–11:00 h, as it was described above. Allnimals (Control and CMS) were deprived of food and water for aeriod of 14-hours before the sucrose intake test.

In addition, the ratio of sucrose intake per body weight wasalculated in order to examine if the CMS effect on sucrose intakeas related to respective changes in body weight.

OF test: Spontaneous OF activity was measured before thetart of CMS (week 0) and at the beginning (Monday morning) ofeeks 3 and 6 of the CMS procedure. The rats were transferred

o the test room one hour before testing for acclimatization. Oneat at the time was introduced into a transparent plastic OF cage40�40�40 cm) and its behavior was recorded during a 5-minbservation period. An observer recorded the horizontal and verticalctivity, using a previously used registration program (Spruijt andispen, 1984; Antoniou et al., 1998). In particular, the frequency anduration of moving (walking on all four feet) and rearing (body in-lined vertically with hindpaws on the floor of the activity cage andorepaws on the wall of the cage) were registered.

Neurochemical measurements. Control and CMS rats wereilled by decapitation, under basal a.m. conditions, 24 h after theast sucrose test (week six of CMS procedure). Brains were thenapidly removed and discrete brain regions, such as hippocam-us, hypothalamus and prefrontal cortex were dissected on ice,eighted, homogenized and deproteinized in 500 �l of 0.2 Nerchloric acid (Merck KgaA, Darmstadt, Germany) solution con-aining 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA (both by Riedel-deaën AG, Seelze, Germany). The homogenate was centrifuged at4,000 r.p.m. for 30 min in 4 °C and the supernatant was stored at80 °C. Analysis was performed by high-performance liquid chro-atography with an electrochemical detector, as previously de-

cribed by Sharpe et al., 1987 with some minor modificationsPapadopoulou-Daifotis et al., 1995). All samples were measuredithin one month after homogenization. Previous studies havehown that all monoamines remain stable up to one month fol-owing homogenization (Kilpatrick et al., 1986).

Reverse-phase ion pair chromatography was used to assay in

able 1. Schedule of CMS protocol

Weekly CMS protocol

onday 10:00 h Cage cleaning followed by no stressonday 20:00 h Food and water deprivation for 14 hoursuesday 10:00 h Sucrose test, followed by food or water

deprivation for 10 hoursuesday 20:00 h Paired housing for 14 hoursednesday 10:00 h Lights switched on and off every 2 h for

10 hoursednesday 20:00 h Soiled cage (250 ml of water was poured

into the sawdust bedding) for 14 hourshursday 10:00 h Cage cleaning, followed by water

deprivation for 10 hourshursday 20:00 h Paired housing for 14 hoursriday 10:00 h Stroboscopic illumination in darkness for

10 hoursriday 20:00 h Food deprivation for 14 hoursaturday 10:00 h Tilting of the cages backwards (45 degrees)

for 10 hoursaturday 20:00 h Cages were put back in straight position/

followed by no stressunday 10:00 h Stroboscopic illumination in darkness for

10 hoursunday 20:00 h Soiled cage (250 ml of water was poured

into the sawdust bedding) for 14 hours

ll samples serotonin (5-HT) and its metabolite 5-hydroxyin-

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oleatic acid (5-HIAA). The mobile phase consisted of an aceto-itrile (Merck KgaA, Darmstadt, Germany) -50 mM phosphateuffer (10.5:89.5) pH 3.0, containing 300 mg/l 5-octylsulfate so-ium salt (Merck KgaA, Darmstadt, Germany) as the ion-paireagent and 20 mg/l Na2EDTA (Riedel-de Haën AG, Seelze,ermany). Reference standards were prepared in 0.2 N perchloriccid solution containing 7.9 mM Na2S2O5 and 1.3 mM Na2EDTAboth by Riedel-de Haën AG, Seelze, Germany). The sensitivity ofhe assay was tested for each series of samples using externaltandards. Assays were performed on a BAS-LC4B HPLC systemith an amperometric detector. The working electrode was glassyarbon; the columns were Thermo Hypersil-Keystone, 150�.1 mm 5 � Hypersil, Elite C18 (Thermo Electron, Cheshire, UK).he HPLC system was connected to a computer, which was used

o quantify all compounds by comparison of the area under theeaks with the area of reference standards with specific HPLCoftware (Chromatography Station for Windows). The limit of de-ection was 1 pg/27 �l (injection volume). The turnover ratio of-HIAA/5-HT is considered to be an index of the activity of theells that cause release of the 5-HT, re-uptake and metabolism to-HIAA (Commissiong, 1985).

Corticosterone measurements. Trunk blood was collecteduring decapitation and samples were centrifuged at 4000 r.p.m.or 30 min. The serum was collected and frozen at �80 °C untilse. Corticosterone levels were determined by using a standardadioimmunoassay kit (ICN Biomedicals, Costa Mesa, CA, USA).he inter- and intra-assay coefficients of variation were both 8%.

xperiment 2

CMS plus FST (CMS�FST). Groups of male (N�13) andemale (N�11) rats were subjected to CMS, or served as controls,s it was described above (see also Table 1) and 24 h after thend of CMS procedure they were subjected to the FST. Thisxperiment was designed in order to investigate if an alteredubstrate induced by exposure to CMS had an effect on theesponse of male and female rats to a short-term stressful proce-ure, such as FST.

