environmental enrichment duration differentially affects behavior

Environmental Enrichment Duration Differentially Affects Behavior and Neuroplasticityin Adult Mice

Marianne Leger1, Eleni Paizanis1, Kwamivi Dzahini2, Anne Quiedeville1, Valentine Bouet1, Jean-Christophe Cassel2,Thomas Freret1, Pascale Schumann-Bard1 and Michel Boulouard1

1Normandie Universités, Université de Caen Basse-Normandie, Groupe Mémoire et Plasticité Comportementale (GMPc), EA 4259,F-14032, Caen, France and 2Laboratoire de Neurosciences Cognitives et Adaptatives, UMR 7364, Université de Strasbourg, CNRS,Faculté de Psychologie, Groupe de Recherche NeuroMem 2905, 12 rue Goethe, F-67000 Strasbourg, France

Address correspondence to Michel Boulouard, Normandie Universités, Université de Caen Basse-Normandie, Groupe Mémoire et Plasticitécomportementale (GMPc), EA 4259, F-14032, Caen, France. Email: [email protected]

Environmental enrichment is a powerful way to stimulate brain andbehavioral plasticity. However the required exposure duration toreach such changes has not been substantially analyzed. We aimedto assess the time-course of appearance of the beneficial effects ofenriched environment. Thus, different behavioral tests and neurobio-logical parameters (such as neurogenesis, brain monoamines levels,and stress-related hormones) were concomitantly realized after dif-ferent durations of enriched environment (24 h, 1, 3, or 5 weeks).While short enrichment exposure (24 h) was sufficient to improveobject recognition memory performances, a 3-week exposure wasrequired to improve aversive stimulus-based memory performancesand to reduce anxiety-like behavior; effects that were not observedwith longer duration. The onset of behavioral changes after a3-week exposure might be supported by higher serotonin levels inthe frontal cortex, but seems independent of neurogenesis phenom-enon. Additionally, the benefit of 3-week exposure on memory wasnot observed 3 weeks after cessation of enrichment. Thus, the3-week exposure appears as an optimal duration in order to inducethe most significant behavioral effects and to assess the underlyingmechanisms. Altogether, these results suggest that the duration ofexposure is a keystone of the beneficial behavioral and neurobio-logical effects of environmental enrichment.

Keywords: Anxiety and depressive like-behavior, enriched environment,long-term memory, neurogenesis, serotonin

Introduction

Environmental enrichment consists of complex housing condi-tions that enhance sensory-motor activities, social interactions,cognitive performances, and stimulate brain plasticity (Rosenz-weig 1966). The enriched condition (EC) is well known toprevent age-related cognitive decline in rodents (Freret et al.2012) and to alleviate those occurring in models of neurode-generative disorders (for review, see Nithianantharajah andHannan 2006; van Praag et al. 2000). Even when applied inhealthy adult rodents, EC globally leads to an improvement ofboth spatial (Kempermann et al. 1998; Huang et al. 2007) andnon-spatial (Rampon et al. 2000; Tang et al. 2001; Bruel-Jungerman et al. 2005) memory performances. EC also exertsanxiolytic-like (Benaroya-Milshtein et al. 2004; Galani et al.2007) and antidepressive-like effects (Hattori et al. 2007;Llorens-Martín et al. 2007; Xu et al. 2009), despite controversialresults (Hattori et al. 2007; Silva et al. 2011). Indeed, recentlyreviewed by Redolat and Mesa-Gresa (2012), numerous var-ieties of EC paradigms are available in the literature, possiblyleading to a high heterogeneity of the results. Nevertheless,

while beneficial effects on cognition have been reported bothafter short (several hours per day) (Rampon et al. 2000; Tanget al. 2001; Bruel-Jungerman et al. 2005) and long (weeks tomonths) (Kempermann et al. 1998; Huang et al. 2007; Leger,Bouet et al. 2012; Leger, Quiedeville et al. 2012) EC exposure,the comparison of increasing EC durations remains lacking.

The neurobiological changes that may underlie the behav-ioral plasticity induced by EC have been extensively studied.Many reports show an increase in cortical weight and thickness(Diamond et al. 1964; Bennett et al. 1969) as well as in regionalhippocampal volume (Kempermann et al. 1997) in rodentshoused in EC. Some modifications affecting various growthfactors such as brain-derived neurotrophic factor (BDNF)(Ickes et al. 2000) and neurotransmitters have also been ob-served. For example, higher serotonin and noradrenalin con-centrations in hippocampus have been reported in EC animals(Galani et al. 2007; Brenes et al. 2009). It is important to under-line that these neurotransmitters are known to be cruciallyinvolved in anxiety, depressive behavior and memory (seefor reviews, Buhot 1997; Pardon 2010). Hippocampal neuro-genesis and synaptogenesis enhancement have also beenproposed as changes supporting the behavioral effects of EC(Kempermann et al. 1997; Bruel-Jungerman et al. 2005).However, literature data suffer from heterogeneity regardingthe EC paradigms leading to such beneficial effects. Thus, theaim of this study was to assess to what extent the duration ofEC affects the behavior and whether these behavioral changesare associated with neurobiological modifications. To this end,several neurobiological parameters were assessed, especiallyhippocampal neurogenesis and brain monoaminergic neuro-transmitter concentrations. By these investigations, our workextends our knowledge on the onset and duration of behavioraland brain modifications occurring during EC and thus contributesto the understanding of the neurobiological mechanismsinvolved in behavioral effects of EC.

Materials and Methods

AnimalsNaval Medical Research Institute (NMRI) male mice aged 10 weeks atthe beginning of the experiments were used (local breeding facility, F1from Centre d”Élevage Roger Janvier, Le Genest, France). All animalswere maintained in a room with reversed 12 h light–dark cycle (20:00–8:00), regulated temperature (21 ± 1 °C) and humidity (55 ± 10%).Water and food were available ad libitum. All experiments werecarried out in accordance with the European Communities Council Dir-ective (2010/63/UE) regarding the care and use of animals for experi-mental procedures, and approved by the regional ethical committee

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Cerebral Cortex November 2015;25:4048–4061doi:10.1093/cercor/bhu119Advance Access publication June 5, 2014

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(Comité d’Ethique NOrmandie en Matière d’EXpérimentation Animale,CENOMEXA; agreement number: 01-10-09/17/07-12).

Environmental EnrichmentFor each experiment, mice were subjected to standard condition (SC)or EC. SC mice were maintained in transparent polycarbonate cages(42 × 29 × 15 cm3, 6 mice per cage) containing nesting material and acardboard tube. As already described (Bouet et al. 2011; Leger, Bouetet al. 2012), EC mice were continuously housed in environmental en-richment that consists in large polycarbonate cages (80 × 60 × 60 cm3,12 mice per cage), provided with various objects of different shapes,sizes, colors, textures, and material (wood, plastic, and metal) and alarge running wheel. Most of the objects and their locations wererenewed twice a week to ensure novelty. During the cage cleaning,some objects were neither changed nor cleaned. At each cage cleaningand for each housing condition, a part of the nesting material from theprevious cage was placed in the clean one to limit inter-individual maleagonistic behaviors (Van Loo et al. 2003). Mice were placed continu-ously in SC (n = 12) or EC (n = 12) for a duration of 24 h, 1 week, 3weeks, or 5 weeks before the start of the behavioral assessment andmaintained in the same conditions during the behavioral experiments.For the object recognition test experiments, mice were also housed for6 weeks in EC.

