Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 1153–1160

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Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

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Environmental enrichment improves cognitive deficits in SpontaneouslyHypertensive Rats (SHR): Relevance for Attention Deficit/HyperactivityDisorder (ADHD)

Fabrício A. Pamplona, Pablo Pandolfo, Robson Savoldi, Rui Daniel S. Prediger, Reinaldo N. Takahashi ⁎Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil

Abbreviations: SHR, Spontaneously Hypertensive RHyperactivity Disorder; WIS, Wistar; SE, Standardenvironment.⁎ Corresponding author. Departamento de Farmaco

versitário Trindade, 88049-900, Florianópolis, SC, Brafax: +55 48 3337 5479.

E-mail address: takahashi@farmaco.ufsc.br (R.N. Tak

0278-5846/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.pnpbp.2009.06.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 March 2009Received in revised form 8 June 2009Accepted 14 June 2009Available online 21 June 2009

Keywords:Attention Deficit/Hyperactivity Disorder(ADHD)Environmental enrichmentMorris water mazeObject recognitionSocial recognitionSpontaneously Hypertensive Rats (SHR)

The interaction between genes and environment seems to be relevant for the development of AttentionDeficit/Hyperactivity Disorder (ADHD), one of the most prevalent childhood psychiatric diseases. Theoccurrence of ADHD is typically associated with poor academic performance, probably reflecting learningdifficulties and/or cognitive impulsiveness. The inbred Spontaneously Hypertensive Rats (SHR) strain hasoften been considered as an animal model of ADHD, since they ‘naturally’ display the main ADHDsymptomatology. Although pharmacological agents improve SHR's cognitive deficits, little is known aboutthe involvement of environmental factors in SHR disabilities and to what extent ‘protective’ non-pharmacological factors may be considered as strategy for ADHD prevention. Here we investigated whetherthe rearing environment during neurodevelopment may counteract later cognitive deficits presented byadult SHR. Wistar (WIS) rats were also used to investigate whether the putative effects of environmentalenrichment depend on a specific genetic background. The animals were reared in enriched environment (EE)or standard environment (SE) from the post-natal day 21 until 3 months of age (adulthood) and tested forcognitive and non-cognitive phenotypes. EE improved SHR's performance in open field habituation, watermaze spatial reference, social and object recognition tasks, while non-cognitive traits, such as nociceptionand hypertension, were not affected by EE. Response of WIS rats was generally not affected by the present EE.These results show that the general low cognitive performance presented by SHR rats strongly depends onthe rearing environment and they may suggest modifications of the familial environment as a putativepreventive strategy to cope with ADHD.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

The interaction between environmental and genetic factors seemsto be relevant for the development of one of the most prevalentchildhood psychiatric diseases, namely Attention Deficit/Hyperactiv-ity Disorder (ADHD) (Brookes et al., 2006; Thapar et al., 2005). ADHDaffects approximately 5–10% of school-aged children in U.S.A. andother countries (American-Academy-of-Pediatrics, 2000; Faraoneet al., 2003) and its core symptoms are motor overactivity andinattention, although the clinical presentation of the disease is muchmore heterogeneous and involves complex motor and cognitiveprocesses, such as impulsivity and social inabilities (Barkley andBiederman, 1997; Himelstein et al., 2000; Taylor et al., 2004; Wilens

ats; ADHD, Attention Deficit/environment; EE, Enriched

logia CCB-UFSC, Campus Uni-zil. Tel.: +55 48 3721 9491;

ahashi).

l rights reserved.

and Dodson, 2004). The occurrence of ADHD is typically associatedwith poor academic performance, probably reflecting learningdifficulties and/or cognitive impulsiveness (Mannuzza et al., 1993;Wilens and Dodson, 2004). Pharmacotherapy using psychostimulantmedications, such as methylphenidate, is rigorously the mostaccepted pharmacological treatment for ADHD patients (Krauseet al., 2002). The diagnosis and treatment of ADHD patients normallyoccur during childhood/adolescence (Wilens and Dodson, 2004), butlittle is known about the persistent effects of treatment withpsychostimulants during childhood (Wilens and Dodson, 2004).Arguably, early post-natal treatment with psychotropic drugs exposesindividuals to the threat of long-term neurodevelopmental conse-quences, which may overcome the drug's therapeutic effects (Ander-sen and Navalta, 2004). In face of this risk, the search for alternative/complementary non-pharmacological therapeutic approaches forADHD is a field of high interest.

