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Behavioural Brain Research 187 (2008) 462–472

Research report

Individual responses to novelty are associated with differencesin behavioral and neurochemical profiles

K. Antoniou a,b,∗,1, G. Papathanasiou b,1, E. Papalexi c, T. Hyphantis d,G.G. Nomikos e, C. Spyraki b, Z. Papadopoulou-Daifoti b

a Department of Pharmacology, Medical School, University of Ioannina, 45110 Ioannina, Greeceb Department of Experimental Pharmacology, Medical School, University of Athens, 11527 Athens, Greece

c Department of Histology and Embryology, Medical School, University of Athens, 11527 Athens, Greeced Department of Psychiatry, Medical School, University of Ioannina, 45110 Ioannina, Greece

e Amgen Inc., Cambridge Research Center, Neuroscience, Cambridge, MA 02139 USA

Received 8 August 2007; received in revised form 9 October 2007; accepted 11 October 2007Available online 22 October 2007

Dedicated to the memory of C. Spyraki.

bstract

Experimental animals can be differentiated on the basis of their horizontal or vertical activity to high responders (HR) and low responders (LR)pon exposure to a novel environment. These individual differences have been associated with behavioral and neurobiological differences in aumber of experimental procedures used for studying sensitivity to psychostimulants, anxiety, depression, and cognitive function. In the presenttudy, we differentiated the rats to HR and LR based on their vertical activity upon exposure to a novel environment. Additionally, we ascertainedhether HR and LR rats differ in a battery of tests such as passive avoidance (PA), object recognition (OR), and the water-maze (WM) that provide

ndices for cognitive function and the forced swim test (FST), an animal model of affective responsivity and antidepressant-like activity. Potentialifferences in neurochemical indices between the two phenotypes were also examined. HR rats displayed impaired non-spatial object recognitionemory, but enhanced spatial performance, as compared to LR rats. FST induced “depressive-like” symptoms in both phenotypes that were

ifferently manifested in HR versus LR rats. Neurochemical findings revealed distinct differences in serotonergic and dopaminergic activity in thetriatum and the prefrontal cortex of HR as compared to LR rats. The above results show that HR and LR rats exhibit important differences in a batteryf tests related to cognitive performance or affective responsivity, which may be associated with differences in certain neurobiological parameters.

2007 Elsevier B.V. All rights reserved.

eywords: Open-field activity; High responders; Low responders; Rats

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Abbreviations: 5-HIAA, 5-hydroxyindoleacetic acid (5-HIAA); 5-HT, sero-

nin; ANOVA, analysis of variance; D, discrimination index; DA, dopamine;-amp, d-amphetamine; DOPAC, 3,4-dihydroxyphenylacetate; FST, forcedwim test; grm, grooming; H, habituation index; HPA, hypothalamic-pituitary-drenal; HPLC, high-performance liquid chromatography; HR, high responders;VA, homovanillic acid; LR, low responders; mov, moving; OR, object recog-ition; PA, passive avoidance; rr, rearing; scr, scratching; sna, sniffing air; sni,niffing; std, standing; WM, water-maze.∗ Corresponding author at: Department of Pharmacology, Medical School,niversity of Ioannina, 45110 Ioannina, Greece. Tel.: +30 26510 97570; fax:30 26510 97859.

E-mail address: kantoniu@cc.uoi.gr (K. Antoniou).1 These authors contributed equally.

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Experimental animals can be differentiated on the basis ofheir horizontal or vertical activity to high responders (HR) andow responders (LR) upon exposure to a novel environment5,38,39,54,55]. These individual differences may be associatedith behavioral or neurobiological differences in experimen-

al procedures used for assessing sensitivity or vulnerability tosychostimulants or with respective differences in the animals’eaction to the testing conditions [1,10,19,39,41,46].

Several studies have reported behavioral and/or neurobi-logical differences in both phenotypes when employed to

xperimental tasks related to stress, anxiety, emotional reac-ivity, depression and cognitive function [6,23,26,40,42,53,54].ome of these studies have shown that HR rats are more anxiousr hyper-responsive in stress-related tasks as compared to LR

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ats [6,31,40,41]. This notion has been challenged given thatR most likely show an increased responsiveness to stress not

oupled with a respective increase in their anxiety-like profile,s compared to LR rats [26,53]. Furthermore, Kabbaj et al. [25]ave reported that basal differences in gene expression of keytress-related molecules such as hypothalamic mRNA CRHevels, and hippocampal expression of glucocorticoid (GR) butot mineralocorticoid receptors (MR) may play an importantole in individual differences following exposure to stress andovelty. However, there have not been any substantial differ-nces between HR and LR rats in relation to anxiety responsessing various tasks such as the plus-maze and object-buryingest [23]. Interestingly, White et al. [62] have reported some dif-erences between HR and LR rats concerning their anxiety-likeeactivity, but these differences were dependent on the specificnimal anxiety model used. In particular, HR rats displayed lessnxiety-like behavior, as deduced by the elevated plus maze andhe defensive withdrawal test than LR, but the opposite effectsere observed using the acoustic startle-induced vocalization

est. In addition, HR did not exhibit any major differences compared to LR rats in the depression-like reactivity aseflected by the forced swim test [23,62].

