Neurobiology of Learning and Memory 106 (2013) 268–273

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Neurobiology of Learning and Memory

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The dopamine D1 receptor agonist SKF 38393 improves temporalorder memory performance in maternally deprived rats

1074-7427/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nlm.2013.10.005

⇑ Corresponding author. Address: INRA, Micalis UMR 1319, équipe AMIPEM,Domaine de Vilvert, bâtiment 440, 78350 Jouy-en-Josas, France. Fax: +33 1 34 65 2462.

E-mail address: laurent.naudon@jouy.inra.fr (L. Naudon).

Stéphanie Lejeune a,b,c, Nathalie Dourmap d, Marie-Pascale Martres a,b,c

Bruno Giros a,b,c,e, Valérie Daugé a,b,c, Laurent Naudon a,b,c,⇑a INSERM, UMRs 952, Physiopathologie des Maladies du Système Nerveux Central, 9 quai Saint Bernard, F-75005 Paris, Franceb CNRS, UMR 7224, Physiopathologie des Maladies du Système Nerveux Central, 9 quai Saint Bernard, F-75005 Paris, Francec UPMC Univ. Paris 06, Physiopathologie des Maladies du Système Nerveux Central, F-75005 Paris, Franced Equipe Région Haute-Normandie – INSERM, ERI 28, Endothélium microcirculatoire cérébral et Lésions du système nerveux néonatal (NeoVasc), IRIB, Université de Rouen,Faculté de Médecine-Pharmacie, 22 Bd Gambetta, 76183 Rouen Cedex 1, Francee Douglas Hospital Research Center, Department of Psychiatry, McGill University, 6875 Boulevard Lasalle Verdun, Montreal, QC, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 August 2013Accepted 11 October 2013Available online 17 October 2013

Keywords:Maternal deprivationPrefrontal cortexHPLCMicrodialysisD1 receptor autoradiographyTemporal order memory task

Previously, we showed that maternal deprivation (MD) (3 h/day, postnatal-day 1–14) impaired the per-formance at adulthood in the object temporal order memory task (TMT) that principally implicates themedial prefrontal cortex (mPFC). Dopamine (DA) transmission in the PFC may play a critical role in theachievement of the TMT. Here, to investigate whether MD could results in dysfunction of the DA systemin the mPFC, we assessed in this region the tissue contents and extracellular levels of DA and its metab-olites, as the density of D1 receptor. Besides we examined whether an agonist of the DA receptor D1, theSKF38393, could have a beneficial effect on the performance of deprived (D) rats in the TMT. We observedthat MD induced a significant reduction of the extracellular level of DOPAC in the mPFC and in the densityof the D1 receptor in the anterior cingulate cortex, a sub-region of mPFC. On the other hand, we observedthat an acute systemic injection of a D1 receptor agonist, SKF38393, was effective to correct the memorydeficiency of D rats in the TMT, when administered before the retrieval phase. We showed that a stresssuffered by rats during the perinatal period led to dysfunction of the adult DA system, possibly triggeringgreater vulnerability to cognitive and mood disorders. Interestingly, an acute administration of a D1receptor agonist in adulthood was sufficient to improve the deficit in the temporal memory. A betterunderstanding of this phenomenon would permit the development of treatments adapted to patientswith a history of early traumatic experiences.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Early life stress and childhood maltreatment increases the riskof developing psychopathology accompanied by reduced cognitivefunction in later life (Teicher et al., 2003). For the infant, the inter-action with his mother is the most important environmental factor,since a variety of his physiological systems responds to specific ele-ments of this interaction. Maternal separation is a commonly usedmodel of early life neglect.

Our model of maternal deprivation (MD), a variant of maternalseparation, consists of a daily separation of newborn Long-Evanspups from their mother and from their littermates for 3 h perday from postnatal days 1 to 14. In adulthood, maternally deprived(D) rats are compared with animal facility rearing (AFR) rats, which

have experienced human intervention for animal care (Pryce &Feldon, 2003). We have shown that MD leads in male rats to differ-ent type of impairments or neurobiological alterations. An en-hanced anxiety and reactivity to stress was observed as well asan increased preference for sucrose, a hypersensitivity to therewarding effect of morphine, a morphine dependence, a hyposen-sitivity of the effect of a dopamine D3 receptor agonist, deficits incognitive flexibility and an exaggerated synaptic plasticity in themedial prefrontal cortex (mPFC) (Baudin et al., 2012; Mourlonet al., 2010; Vazquez, Farley, Giros, & Daugé, 2005; Vazquez, Giros,& Daugé, 2006; Vazquez, Penit-Soria, Durand, Besson, Giros et al.,2005, 2007).