Among the several genetic and drug-induced animal models ofADHD available, the inbred Spontaneously Hypertensive Rats (SHR)strain has often been considered the most validated model, since they‘naturally’ display the main ADHD symptomatology, including

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hyperactivity, impulsivity and poorly sustained attention (Russell,2007; Sagvolden et al., 2005). Although SHR's hyperactivity has beenquestioned – and almost exclusively appears in response to noveltyand in comparison to Wistar–Kyoto rats (Ferguson and Cada, 2003) –the cognitive deficits of SHR have been extensively reported in severalbehavioral paradigms by our group (Pires et al., 2009; Prediger et al.,2005a,b) and others (De Bruin et al., 2003; Kantak et al., 2008;Nakamura-Palacios et al., 1996; Sagvolden and Xu, 2008). For thisreason, the SHR strain is of interest in the study of neurobiologicalchanges involved in ADHD and for testing of new therapeuticinterventions for cognitive dysfunctions (Takahashi et al., 2008).Although different pharmacological approaches have been success-fully tested in SHR, and therefore suggested as alternatives for ADHDtherapy, relatively little is known about the involvement of environ-mental factors in ADHD-like symptomatology presented by this ratstrain and to what extent ‘protective’ non-pharmacological factorsmay be considered as preventive strategies for such behavioralabnormalities.

One potential non-pharmacological approach to neurologicaldisorders is environmental enrichment, which induces beneficialeffects against cognitive dysfunctions (Kempermann et al., 1997; vanPraag et al., 2000). In a broad sense, environmental enrichment is anexperimental paradigm in animals which supposedly parallel physi-cal, social and intellectual activity of humans. These includeencouragements to perform physical exercise (e.g., running wheels),exposure to a variety of active and passive sensorial stimuli (e.g., toysand colored objects), opportunity to forage for food and water (e.g.changing the location of eating and drinking places) and increasedsocial contact. Long-term exposure to an enriched environment (EE)stimulates neurogenesis, elicits neuroprotective responses andinduces synaptic structural changes that enhance learning andmemory (Kempermann et al., 1997; van Praag et al., 2000). However,little is known about the effects of EE in the symptomatology of ADHD.

Therefore, the aim of the present study was to investigate whetherthe exposure to EE during neurodevelopment may prevent latercognitive deficits presented by adult SHR. The animals have been alsotested for a non-cognitive behavioral trait (nociception) and aphysiological measure (arterial pressure). Wistar (WIS) rats werealso included in the present study as a 'normal' heterogenic outbredstrain, to investigate whether the effects of EE depend on a specificgenetic background.

2. Methods

2.1. Subjects

WIS and SHR rats from our own colony were separated from theirmother at the age of 21 days and themale pups were reared in EE or instandard conditions (see below). They were maintained undercontrolled temperature (23±1 °C), with a 12 h light cycle (lights on7:00 a.m.) and free access to food and water. Male rats of both strainswere tested during adulthood (3 months old), weighing about 280–340 g (WIS) or 250–300 g (SHR). Juvenile male rats of these samestrains (approximately 1 month old, 100–150 g) were kept in groupsof 10 per cage and served as social stimuli for the adult rats of thecorrespondent strain in the social recognition task. The proceduresused in the present study follow the rules of the UFSC EthicsCommittee on the Use of Animals and respect international guidelineson animal care. The authors further attest that all efforts weremade tominimize the number of animals used and their suffering.