Behavioral ExperimentsMice were placed in the experimental room 30 min before the begin-ning of the behavioral experiments. To minimize the influence of testson each other, 4 different cohorts were used (Fig. 1A). A cohort wasused to assess the spontaneous locomotor activity, a second one usedfor the anxiety and depressive-like behavioral tests, and the 2 otherswere used for long-term memory tests (passive avoidance and objectrecognition). The analyses were conducted by an experimenter blindto the housing condition.

Spontaneous Locomotor Activity Test

ActimetryThe apparatus consisted of a photoelectronic actimeter (APELAB®)that automatically counts the number of interruptions of 2 perpendicu-lar infrared light beams of an individual box (Boissier and Simon1965). As previously described (Leconte et al. 2012), the number oflight beams interruptions was recorded during a single session of 30min and used as an index of spontaneous locomotor activity.

Anxiety-Like Behavior TestsTwo tests were used to measure anxiety-like behavior. Both are basedon the innate preference of mice for dark and confined compared withlight and open spaces. The light–dark box test and the elevated-plus-maze test were performed in the morning (9:00–12:00) and in the after-noon (14:00–17:00) of the first day of experiments, respectively.

Light–Dark BoxThe apparatus (LETICA LE 810, Bioseb®, France) consisted of 2 com-partments separated by a squared opening (Costall et al. 1989). Asalready described (Leconte et al. 2012), the mouse was placed in thecenter of the illuminated white compartment (28 × 27 × 27 cm, 900lux) and left to freely explore the apparatus for 5 min. The latency toenter the dark compartment (17 × 27 × 27 cm, 100 lux, red light), thenumber of transitions between the compartments and the time spent ineach compartment were collected. The percentage of time spent in thelit compartment was used as an index of anxiety-like behavior.

Elevated-Plus MazeThe apparatus was elevated 40 cm above the floor and consisted of 2opposite white open arms (30 × 5 cm, 40 lux) crossing 2 black closedarms (30 × 5 × 15 cm, 10 lux) with a central platform (5 × 5 cm) (Lister1987). As already described (Leconte et al. 2012), the mouse wasplaced onto this platform, facing an open arm, and left to explore the

apparatus for 5 min. The total number of entries and the time spent inthe different arms were collected. The percentages of number ofentries and time spent in the open arms were used as an index ofanxiety-like behavior.

Depressive-Like Behavior TestsTail suspension and forced swimming tests are based on the immobileposture adopted by mice when exposed to an inescapable stressor.Changes in immobility time are considered to reflect changes in mood(Porsolt et al. 1977; Steru et al. 1985). These tests have been widelyused to detect potential antidepressant effects of drugs. Both testswere conducted in the morning and in the afternoon of the second dayof experiments (i.e., the day following the anxiety-like behavior tests),respectively.

Tail Suspension TestThe apparatus consisted of a support elevated 30 cm above the floor(10 lux). The mouse was suspended by the tail using an adhesive tape(at 1.5–2 cm from the tip). The latency for the first period of immobilityand the immobility time during the last 4 min of the whole 6 mintesting period were collected as already described (Leconte et al.2011).

Forced Swimming TestThe apparatus consisted of a transparent glass cylinder (30-cm height,15-cm diameter) containing water (20-cm deep) at 25 ± 1 °C. Themouse was placed in the water and the immobility time, the time ofswimming and the time of climbing were collected during the last 4min of the 6 min testing period as already described (Aguila et al.2007). The latency for the first immobility was also recorded.

Long-TermMemory TestsTwo memory tests were performed in 2 sessions separated by a 24h-intersessions interval (24 h-ISI).

Passive AvoidancePassive avoidance behavior reflects the ability of an animal to inhibitan innate preference for a dark and confined space after a single associ-ation with an aversive stimulus (Jarvik and Kopp 1967). As describedbefore (Leger, Bouet et al. 2012), the apparatus consisted of 2 compart-ments with an electric grid floor, separated by an automated slidingdoor (LETICA LE 812, Bioseb®, France). For the acquisition session,the mouse was placed in the large illuminated white compartment (20× 21 × 20 cm, 1000 lux), with the door closed. After 30 s, the dooropened and latency to enter into the small dark compartment (7.3 ×7.5 × 14 cm, 10 lux) was measured (maximum latency: 50 s). Immedi-ately after entering, the door was closed and the mouse received an in-escapable electric foot shock (0.4 mA; 2 s) before being placed back inits home cage. For the test session (24 h-ISI), the experimental proced-ure was the same as for acquisition except no electric shock was used(the maximal length of this test session was 300 s). Because differencesin latencies during the acquisition session were found between groups,data were normalized as previously described (Leger, Bouet et al.2012). The ratio of acquisition versus test session latencies to enter thedark compartment was used as an index of memory performance.

Object Recognition in Y-MazeObject recognition test was performed in Y-maze as previously de-scribed (Leger, Quiedeville et al. 2012). The apparatus consisted of agray plastic Y-maze with 3 arms (33 × 8 × 16 cm each). During the pres-entation session, 2 identical objects were placed in the distal thirdareas of 2 arms. The mouse was placed in the starting arm containingno object, facing the experimenter, for free exploration (maximum: 10min). When a criterion of 20 s of total exploration of both objects wasachieved, the mouse was returned to the home cage. During the testsession (24 h-ISI), a copy of the familiar objects and a novel objectwere placed in 2 arms of the maze. As previously, the mouse wasallowed to freely explore the 3 arms until the criterion of 20 s total

Cerebral Cortex November 2015, V 25 N 11 4049


object exploration time was reached. The nature of the objects (Lego®

vs. Falcon®) and the position of the novel object (left vs. right) wererandomized among animals. As previously described (Leger et al.2013), object exploration was considered when the mouse sniffed theobject with the nose directed towards the object at a distance <2 cm.The time of exploration of each object was manually collected andused as an index of memory performances by comparison with thechance level (10 s). Mice failing to reach the criterion within 10 min forthe presentation or the test session were excluded from the analysis.