During the last decade, several studies using different behav-ioural tasks have shown that maternal separation can lead tomemory impairments in adult rat. This was demonstrated withthe novel object recognition task (Aisa, Tordera, Lasheras, Del Rio,& Ramirez, 2008; Hulshof et al., 2011), the Morris water maze task(Aisa, Tordera, Lasheras, Del Rio, & Ramirez, 2007; Hui et al., 2011;Huot, Plotsky, Lenox, & McNamara, 2002; Mello, Benetti,

S. Lejeune et al. / Neurobiology of Learning and Memory 106 (2013) 268–273 269

Cammarota, & Izquierdo, 2009; Uysal et al., 2005; Zhu et al., 2010)and the radial arm maze task (Sandstrom & Hart, 2005). Inaddition, we have recently demonstrated that rats subjected toMD performed poorly in the object temporal order memory task(TMT) (Baudin et al., 2012). This task, adapted from the object rec-ognition task by Mitchell and Laiacona (1998), assesses the abilityto remember the temporal order of appearance of items and thusdiscriminate objects encountered at different times in the past.

In rat, several studies provided support that the mPFC is in-volved in memory for temporal order. It was initially shown thatmPFC lesions induced memory deficits on tests of temporal ordermemory for spatial locations (Chiba, Kesner, & Reynolds, 1994;Kesner & Holbrook, 1987). Moreover, performance in the TMTrelies critically on the mPFC and its networks as demonstratedby inactivation or lesion of the mPFC (Barker, Bird, Alexander, &Warburton, 2007; Hannesson, Howland, & Phillips, 2004; Mitchell& Laiacona, 1998). On the other hand, experimental data suggestthe involvement of dopamine (DA) transmission in the object tem-poral order memory. Indeed, administration of an agonist of the D1receptor before the memory testing trial of the TMT improves theabilities to retrieve previously acquired temporal information(Hotte, Naudon, & Jay, 2005). In the present study, we investigatedwhether MD resulted in changes in the contents of DA and itsmetabolites in the mPFC, as well as in their extracellular levelsquantified after microdialysis and in the density of D1 receptors.Finally, we investigated whether the D1 agonist SKF 38393 admin-istered before the retrieval phase in the TMT, could have a benefi-cial effect on the performance of D rats.

2. Material and methods

2.1. Maternal deprivation procedure

Experimental procedure and animal care were performed inaccordance with local committee guidelines and the EuropeanCommunities Council Directive of November 24, 1986 (86/609/EEC). Two cohorts of 20 and 26 pregnant Long-Evans rats (Janvier,Le Genest St. Isle, France) were received on day 14 of gestation. Thedams gave birth 1 week after inclusion. MD was performed as pre-viously described (Baudin et al., 2012; Mourlon, Naudon, Giros,Crumeyrolle-Arias, & Daugé, 2011; Mourlon et al., 2010). On thepostnatal day 1, litters were cross-fostered culled to eight to twelvepups, half females-half males randomly chosen. Neonates of thematernal deprivation group were individually placed in tempera-ture (30–34 �C) and humidity-controlled cages. D pups were iso-lated 3 h daily (2 pm–5 pm) from days 1 to 14. AFR pupsremained with their mothers during this period and received nospecific handling other than changing the bedding in their cagesonce a week. On day 22, pups were weaned and housed in groupsof two or three. Only male rats were included in the study and eachindividual has been used only for one of the biochemical quantifi-cations or for the TMT experiment. All the experimentations wereperformed on rats from the first cohort, with the exception of themeasurement of extracellular DA that was performed on rats fromthe second cohort.