2.2. Environmental enrichment

From the post-natal day 21 until the age of 3 months (andthroughout behavioral testing), WIS and SHR rats were maintained ingroups of 7–10 animals in an enriched environment (EE) or in groups

of 4–5 animals in each of two cages of standard environment (SE). TheEE consisted of two specially designed plastic cages (46×33×23 cm)with a wire mesh cover and connected to each other by a plastic tube(10 cm diameter, 20 cm long). These EE cages were equipped with aset of plastic tubes, a running wheel, wood shelter and small colorfultoys, providing multimodality stimulation (Nithianantharajah andHannan, 2006; van Praag et al., 2000). Twice a week the EE wascleaned and the objects inside were rearranged to enhance novelty.The SE consisted of a standard plastic cage (42×34×17 cm) with awire mesh cover. Rats in EE and SE groups were subjected to the samecleaning and handling procedures and reared in parallel in two cagesetups. EE rats were transferred to a clean collective cage right aftertesting and back to the EE by the end of a given experimental session.

2.3. Behavioral tests

The behavioral tests were conducted in three independent cohortsof WIS and SHR rats, reared at the same time in two sets of standard(SE) and enriched (EE) cages. One group was tested in the open fieldhabituation and water maze tasks, the second group in the objectrecognition and hot-plate tests and the third one was used for socialrecognition task and arterial pressure measurements. Tests were runin the order listed, from the least aversive to the most aversive, tominimize the chance that behavioral responses weremarkedly alteredby prior test history (McIlwain et al., 2001). Behavior was monitoredthrough a video camera positioned above the apparatuses and theimages were analyzed online, in an adjacent room, by an experiencedexperimenter who was unaware of the strain and experimental groupof the animals tested.

2.3.1. Open field habituation taskThe open fieldwasmade of white-paintedwood, with awhite floor

of 100×100 cm (divided by black lines into 25 squares of 20×20 cm)and 40 cm-high white walls. The experiments were conducted in asound-attenuated room under low-intensity light (10 lx). In thehabituation task, the animals were allowed to explore the open fieldfor 10 min in two consecutive days. The number of squares crossedwas registered as an index of general activity (Rodgers, 1997). Theapparatus was cleaned with ethanol solution (10% v/v) and dried withpaper towels after each trial in order to avoid odor impregnation.

2.3.2. Water maze spatial reference taskThewatermazewasmade of black painted fiberglass,1.70 m inside

diameter, 0.8 m high, and filled to a depth of 0.6 m with watermaintained at 25 °C. The target platform (10×10 cm) was made oftransparent Plexiglas and submerged 1–1.5 cm beneath the surface ofthe water in the center of one quadrant (approximately 35 cm fromthe wall). The animals were subjected to a spatial reference task of thewater maze following a protocol previously established in ourlaboratory (Takahashi et al., 2008). The training session consisted ofsix consecutive trials during which the animals were left in a startingpoint in the tank and allowed to swim freely to the submergedplatform. The animal was allowed to remain on the platform for 10 safter escaping to it and was then removed from the tank for 20 sbefore being placed at the starting point for the next trial. Theplatform was located in a fixed position throughout the trainingsession, whereas the starting point was different for each trial. Thetime necessary to reach the platform (escape latency) was registeredfor each training trial and animals that did not find the platformduring a period of 60 s were gently guided to it. The test session wascarried out 24 h later and consisted of a single probe trial where theplatformwas removed from the pool and each animal was allowed toswim for 60 s in the maze. The time spent in the correct quadrant (i.e.where the platformwas located on the training session) was recordedand analyzed as percentage of the total time.

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2.3.3. Social recognition taskThe social memory was assessed with the social recognition task

originally described by Dantzer et al. (1987) and previously used inour laboratory (Prediger et al., 2005a). Adult rats from both the SE andEE groups were individually housed for 7 days in standard plasticcages and the juvenile rats used as social stimuli were isolated for20 min before the experiment. The social recognition task consisted oftwo consecutive 5-min presentations to the same juvenile rat,separated by an interval of 30 min, during which the juvenile ratswere kept in individual cages. The experimental adult animals