A 3-week exposure seemed to be a crucial duration to achieve bene-ficial effect in most behavioral tests made. Besides, among those tests,the object recognition test was the more sensitive to EC exposure (re-vealing performances modifications even after a short duration). Thus,we aimed to assess whether this 3-week exposure beneficial effectcould persist after a similar time of EC interruption (i.e., 3 weeks). Forthis specific study, 3 additional groups were thus included (n = 12 pergroup; Fig. 1A). The first group was exposed to 3 weeks in EC and re-placed in SC for 3 additional weeks. As control groups for this study, 2

Figure 1. Experimental design. (A) After 24 h, 1 week, 3 weeks, or 5 weeks in either SC (n= 12 per duration) or EC (n= 12 per duration), behavioral experiments were conductedduring 2 consecutive days (D1 and D2). The first cohort was used to assess the spontaneous locomotor activity while the second cohort was used to assess anxiety anddepressive-like behaviors (light–dark box [LD], elevated plus maze [EPM], tail suspension test [TST] and forced swimming test [FST]). The third and fourth cohort were used toassess the long-term memory through the passive avoidance (PA) test and the object recognition test performed in a Y-maze (OR). (B) Immunohistochemical experiments wereconducted after 1, 3, or 5 weeks in either SC or EC. For the DG cell proliferation study, mice of each condition (n=6 SC and n= 6 EC) were injected with one singly BrdU dose(75 mg/kg) 2 h before killing. To assess the newborn cell survival, mice of each condition (n=6 SC and n= 6 EC) were injected with 2 daily BrdU doses (50 mg/kg) the first 4 dayof housing and killed after 3 or 5 weeks of housing exposure. (C) Neurochemical changes were assessed after 1, 3, or 5 weeks in either SC or EC. Brain regions of interest werecollected at the end of the housing period (BLA, baso-lateral amygdala; FrC, frontal cortex; Hpp, hippocampus). (D) Stress-related hormones were measured after 1, 3, or 5 weeks ineither SC or EC. Blood samples were collected at the end of the housing period, (1) either in resting condition, (2) or in stressed-induced condition (FST).

4050 Duration of Enriched Condition on Brain and Behavioral Plasticity • Leger et al.


other groups were placed either in EC or in SC for 6 weeks beforeobject recognition memory assessment.

Neurobiological ExperimentsTo avoid the potential influence of behavioral testing on neurobiologic-al parameters, 3 other cohorts of mice were used for immunohisto-chemistry (Fig. 1B), measures of monoamine concentrations (Fig. 1C)and endocrine response (Fig. 1D).

Immunohistochemistry

Cell ProliferationTo assess the influence of EC duration (i.e., 1 week, 3 weeks, and 5weeks) on hippocampal cell proliferation, mice (n = 6 SC and 6 EC foreach housing duration) were injected intraperitoneally with a singledose of 5-bromodeoxyuridine (BrdU, 75 mg/kg) on the last day ofhousing, 2 h before killing (Paizanis et al. 2010) (Fig. 1B).

Mice were deeply anesthetized with an intraperitoneal injection ofpentobarbital (120 mg/kg) and transcardially perfused with phosphatebuffered saline (PBS 0.1 M) supplemented with sodium nitrite (0.1%)followed by a 4% paraformaldehyde solution. The brains were thenremoved and postfixed for 12 h in the same fixative solution beforebeing coronally sectioned (40 µm thickness) on a vibratome apparatus(Vibratome VT 1000S, LEICA). Free-floating sections were then storedat −20 °C in cryoprotective solution (30% glycerol, 30% ethyleneglycol, 40% PBS) until immunohistochemistry was processed (Paizaniset al. 2010).

Briefly, series of 1 in 4 free-floating sections were rinsed in PBS 0.1M before being incubated in 3% H2O2/10% methanol/PBS 0.1 M toquench endogenous peroxidases. After 5 min, they were rinsed and in-cubated in 2 M HCl/0.1% Triton X-100/PBS 0.1 M (30 min; 37 °C) to de-nature the DNA; pH was adjusted by incubation in 0.1 M sodiumtetraborate buffer (30 min; pH 8.6). After several washes, sections wereincubated for 1 h in 5% normal rabbit serum/0.1% Triton X-100/PBS0.1 M before overnight incubation at 4 °C in the same solutionsupplemented with the primary antibody (monoclonal rat IgG anti-BrdU, 1:100, AbCys, France). After several washes, sections were incu-bated for 2 h at room temperature with the secondary antibody (bioti-nylated rabbit anti-rat IgG, 1:200, Vector Laboratories, France) andstained using ABC staining system (Vectastain ABC, Elite kit, Vector La-boratories, France). Peroxidase activity was detected by incubation ofsections in 0.05% diaminobenzidine/0.01% H2O2/PBS 0.1 M for 5 min.Finally, free-floating sections were rinsed, mounted on gelatinizedslices, dehydrated and coverslipped with R.A. Mounting Medium™(Richard-Allan Scientific, Kalamazoo, MI, USA).

Quantification of hippocampal BrdU-immunopositive cells of thegranular cell layer (GCL) and adjacent subgranular zone of the dentategyrus (DG) (2-cell body-wide zone along the border between the GCLand the hilus) was performed in a blind manner on an Olympus micro-scope (×20–×40 objectives). GCL surface areas were measured withImageJ 1.6® software. Results were expressed by the total number ofhippocampal BrdU-immunopositive cells (total number of BrdU-stainedcells × 4). GCL volume was calculated by multiplying the sum of surfaceareas by 160 µm.

Newborn Cells SurvivalTo assess the influence of EC duration on newborn hippocampal cellsurvival, mice (n = 6 SC and 6 EC for each housing duration) weretreated with BrdU at the beginning of housing. Two daily injections ofBrdU (50 mg/kg) every 12 h were administered during the first 4 daysof EC or SC (Tashiro et al. 2007). After 3 and 5 weeks of housing, micewere killed as described above and brains were removed and sectionedas previously described (Paizanis et al. 2010) (Fig. 1B).

A first series of one in 4 free-floating sections was immuno-stainedand analyzed as described before.

A second series of 1 in 4 free-floating sections was double-stainedfor BrdU and neurospecific nuclear protein (NeuN) using rat anti-BrdU(monoclonal rat IgG, 1:100, AbCys, France) and mouse anti-NeuN (1:500,Chemicon International, Millipore, France) antibodies. Immunofluorescence

was revealed with the secondary antibody (biotinylated rabbit anti-ratIgG, 1:200, Vector Laboratories, France) coupled with streptavidin-Alexa Fluor 488 (1:1600, Molecular Probes, USA) and an Alexa Fluor568 goat anti-mouse (1:1600, Molecular Probes, USA). Free-floatingsections were finally mounted on gelatinized slices, dehydrated andcoverslipped with Fluoromount-G solution (Clinisciences, France).The neuronal phenotype was determined by analyzing 100 BrdU-positive cells per mouse labeled both by BrdU and NeuN staining withan Olympus Fluoview FV 1000 microscope in the entire z-axis (reso-lution of 1 µm/pixel), using a step interval of 0.03 µm (×60 water-immersion objective; Olympus Fluoview Ver. 3.1.® software).

NeurochemistryAt the end of each housing condition exposure for which the main be-havioral effects of EC were observed (1, 3, and 5 weeks), mice werekilled by cervical dislocation (n = 10 per housing condition) (Fig. 1C).The brains were rapidly removed and dissected on a cold plate. Thefrontal cortex and the hippocampus were rapidly isolated, weighedand stored at −80 °C for further analysis. Considering the neuronalactivity changes induced by a 3-week EC exposure in such brainstructures (Leger, Bouet et al. 2012; Leger, Quiedeville et al. 2012), thebaso-lateral amygdala was additionally isolated for the 3-week exposedanimals. Using high-performance liquid chromatography (HPLC), theconcentrations (pg/mg of wet tissue) of serotonin (5-HT), dopamine(DA), and their metabolites, that is, 5-hydroxyindolacetic acid (5-HIAA),3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)were quantified.