Behavioral measurements in FST. For the FST, rats werendividually placed in a cylindrical tank measuring 60�38 cm. Theank was filled with water (24�1 °C) at a height of 40 cm. Thenimals were forced to swim for a 15-min period (pre-test) and4 h later were subjected to a 5-min swimming session (test)Porsolt et al., 1977). Following FST sessions, the rats wereemoved from the tank, carefully dried in heated cages and theneturned to their home cages until decapitation. The total durationf floating and swimming were measured during the first 5 min ofach FST session (pretest and test). Rats were considered tohow floating (immobility) when they did not struggle, while madenly those movements necessary to keep their heads above theater. Swimming included vigorous movements of the wholeody, active swimming in circles and climbing on the walls of theank. Increased passive behavioral responses in FST such asmmobility and decreased active behaviors like swimming or strug-ling, are thought to be a clear indication of “depressive-like”ymptomatology (Detke et al., 1995; Lopez-Rubalcava and Lucki,000).

Physiological and neurochemical measurements. The ratsere decapitated 20 min after the 2nd FST session, their brainsere rapidly removed and the hippocampus, hypothalamus andrefrontal cortex were isolated on ice. Trunk blood samples werelso collected 20 min after the 2nd FST session, under a.m.onditions. Levels of 5-HT and 5-HIAA in the different brain areass well as corticosterone serum levels were assayed as describedbove. Body weight was weekly taken during CMS and following

ST application. a

tatistics

Experiment 1. Statistical analysis of sucrose intake, bodyeight and behavioral measurements from the OF was performedsing repeated analysis of variance (ANOVA), with two between-ubjects factors: sex (male versus female) and exposure to CMSControl versus CMS) and one within-subject factor of time (week–6). Greenhouse-Geisser corrections were used when needed.eparate repeated ANOVAs and one-way ANOVAs were per-

ormed, when needed, in order to elucidate specific differencesetween groups.

Regarding neurochemical measurements and corticosteroneata a two-way ANOVA with two between-subjects factors: sexmale versus female) and exposure to CMS (control versus CMS)as used. Separate one-way ANOVAs were performed, wheneeded, in order to elucidate specific differences between groups.

Experiment 2. In order to estimate the impact of CMS pro-edure on the changes induced by FST application in both sexes,he following statistical analyses were performed. A repeatedNOVA was used, with two between-subjects factors: sex (maleersus female) and exposure to CMS (Control�FST versusMS�FST) and one within-subjects factor of session (pretest vs.

est), for behavioral measurements from FST. Greenhouse-Geis-er corrections were used when needed. Separate repeatedNOVAs and one-way ANOVAs were performed, when needed,

n order to elucidate specific differences between groups.A two-way ANOVA with two between-subjects factors: sex

male versus female) and exposure to CMS (Control� FST versusMS�FST), was performed for the neurochemical measurementsnd corticosterone data. Separate one-way ANOVAs were per-ormed, when needed, in order to elucidate specific differencesetween groups.

RESULTS

xperiment 1

valuation of the phases of estrous cycle in female ratsuring the last week of CMS showed an alteration ofstrous cycle in all CMS rats and in 50% of isolated controlats. In particular, the estrous cycle was desynchronizednd did not have the normal duration of 4–5 days.

Body weight. For purposes of better comparison be-ween groups a transformation of the data derived fromody weight measures was performed. In particular, theeight of week 0 has been considered as the basis ofomparison at 100, so that the weight of weeks 1, 2, 3, 4,and 6 could be expressed as the respective percentage

%) changes.Fig. 1 shows that male (A) rats exposed to CMS in-

reased their body weight over the six experimental weeks tosignificantly slower rate than their controls. No differences

re depicted between control and CMS female rats (1B) inerms of % weight gain over the experimental period.

A two way repeated ANOVA with sex and CMS asndependent factors and time as repeated factor revealedn effect of time [F(6,144)�118.754; P�0.001], an effect ofMS [F(1,24)�6.989; P�0.014], an interaction of time withMS [F(6,144)�4.446; P�0.001] and time with sex andMS [F(6,144)�13.647; P�0.001] on the % body weight.eparate one-way repeated ANOVA with CMS as indepen-ent factor revealed a time effect [F �144.528; P�0.001]

(6,66)

nd an interaction of time and CMS [F(6,66)�15.355;

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�0.001] on the % body weight of male rats. One wayNOVAs revealed a statistical significant decrease in theercent weight gain between CMS and control male ratsF(1,12)�7.046; P�0.022; F(1,12)�9.268; P�0.011; F(1,12)�6.646; P�0.002; F(1,12)�20.026; P�0.001; F(1,12)�18.306;�0.001, for weeks 2–6 respectively]. The same analysis for

emale rats revealed only a time effect [F(6,78)�38.101;�0.001] on the % body weight.