Fig. 1. Effects of environmental enrichment on long-term spatial memory abilities of Wistarthe post-natal day 21 to 3 months of age (adulthood) in standard environment (SE) or enrichtask was performed in 2 consecutive 10-min OF sessions, with 24 h interval. The number of sqspatial reference task was performed in one 6-trials training session, when the animals learrecorded as an index of learning. (E, F) A 1-min test of spatial memory was conducted 24 h latspent in the quadrant where the platformwas placed during the training sessionwas recordeand C–F: (WIS SE n=7;WIS EE n=8, SHR SE n=7, SHR EE n=7). Data are presented as mea(Duncan's post-hoc test).

remained in the home cage, where they were isolated beforehand.The time spent by the adult rat investigating the juvenile (sniffing,grooming or touching) was recorded during each exposure. The short-term social recognition memory is expressed as a reduction in theinvestigation time during the second exposure compared to the first.

2.3.4. Object recognition taskThe object recognition task was conducted in the open field in

three distinct phases: habituation, sample and discrimination (Enna-ceur and Delacour, 1988). The habituation phase consisted of

(WIS) and Spontaneously Hypertensive Rats (SHR). WIS and SHR rats were reared fromed environment (EE) and tested in cognitive tasks. (A, B) The open field (OF) habituationuares crossed was recorded as an index of general activity. (C, D) Thewater maze (WM)ned to find a submerged platform. The time to find the platform (escape latency) waser in thewatermaze. The platformwas removed and the percentage of time the animalsd as an index of memory. A–B: (WIS SE n=7;WIS EE n=8, SHR SE n=7, SHR EE n=7),n±S.E.M. +pb0.05 compared to the 1st OF session, ⁎pb0.05 compared to the SE group

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exploration of the open field for 10 min in two consecutive days. In thesample phase, 24 h later, two identical objects (A1 and A2, cubes)were placed in opposite corners of the open field for 3 min of freeexploration. The rats stayed for 30 min in the home cages before startof the discrimination phase. Then, objects A1 and A2 were replaced byan identical copy of the familiar object (A3) and a novel object (B) for3 min of exploration. Object locations were counterbalanced. Explora-tion of an object was defined as directing the nose to the object at adistance of less than 2 cm and/or touching it with the nose (Ennaceurand Delacour, 1988). The following measures were considered: totalexploration time (A1+A2) in the sample phase and discriminationindex (B−A3/B+A3) in the discrimination phase.

Fig. 2. Effects of environmental enrichment on short-term recognition memory abilities of Wfrom the post-natal day 21 to 3 months of age (adulthood) in standard environment (SE) or(SR) task was performed in 2 consecutive 5-min presentations of a juvenile rat, with 30 minrecognition (OR) task was performed in 2 consecutive phases, with 30 min interval. (C, D) In tinvestigation time was recorded (A1+A2). (E, F) In the discrimination phase, another identwas calculated as the percentage of investigation time of (B−A3)/(B+A3). A–B: (WIS SE nSHR SE n=7, SHR EE n=7). Data are presented as mean±S.E.M. +pb0.05 compared to th

2.3.5. Hot-plateThe hot-plate test was performed as previously established in our

laboratory (Vendruscolo et al., 2004). Briefly, the hot-plate (Insight,Brazil) was maintained at 52.2±0.5 °C and the rats were placed in anacrylic cylinder on the heated metal surface. Time between placementand hind paw licking or jumping (whatever occurred first) wasrecorded as nociceptive latency (s).

2.4. Blood pressure measure

The arterial blood pressure (mm Hg) was measured using aprotocol previously described (Prediger et al., 2005b). Briefly, under

istar (WIS) and Spontaneously Hypertensive Rats (SHR). WIS and SHR rats were rearedenriched environment (EE) and tested in cognitive tasks. (A, B) The social recognitioninterval. The investigation time was used as an index of recognition memory. The objecthe sample phase 2 identical objects (A1, A2) were presented to the animals and the totalical object (A3) and one novel object (B) were presented and the discrimination index=9; WIS EE n=10, SHR SE n=9, SHR EE n=8), and C–F: (WIS SE n=7; WIS EE n=7,e 1st SR session, ⁎pb0.05 compared to the SE group (Duncan's post-hoc test).