Although adult animals show phenotypic differences relatedo their reaction to novelty or to their specific responses recordedn various testing conditions, the potential association of theseifferences with tentative differences in attention, learning oremory procedures cannot be excluded. Interestingly, Cools et

l. [9] have suggested that some differences between HR and LR,n the response to a simple four-arm radial maze, are not due sim-ly to differences in motor activity, but to subtle yet importantifferences in the mode of learning. Moreover, our recent results5] have shown that a phenotypic distinction between HR and LRats probably reflects quantitative and qualitative differentiationshat characterize their respective reaction to an experimen-al procedure and subsequently the specific type of a neededehavioral response in the respective task. On the other hand,inor phenotypic differences were yielded between HR andR rats in some tasks related to learning and memory function

6,38].It seems that despite the substantial work on the study

f individual differences, inconsistent findings provide limitedlarification on the respective behavioral variations. Notably,istinct differences probably predict respective phenotypes inubsequent behavioral paradigms related to drug abuse, anxiety,epression, or cognition, but these differentiations are depen-ent on both the screening procedure and the specific modelsed. There are few studies that have focused on differencesn various behavioral and neurobiological responses using aet of experimental tasks, following a distinct classification ofhe rats under study. Spontaneous open filed activity includes

variety of responses that could be interpreted as indicesf exploration, arousal, locomotion, anxiety and emotional-ty. In particular, increased locomotion (horizontal, forward

r ambulatory activity) and vertical activity (rearing) mainlyharacterize the rat’s behavioral response to novelty. Rear-ng behavior reflects most of the aforementioned aspects ofpontaneous open field activity and has been used apart from

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Research 187 (2008) 462–472 463

ocomotion as a valuable variable for the rats’ phenotypic clas-ification [5,23,38,55]. Thus, in the present study, the rats wereifferentiated into HR and LR, based on the counts of verticalctivity during their exposure to a novel environment. More-ver, we ascertained whether HR and LR rats differ in a batteryf tests such as passive avoidance (PA), object recognitionOR), and the water-maze (WM) that provide indices for dif-erent aspects of learning and memory function. In addition, aepression-like reactivity using the behavioral responses in theorced swim test (FST) was examined in both groups of rats.

oreover, possible basal neurochemical differences betweenhe two phenotypes regarding dopaminergic and serotonergicrain function were examined by measuring tissue levels ofhe monoamines and their metabolites in distinct regions of therain.

. Materials and methods

Male Wistar rats (inbred in the Pasteur Institute of Athens), aged 70–90ays and weighing 250–300 g, at the beginning of the experiments weresed. The animals were housed in groups of six or seven in plastic cages57 cm × 35 cm × 20 cm) with food and water available ad libitum, under con-rolled laboratory conditions, i.e. 12-h light/dark with lights on at 06:00 h and aonstant temperature of 21 ± 1 ◦C. All rats were accustomed to our laboratoryonditions for a 2-week period. They were gently handled twice per week andheir weight was routinely checked once per week.

All animal experiments were reviewed and approved by the local committeend all studies have been carried out in accordance with the National Institutef Health Guide for the Care and Use of Laboratory Animals (NIH Publicationso. 80-23; revised 1996). Efforts were made in order to use a minimal numberf rats necessary for experiments and reduce their suffering.

.1. Open field behavior—response to novelty

The behavioral testing was performed between 08:00 and 16:00 h. The ratsere initially accustomed to the experimental room, for 1 h prior to the experi-ent. The rats were then introduced into the testing cage, a transparent plastic

pen field cage (40 cm × 40 cm × 40 cm) and behavior was recorded for a 15-in observation period. In brief, an observer recorded the behavioral responses,

sing a registration program based on Spruijt and Gispen [52]. The frequencynd duration of each behavioral response were recorded at the end of each 15-in observation period [2–5]. The duration of this session was chosen becauseshort exposure to a novel environment captures clearly the reactivity of the

ats to novelty [5,55,62]. Pilot studies in our laboratory have shown that therst 15-min period of novelty is the most important period for screening theat’s reactivity. After the 15-min period the rats become habituated, they are notoving or sniffing around while their rearing behavior is declined. Thus, the

ollowing behavioral responses were recorded: standing (std) on all four feet,ssentially motionless and not sniffing; moving (mov), walking on all four feet;niffing (sni), not moving but sniffing parts of the walls or floor of the appa-atus; rearing (rr), body inclined vertically with hindpaws on the floor of thectivity cage and forepaws on the wall of the cage; grooming (grm), washinghe face or any other body part with the forepaws; scratching (scr), raising ofindpaw to touch any body part; sniffing air (sna), rearing (rr) in the open fieldrea of the activity cage. Then, all rats (n = 130) were ranked using the fre-uency of rr and sna and the upper half (score above the median) of the animalsas designated as HR (n = 65), while the lower half of animals (score below

he median) was indicated as LR rats (n = 65). The summation of rr and snaesponses reflect vertical activity, which has been used apart from locomotion

s a reliable criterion for assignment of rats into groups during their exposure toovelty [5,55]. After designation to HR/LR groups according to the aforemen-ioned criterion, the rats were left undisturbed (except for some gentle handling)or a 10-day period. Subsets of these rats were then employed for testing at theA, the OR, the WM or the FST. The coders were blind to rat classification.

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nother subset of rats was sacrificed and dopaminergic and serotonergic activ-ty was assessed in various brain regions. HR and LR rats were matched andivided in each subset of rats based on the median of vertical activity as previ-usly described. Thus, each experiment contained similar distribution of HR andR rats.