Sucrose intake and relative sucrose intake. Fig. 2hows that CMS affected sucrose consumption mainly inale (A), but also to some extent in female (B) rats.pecifically sucrose consumption was lower in all weeks

ollowing week 1 in male CMS rats compared with controls,hile this decrease was evident only in weeks 1 and 4 in

he female CMS rats.In particular, a two way repeated ANOVA with sex and

MS as independent factors and time as repeated factorevealed an effect of CMS [F(1,23)�22.664; P�0.001], anffect of sex [F(1,23)�6.374; P�0.019] and an interaction ofime with CMS [F(6,138)�2.361; P�0.033] on sucrose in-ake. In particular male CMS rats exhibited significantlyess sucrose intake in all weekly tests (weeks 1–6) com-ared with controls [F(1,12)�10.28; P�0.008, F(1,12)�4.65;�0.05, F(1,12)�5.43; P�0.04, F(1,12)�5.101; P�0.045,

(1,12)�26.31; P�0.001, F(1,12)�5.441; P�0.04, for weeks–6 respectively] (Fig. 2A). This effect in female rats wasvident only in two of seven sucrose tests [F(1,13)�15.102;

ig. 1. Effect of CMS exposure on body weight of male (A) and femalemale control and CMS rats (% of basal body weight at week 0). Basa36�6.8 for female controls and 230�7.8 for female CMS rats. *, **, *

of basal body weight of control and CMS male rats per week.

ig. 2. Effect of CMS exposure on sucrose intake of male (A) and fem, ,

nd female control and CMS rats. * ** *** (P�0.05, P�0.01, P�0.000, respect

ats per week.

�0.002; F(1,13)�5.433; P�0.038, for weeks 1 and 4 re-pectively] (Fig. 2B). Moreover, a time-dependent increase

n sucrose intake was seen only in control male and femaleF(6,36)�3.723; P�0.048 and F(6,36)�2.135; P�0.073, re-pectively], but not in CMS male and female rats.

A two way repeated ANOVA with sex and CMS as inde-endent factors and time as repeated factor revealed anffect of time [F(6,138)�2.197; P�0.047], an effect of CMS

F(1,23)�10.349; P�0.004], an effect of sex [F(1,23)�56.338;�0.001] and an interaction of time with CMS [F(6,138)�.214; P�0.045] on relativesucrose intake (sucrose intakeivided with body weight). The differences in relative su-rose intake were similar to those for sucrose intake, soeparate analyses and data are not presented here.

OF test. Fig. 3A shows that male control rats de-reased their moving behavior in weeks 3 and 6 as com-ared with week 0. The decrease in the moving behavior inale CMS rats was apparent only at week 6. Fig. 3B

hows that in female rats CMS had a more pronouncedffect as a decrease in the moving behavior was seen inoth weeks 3 and 6 in comparison with week 0. Controlemale rats also reduced their moving behavior, but thisecrease did not reach significance.

Those observations were confirmed by two way re-eated ANOVA with sex and CMS as independent factorsnd time as repeated factor that revealed an effect of timeF(2,38)�17.603; P�0.001] and an interaction of time and

. Each value represents the mean�S.E. for weeks 0–6 for male andeight in g was 351�9.8 for male controls, 352�12 for male CMS rats,5, P�0.01, P�0.000, respectively) significant difference between the

ats. Mean values�S.E. for sucrose intake (g) in weeks 0–6 for male

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ex and CMS [F(2,38)�6.907; P�0.003] for the duration ofoving. Separate repeated ANOVA revealed a time effect

or control males [F(2,8)�9,713; P�0.007]. In addition, aime effect was also revealed for the duration of moving ofMS males [F(2,8)�21.456; P�0.001], but this effect wasue to decrease of moving duration only in week 6F(1,9)�5.966; P�0.04, in comparison with controls] (Fig.A). Additionally, there was a statistical significant de-rease of moving duration over time in CMS femalesF(2,12)�10.463; P�0.002] (Fig. 3B).

The profile of the duration of rearing of male and fe-ale CMS rats shown in Fig. 3C and D respectively is quite

imilar to that one of moving. Thus, CMS induced a signif-cant decrease in rearing behavior of males at week 6 inomparison with week 0 (Fig. 3C); while in females thisecrease was obvious at both weeks 3 and 6 (Fig. 3D). Inhe same way, two way repeated ANOVA with sex andMS as independent factors and time as repeated factor

evealed an effect of time [F(2,38)�13.210; P�0.001], anffect of sex [F(1,19)�8.724; P�0.008] and an interaction of

ime with sex and CMS [F(2,38)�4.401; P�0.019] for theuration of rearing. Further repeated ANOVA revealed thathe duration of rearing was decreased over time onlyn CMS male and female rats [F(2,8)�8.572; P�0.01;

(2,12)�22.076; P�0.001, respectively] (Fig. 3C, D). Nev-rtheless, the decrease in the duration of rearing in CMSales compared with their controls was significant only ineek 6 [F(1,9)�10.995; P�0.011], while the same effectas observed for female CMS rats already from the week

ig. 3. Effect of CMS exposure on moving duration in male (A) and feale and female control and CMS rats �� (P�0.01) significant differenale and female rats, respectively. Effect of CMS exposure on rearing d

or weeks 0, 3 and 6 for male and female control and CMS rats �, ���

etween weeks 3, 6 and week 0 of CMS male and female rats, respe

[F(1,12)�4.860; P�0.05]. r

Interestingly, the duration of rearing was higher in fe-ale control rats compared with male controls in weeks 0

start of the experiment) and 3 [F(1,10)�6.207; P�0.034;

(1,10)�7.778; P�0.021, respectively].The frequency of the recorded behavioral variables

ent in parallel with the duration of the respective behav-ors, thus statistics and data are not presented here.