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deep ketamine/xylazine anesthesia a polyethylene catheter wasinserted into the right carotid artery for recording of arterialpressure. After 30 min of stabilization, systolic and diastolic arterialblood pressures were recorded for 60 min. Data were recorded at a10 s sampling rate with a Digit-Med BP Analyser system (Model190) connected to a Digit-Med System Integrator (Model 200;Louisville, KY).

2.5. Statistical analysis

All data were checked for normality of frequency distributionwiththe Kolmogorov–Smirnov test. The statistical comparison of resultswas carried out using one- or two-way ANOVA with environment,objects and trial/session/juvenile presentation (repeated measures)as independent variables. Following significant ANOVAs, differencesbetween groups were evaluated by the Duncan's post-hoc test. Allvalues are expressed as means±S.E.M. and the accepted level ofsignificance for the tests was pb0.05. All tests were performed usingthe Statistica 6.0 software package.

3. Results

Data of each strain were individually analyzed because ourobjective was to investigate the effects of environmental enrichmentin one strain and the other. A direct comparison between the presentcontrol groups (SE) of each strain replicated the response pattern

Fig. 3. Effects of environmental enrichment on non-cognitive traits ofWistar (WIS) and Spont21 to 3 months of age (adulthood) in standard environment (SE) or enriched environment (Etop of a hot-plate 52.2±0.5 °C and the latency to lick the hind pawor jump (whatever occurrein the right carotid artery and themean arterial blood pressurewas recorded for 60 min. A–B:n=5, SHR SE n=5, SHR EE n=5). Data are presented as mean±S.E.M. No significant differ

previously observed in our experimental settings (Pamplona et al.,2007; Pandolfo et al., 2007; Pires et al., 2009; Prediger et al., 2005a,b;Takahashi et al., 2008; Vendruscolo et al., 2004), indicating overallhyperlocomotion in the open field [F(1,12)=10.22, pb0.01 for strain;F(1,12)=8.12, pb0.01 for session], deficit of long-termmemory in thewater maze (% time in platform quadrant; t=2.24, pb0.05), increasedinvestigation in the first session (t=−4.59, pb0.001) and short-termmemory deficit [F(1,16)=49.49, pb0.0001 for strain; F(1,16)=11.88,pb0.01 for session] in the social recognition test, increased investiga-tion (t=−3.68, pb0.01) and a trend towards short-term memorydeficit (t=1.89, p=0.08) in the object recognition test, as well ashyponociception in the hot-plate test (t=−3.19, pb0.01) andhypertension (t=−2.81, pb0.05) for SHR compared to WIS rats.

3.1. Effects of environmental enrichment in the performance of WIS andSHR rats in learning and memory tasks

Fig. 1(A, B) shows the number of squares crossed (locomotoractivity) exhibited in the open field habituation task. Two-way ANOVA(session vs. environment) of the total locomotion revealed significanteffects of session [F(1,13)=9.21, p=0.01] and environment [F(1,13)=10.13, pb0.05] forWIS rats (Fig.1A) and significant effects of session [F(1,12)=26.98, pb0.001], environment [F(1,12)=40.36, pb0.0001]and session vs. environment interaction [F(1,12)=5.09, pb0.05] forSHR (Fig. 1B). Post-hoc comparisons indicated that EE reduced thelocomotor activity of WIS and SHR in the 1st open field session

aneouslyHypertensive Rats (SHR).WIS and SHR ratswere reared from the post-natal dayE) and tested for pain sensitivity and blood pressure. (A, B) The animals were placed ondfirst)was recorded as nociceptive latency. (C, D) Anesthetized animalswere cannulated(WIS SE n=7;WIS EE n=7, SHR SE n=7, SHR EE n=7), and C–F: (WIS SE n=4;WIS EEences have been found.

1158 F.A. Pamplona et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (2009) 1153–1160

(pb0.05) (Fig. 1A, B). Moreover, while WIS rats (regardless of theenvironment) displayed habituation to the open field apparatus (i.e.,reduced locomotion in the 2nd open field session compared to the 1st)(p≤0.05), the habituation responsewas only observed in SHR animalsfrom the EE group (Fig. 1A, B).