The tissue samples were homogenized for 30 min in 100 µl of anextraction solution (pH = 3) constituted the mobile phase supplemen-ted with perchloric acid 0.1 M. The samples were then centrifuged(10 000 rpm, 10 min), the supernatant was isolated and centrifugedagain (10 000 rpm, 5 min). A sample volume of 20 µl of the super-natant was injected. The apparatus consisted of a Waters Series system(Waters Corporation®, Milford, MA, USA) composed of an online de-gasser and a binary pump (Waters 1525) coupled with an electrochem-ical detector (Waters 2465: ISSAC reference, VT-03 Glassy carbon WE).A reverse phase column (Perkin Elmer, USA: C18 Brownlee® RP5, 5µm pore size, 2.1 mm in diameter and 220 mm long) was used at 33 °Cand a mobile phase (adapted from Dzahini et al. 2010) was delivered at0.5 mL/min flow rate. The mobile phase constituted of 2 mM KCl, 50mM KH2PO4, 0.1 mM ethylenediaminetetraacetic acid (EDTA)–Na2,0.28 mM sodium octyl sulfate and 6% methanol (pH = 4.5). The analyt-ical system’s best sensitivity average value was 3 pg/10 µl with asignal/noise ratio of 3:1. Specific limits of detection were 490 fmol forDOPAC, 530 fmol for DA, 446 fmol for 5-HIAA, 560 fmol for HVA and496 fmol for 5-HT. Using a standard of 5 and 50 pg and performing theanalysis 5 subsequent times, repeatability was found to be better than4% relative standard deviation for peak area and the method showsgood linearity for all analytes (correlation coefficients > 0.991). Chro-matogram analysis was obtained with Waters eSATIN and Breeze2®

data acquisition software. The neurochemistry experiments were per-formed in a double blind design.

Stress-Related Hormones MeasurementAt the end of 1, 3, and 5 week housing durations, half of the mice wereexposed to the forced swimming test (same protocol as describedbefore). Immediately after this stress procedure (n = 6 per housing con-dition) or after resting condition (n = 6 per housing condition), mice weredecapitated and trunk blood samples were collected (between 10:30 and12:00) in EDTA-coated tubes (adapted from Keeney et al. 2006) (Fig. 1D).Samples were rapidly centrifuged (5 min at 4 °C) and plasma supernatantswere isolated and stored at −20 °C until radioimmunoassay procedure.Corticosterone plasma concentration was determined with a radio-immunoassay kit (ImmuChem™ Double antibody, Corticosterone-125Ikit, MP Biomedicals, LLC, Orangeburg, NY, USA).

The average assay sensibility was 7.7 ng/mL and corticosteronelevels were determined in duplicates with an intra-assay variation coef-ficient of 4.4%. All samples were run together in a single assay to avoidinter-assay variability.



Statistical AnalysesStatistical analyses were processed with Statview® 5.0 software. For allexperiments, we checked with an analysis of variance that SC mice pre-sented similar behavioral performances, whatever the duration of ex-posure (data not presented). To compare the behavioral performancesbetween the housing conditions, analysis of variance (ANOVA) and,when appropriate, post hoc Bonferroni/Dunn test were used to reduceType I errors. Because 4 comparisons were made (comparisonbetween respective same duration of SC and EC housing, i.e., 24 h, 1week, 3 weeks, and 5 weeks), the significance level was adjusted to P =0.0125 for the behavioral experiments. In the object recognition test, aunivariate t-test was used to compare the exploration time of novelobject with the chance level of exploration (10 s). The comparisonbetween the 5 housing durations (24 h, 1, 3, 5, and 6 weeks) was donewith a post hoc Bonferroni/Dunn test and the significance level wasadjusted to P = 0.01. For the neurochemistry analysis, because 3 com-parisons were made (comparison between respective same durationof SC and EC housing, i.e., 1, 3 and 5 weeks), the significance levelwas adjusted to P = 0.0167. Values are expressed as means ± SEM. Fordata that were not normally distributed (ratio of latency betweenthe acquisition and the test sessions in the passive avoidance test) orobtained from small sample sizes (total number of BrdU-stainedcells and GCL volumes, corticosterone levels), results are expressedas median ± quartile and analysis was performed with the non-parametric Kruskal–Wallis test followed when appropriate with aMann–Whitney U test. A significant difference was considered whenthe P value was <0.05.

Results

EC Decreases Spontaneous Locomotor Activity From the1-Week ExposureAnalysis of 30-min locomotor activity showed a significant re-duction in the number of light beams interruptions in EC com-pared with SC mice at all exposure times from the 1-weekhousing duration upwards (ANOVA: F7,88 = 5.73, P < 0.001,Bonferroni/Dunn test P < 0.0025 for the 1- and 5-week expos-ure; P < 0.0125 for the 3-week exposure) but not with theshortest exposure of 24 h (Fig. 2).

Exposure to EC Does not Modify the Anxiety-Like Behaviorin the Light–Dark Box TestThe number of transitions between the 2 compartments didnot differ between groups (data not shown, ANOVA: F7,88 = 1.03,P > 0.05), suggesting no modulation of exploratory and/or

spontaneous activity. There was no difference between groupsfor the percentage of time spent in the aversive lit compartment(ANOVA: F7,88 = 1.21, P > 0.05). However, EC mice entered sig-nificantly more rapidly in the dark compartment than SC ones(ANOVA: F7,88 = 4.30, P < 0.001, Bonferroni/Dunn test: P <0.0125 for the 3- and 5-week exposure) (Fig. 3A).

The 3-Week EC Exposure Exerts Anxiolytic-Like Effects inthe Elevated-Plus-Maze TestThe total number of entries in the closed arms did not differbetween groups (data not shown, ANOVA: F7,88 = 2.13, P < 0.05;followed by a Bonferroni/Dunn test that did not reveal differ-ence between groups), suggesting no modulation of exploratoryand/or spontaneous activity by EC in the elevated-plus maze. Asignificantly higher percentage of time spent and entries in theopen arms was observed in mice exposed to the 3-week EC dur-ation compared with the corresponding SC group (ANOVA:F7,88 = 4.57, P < 0.001 and F7,88 = 2.62, P < 0.05, respectively, P <0.0025 and P < 0.00025, respectively) (Fig. 3B).

EC Does not Influence Depressive-Like BehaviorThere was no effect of EC on the immobility time or on thelatency for the first immobility, in the tail suspension test(ANOVA: F7,87 = 1.77, P > 0.05 and F7,87 = 1.85, P > 0.05, respect-ively), or in the forced swimming test (ANOVA: F7,87 = 1.44,P > 0.05 and F7,87 = 1.13, P > 0.05, respectively). This is comple-mented by the absence of difference in time spent to swim andto climb between groups in the forced swimming test (ANOVA:F7,87 = 1.47, P > 0.05 and F7,87 = 0.63, P > 0.05, respectively) (datanot shown).