Corticosterone levels. Table 2A shows that CMS in-uced a significant increase in corticosterone levels ofemale rats but not of male rats. Notably, there was no sexffect on corticosterone levels in control rats.

Two way ANOVA with CMS and sex as factors re-ealed a tendency for an effect of CMS [F(1,35)�3.733;�0.063] on corticosterone levels. Separate one wayNOVA revealed a significant CMS effect on the cortico-terone levels of female rats [F(1,21)�5.866; P�0.025].

Neurochemical results. Hippocampus: Table 3Ahows that CMS decreased significantly the 5-HT turnoveratio only in female rats. This decrease appears to be dueo an increase (although non-significant) of 5-HT levels.

Two way ANOVA, with CMS and sex as factors, re-ealed an effect of CMS on 5-HT levels and subsequentlyn 5-HT turnover ratio (5-HIAA/5-HT) [F(1,13)�6.037;�0.021; F(1,13)�5.465; P�0.027, respectively]. In addi-

ion, it revealed a significant effect of sex on 5-HT levelsF(1,27)�6.477; P�0.014] and a tendency for an interactionetween CMS and sex [F(1,27)�3.223; P�0.07]. Subse-uent one way analyses revealed that the 5-HT turnover

rats in weeks 0, 3, and 6. Mean values�S.E. for weeks 0, 3 and 6 formoving duration between weeks 3, 6 and week 0 of control and CMSmale (C) and female (D) rats in weeks 0, 3, and 6. Mean values�S.E., P�0.000, respectively) significant difference of the rearing duration

male (B)ce of theuration in

atio (5-HIAA/5-HT) was decreased following CMS appli-

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ation, only in female rats [F(1,13)�4.402; P�0.043] (TableA). In addition, 5-HT levels were higher in female CMSats compared with male CMS rats and 5-HIAA/5-HT wasower in female CMS rats compared with males [F(1,13)

6.037; P�0.021; F(1,13)�5.465; P�0.027, respectively]Table 3A).

Hypothalamus: Table 4A shows that CMS decreased-HIAA levels only in female rats [F(1,13)�4.693; P�0.05].t is also apparent that the 5-HT and 5-HIAA levels areower in all groups of female rats as compared with their

ale counterparts. These differences however do not haven impact on the 5-HT turnover ratio.

Two way ANOVA with CMS and sex as factors re-ealed an effect of sex on 5-HIAA and 5-HT levelsF(1,27)�27.605; P�0.001; F(1,27)�21.076; P�0.001, re-pectively]. Subsequent one way analyses for sex differ-nces revealed that 5-HIAA and 5-HT levels were higher inale rats compared with females, irrespective of CMS appli-

ation [F(1,12)�12.320; P�0.005; F(1,13)�16.43; P�0.002;

(1,12)�22.795; P�0.001; F(1,13)�9.74; P�0.009, for 5-HIAAnd 5-HT levels of control and CMS rats, respectively]Table 4A).

Prefrontal cortex: Table 5A shows that neither CMSor sex had an effect on the serotonergic activity of therefrontal cortex. That was confirmed by two way ANOVAith CMS and sex as factors, which revealed no effect ofex or CMS.

able 2. Corticosterone levels

. cms Male control Mal

orticosterone (ng/mL) 97.3�30 116

. cms�fst Male control�FST Mal

orticosterone (ng/mL) 487�53 649

A. Effect of CMS on corticosterone levels of male and female rats ((P�0.05) significant differences between control and CMS female rapplication (mean values�SE). † (P�0.05) significant difference betw

able 3. Neurochemical analysis of serotonergic neurotransmission in

Hip

. cms 5-H

ale Control 0.4CMS 0.3

emale Control 0.4CMS 0.5

. cms�fst 5-H

ale Control�FST 0.6CMS�FST 0.8

emale Control�FST 0.5CMS�FST 0.6

A. Levels of 5-HT, 5-HIAA (�g/g tissue), as well as serotonergic turocedure (mean values�SE). B. Levels of 5-HT, 5-HIAA (�g/g tissueoth sexes following CMS procedure plus FST application (mean valu(P�0.05) significant differences between Control and CMS rats, # (P

rocedure.

xperiment 2

Body weight. Chronic exposure to CMS induced spe-ific body weight changes that have been previously de-cribed in experiment 1. FST application did not induceny changes in the body weight probably due to the shorturation of this experimental procedure. Apparently, ratshat were exposed to CMS plus FST exhibited body weighthanges that were similar to those observed during CMSrocedure in experiment 1 (see Fig. 1A, B).

Behavior in FST. Fig. 4A and B show that CMS fe-ale rats floated less and swam more as compared with

ontrol female rats during the test session. Male CMS ratswam less during the pretest session than controls, whilehey did not increase their floating duration nor they de-reased their swimming duration in the test session com-ared with pretest.