Fig. 1 (C, D) shows the escape latencies exhibited during thetraining session of the water maze spatial reference task. Two-wayANOVA (environment vs. trials) revealed significant effect of trial forboth WIS [F(5,65)=17.77, pb0.0001] and SHR [F(5,60)=12.20,pb0.0001]. However, it indicated a significant effect of environmentfor SHR [F(1,12)=47.62, pb0.0001], but not for WIS [F(1,13)=3.15,p=0.10]. There was no significant effect of environment vs. trialsinteraction for WIS [F(5,65)=0.11, p=0.99] or SHR [F(5,60)=2.04,p=0.09]. Post-hoc comparisons indicated that the EE improved theperformance of SHR during the training trials of the water maze task,as indicated by a significant reduction in the escape latency to find theplatform across the trials in comparison to SE group (Fig. 1D).

Fig. 1 (E, F) shows the percentage of time spent in the platformquadrant during the probe test of thewater maze spatial reference task.One-way ANOVA (environment) revealed significant effect of environ-ment for SHR [F(1,12)=5.12, pb0.05], but not for WIS [F(1,13)=1.62,p=0.22]. Subsequent post-hoc comparisons indicated that EE selec-tively improved the spatialmemoryof SHR, promotingan increase in thepercentage of time spent in the quadrant where the platform waslocated during the training session (Fig. 1F).

Fig. 2(A, B) shows the investigation time exhibited in the socialrecognition task. Two-way ANOVA (environment vs. juvenile presenta-tion) revealed significant effect of juvenile presentation [F(1,17)=25.57,pb0.0001], but not of environment [F(1,17)=0.87, p=0.36] for WIS.Moreover, it revealed a significant effect of presentation [F(1,15)=55.47,pb0.0001], environment [F(1,15)=63.19, pb0.0001] and of the envir-onment vs. juvenile presentation interaction for SHR [F(1,15)=4.36,pb0.05]. Subsequent comparisons indicated that the SHR SE grouppresented a significantly higher investigation time in the first juvenilepresentation compared to WIS SE group, with no significant reductionduring the second exposure. Moreover, post-hoc comparisons indicatedthat the EE reversed the social memory impairment in SHR, since theSHR EE animals showed a reduction on investigation time during thesecond juvenile exposure (Fig. 2B).

Fig. 2 (C–F) illustrates the performance of rats during the sampleand discrimination phases of the object recognition task. One-wayANOVA (environment) of the investigation time in the sample phaserevealed that EE reduced the investigation time for bothWIS [F(1,12)=6.20, pb0.05] and SHR [F(1,12)=29.60, pb0.001] (Fig. 2C, D).Moreover, one-way ANOVA (environment) of the discriminationphase revealed that EE improved the discrimination index for SHR [F(1,12)=4.97, pb0.05], but not for WIS [F(1,12)=0.46, p=0.51](Fig. 2E, F).

3.2. Effects of environmental enrichment in non-cognitive traits of WISand SHR

Fig. 3(A, B) shows thenociceptive latencies exhibited in thehot-platetest. One-way ANOVA (environment) revealed no significant effects ofenvironment for WIS [F(1,12)=0.05, p=0.83] or SHR [F(1,12)=0.00,p=0.99] in the pain sensitivity. Additionally, Fig 3 (C, D) shows thatEE did not alter the mean arterial pressure of WIS [F(1,7)=3.47,p=0.10] or SHR [F(1,8)=0.31, p=0.59].

4. Discussion

The present results show that the general low cognitive perfor-mance presented by SHR rats strongly depends on the rearingenvironment. SHR rats reared from the post-weaning period toadulthood in a stimulating environment, full of novelty, physicalexercise and social contact (i.e., EE) displayed a remarkable better

performance in short- and long-term cognitive tasks, despite nochange in non-cognitive characteristics, such as pain sensitivity andblood pressure. The pro-cognitive effects of EE were observed in theSHR rats, but not in the WIS rats, suggesting that impoverished non-stimulating rearing conditions is detrimental to cognitive functions onindividuals with a specific (yet undetermined) genetic background,but maybe not on a general heterogeneous population. One limitationof the present study is that our analysis was restricted to learning andmemory abilities, whereas the investigation must be expanded toother ADHD-like symptoms presented by SHR.