The 3-Week EC Exposure Improves the Memory Based onAversive StimulusBecause mice exposed to 3 and 5 weeks of EC entered the darkcompartment significantly more rapidly compared with SCmice in the light–dark box test, we checked whether this be-havior occurred in the passive avoidance, since at the acquisi-tion session, the mouse also has to enter a dark compartment.A significant difference in the latency to enter in the dark com-partment was found between the 3-week SC (23.7 s ± 4.4) andEC (8.9 ± 2.8) groups (data not shown, ANOVA: F7,85 = 2.43,P < 0.05, Bonferroni/Dunn test: P < 0.0025). The ratio of laten-cies test/acquisition was significantly higher in mice exposedto the 3-week EC duration indicating better memory perform-ance compared with the SC group (Kruskal–Wallis test: H =17.12, P < 0.05, Mann–Whitney U test: Z = -2.21, P < 0.05, forthe 3-week EC exposure) (Fig. 4A).

Whatever the Housing Duration, EC Improves ObjectRecognition Memory but This Effect Does not PersistAfter Return to SCDuring the presentation session, no difference in the time toexplore the 2 objects was observed, whatever the group con-sidered (data not shown, univariate t-test: t116 = 0.90, P > 0.05).During the test session, the time spent to explore the novelobject was overall significantly higher than chance (10 s) (uni-variate t-test: t116 = 5.63, P < 0.001; Fig. 4B). While SC mice glo-bally did not discriminate the novel object compared with thechance level (univariate t-test: t55 = 0.47, P > 0.05), EC mice sig-nificantly discriminated the novel object, at all housing dura-tions (univariate t-test: t11 = 2.41, P < 0.05 and t11 = 2.62,

Figure 2. Effect of EC duration (24 h, 1, 3, and 5 weeks; n= 12 per group) onspontaneous locomotor activity compared with SC. Data are the mean (±SEM)number of light beam interruptions. A significant reduction in light beams interruptionswas observed in EC groups exposed to 1, 3, and 5 weeks compared with respectiveSC groups (Bonferroni/Dunn test, *P< 0.0125, **P<0.0025).



P < 0.05 for the 5- and 6-week exposure, respectively, t9 = 5.06,P < 0.01 for the 24 h exposure, t6 = 4.82 and t9 = 6.19, P < 0.001for the 1- and 3-week exposure, respectively). By contrast, the3-week exposed group that had been transferred for 3 weeksto SC did not discriminate the novel object (univariate t-test:t11 = 1.42, P > 0.05). Moreover, a significantly higher explor-ation time of the novel object was observed in mice exposed toEC from 1 week up to 5 weeks compared with the respectiveSC groups (ANOVA: F10,106 = 5.18, P < 0.001, Bonferroni/Dunntest: P < 0.002 for the 1- and 3-week exposure and P < 0.01 forthe 5- and 6-week exposure).

EC Does not Modify Hippocampal Cell ProliferationWhatever the housing duration, EC did not modify the GCLvolume (Kruskal–Wallis test: H = 5.61, P > 0.05). Besides, therewas no difference in the total number of BrdU-immunopositivecells between EC and SC groups (Kruskal–Wallis test: H =11.10, P < 0.05, Mann–Whitney U tests: P > 0.05; Fig. 5A).

Exposure to 3 and 5 Weeks of EC Promotes the NeuronalPhenotype of Surviving CellThere was no difference in GCL volume between groups (datanot shown, Kruskal–Wallis test: H = 2.90, P > 0.05). Concerning

Figure 3. Effect of EC duration on anxiety-like behavior compared with SC. (A) Effect of EC duration (24 h, 1, 3, and 5 weeks; n=12 per group) on anxiety-like behavior in the light–darkbox test. Data are the mean (±SEM) percentage of time spent in the lit compartment (graphic on the left) and the latency to enter into the dark compartment (graphic on the right).There was no difference in the percentage of time spent in the lit compartment between groups. A significantly lower latency to enter in the dark compartment was observed after the3- and 5-week EC exposure (Bonferroni/Dunn test, *P<0.0125). (B) Effect of EC duration (24 h, 1, 3, and 5 weeks; n=12 per housing condition) on anxiety-like behavior in theelevated-plus-maze test. Data are the mean (±SEM) percentage of time spent (graphics on the left) and percentage of entries (graphics on the right) in the open arms. Mice exposed3 weeks in EC significantly spent more time and showed a higher percentage of entries in the open arms (Bonferroni/Dunn test, **P<0.0025; *** P<0.00025).

Figure 4. EC duration differentially improved memory based on aversive stimulus and object recognition memory (24 h-ISI) compared with SC. (A) Effect of EC duration (24 h(n= 12 per group), 1 week (n=12 per group), 3 weeks (n=12 SC; n=10 EC) or 5 weeks (n=12 SC; n=11 EC)) on passive avoidance performance. Data represent theboxplot of the ratio test/acquisition latencies measured in the passive avoidance test. A significantly higher ratio was found for the 3-week EC compared with SC duration (Mann–Whitney U test: *P<0.05). (B) Effect of EC duration (24 h [n=12 SC; n=10 EC], 1 week [n= 11 SC; n= 7 EC], 3 weeks [n= 10 SC; n= 10 EC], 5 weeks [n= 11 SC;n=12 EC], 6 weeks [n= 10 SC; n= 12 EC] and 3 weeks of EC followed by 3 weeks of SC [n= 12]) on object recognition performance in Y maze. Data are the mean (±SEM)exploration time of the novel object during the test session. Only EC mice significantly discriminated the novel object compared with chance level (10 s) (univariate t-test:§P< 0.05; §§P< 0.01; §§§P<0.001). EC mice presented higher exploration time of the novel object compared with SC ones, from 1 week of exposure (Bonferroni/Dunn test,*P< 0.01; **P<0.002).



the newborn cell survival, only the 5-week EC exposure signifi-cantly enhanced the cell survival compared with the respectiveSC group (Kruskal–Wallis test: H = 9.22, P < 0.05, Mann–Whitney U tests: Z =−1.12, P > 0.05, and Z =−2.36, P < 0.05,for the 3- and 5-week exposure, respectively) (Figs 5B and 6A).Confocal immunofluorescence analysis revealed a significantlyhigher number of positive cells coexpressing BrdU and NeuN,after both 3 and 5 weeks of EC, compared with SC groups(Kruskal–Wallis test: H = 16.13, P < 0.01, Mann–Whitney Utests: Z =−2.25, P < 0.05, and Z =−2.88, P < 0.01, for the 3- and5-week exposure, respectively) (Figs 5C and 6B).