A two way repeated ANOVA for the duration of floatingehavior with sex and CMS as independent factors andession as repeated factor revealed a significant effect ofession [F(1,,21)�36.17, P�0.001] and an interaction be-ween session and sex [F(1,21)�7.44, P�0.014]. Separateepeated ANOVA analysis revealed that control femalesncreased their duration of floating between test and pre-est session [F(1,5)�17.82, P�0.05]. In addition, floatinguration was increased in test compared with pretest ses-

Female control Female CMS

108�33 207�25*

ST Female control�FST Female CMS�FST

608�111 725�75

lues�SE).ticosterone levels in male and female rats exposed to CMS plus FST

trol�FST and CMS�FST male rats.

ocampus

s

5-HIAA 5-HIAA/5-HT

0.30�0.027 0.74�0.0430.30�0.020 0.83�0.0530.35�0.016 0.77�0.049

# 0.35�0.021 0.64�0.038#*

5-HIAA 5-HIAA/5-HT

0.34�0.024 0.55�0.0620.41�0.04 0.49�0.0290.32�0.011 0.62�0.0730.41�0.10 0.59�0.043

tio (5-HIAA/5-HT) in the hippocampus of both sexes following CMSl as serotonergic turnover ratio (5-HIAA/5-HT) in the hippocampus of.ignificant differences between male and female rats exposed to CMS

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C. Dalla et al. / Neuroscience xx (2005) xxx 7

ARTICLE IN PRESS

ion in female rats previously subjected to CMS procedureF(1,5)�42.99, P�0.001]. Floating behavior was lower inemale CMS rats compared with their controls during theest session [F(1,11)�5.31, P�0.05] (Fig. 4A).

A two way repeated ANOVA for the duration of swim-ing behavior with sex and CMS as independent factorsnd session as repeated factor revealed a significant effectf session [F(1,21)�21.78, P�0.001], an interaction be-

ween session and sex [F(1,21)�5.32, P�0.033] and annteraction between session and CMS [F(1,21)�5.32,�0.034]. Separate repeated ANOVA analysis revealed aecrease in the duration of swimming in male and femaleontrol rats between test and pretest session [F(1,4)�31.9,�0.005; F(1,5)�46.84, P�0.021, respectively] (Fig. 4B).

n addition, swimming duration was decreased in test com-ared with pretest session in female rats previously subjectedo CMS procedure [F(1,5)�17.82, P�0.008] (Fig. 4B).

able 4. Neurochemical analysis of serotonergic neurotransmission in

Hyp

. cms 5-HT

ale Control 3.15CMS 3.38

emale Control 2.33CMS 2.1

. cms�fst 5-HT

ale Control�FST 1.74CMS�FST 2.18

emale Control�FST 1.01CMS�FST 0.96

A. Levels of 5-HT, 5-HIAA (�g/g tissue), as well as serotonergic turocedure (mean values�SE). B. Levels of 5-HT, 5-HIAA (�g/g tissueoth sexes following CMS procedure plus FST application (mean valu(P�0.05) significant differences between Control and CMS female retween male and female control rats, male and female CMS rats, mapplication.

able 5. Neurochemical analysis of serotonergic neurotransmission in

Pre

. cms 5-H

ale Control 1.2CMS 1.3

emale Control 1.4CMS 1

. cms�fst 5-H

ale Control�FST 1.1CMS�FST 1.0

emale Control�FST 0.8CMS�FST 0.9

A. Levels of 5-HT and 5-HIAA (�g/g tissue), as well as serotonergic turocedure (mean values�SE). B. Levels of 5-HT and 5-HIAA (�g/gortex of both sexes following CMS procedure plus FST application (m

ontrol rats exposed to FST.

The duration of swimming in male CMS rats was shorterompared with their controls during the pretest sessionF(1,12)�5.05, P�0.046], while in CMS female rats the dura-ion of swimming was longer compared with their controlsuring the test session [F(1,8)�5.32, P�0.05] (Fig. 4B).

In general, female rats exhibited higher duration ofwimming compared with males during the pretest ses-ion, irrelevant of CMS exposure [F(1,10)�10.95, P�0.013;

(1,13)�14.10, P�0.003, for control and CMS rats, respec-ively] (Fig. 4B).

Neurochemical measurements. Hippocampus: TableB shows that there was no statistical significant difference

n hippocampus between control rats subjected to FST andats subjected to CMS and FST. That was confirmed bywo way ANOVA with CMS and sex as factors that did noteveal any statistically significant CMS or sex effect on-HIAA, 5-HT levels and 5-HIAA/5-HT ratio (Table 3B).

othalamus

s

5-HIAA 5-HIAA/5-HT

0.99�0.07 0.32�0.020.98�0.08 0.31�0.030.72�0.04## 0.32�0.030.54�0.07*## 0.26�0.02

5-HIAA 5-HIAA/5-HT

0.74�0.03 0.40�0.030.72�0.03 0.34�0.020.50�0.024## 0.53�0.070.53�0.068# 0.56�0.047##

tio (5-HIAA/5-HT) in the hypothalamus of both sexes following CMSl as serotonergic turnover ratio (5-HIAA/5-HT) in the hypothalamus of.