The fact that EE did not alter the cognitive performance ofWIS rats inthe present results might be interpreted as if the rearing environmentplayed little role on the formation of ‘normal’ cognitive abilities. In fact,although the benefits that EE canproduce on the brainplasticity arewelldocumented, most of the literature showing unequivocal behavioralbenefits of EE in rodents has been obtained in cognitively-impairedanimals (e.g. aging rodents and experimental models of neurodegen-erative diseases) (for review see Nithianantharajah and Hannan, 2006;van Praag et al., 2000). However, it must be conceded that some authorsreported cognitive enhancement inWIS rats exposed to EE (Leggio et al.,2005). In thepresent experiments,we intentionally used task settings inwhichwe knew by experience thatWIS ratswould present a reasonableperformance, as opposed to SHR rats, which reduced the possibility offurther mnemonic improvement for WIS due to ceiling effect. Thisquestionwould likely be addressed by usingmore demanding cognitivetasks, such as more elaborated maze tasks (radial arm mazes, watermaze reversal task), complex operant schedules of reinforcement orlonger delays between training and test (Leggio et al., 2005). Anotherpossibility might be that a more intense enrichment protocol (forinstance, by increasing novelty evenmore by changing objects on a dailybasis)would be necessary to induce positive cognitive effect inWIS rats.Nevertheless, as EE affected the impact of novelty on rat's behavior,irrespective of the genetic background – as already reported (Bezardet al., 2003) – this may be taken as an evidence that our enrichmentprotocol induced neurodevelopmental alterations in WIS rats as well,turning unlikely that a more intense enrichment protocol was required.The mentioned reduced response to novelty was consistently observedas reduced locomotion on the open field and reduced investigation onthe 1st trial of the social and object recognition tests.

In the recent decades,manyefforts havebeenmade to understand thegenetic and pharmacological factors contributing to brain function anddysfunction, while the detailed exploration of environmental factors hasbeen kept relatively aside. Although the classic studies already suggestedthat animals living in a complex and stimulating environment presentimproved cognition (Hebb, 1947) accompanied by neurochemical andanatomical changes (Bennett et al., 1964), the interaction of environ-mental factors with genetics and pharmacology deserves a moreprofound investigation. One of the most popular strategies to depict therole of environment on animal behavior is the so-called environmentalenrichment. This term refers to housing conditions, either home cages orexploratory chambers, that provide higher sensory, cognitive and motorstimulation compared to standard laboratory housing conditions(Nithianantharajah and Hannan, 2006). This leads to a way morestimulating rearing environment, often with increased social contactthrough enhanced number of individuals sharing the cages and theinherent challenges of ever-changing social hierarchy networks. Enrich-ment objects generally vary in composition, shape, size, texture, smell,color, etc. and are constantly re-positioned inside the cage, either by theanimals or experimenters: novelty is one important component to keepEE being stimulating. Another important aspect is whether or not the EEprovides access to runningwheels (for rodents), since voluntary physicalexercise has alone beneficial effects on the brain (for comprehensivereviews, van Praag et al., 2000; Will et al., 2004). The time the animalsspent in the EE is another variable and there are studies exposing theanimals for long periods (days, weeks) to such environments, whileothers provide short (hours) repeated exposures. Finally, the age when

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the animals are exposed to enriched housing conditions plays anotherimportant role. If EE starts early in life, it may have additional effectscompared to EE starting during adulthood. Theremight hypothetically beneurodevelopment critical periods of maturation during which environ-mental enrichment would exert its maximum effects.