.2. Passive avoidance test

A subset (n = 24, HR = 12, LR = 12) of these rats was used for PA testxperiments. PA was conducted as described earlier by Misane and Ogren32,33]. A standard shuttle box (Ugo Basile, Comerio-Varese, Italy), withwo communicating (7 cm × 7 cm sliding door built in the separating wall)ompartments of equal size and a stainless-steel bar floor was used. Theight-hand compartment (shock compartment) was painted black to obtain aark chamber. The left-hand compartment was illuminated by a bulb (24 V;W) installed on the top Plexiglas cover.

PA training was conducted in a single session (day 1) during the light phase of12-h day/night cycle (09:00–16:00). Each rat was placed in the light compart-ent with no access to the dark compartment and allowed to explore for 2 min.hen 2 min expired, the sliding door was automatically opened by pressing a

edal and the rat was allowed to cross over into the dark compartment. Once theat had entered the dark compartment, the sliding door was automatically closednd an inescapable, constant current, scrambled shock (5 s, 0.6 mA) was deliv-red through the grid floor. Latency to cross into the dark compartment (trainingatency) was recorded. If a rat failed to move into the dark compartment within00 s (cut-off latency), the door was reopened and the rat was gently moved intohe dark compartment by the experimenter, where it received footshock. Follow-ng training, the rat was immediately removed from the PA apparatus. Retentionerformance was examined 24 h after training (day 2). The animal was placed inhe light (safe) compartment, with access to the dark compartment (within 15 s)or a period of 300 s. The latency to enter the dark compartment with all foureet (retention latency) was automatically measured. If the rat failed to enter theark compartment within 300 s, it was removed and assigned a maximum testatency score of 300 s.

.3. Object recognition test

A subset (n = 30, HR = 15, LR = 15) of rats was used for OR test experiments.ats were subjected to the object recognition task, according to the protocolescribed by Ennaceur and Delacour [18] with minor modifications. Thisaradigm has been widely used as a non-spatial paradigm that reflects workingemory, based on spontaneous exploratory activity [37]. The task used consists

f a 2-day habituation period. The rat was placed in the arena without any objectsor 2× 3 min trials on the first and second day, with a 60 min interval between tri-ls. On the testing day (third day), each rat was subjected to 2× 3 min trials in theresence of two objects. During Trial 1 (T1), two identical objects were placedn the arena and the time spent by the animal for objects exploration (rat nose atdistance <2 cm from the object) was recorded. During Trial 2 (T2), following60 min interval, one of the objects was replaced by a new, different one. The

ime spent exploring the familiar (F) and the novel (N) object was recorded and aiscrimination index (D) was calculated as (N − F)/(N + F). This ratio representshe difference in exploration time between the two objects during the Trial 2 (T2),xpressed as a proportion of the total time spent exploring these two objects. Inddition, an index of habituation (H) to the familiar object, H = T1/2 − F waslso calculated. This index measures the difference between an average timepent in exploring one of the identical objects during Trial 1 (T1) and the timepent exploring the familiar object (F) during the Trial 2. The discriminationatio and the index of habituation are related variables and widely used for thestimation of the rats’ performance in the OR paradigm [18,43]. A dark glassottle and a plastic shaker filled with water were the different objects used inuplicates.

.4. Water-maze test

A subset (n = 20, HR = 10, LR = 10) of the rats was used for the Morris WMaradigm with hidden platform, which is a valid test for investigating spatialearning and memory procedures. It is worthy to note that the rat performance

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Research 187 (2008) 462–472

uring the memory procedure of this paradigm is dependent on procedural asell as working memory function [59]. The water-maze was a circular galva-ized pool 140 cm in diameter and 50 cm deep, filled with opaque water (24 ◦C)o a depth of 30 cm. The pool was divided in four equal quadrants. A plat-orm (13 cm × 13 cm) was placed in the center of one of the quadrants (targetuadrant), 2 cm beneath the water surface. The present procedure consisted of2-day period, the learning and the memory test, respectively, as described bye Quervain et al. [15] with some minor modifications. During the first day ofhe WM paradigm (learning test), the animals were trained to find the positionf the fixed, hidden platform and escape onto the platform, in a total numberf eight trials. The rats were placed into the water, facing the wall of the tank,equentially from four different entry points, equally set around the pool. Allats were subjected to the four entry points, following the same order whileach entry was repeated twice. Each trial lasted 90 s and when a rat escapednto the platform, it was left there for 20 s, to allow orientation to visual cuesn the room, and was then placed for 30 s in a dry holding cage, ready to pro-eed to the next trial after that. If a rat failed to locate the platform within the0 s trial, it was gently guided to it by the experimenter. By the end of theraining session all animals returned to their home cages. The following day,he rats were given a retention trial in the absence of the platform (memoryest). A single 60 s trial was applied in which the rat was put into the water inposition equally distanced from the imaginary target and opposite quadrants

28]. The parameters recorded were escape latency, total distance swum andean velocity during training test, as well as time spent and mean velocity in

ach quadrant, during the memory test (EthoVision V1.90, Noldus, Wageningen,he Netherlands).