### (P�0.05, P�0.01, P�0.000, respectively) significant differencesmale FST rats and male and female rats subjected to CMS plus FST

rontal

ortex

5-HIAA 5-HIAA/5-HT

0.44�0.036 0.35�0.0140.46�0.040 0.35�0.0380.54�0.023 0.39�0.0400.53�0.060 0.33�0.030

5-HIAA 5-HIAA/5-HT

0.43�0.067 0.35�0.0370.35�0.020 0.35�0.042

# 0.39�0.039 0.47�0.0350.38�0.039 0.43�0.057

tio (5-HIAA/5-HT) in the prefrontal cortex of both sexes following CMSs well as serotonergic turnover ratio (5-HIAA/5-HT) in the prefrontal

es�SE). # (P�0.05) significant differences between male and female

the hyp

othalamu

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C. Dalla et al. / Neuroscience xx (2005) xxx8

ARTICLE IN PRESS

Hypothalamus: Table 4B shows that there was notatistical significant difference in hypothalamus betweenontrol rats subjected to FST and rats subjected to CMSnd FST. Notably, the 5-HIAA and 5-HT levels were higher

n all males compared with females.Two way ANOVA with CMS and sex as factors did not

eveal any statistically significant CMS effect on 5-HIAA,-HT levels and 5-HIAA/5-HT ratio (Table 4B). Nevertheless,

he same analysis revealed a sex effect on 5-HIAA, 5-HTevels and 5-HIAA/5-HT ratio [F(1,24)�20.056, P�0.001;

(1,24)�51.291, P�0.001; F(1,24)�12.15, P�0.003, respec-ively]. Subsequent analyses revealed that 5-HIAA and 5-HTevels were higher in male compared with female rats irre-pective of exposure to CMS [F(1,10)�20.87, P�0.004;

(1,13)�8.69, P�0.013; F(1,10)�16.72, P�0.006; F(1,13)�3.75, P�0.001, for controls plus FST and CMS plus FST,espectively]. In addition, 5-HIAA/5-HT ratio was lower inales subjected to CMS plus FST compared with their cor-

esponding females [F(1,12)�14.49, P�0.003] (Table 4B).Prefrontal cortex: Table 5B shows that there was no

tatistical significant difference in prefrontal cortex be-ween control rats subjected to FST and rats subjected toMS and FST. Two way ANOVA with CMS and sex as

actors did not reveal any statistically significant CMS ef-ect on 5-HIAA, 5-HT levels and 5-HIAA/5-HT ratio (TableB). Nevertheless, the same analysis revealed a sex effectn 5-HT levels [F(1,24)�4.375, P�0.05]. Subsequent anal-ses revealed that 5-HT levels were higher in male controlats subjected to FST compared with their correspondingemales [F(1,10)�7.577, P�0.028] (Table 5B).

Corticosterone levels. Table 2B shows that cortico-terone levels were higher in males subjected to CMSlus FST compared with controls subjected only to FSTF(1,12)�5.587, P�0.038]. Similar increase was observedn female rats which was not statistically significant (TableB). That was confirmed by, two way ANOVA with sex andMS as factors that revealed a statistical significant effect

ig. 4. (A) Effect of CMS exposure on floating duration during pretest a, �� (P�0.05, P�0.01, respectively) statistically significant differencreviously subjected to CMS procedure. * Statistically significant differehe test session of FST application. (B) Effect of CMS exposure on swemale rats (mean values�S.E.). �, �� (P�0.05, P�0.01, respectiveontrol rats, female control rats and female rats previously subjected toales previously subjected to CMS during the pretest session and bef FST application. #, ## (P�0.05, P�0.01, respectively) statistically signhe pretest session.

f CMS on corticosterone levels [F(1,22)�4.287, P�0.05]. c

DISCUSSION

he present study shows that the CMS impact on behav-oral parameters, as well as on serotonergic activity andorticosterone levels is sex dependent. In particular, fe-ale rats appear to be more vulnerable in application ofMS than males, since their motor activity was affected togreater extent than in males; sucrose intake was dis-

upted; serotonergic activity in hippocampus and hypothal-mus was decreased; corticosterone levels were in-reased and estrous cycle was desynchronized. Mean-hile, male rats in response to the current CMS protocolxhibited only behavioral changes, while they show noiochemical alterations.

Nevertheless, when examining the impact of CMS onST, females seem to cope better with the novel stressorFST) and they exhibit higher hypothalamic serotonergicctivity than corresponding males. On the other hand,ales in response to an additional stressor are more af-

ected by CMS exposure and exhibit the “depressive-like”ymptomatology in the FST earlier, along with higher cor-icosterone levels.