It is interesting to observe that our ‘life-long’ environmentalenrichment improved the performance of SHR rats on both short- andlong-term memory tasks. EE groups displayed consistent better perfor-mance than SE groups in the open field habituation task, water mazespatial reference task, social recognition task andobject recognition tasks,each one of them with particular sensorial characteristics and physicaldemands. The better performance of SHR on these distinct mnemonictasks suggests a functional improvement in multiple brain sites, as theinteraction among distinct neural systems, as well as cellular changeswithin specific regions supports memory formation (McGaugh, 2000).One may highlight the likely participation of the prefrontal cortex –

whose function is somewhat deficient in the SHR strain (Heal et al., 2008;Li et al., 2007) – and hippocampus, which are highly involved inworkingmemory and in the formation of long-term spatial memories (open fieldhabituation, water maze reference task) and in the discrimination usingshort-term sampling (object and social recognition) (Dalley et al., 2004;Izquierdo et al., 2006). The perirhinal and entorhinal cortices are alsoimportant brain sites for stimuli discrimination, while the perirhinalcortex is involved in object recognition (Norman and Eacott, 2004) theentorhinal cortex is important in the social recognition task (Sanchez-Andrade et al., 2005). The amygdala complex might have played a role,especially in the consolidation of mnemonic tasks with higher emotionalcontent (McGaugh, 2000). Moreover, it is important to emphasize thathippocampus and enthorhinal cortex may play different roles during thedynamic stages ofmemory formation, which involves parallel processingof short- and long-term memory (Izquierdo et al., 1998). As we did notquantify the rats' swim speed in thewatermaze, the possible influence ofEE on this parameter cannot be ruled out. Rats from both groupsdisplayed comparable results in other non-cognitive traits expressedunder genetic influences, such as hot-plate pain sensitivity and meanarterial blood pressure. Arguably, this means that the present environ-mental modifications specifically affected certain behavioral character-istics linked to cognitive performance in the SHR strain, rather thanmodifying the phenotype of these animals as a whole. It could behypothesized that EE maintained the original ‘genetic’ characteristics ofthe strain, while improved the ability of these animals to change thesynaptic function depending on experience (i.e. better synaptic plasti-city), which constitutes a very interesting field that requires additionalresearch. For instance, there are reports that EE enhances learning andmemory, which relates to a variety of effects on neuronal synapticplasticity at certain brain sites (Moser et al., 1997; Rampon et al., 2000;Tang et al., 2001). This is in line with the concept that higher cognitiveperformance may be one predictive factor of protection against thedevelopment of psychiatric diseases, as depicted in the Theory ofCognitive Reserve (Barnett et al., 2006). The idea is that the higher theneuronal/synaptic density, the higher will be the number of healthysynapses prior to a given pathology. If this ‘synaptic reserve’ is sufficient,little or no loss of cognitive function will be observed; conversely, if thereserve is low, the threshold atwhich clinicalmanifestations occurwill beeasily reached (Barnett et al., 2006). In summary, the term refers to theability of an individual to cope or compensate a given brain insult byrecruitment of an alternative set of neurons to function. This perspectiveis somewhat pioneering in ADHD research and the clinical relevance of itmay be assessed by examining epidemiological indices of educationgrade, or psychological indices of cognitive functionality in associationwith the severity of ADHD symptoms.

5. Conclusion

To the best of our knowledge, this is the first report showing therelationship between exposure to environmental factors during

neurodevelopment and occurrence of ADHD-like symptomatology inlaboratory animals, as far as SHR rats are concerned as an animalmodel. We hope that these preliminary positive results encouragefurther studies in the field and foster the translation of these findingsinto clinical practice. Thus, modifications of the familial environmentcould be considered as a putative preventive strategy to ADHD. Thisapproach may (or not) be used in combination with conventionaldrugs. Additional studies should also investigate whether such non-pharmacological manipulations may prove of therapeutic utility onceADHD symptoms appear.

Acknowledgements

This work was supported by grants from the Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), the Coordenacãode Aperfeiçoamento de Pessoal de Nível Superior (CAPES), thePrograma de Apoio aos Núcleos de Excelência (PRONEX), and theFundação de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC),all of Brazil. The authors are grateful to Dr. Jamil Assreuy and Dr. DanielFernandes for their expert assistance in blood pressure measure-ments. FAP and PP are supported by scholarships from CNPq-Brazil.RNT and RDSP are supported by research fellowships from CNPq-Brazil.

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