.5. Forced swim test

Rats (n = 15, HR = 7, LR = 8) were individually placed in a cylindricalank measuring 60 cm height × 38 cm width. The tank was filled with water24 ± 1 ◦C) at a height of 40 cm and water was changed after each session. Thenimals were forced to swim for a 15-min period (pretest) and 24 h later wereubjected to a 5-min swimming session (test) [44]. The total duration of floatingimmobility), swimming, head swinging and climbing periods was measuredor the first 5 min of each session (pretest and test). Rats were considered tohow immobility (floating) when they floated without struggling and makingnly those movements necessary to keep their heads above the water. Swimmingas recorded when they actively swam around in circles. Head swinging was

ecorded when the rats exhibited headshake responses indicative of searchingor an escape. Climbing was considered when the rats were climbing at thealls of the cylinder. It has been previously shown that the aforementionedehavioral parameters such as floating, swimming and climbing are sensitive tontidepressants [12,16]. Following swimming sessions, the rats were removedrom the tank, carefully dried in heated cages and then returned to their homeages.

.6. Neurochemical analyses

After assignment to LR/HR groups, the rats (n = 25, HR = 12, LR = 13) wereeft undisturbed for 4–5 days. They were then sacrificed, their brain was rapidlyemoved and discrete brain regions were dissected (hippocampus, hypothala-us, nucleus accumbens, dorsal striatum, prefrontal cortex and amygdala). Aftereighing, the dissected tissue was homogenized and deproteinized in 500 �lf 0.2N perchloric acid solution (Merck KgaA, Darmstadt, Germany) contain-ng 7.9 mM Na2S2O5 and 1.3 mM Na2EDTA that were both purchased fromiedel-de-Haen AG (Seelze, Germany). The homogenate was centrifuged at4,000 rpm for 30 min and the supernatant was stored at −80 ◦C. The analyti-al measurements were performed by high-performance liquid chromatographyHPLC) with an electrochemical detector, as described by Sharp et al. [51]nd Papadopoulou-Daifotis et al. [36] with some minor modifications [13,17].eversed phase ion pair chromatography was used in all analyses of dopamine

DA), 3,4-dihydroxyphenylacetate (DOPAC), homovanillic acid (HVA), sero-onin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). The mobile phaseonsisted of an acetonitrile (Merck) and 50 mM phosphate buffer (10.5:91.5)H 3.0, containing (300 mg/l) 5-octylsulfate sodium salt (Merck) as the ion-paireagent and (20 mg/l) Na2EDTA (Riedel-de Haen AG). Reference standards

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ere prepared in 0.2N perchloric acid solution containing 7.9 mM Na2S2O5

nd 1.3 mM Na2EDTA. The sensitivity of the assays was always tested usingxternal standards. The HPLC system consisted of a BAS-LC4B with an amper-metric detector. The working electrode was glassy carbon; the columns wereypersil, Elite C18 150 mm × 2.1 mm, 5 �m (Thermo Electron, Cheshire, UK)

nd the HPLC system was connected to a computer. Samples were quantifiedy comparison of the area under peaks with those of reference standards usingn HPLC software (Chromatography Station for Windows). Additionally, theurnover ratios of DA (DOPAC/DA, HVA/DA) and 5-HT (5-HIAA/5-HT) werelso calculated, in order to provide a better evaluation of dopaminergic anderotonergic functions [8,11].

.7. Statistical analyses

One-way analysis of variance (ANOVA) was used for the comparisonetween HR and LR rats concerning all the behavioral responses registereduring their reaction to the open field.

Specifically, one-way repeated ANOVA with novelty as “between-subjectsactor” (HR versus LR rats) and session as “within-subjects factor” (trainingatency versus retention latency) was used in the PA test. Separate analyses wereonducted when appropriate.

One-way ANOVA was used for comparison between HR and LR rats con-erning discrimination index (D) and habituation index (H) in the OR task.

For the analysis of the WM training data, a repeated one-way ANOVA wassed. Novelty was considered as “between-subjects factor” and trial as “within-ubjects factor” for all available variables: escape latency scores, mean velocitiesnd total distances swum registered during the 8 repeated trials. In addition, one-ay ANOVA with novelty as factor (HR versus LR) was conducted to estimateossible differences for all available variables (time spent in target or oppositeuadrant, mean velocity, total distance swum) registered during the memoryetrieval test.

A repeated ANOVA was used for FST data, with between-subjects factorovelty (HR versus LR) and within subjects factor, the session (pretest versusest). Separate analyses were performed when appropriate.

For neurochemical measurements, a one-way ANOVA was used for com-arison between HR and LR rats.

For post hoc comparisons, on factorial and repeated measures ANOVAs,onferroni’s tests were used for adjustment and control of the type I error rate.

Significance was accepted when p < 0.05.

. Results

.1. Open field behavior

As expected, there were statistically significant differ-nces between HR and LR rats concerning the frequencynd duration of rearing [F(1,125) = 99.45;45.8, p < 0.001] andniffing-air [F(1,125) = 66.5;68.8, p < 0.001] behavior. Further-ore, HR rats exhibited increased frequency and dura-

ion of moving [F(1,125) = 39.3;32.1, p < 0.001] and sniffingF(1,125) = 49.5;13.9, p < 0.001], but decreased duration of stand-ng [F(1,125) = 64.8, p < 0.001], as compared to LR rats (Fig. 1).

.2. Passive avoidance test

There was not any statistically significant difference in PAerformance between HR and LR (Fig. 2). One-way ANOVAith repeated measures did not reveal any difference concern-

ng training or retention latency scores between HR and LR

ats, although there was a tendency for lower training andigher retention latency scores in HR, as compared to LR ratsFig. 2). On the other hand, this analysis revealed a sessionffect [F(1,22) = 40.8, p < 0.001] and separate repeated analyses

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Research 187 (2008) 462–472 465

howed an increase in retention latency scores for HR and LRats [F(1,12) = 21.64;19.5, p < 0.001, respectively] (Fig. 2).