MS-induced behavioral changes in males andemales

s expected control male and female rats increased theironsumption of sucrose solution over time. On the con-rary, rats exposed to CMS appeared to have a stableonsumption of sucrose solution over all seven weeklyessions. Similar findings were also revealed by analysisf relative sucrose intake (sucrose intake/body weight)

n order to ensure lack of interference of body weighthanges on sucrose intake (Matthews et al., 1995; Forbest al., 1996). Interestingly, male CMS rats exhibited a slowate of weight gain, in agreement with previous findingsD’Aquila et al., 1997; Bielajew et al., 2003; Konkle et al.,003), while females exhibited the same weight gain as

ssion of FST application in male and female rats (mean values�S.E.).n pretest and test session in female control rats and in female ratseen control females and females previously subjected to CMS, duringuration during pretest and test session of FST application in male andically significant difference between pretest and test session in malecedure. * Statistically significant difference between control males andntrol females and females subjected to CMS during the test sessionfference between male and female control, as well as CMS rats during

nd test see betweence betw

imming dly) statistCMS protween coificant di

ontrols.

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C. Dalla et al. / Neuroscience xx (2005) xxx 9

ARTICLE IN PRESS

These findings show that mainly in males and to somextent in females following CMS, the rewarding value ofucrose solution is disrupted. Nevertheless, sex differ-nces in sucrose intake can be attributed to baseline dif-

erences, as female controls exhibited higher sucrose con-umption and a more erratic increase than males. Theseaseline differences probably do not reflect a differentiatededonic responsiveness, but are possibly mediated by theffects of sex steroids on taste and/or ingestive responsesHamilton and Timmons, 1976; Clarke and Ossenkopp,998; Curtis et al., 2004).

Meticulous studies have reported decrease sucrosentake/preference in male and female rats following CMSWillner et al., 1987; D’Aquila et al., 1997; Benelli et al.,999; Kioukia et al., 2000; Duncko et al., 2001b; Papp etl., 2002; Bekris et al., 2005), but this decrease has noteen replicated conclusively (Harro et al., 2001; Haidkindt al., 2003; Konkle et al., 2003). Nevertheless, the resultsf the present study along with that by Konkle et al. (2003)uggest that the CMS-induced anhedonia could be alsoeflected by the inability of CMS exposed rats to increaseheir sucrose consumption over time.

Exposure of male and female rats to CMS resulted inn overall decrease in rearing behavior, indicating a CMS-

nduced decrease in exploratory activity in accordance withHarro et al., 1999). Nevertheless, the decreased rearingehavior appeared earlier in female rats as compared withales and maintained low until the end of CMS. Mean-hile, CMS females exhibited reduced moving behaviorver time, while CMS males displayed decreased movingehavior during week 6. It seems that CMS affected hori-ontal activity as deduced by moving behavior in malesnd females, but this influence was more prominent inemales. Our results are in line with Kennett et al. (1986)ho suggested that females are more vulnerable in testsodeling depression since they maintain reduced activ-

ty in the OF test after exposure to repeated restrainttress.

MS-induced alterations in HPA and HPG axis

orticosterone levels were increased only in female ratsollowing 6 weeks of CMS, indicating that females areore vulnerable in the dysregulation of the HPA axis afterxposure to chronic stress. The different response inales most probably indicates their hormonal adaptation

o chronic stressful exposure. This view is supported byeports that corticosterone levels are elevated in male ratsfter 3 weeks of exposure to CMS (Bielajew et al., 2002),ut are normal (Willner et al., 1987; Stout et al., 2000;ilberman et al., 2002; Gronli et al., 2004) or slightly in-reased after longer application of CMS (Konkle et al.,003). Based on previous findings which have shown thatex steroids modulate adrenocortical and behavioral re-ponse to stress via their organizational and activationalffects (Patchev and Almeida, 1998; Rachman et al., 1998;arandrea et al., 2002; Bowman et al., 2003; Dalla et al.,004), we propose that the sex differences in HPA axisollowing CMS are associated with the differential action of

ex steroids in females and males (progesterone, estro- f

ens vs. androgens). However, in our control rats thereas no sex effect on corticosterone levels, probably due to

solation housing that has been considered as a key factoror male and female rodents (Palanza, 2001).

Moreover, CMS exposure caused a desynchronizationf estrous cycling in female CMS rats, suggesting a dys-egulation of HPG axis. Surprisingly, estrous cycle waslso altered in 50% of control rats, probably due to isolationousing that is considered to be stressful for femalesPalanza, 2001; Konkle et al., 2003; Westenbroek et al.,003). Similar to our findings, other investigators have alsoeported prolongation of estrous cycling in female rats afterxposure to CMS (Konkle et al., 2003; Grippo et al., 2005).

The dysregulation of HPG axis and specific behaviorallterations may be related to likely changes in the cyclicalecretion of estrogens and progesterone in CMS females.n the other hand, the lack of increased corticosterone

evels along with specific behavioral alterations in CMSales suggests that the stress paradigm either leaves

esticular function intact or that the male’s behavior is lessependent on testicular hormones. Thus, the differentiatedole of sex hormones on HPA/HPG alterations and on theehavioral responses induced by CMS exposure remainso be elucidated.

MS-induced alterations in serotonergic activity

resent findings show that CMS induced a decreasederotonergic activity, due to decreased serotonergic ratio5-HIAA/5-HT) in hippocampus and decreased 5-HIAA lev-ls in the hypothalamus of female rats. The aforemen-ioned results are in accordance with previous findingsbserved in female rats following FST (Drossopoulou etl., 2004) or repeated restraint stress (Kennett et al., 1986;owman et al., 2003).