The 3-Week EC Exposure Enhances SerotoninConcentration in the Frontal CortexBrain monoamines analysis revealed a global significant effectof EC duration (1, 3 and 5 weeks) compared with SC on sero-tonin concentration in the frontal cortex (Table 1) but not inthe hippocampus or the baso-lateral amygdala (data notshown). A significantly lower 5-HIAA concentration (−30%)was found in mice exposed to 1 week of EC, without modifica-tion of the 5HT concentration (ANOVA: F5,54 = 8.54, P < 0.001and F5,54 = 5.89, P < 0.001, Bonferroni/Dunn test: P < 0.0001and P > 0.0167, respectively). This was associated with a sig-nificant decrease in the serotonergic turnover (5-HIAA/5HT;−31%) (ANOVA: F5,54 = 11.93, P < 0.001, Bonferroni/Dunn test:P < 0.0002). After a 3-week exposure to EC, a significantlyhigher 5HT concentration was observed (+29%), as well as atrend to higher 5-HIAA concentration (ANOVA: F5,54 = 5.89,

P < 0.001 and F5,54 = 8.54, P < 0.001, Bonferroni/Dunn test:P < 0.0167 and P = 0.0338, respectively) without modificationof the serotonergic turnover. There was no effect of EC on theother monoamine and metabolite concentrations in the frontalcortex (data not shown, ANOVA: F5,54 = 1.25, P > 0.05 for theDA, F5,54 = 1.09, P > 0.05 for the DOPAC, F5,54 = 1.12, P > 0.05for the HVA).

EC Neither Modifies Basal nor Stress-Induced PlasmaCorticosterone LevelsRadioimmunoassay revealed in both groups a significant en-hancement of the corticosterone levels under stress-inducedcondition (forced swimming test exposure) compared with thebasal condition (Kruskal–Wallis test: H = 33.70, P < 0.001;Mann–Whitney U tests: Z =−3.44, P < 0.001 in SC groups, andZ =−4.05, P < 0.001 in EC groups). However, there was noeffect of EC on these corticosterone levels neither in basal con-dition (Kruskal–Wallis test, P > 0.05; Fig. 7A), nor in stress-inducedcondition (Kruskal–Wallis test, P > 0.05; Fig. 7B), comparedwith SC group.

Discussion

The aim of this study was to assess how the duration of ECaffects the behavioral performances and whether these behav-ioral changes are related to neurobiological modifications. Asreported by others (Rampon et al. 2000; Tang et al. 2001;Huang et al. 2007; Leger, Bouet et al. 2012), among the main

Figure 5. Effect of EC duration on DG cell proliferation (A), cell survival (B), and neuronal phenotype (C) compared with SC. Data are expressed as boxplots. (A) No differencebetween the number of BrdU-stained proliferative cells (left) and of GCL volume (right) was observed between groups, whatever the housing duration (1 week (n= 5 per group), 3weeks (n= 6 SC; n=4 EC), and 5 weeks (n=6 per group)). (B) A significantly higher number of BrdU-stained survival cells was detected in mice exposed to 5 weeks of ECcompared with SC corresponding group (Mann–Whitney U test: *P< 0.05; n= 6 per group). (C) A significantly higher number of Brdu–NeuN-stained cells was observed in ECboth after the 3- and 5-week exposure compared with respective SC groups (Mann–Whitney U test: *P< 0.05; **P<0.01; n= 6 per group).



behavioral effects, we observed a promnesiant effect of EC on 2kinds of memory, either based on an aversive stimulus (passiveavoidance) or on recognition memory (object recognition). Wealso found a reduced spontaneous locomotor activity, a behav-ioral change probably related to a faster habituation to noveltythat we identified to occur after the 1-week exposure, and ananxiolytic-like effect in the elevated-plus maze. These cognitiveeffects may be related to neurobiological changes induced byEC. Indeed, serotonergic changes in the frontal cortex and hip-pocampal cell survival associated with a neuronal phenotype in-crease were detected. Finally, we might suppose that NMRI mice

are less sensitive to the antidepressive-like effect of environmen-tal modulation observed in despair-like paradigms (Hattori et al.2007; Llorens-Martín et al. 2007) as no effect of EC on thedepressive-like behavior was detected in both tests used.

In accordance with recent investigations in rats (Birch et al.2013), our findings highlight the importance of the duration ofexposure in EC-related effects. While short EC exposure (24 h)was sufficient to improve object recognition memory, the en-hancement of hippocampal cell survival required longer EC ex-posure (5 weeks). Moreover, the anxiolytic-like effect and thememory improvement in the passive avoidance test induced by

Figure 6. Microphotographs of BrdU-stained cells survival in the GCL of the DG. (A) A higher number of BrdU-stained cells survival was observed in mice exposed to 5 weeks in ECcompared with respective SC mice. (B) Representative double-stained for BrdU (green) and the neuronal marker NeuN (orange) after a 3-week EC exposure compared withcorresponding SC group.



EC were detected after the 3-week exposure, a duration afterwhich an increase in serotonin concentration was observed inthe frontal cortex. The potential relationship between thetiming of appearance of the neurobiological and the behavior-al effects are now discussed.

EC Does not Modulate the Plasma Corticosterone LevelsWhatever the duration of exposure, EC did not modify theplasma corticosterone concentration compared with SC mice,neither in resting, nor in stress-induced condition. Becauseboth agonistic behavior and stressful situation are known toinduce higher corticosterone levels (Haemisch and Gartner1994; Haemisch et al. 1994; Von Holst 1998), the absence of

corticosterone levels changes could reflect a less stressful effectof the EC in NMRI mice, as since reported in a docile inbredstrain (Marashi et al. 2004). Besides, it has been demonstratedthat the antidepressive-like effects of EC required glucocorti-coids elevation in mice (Xu et al. 2009). The unchangeddepressive-like behavior observed between our groups maythus be underlain by the absence of hyperactivity of the hypo-thalamic–pituitary–adrenocortical axis.

EC Affects Hippocampal Neuronal Survival From the3-Week Exposure DurationHippocampus-dependent memory training has been involvedin modifications of survival and incorporation of immaturenewborn cells in neuronal networks (for review, see Zhaoet al. 2008). To isolate the effects of EC on neurogenesis fromthose potentially related to the memory test, we explored theeffects of EC on neurogenesis in animals that did not experi-ence any task training. In opposition to the higher cell prolifer-ation reported after short EC exposures as used herein, that is,1 week (Steiner et al. 2008; Llorens-Martín et al. 2010), we didnot observe a modification of DG cells proliferation, whateverthe EC duration. In these studies, however, BrdU was injectedat the beginning of the EC, which could be more stimulatingthan the end of the exposure, and explain the discrepancy withour results. By contrast, we report a higher number ofBrdU-stained surviving cells, which reached the significancelevel after the 5-week EC duration, as similarly found afterlonger exposure periods ranging from 6 weeks to 6 months(Kempermann et al. 1998; Brown et al. 2003; Rossi et al. 2006;Llorens-Martín et al. 2010). In agreement with other studies(Kempermann et al. 1998; Tashiro et al. 2007), a significantlyhigher percentage of surviving cells displaying the neuronalphenotype was observed in EC mice compared with SC, aneffect that we identified from the 3-week EC exposure. Thus,we show for the first time that the minimal duration of EC ex-posure of 3 weeks is necessary to induce neurogenesischanges in mice. While the 3-week EC exposure resulted in ahigher proportion of new neurons, the neuronal survival isprolonged specifically after 5 weeks of EC. This indicates aduration-dependent effect of EC on neurogenesis, an observa-tion similar to the results described by Birch et al. (2013)in rats. Because of the nature of the EC that we chose (socialinteractions, object interactions, and exercise), it is difficultto determine here the role of each aspect of the EC on theneurogenesis changes observed. Considering the recent workspublished, the running activity definitely plays a major contrib-uting role on hippocampal neurogenesis increase (Marlattet al. 2013) as well as neurotrophine levels enhancement suchas BDNF (Kobilo et al. 2011; Vivar et al. 2013). Despite no re-cording of the running activity in our study, the large runningwheel used in our experiment allowed access to several miceat the same time. The physical activity may thus largelybe involved in the neurogenesis stimulation. Interestingly, asstress-induced release of glucocorticoid hormones (i.e., cor-ticosterone) is known to impair the neurogenesis (Lucassenet al. 2010; Schoenfeld and Gould 2013), the beneficial effectsof EC reported here on neurogenesis occurred without anycorticosterone changes. Our results tend to confirm those fromXu et al. (2009) demonstrating that the enhanced rate ofadult neurogenesis was maintained in mice exposed to EC in-dependently of the prevention of elevated corticosterone