.3. Object recognition task

HR rats displayed a lower discrimination index (D), but aigher habituation index (H) as compared to LR rats (Fig. 3).ne-way analysis revealed a decreased ability to discrimi-ate (index D) between novel and known objects [F(1,28) = 5.7,< 0.02] for HR as compared to LR rats (Fig. 3). In addition,

he index H, which reflects habituation to the object, was highern HR as compared to LR rats [F(1,25) = 4.7, p < 0.05] (Fig. 3).

oreover, time spent exploring the familiar and novel objectT2) was not different between both groups of rats, while timepent by the animal for exploration of identical objects (T1)as increased in HR in comparison with LR rats [F(1,29) = 6.2,< 0.01] (Fig. 3).

.4. Water-maze test

HR displayed enhanced spatial memory in the WM task asompared to LR rats (Fig. 4). In the training (learning) session ofhe water-maze, one-way repeated ANOVA revealed only a trialffect [F(7,112) = 9.7, p < 0.001] for escape latency. In particular,scape latency was decreased over time in both HR and LRats. Subsequent analysis showed that HR displayed a shorterscape latency score as compared to LR rats in trial 2 (p < 0.05)Fig. 5A). This difference was also observed in Trial 3 but itid not reach any statistical significance (Fig. 5A). The samenalysis revealed that total distance swum was decreased overrials in both HR and LR rats [F(7,112) = 4.87, p < 0.001]. Similaro escape latency, HR rats have shown decreased total distancewam as compared to LR rats in trial 2 (p < 0.05) and trial 3, but itid not reach significance (Fig. 5B). Mean velocity was changedver trials in both HR and LR rats [F(7,112) = 3.02, p < 0.01]. Inarticular, HR rats exhibited higher mean velocity than LR ratsn trial 6, 7 and 8 but this increase was statistically significantnly in trial 6 (p < 0.05) (Fig. 5C).

During the memory retrieval test, one-way ANOVA revealedhat HR rats spent more time in the target quadrant as comparedo LR rats ([F(1,17) = 13.05, p < 0.001] (Fig. 4A). Additionally,R rats spent more time in the target quadrant as compared to

he opposite quadrant [F(1,17) = 19.14, p < 0.001], an effect thatas not observed in LR rats (Fig. 4A). Total distance swam in

arget as well as in opposite quadrant was not different betweenR and LR rats (data not shown). HR rats displayed higherean velocity in the opposite quadrant as compared to LR rats

F(1,17) = 5.1, p < 0.05) (Fig. 4B). Moreover, mean velocity inhe opposite quadrant was higher than that observed in the targetuadrant only in HR rats [F(1,17) = 6.29, p < 0.05] (Fig. 4B).

.5. Forced swim test

HR and LR rats showed a “depressive-like” behavior in theST paradigm which was differently profiled between eachhenotype (Fig. 6). Repeated measures ANOVA revealed aession effect [F(1,13) = 17.9, p < 0.001], on floating duration.

466 K. Antoniou et al. / Behavioural Brain Research 187 (2008) 462–472

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ig. 1. Mean values ± S.E. of the frequency and duration of rearing, sniffing-aled apparatus. ***p < 0.001 (HR vs. LR rats).

n particular, separated repeated ANOVA analyses revealed anncrease in floating behavior in the test session as compared withhe pretest session, in both HR and LR rats [F(1,7) = 10.2;8.1,< 0.01; p < 0.05, respectively]. Repeated measures ANOVA

evealed a session effect [F(1,13) = 6.1, p < 0.05], on swim-

ing duration. Separate repeated ANOVA analyses revealeddecrease in swimming duration [F(1,7) = 7.1, p < 0.03] only

or HR rats (Fig. 6). Repeated measures ANOVA revealed aession effect [F(1,13) = 5.9, p < 0.05], on head swinging dura-

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ffing, moving and standing in HR and LR rats following exposure to the open

ion. Separate repeated ANOVA analyses revealed a decrease inead swinging duration [F(1,8) = 11.3, p < 0.01] only for LR ratsFig. 6).

Similar analyses did not reveal any statistically significantffects on climbing behavior. Interestingly, separate one-way

NOVA with novelty as between factor for pretest and test

essions, revealed decreased head swinging and increased swim-ing behavior in HR as compared to LR rats during the pretest

ession of FST [F(1,14) = 5.9;6.1, p < 0.05] (Fig. 6).

K. Antoniou et al. / Behavioural Brain Research 187 (2008) 462–472 467

Fig. 2. Passive avoidance test. Mean values ± S.E. of the training latency (T1)and retention latency (T2) scores in LR and HR rats. ***p < 0.001 (retentionlatency score vs. training latency score in LR and HR rats, respectively). Fig. 4. Water-maze test. Mean values ± S.E. of time spent in quadrants (A) and

mean velocity in quadrants (B) of LR and HR dats during the memory testof water-maze paradigm. *p < 0.05; ***p < 0.001 (HR vs. LR rats). +p < 0.05;+++p < 0.001 (target vs. opposite quadrant in HR rats).

Fig. 3. Object recognition task. Total exploration time of trial 1 (T1) and trial 2 (T2) displayed by LR and HR rats (A). Discrimination index (D) performance of LRand HR rats in the trial 2 of the testing day (B). Habituation index (H) performance of LR and HR rats (B). Data are expressed as mean values (±S.E.) *p < 0.05;**p < 0.01 (HR vs. LR rats).