The decreased serotonergic status in hippocampusnd hypothalamus of females underlies the sexual dimor-hism observed in stress models of depression and isrobably directly or indirectly associated with respectivehanges in HPA/HPG axis. It is known that 5-HT, by acti-ating 5-HT1A receptors in the hippocampus, causes hy-erpolarization (Hoyer et al., 2002), suppression of hip-ocampal output and subsequently disinhibition of HPAxis (Joels and De Kloet, 1992; De Kloet et al., 1998;eijer et al., 1998). On the other hand, the decreased

erotonergic activity in CMS females could have resultedrom changes in serotonergic function, mediated by alteredorticosteroid (Chalmers et al., 1993; Lopez et al., 1998;eitch et al., 2003; Froger et al., 2004; Grippo et al., 2005)nd/or sex steroid (Fink et al., 1998; Sumner et al., 1999;aap et al., 2000; Zhou et al., 2002) availability.

In contrast with females, males did not respond to CMSith changes in serotonergic activity, probably because of

heir better adaptation to the stress as suggested by theirnchanged corticosterone levels. This finding is not in lineith our previous results (Bekris et al., 2005) or resultserived from other studies (Gamaro et al., 2003; Li et al.,003), but it could be attributed to differences in the regi-ent of CMS used in the various studies. As a matter of

act, the present CMS protocol included less severe and

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C. Dalla et al. / Neuroscience xx (2005) xxx10

ARTICLE IN PRESS

horter in duration stressors, as compared with the previ-us CMS protocol used in our experiments.

Concerning controls several differences in the 5-HTnd/or 5-HIAA tissue levels were observed between malend female rats, especially in the hypothalamus whicheflect respective differences in the 5-HT synthesis, re-ease and metabolism and probably indicate a sex differ-ntiated basal serotonergic function.

MS impact on FST performance in males versusemales

MS females produced a lower duration of passive behav-or (floating) during the second session of FST comparedith control females, a finding that could be attributed to

he manifestation of a better coping strategy. Alternatively,his finding could be attributed to an impairment of memoryn CMS females, since the existence of increased floatingehavior in the second FST session could also reflectome learning or memory properties (Thierry et al., 1984;orsini and Meli, 1988). Nevertheless, our CMS females

ncreased their floating behavior between the two tests ofST, though in a lower extent than controls.

Opposing to females, CMS males exhibited reducedehavioral activity during pretest session that was not

urther decreased after exposure to the second session,ndicating an impact of CMS on FST performance. Biela-ew et al., 2003 described that male Long Evans ratsxposed to CMS performed less active and longer passiveehaviors in the test session of FST, while females did notiffer from controls. In addition, several studies reported

ncreased immobility in FST after exposure of male rats tohronic stress regimen (Garcia-Marquez and Armario,987; Molina et al., 1994), although opposing results haveeen reported (Haidkind et al., 2003).

Based on these findings it could be suggested thathere is a sex differentiation when rats are subjected toombination of different stressful paradigms.

ombination of CMS and FST impact onorticosterone blood levels and serotonergic activityf males and females

orticosterone levels were statistically higher only in malesxposed to CMS and FST, but not in males only exposed

o FST, suggesting that CMS history induced a potentiatedPA axis response. Part of these differences may bettributed to the respective actions of sex steroids inR/MR receptors of limbic areas (Patchev and Almeida,998; Karandrea et al., 2000; Karandrea et al., 2002), andur findings may be linked with the earlier expression ofdepressive-like” symptomatology in CMS males tested inhe FST paradigm, as well as with lower hypothalamicerotonergic activity, as deduced by 5-HT ratio, in malesxposed to CMS and FST compared with correspondingemales.

Nevertheless, serotonergic activity was similar in ratsubjected either to FST alone, or to FST after CMS. Other

nvestigators also found no changes in serotonergic activ-ty in male rats subjected to CMS followed by FST (Harro

t al., 2001; Haidkind et al., 2003). Taking into consider-

tion that CMS exposure decreased serotonergic activityn female rats; it could be argued that FST had no addi-ional effect on the already established CMS neurochem-cal profile.

oncluding remarks

n light of our findings, specific changes in HPA and HPGxis indices along with altered serotonergic activity in hip-ocampus and hypothalamus underline the sex differenti-tion in models of depression. Although these findingshow a higher vulnerability of females to stressful condi-ions, different compensatory mechanisms of femalesrobably are developed when they are exposed to addi-ional stressors. Minimally, our data point out that whenxamining stress-induced effects in animal models of de-ression, both sex differences and combination of stressfulrocedures should be carefully considered.

Present findings of this study could be relevant toexual dimorphism observed in affective disorders and theourse of related illnesses. Future experimental studiesre needed in order to further elucidate the role of gendernd sex steroids on the response of HPA axis to combinedtressful conditions and on the neurobiological substrate ofepression.

cknowledgments—The authors would like to thank Professor C.pyraki for her critical comments on the manuscript. This workas supported in part by the General Secretariat of Research andechnology (GSRT) of Greece (PENED01, 01ED82).

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(Accepted 26 June 2005)