Table 1Effects of the EC duration (1, 3, and 5 weeks) on serotonergic concentrations in the frontal cortex(pg/mg wet tissue) compared with SC

Monoamines Group 1 Week 3 Weeks 5 Weeks

5HT SC 72.8 ± 7.1 78.2 ± 4.5 63.1 ± 4.5EC 74.0 ± 5.7 101.3 ± 8.4* 65.6 ± 4.2

5-HIAA SC 399.5 ± 15.2 300.8 ± 18.3 371.9 ± 17.0EC 281.0 ± 13.7*** 349.3 ± 17.1 373.9 ± 17.8

5-HIAA/5HT turnover SC 5.8 ± 0.5 3.9 ± 0.2 6.0 ± 0.4EC 4.0 ± 0.4*** 3.6 ± 0.2 5.8 ± 0.2

Note: Values are means ± SEM, ANOVA followed by a post hoc Bonferroni/Dunn test,*P< 0.0167, ***P< 0.0002 compared with the corresponding SC group (n= 10 per group).

Figure 7. Effect of EC duration on corticosterone levels under basal condition ofstress-induced condition (FST test) compared with SC. Data are expressed asboxplots. (A) No difference of corticosterone levels was observed between groupsunder basal condition (1 week [n=5 SC; n= 6 EC], 3 weeks [n= 5 SC; n= 6 EC],and 5 weeks [n= 6 per group]). (B) No difference of corticosterone levels wasobserved between groups under stress-induced condition (1 week [n= 5 SC; n=6EC], 3 weeks [n= 6 per group], and 5 weeks [n=5 SC; n=6 EC]).



release. In rats exposed to EC, elevated hippocampal BDNFlevels negatively correlated with corticosterone levels (Bakoset al. 2009). Considering the importance of BDNF for thenewborn cell survival and differentiation in the DG (Sairanenet al. 2005; Taliaz et al. 2010), it would be relevant to further in-vestigate how much the BDNF may be involved in the behav-ioral, neurochemical and neurogenesis changes induced byEC.

EC Exposure Transiently Affects SerotoninConcentration in the Frontal CortexThe monoamine levels were measured in 3 brain regionsknown to take part in the beneficial effects of EC on memory,namely the hippocampus, the frontal cortex and the baso-lateral amygdala (Leger, Bouet et al. 2012; Leger, Quiedevilleet al. 2012). To our knowledge, the brain monoamine levelsfollowing different EC durations have never been investigated.Here, we report a transient serotonergic modulation in thefrontal cortex after a 3-week EC exposure, suggesting that com-pensatory mechanisms leading to normalization of brain sero-tonin levels appear after longer exposure. This could explainthe absence of serotonergic changes observed in mice exposedto a longer duration of EC, that is, 5 weeks in our experimentsor 40 days in a previous study (Naka et al. 2002). Thus, EC aswell as novelty exposure protocols (Miura et al. 2002; Antoniouet al. 2008; Ford et al. 2008), modulate the serotonergic systemwith a regional selectivity as there was no change in the hippo-campus, or the baso-lateral amygdala.

EC Exposure Transiently Affects Anxiety-Like Behaviorin a Task-Dependent MannerOur main findings concern the transient anxiolytic-like effectin the elevated-plus maze induced by 3 weeks of EC but not bylonger exposure (5 and 8 weeks, data not presented). If thisanxiolytic-like effect has already been described in the litera-ture after longer EC duration (5–6 weeks) (Benaroya-Milshteinet al. 2004; Galani et al. 2007), it is the first time that it is shownto be transient. This emotional response does not seem to berelated to a hypothalamic–pituitary–adrenocortical axis re-sponse. Indeed, EC had no effect on plasma corticosteroneconcentration under resting or stress conditions. Interestingly,the control of emotions, and especially anxiety, has beenshown to be supported by the prefrontal cortex (Griebel 1995;Buhot 1997; Gross et al. 2002), a brain region receiving adense serotonin innervation (Azmitia and Segal 1978). Consid-ering their similar kinetics of onset, the anxiolytic-like effectsinduced by 3 weeks of EC could thus be supported by theserotonergic adaptation observed in the frontal cortex. This hy-pothesis could be further studied by focusing on the serotoner-gic changes induced by EC, not only on basal condition, butalso during the elevated-plus-maze test.

By contrast, the anxiety-like behavior was not modified byEC in the light–dark box test, an effect already observed byothers (Augustsson et al. 2003; Chourbaji et al. 2005), that wefound to be independent of the exposure duration. The shorterlatency to enter the dark compartment found after the 3 and5-week exposure to EC may be related to a faster habituation tonovelty, as also reported by Chourbaji et al. (2005) after similarexposure duration. Because of its openness and elevation, theelevated-plus-maze is probably more aversive than the light–

dark box and could facilitate the apparent anxiolytic-like effectinduced by EC.

Aversive Stimulus-Based Memory and ObjectRecognition Memory are Differentially Affected by theDuration of EC ExposureThe major effects of the 3-week EC exposure on anxiety areassociated with an improvement in memory performancesassessed in the passive avoidance test. Again, such a beneficialeffect was transient and disappeared after 5 weeks of expos-ure. Because the test requires the use of an aversive stimulus,one could suppose that the reduced anxiety-like behavior eli-cited by the 3-week EC exposure was related to the memoryimprovement. However, such a hypothesis does not seem tobe supported by the pharmacological studies reporting thatanxiolytic drugs produce memory impairment in the passiveavoidance test (Nabeshima et al. 1990; Lelong-Boulouard et al.2006). Moreover, no changes in anxiety-like behavior were ob-served when assessed in the light–dark box which is a testingcondition very close to the passive avoidance test.