468 K. Antoniou et al. / Behavioural Brain

Fig. 5. Water-maze test. Mean values (±S.E.) of the escape latency (A), totaldcv

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istance swum (B) and mean velocity (C) of LR and HR rats over the eightonsecutive trials of the training session in the water-maze test. *p < 0.05 (LRs. HR rats).

.6. Neurochemical measurements

HR rats displayed lower 5-HT turnover ratio in the prefrontalortex, while they displayed higher tissue concentrations ofA, HVA, 5-HIAA and 5-HT turnover ratio in the striatum,s compared to LR rats (Table 1). Moreover, higher tissue con-entrations of DOPAC in the hypothalamus were observed inR in comparison with LR rats (Table 1). Specifically, in therefrontal cortex, a one-way ANOVA revealed reduced 5-HTurnover ratio (5-HIAA/5-HT), which is an index of serotoner-ic activity, in the HR as compared with LR rats [F = 4.5,

(1,24)< 0.05] (Table 1). There were not any statistically signifi-ant differences in dopaminergic or serotonergic activity in theucleus accumbens between HR and LR rats. A tendency for

ig. 6. Forced swim test. The duration of climbing, head swinging, swimming and florced swim test procedure are expressed as mean values (±S.E.). *p < 0.05 (HR vs. LR

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Research 187 (2008) 462–472

ncreased DA levels was only observed in HR compared withR rats (Table 1). In the dorsal striatum, an increase in DA andVA levels was observed [F(1,22) = 5.6;6.5, p < 0.05; p < 0.01]

n HR as compared to LR rats (Table 1). In addition, 5-HIAAevels and the 5-HT ratio (5-HIAA/5-HT), were increased in theR compared with LR rats [F(1,21) = 7.5;5.1, p < 0.01; p < 0.05]

Table 1). In the hypothalamus, an increase in DOPAC levels wasbserved [F(1,23) = 6.5, p < 0.05] for HR compared with LR ratsTable 1). Furthermore, a tendency (p = 0.1) for increase of theA ratio (DOPAC/DA) was observed in HR as compared to LR

ats (Table 1). There were not any statistically significant neuro-hemical differences in the studied indices of the hippocampusnd the amygdala in HR compared with LR rats (data nothown).

. Discussion

The present study aimed to identify differences in the behav-oral profiles, using a variety of tests, as well as in neurochemicalndices of male rats exhibiting high and low rearing behaviorpon exposure to a novel open field. HR displayed an increasedocomotor/exploratory activity as compared to LR rats. Interest-ngly, HR rats seemed to display impaired object recognition

emory, but enhanced spatial performance, as compared toR rats. Affective responsivity, i.e., “depressive-like” behav-

or in the FST was differently manifested in HR compared withR rats. Concerning the neurochemical evaluation, distinct dif-

erences in striatal and cortical serotonergic and dopaminergicctivity were observed between the phenotypes.

.1. Behavioural analysis

HR exhibited decreased standing but increased moving andniffing behavior in the novel open field compared with LR rats.pparently, HR also displayed an increase in rearing behavior

s compared to LR rats. These findings are in agreement withur previous findings [5] and with those by Thiel et al. [55] whoave also reported that rats with more rrs also display higherevels of locomotion. Based on the aforementioned studies andn our findings, it is suggested that rats with a high frequencyf rearing behavior display an enhanced motor activity profile

oating responses of LR and HR rats, during the pretest and test session of the), #p < 0.05; ##p < 0.01 (test vs. pretest session in HR and LR rats, respectively).

imilar to the HR profile derived from differentiation of rats onhe basis of locomotor response to novelty [10,39].

HR and LR rats displayed similar performance in the PAask, although a shorter training latency and longer retention

K. Antoniou et al. / Behavioural Brain Research 187 (2008) 462–472 469

Table 1Neurochemical evaluation of dopaminergic and serotonergic activity in the prefrontal cortex, nucleus accumbens, dorsal striatum and hypothalamus of high responders(HR) and low responders (LR) rats

Prefrontal cortex Nucleus accumbens Dorsal striatum Hypothalamus

LR HR LR HR LR HR LR HR

DA 0.26 ± 0.04 0.14 ± 0.01 2.75 ± 0.60 3.98 ± 0.60 15.7 ± 1.60 22.8 ± 2.20* 0.44 ± 0.02 0.48 ± 0.03DOPAC 0.05 ± 0.001 0.04 ± 0.002 1.41 ± 0.33 1.91 ± 0.30 2.89 ± 0.80 4.23 ± 0.90 0.05 ± 0.006 0.08 ± 0.006*HVA 0.05 ± 0.001 0.04 ± 0.002 0.40 ± 0.07 0.44 ± 0.04 0.70 ± 0.20 1.65 ± 0.20** 0.03 ± 0.004 0.02 ± 0.004DOPAC/DA 0.21 ± 0.03 0.28 ± 0.03 0.53 ± 0.08 0.50 ± 0.04 0.19 ± 0.05 0.23 ± 0.04 0.13 ± 0.01 0.16 ± 0.01HVA/DA 0.13 ± 0.02 0.19 ± 0.02 0.17 ± 0.03 0.13 ± 0.02 0.04 ± 0.01 0.08 ± 0.02 0.07 ± 0.009 0.05 ± 0.007

5-HT 1.51 ± 0.22 1.60 ± 0.23 1.19 ± 0.19 1.21 ± 0.23 0.40 ± 0.04 0.54 ± 0.08 2.6 ± 0.15 2.5 ± 0.225-HIAA 0.52 ± 0.04 0.52 ± 0.04 0.65 ± 0.08 0.63 ± 0.09 0.36 ± 0.06 0.80 ± 0.15** 0.87 ± 0.06 0.82 ± 0.065-HIAA/5-HT 0.52 ± 0.04 0.37 ± 0.05* 0.58 ± 0.07 0.62 ± 0.11 0.84 ± 0.16 1.43 ± 0.18* 0.33 ± 0.02 0.34 ± 0.03

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evels of DA, its metabolites (DOPAC and HVA), 5-HT and its metabolite (5-Hnd HVA/DA) and the serotonergic turnover ratio (5-HIAA/5-HT) are expresse

atency was observed in HR rats. The PA task is one of theost frequently used animal models for studying learning andemory processes based on aversive learning [35]. Similar to

ur findings, Borta and Schwarting [6] reported that HR andR rats showed comparable step-in retention latencies, 24 hfter the training procedure, using different shock intensities.otably, these authors observed that HR displayed an impaired

tep-through PA retention, 72 h after the training procedure,specially when using the higher shock intensity. Moreover,o et al. [23] did not report any difference between HR andR rats using the two-way avoidance paradigm. Based on ourndings and on the aforementioned studies, it seems that thesehenotypes display subtle differences in terms of reaction to anversive stimulus or aversive learning procedures.

When the two groups of rats were subjected to the objectecognition test, impairment in object recognition was observedn HR as compared to LR rats. In particular, HR exhibited aigher time for exploration of both identical objects (T1 session),long with a tendency for higher time for exploration of bothovel and familiar objects (T2 session), a higher familiarizationndex (H), but a lower discrimination index (D). This behavioralerformance could be related to the increased motor/exploratoryctivity, observed in this phenotype of rats. However, based onhe higher H index displayed by the HR rats, which could bettributed to their higher exploration time of identical objectsT1), it is assumed that HR rats displayed an enhanced famil-arization to the object as compared to LR rats due to theirxaggerated first (basal) reaction to the object during the T1. Onhe other hand, Pawlak and Schwarting [38] reported that HRats showed higher exploration of the new instead of the familiarbject in the object recognition task. It is worth mentioning thathese authors have not indicated the discrimination index (D) forR and LR rats, while the intertrial interval was shorter as com-ared to that used in the present study. Notably, playground mazelong with object recognition task have been used for assessmentf novelty seeking and categorization of animals as either HR or

R rats [30,34]. Relevant studies have not shown any correlationetween the horizontal activity in an inescapable novel environ-ent and the scores in playground maze [29,30]. However, the

bject recognition task used in the present study has been consid-

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tissue concentration as well as the dopaminergic turnover ratios (DOPAC/DAean values (±S.E.). *p < 0.05; **p < 0.01 (HR vs. LR rats).

red as a non-spatial paradigm based on spontaneous exploratoryctivity that reflects working memory [37,43]. It is worthy to notehat hyperactivity has been associated with deficits in short-term

emory in spontaneously hypertensive rats [45,48]. Therefore,ased on our findings and the aforementioned studies it coulde argued that the higher locomotor response to novelty, mighte contributing to an impaired non-spatial performance.

On the other hand, HR rats showed a better spatial related per-ormance, as revealed in the WM paradigm. Thus, HR rats learno navigate to a hidden platform faster than LR rats, as deducedy escape latency scores and total distance swum in the initialet of trials, during the learning phase of the WM procedure.dditionally, HR rats showed a clear preference for target quad-

ant along with a reduced velocity in this quadrant, comparedith LR rats, during the learning test of WM paradigm. It shoulde pointed out that differences in the learning phase were nots robust as those observed in the memory phase, supportinghe notion by Jakubowska-Dogru et al. [24] that memory acqui-ition and memory performance reflect independent processes.he WM with hidden platform has been used for elucidatingippocampal-dependent spatial learning and memory abilities.n accordance with our findings, HR displayed a better perfor-ance in the radial maze task, albeit of more working errors

elated to memory function as compared to LR rats [22]. Interest-ngly, these authors reported that this differential pattern betweenR and LR rats is based on differences in motivational and

ognitive status. Moreover, Cools et al. [9] have suggested thatifferences between HR and LR rats in response to a simpleour-arm radial maze are not due simply to differences in motorctivity, but to minor yet important differences in the mode ofearning. However, Topic et al. [56] have reported a relation-hip between reactivity to novelty and thigmotaxis in the WMhich may reflect behavioral traits and may bias spatial learn-

ng. This behavioral differentiation is probably characterized byognitive and/or non-cognitive factors, as reported by severaltudies [22,56]. Based on these studies and on our findings it

eems that differences in spatial memory and learning abilitiesetween the HR and LR rats are probably related to their dif-erentiated reaction to novelty and not simply attributed to theirespective differences in motor activity.