Despite the involvement of adult hippocampal neurons inthe expression of contextual fear memory (Saxe et al. 2006;Drew et al. 2010; Denny et al. 2012), it seems that the increasednumber of cells expressing the neuronal phenotype observedfrom the 3-week EC exposure may not fully contribute to thetransient effects of EC on memory based on aversive stimulus.Indeed, the neurogenesis changes were induced from the3-week EC exposure and persisted after the 5-week duration.By contrast, the onset of memory improvement was only ob-served for the 3-week duration and not after longer duration ofexposure in the passive avoidance test. Thus, other neurobio-logical mechanisms may also be involved in the transient bene-ficial effects of EC on memory based on aversive stimulus.Interestingly, we found a transient increase in serotonin levelsspecifically after 3 weeks of EC in the frontal cortex, a brainregion previously reported to be recruited during the passiveavoidance test in EC mice (Leger, Bouet et al. 2012). Theseresults suggest a possible involvement of the serotonergicsystem in the transient beneficial effects of EC on this memory.Further investigations will nevertheless be required to assessthe mechanisms underlying the higher serotonergic releaseobserved in EC, for example, by regarding the evolution of theexpression of serotonergic receptors known to play a role inanxiety and memory (King et al. 2008; Meneses and Liy-Salmeron 2012). Because the prefrontal cortex is particularlyrich in 5-HT1a receptors (Puig and Gulledge 2011), these sero-tonergic receptors may represent a first interesting target. Con-sidering emerging evidence on the role play by other receptorsubtypes on memory processes, such as 5-HT6 (Quiedevilleet al. 2014) and 5-HT7 (Gasbarri and Pompili 2014) receptors,it could also be particularly relevant to assess the influence ofEC duration on the expression of these receptors, that as so farnever been investigated.

Finally, the transient nature of the behavioral and neuro-chemical effects observed after the 3-week EC exposure hasnever been reported before and may be surprising. However,these results complement some other transient effects of ECthat have recently emerged from the literature. For example, adecrease of dendritic arborization was reported as a transientphenomenon in the prefrontal cortex of rats housed for only afew days in EC (Comeau et al. 2010). More recently, a transient



increase in hippocampal cell activity and plasticity in both CA1area and DG was found after a 10-day EC exposure in rats(Eckert and Abraham 2013). Thus, our work provides newsupport to the view of duration-dependent effects of EC expos-ure on both behavioral and neurobiological changes.

When investigating object recognition memory perform-ance, we found a rapid memory enhancement following short-term exposure to EC (24 h). Such early beneficial effects of EChave never been reported before and could be related to thenature of the environmental enrichment used, that is, the ex-posure to various objects. Suggested by others (Birch et al.2013) and supported by our previous findings (Leger, Quiede-ville et al. 2012), this early improvement in object recognitionmemory could be related to a strengthening of the neuronalnetworks involved in novelty processing following renewedobjects exposure. Interestingly, the memory enhancementinduced by EC did not persist after 3 weeks of EC cessation.These results indicate that continuous EC exposure seems tobe required to enhance the object recognition memory. In op-position, the behavioral changes induced in rodents exposedto EC, such as reduced locomotor activity (Amaral et al. 2008),enhanced exploratory activity (Fernandez-Teruel et al. 2002),or anxiolytic-like effects (Friske and Gammie 2005), werefound to persist under SC. Among the neurobiological changesproposed to support the EC-induced object recognitionmemory, one may take into consideration neurogenesis modu-lation. Indeed, some studies support a role for increased prolif-eration and enhanced survival of DG cells in the beneficialeffect of EC on memory, be it in adult (Kempermann et al.1997; van Praag et al. 1999; Bruel-Jungerman et al. 2005) oraged (Kempermann et al. 1998; van Praag et al. 2005) rodentsand especially for hippocampal-dependant spatial memory(Dupret et al. 2008). Interestingly, a higher cell survival in asso-ciation with better object recognition memory performanceswas reported in rats exposed to EC (Bruel-Jungerman et al.2005), a view also supported by the recent works of Birch et al.(2013). After similar EC durations to those we used, Birch andcollaborators found an object recognition memory improve-ment from the 3-week EC exposure in rats and a delayed effecton neurogenesis (i.e., after 6 weeks of EC). We report hereearlier beneficial effects of EC (beginning from 24 h of expos-ure) on the recognition performances. Taken together with theworks of Birch et al. (2013), our study suggests that the dur-ation of EC differentially affects the memory trace prolongation(when SC rodent presents a memory loss) and the memory im-provement (when SC rodent discriminates the novel object).Moreover, the onset of neurogenesis changes observed fromthe 3-week exposure `did not match with the early memory im-provement observed from the 24-h exposure, suggesting theinvolvement of other neurobiological mechanisms in the bene-ficial effects of EC on memory. However, the cell-labeling thatwe used only allowed us to determine the effects of EC on theproliferation and the survival of neurons. Further investigationwould thus be necessary to assess the effects of EC on themorphology and the earlier and intermediate phases ofneuronal maturation. Indeed, it has been shown that the mor-phological alteration of young differentiating doublecortin(DCX)-positive neurons may play a role in the impairmentof spatial memory (Oomen et al. 2010). Because someplasticity-inducing experience such as voluntary exercise accel-erated the maturation of newborn cell (Zhao et al. 2006), thebeneficial effects of EC on memory may be supported by

earlier neurogenesis changes. The precise phase of maturationaffected by EC could thus be determined with the use of othercell markers as well as investigating the morphology of the cell(dendritic branching of DCX-positive cells for example).Finally, the analysis of different subregions of the hippocam-pus would also be of great importance as a delayed acceler-ation of neuronal maturation through a septo-temporal axis ofthe DG was recently observed in mice exposed to running ac-tivity (Piatti et al. 2011).

To conclude, we describe here for the first time, the timecourse of several behavioral and neurobiological changes oc-curring after environmental enrichment in mice. While somebehavioral components, such as memory based on aversivestimulus and anxiety-like behavior, seem to be sensitive to theduration of EC exposure, enhanced object recognition memoryis not or less dependent on EC duration. In addition, the onsetof the behavioral effects of EC does not seem to depend on in-creased survival of newly generated hippocampal neurons. Atthe molecular level, we hypothesize that the onset of behavior-al effects of EC on aversive stimulus-based memory could berelated to specific neurochemical changes, with a particularrole for serotonin in the frontal cortex. Of note, brain activityin this region had already been reported to undergo modula-tions during memory tests in EC mice (Leger, Bouet et al. 2012;Leger, Quiedeville et al. 2012). Taken together, these resultsshow that different behavioral components are not equally sen-sitive to the duration of EC exposure, suggesting that theycould be supported by different neurobiological mechanismson which EC does or does not act with varying time-courses.These peculiarities could partly explain the heterogeneity ofthe results found in the literature as to the effects of EC inrodents.

Funding

Marianne Leger is supported by funding from the French Min-istry of Research.

NotesWe gratefully thank Pauline Delasalle for her contribution to the be-havioral work, Nadege Villain-Naud, Celine Thomasse, Sophie Corvai-sier for their technical assistance, Nicolas Elie and Didier Goux forgiving us access to the confocal microscopy platform, Alexandra Bar-belivien for her contribution to the HPLC work and Lisa Adamson andChloe Piercy for reading the manuscript. Conflict of Interest: Nonedeclared.

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