4 Brain

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70 K. Antoniou et al. / Behavioural

Both HR and LR rats exhibited increased floating behaviornd decreased active behaviors during the test session of FST,roperties that reflect a well-known “depressive-like” behavior.n accordance with Ho et al. [23] and White et al. [62] theseats do not exhibit any essential differentiation in relation toheir “depression-like” status. Notably, HR rats exhibited lesswimming behavior while LR rats displayed less head-swingingehavior during the test session of the FST paradigm. Duringhe first exposure to the water (pretest), HR rats were swimming

ore than LR rats while LR rats were head-swinging more thanR rats. Therefore, HR and LR rats exhibited increased passiveehavior and decreased active behavior in the FST paradigm,ut active behavior was differently manifested in the two phe-otypes in both the pretest and test FST session. HR and LRats developed a different behavioral FST pattern characterizedy a common behavioral state of “despair”, but distinct activeehavioral reactions expressed either during the first or duringhe second exposure to the water.

Taken together, the results of previous studies [14,46,57] asell as our present findings suggest that the two behavioral phe-otypes studied reveal differences in a variety of behavioral tasksue to differential responsiveness to the test conditions and theiversity in the repertoire of the responses studied in behavioralaradigms that assess novelty, habituation, reaction to an aver-ive stimulus or a stressful situation, and cognitive or affectiveeactivity.

.2. Neurochemical analysis

Concerning the neurochemical findings, higher DA and HVAoncentrations in the striatum were found in the HR comparedo the LR rats. Although the exact impact of these differencesn the overall state of dopaminergic neurotransmission in theorebrain is not clear, they are probably related to the behavioralifferences seen between the two phenotypes. Similar to previ-us studies, dopaminergic activity in striatal tissue seems to beecessarily involved in the differentiation of animals exposed tonovel environment [41]. In particular, Piazza et al. [42] have

eported a positive correlation between dopaminergic activityn the nucleus accumbens and motor activity in a novel envi-onment, while this correlation was negative concerning theopaminergic activity in the prefrontal cortex. Moreover, Sai-usa et al. [49] and Van der Elst et al. [58] have suggested anssociation between response to novelty and dopaminergic sta-us in the nucleus accumbens. Similar to our findings, Thiel etl. [55] failed to show a clear increase in dopaminergic functionn the ventral striatum (that includes the nucleus accumbens weampled in our study) of HR rats, but they showed an increasedA function in the neostriatum of these rats. In accordance tour results, Shakil et al. [50] reported an increased DA ratio inhe striatum of HR mice.

A decreased cortical serotonergic activity as well as anncreased striatal serotonergic activity, as reflected by the 5-

IAA/5-HT turnover ratio, was observed in the HR as compared

o LR rats. These results are slightly different concerning the cor-ical serotonergic activity as compared to the respective findingsy Thiel et al. [55], who found reduced 5-HT tissue levels with-

iawp

Research 187 (2008) 462–472

ut any subsequent change in the 5-HT turnover ratio of HR asompared to LR rats. On the other hand, our results are to somextent similar to those observed by Piazza et al. [41], who haveeported overall reduced 5-HT and 5-HIAA tissue levels in therefrontal cortex, the nucleus accumbens and the striatum of HRersus LR rats. Notably, Ray et al. [47] have addressed a linketween 5-HT-related parameters and novelty seeking behavior.t is worthy to add that the differentiation in the FST performanceetween the HR and LR rats could be linked to the aforemen-ioned differences in 5-HT activity between the two groups, sincective performance in the FST paradigm has been associatedith the serotonergic function [27]. Taking into consideration

he involvement of dopaminergic and serotonergic systems inemory and learning processes [7,20,21], it is proposed that the

istinct differences in dopamine and serotonin indices betweenhe two groups of rats may be contributing to the differential non-patial and spatial performance observed in these different phe-otypes. In addition, the role of other neurotransmitters such ascetylcholine, noradrenaline and glutamate cannot be excluded.nterestingly, Thiel et al. [54] have reported that differencesetween HR and LR (designated by rearing behavior) are relatedo differential hippocampal cholinergic activity, an effect that

ay be also associated with respective behavioral differenceseported in the present study. In support of this notion, Zucker-an [60,61] suggested that the personality trait of high sensation

eeking could be based on a hyperactive dopaminergic systemnd a hypoactive serotonergic and noradrenergic system. Nev-rtheless, the differences in the dopaminergic and serotonergicrofile of HR rats observed in the present study as compared torevious studies could be attributed to the different criterion foresignation of the animals, to the timing and context of the neu-ochemical assessments (under basal conditions or upon expo-ure to novelty) as well as to different methodological aspectssed for the detection of neurotransmitters and their metabolites.

In conclusion, the present results further confirm and extendrevious findings, indicating that certain neurobiological param-ters are most likely accountable for the differences in taskerformance/responsivity observed between HR and LR rats.t could be assumed that HR displayed increased motor activity,mpaired non-spatial memory, but enhanced spatial memory andifferent pattern in the manifestation of depressive-like behaviors compareto LR rats. It could be further suggested that distinctehavioral indices in the HR rats, such as increased reaction toovelty, increased exploration period (T1) during the first expo-ure of the object recognition task, increased velocity duringhe WM procedure and increased swimming behavior duringhe pretest session of FST could for the most part be relatedo an altered dopaminergic function. On the other hand, theirifferent final performance in the FST test or other cognitiveelated tasks could be related to differences in the dynamic inter-elationship of various neurotransmitters, including dopamine,erotonin, glutamate, acetylcholine and noradrenaline. In sum,ndividual variations in novelty behavior may reflect differences

n the reaction to specific test conditions related to cognitivend affective function and susceptibility to neurotropic agents,hile these variations may be linked to certain neurobiologicalarameters.

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cknowledgement

This study was partially supported by Empirikion Founda-ion.

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