2.2. Homogenate preparation for HPLC determination

Rats (109–110 days old; AFR, n = 12; D, n = 12) were killed bydecapitation. The mPFC was collected and homogenised with aPotter–Elvehjem type homogenizer in 1 mL ice cold perchloric acid0.1 N containing 0.1% cysteine. The homogenates were centrifuged(10,000g, 10 min at 4 �C). The supernatants were filtered by pres-sure through 0.45-lm filters (Millipore, Ireland) and then storedat �80 �C until use. The pellets were resuspended in NaOH(0.1 M) and used for protein determination.

2.3. Surgery and brain dialysis

Surgery and microdialysis experiments were performed as pre-viously described in Vazquez, Penit-Soria et al. (2005) (slightlymodified).

Surgery: Rats (90–120 days old; AFR, n = 7; D, n = 8) were anes-thetized by i.p. injection of chloral hydrate (400 mg/kg) and unilat-erally implanted with stainless steel cannula guide (CMA 11, CMA/Microdialysis, Phymep, Paris) in the mPFC. The stereotaxic coordi-nates were: anteriority +3 mm, laterality +0.6 mm from bregma,depth �2.8 mm from the skull surface (Paxinos & Watson, 2005).Then the guide cannula was fixed to the skull with dental cementand inox screws. The microdialysis experiment was conducted8–10 days after guide cannula implantation.

Brain dialysis: The evening before the experiment, the rats weretransferred to the experimental cage (40 � 40 � 40 cm) withad libitum access to food and water to habituate the rats to thisnew environment and to the connection system of the dialysis.The next morning, the microdialysis probe (diameter 0.25 mm;CMA 11, CMA/Microdialysis, Phymep, Paris) was inserted throughguide cannula into the mPFC. The freely moving rat was continu-ously perfused with a dialysis buffer: NaCl 140 mM, KCl 4 mM,CaCl2 1.2 mM, MgCl2 1 mM, Na2HPO4 1.9 mM, NaH2PO4 0.1 mM,pH = 7.4, at a rate of 2 lL/min by means of a seringue pump(Precinorm; Infors, Bottmingen, Switzerland) via a channel liquidswivel. After 2 h of perfusion, three samples were collected for20 min each in tube containing 20 lL of perchloric acid 1 M andcysteine 0.1% and maintained in dry ice. The samples were storedat �80 �C until the quantification of DA and its metabolites.

Histological control: After microdialysis experiment, rats werekilled by decapitation. The brains were removed, frozen then cuton a microtome (Leitz, Wetzlat, Germany), and the slices (30 lm)were stained with cresyl violet. The position of the probe was esti-mated according to the atlas of Paxinos and Watson (2005). Probesthat traversed >70% of the mPFC were considered to be placedcorrectly.

2.4. HPLC determination of DA and its metabolites

Levels of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homo-vanillic acid (HVA) and 3-methoxytyramine (3MT) in homogenatesprepared from mPFC or dialysates obtained from mPFC were deter-mined using a reverse-phase ion-pair HPLC system with electro-chemical detection. The HPLC system consisted of a pump(LC10AD VP, Shimadzu, Kyoto, Japan) connected to a C18 reversedphase column Supelcosil (3.0 � 150 mm, 3 lm, Sigma–Aldrich,Bellefonte, PA, USA) coupled to an electrochemical detector (Dec-ade II, Antec, Leyden, Netherlands) with a glassy carbon electrodeset at 0.6 V vs. Ag/AgCl reference electrode. The mobile phase con-sisted in KH2PO4 73.4 mM, methanol 125 mL/L, octan-1-sulphonicacid sodium salt 0.46 mM and Na2EDTA 0.15 mM at pH 3.75. Thissolutions was filtered through 0.45-lm cellulose acetate filters(Millipore) and delivered at a 0.4 mL/min flow rate. Twenty-micro-litre samples were injected into the HPLC system by means of anautomatic device (AS300, Spectra Physics, San Jose, CA, USA). Chro-matograms were recorded and integrated by PC integration Azursoftware (Datalys, Le Touvet, France).

2.5. Autoradiographic labelling of the dopamine D1 receptor

Rats (84–89 days old; AFR, n = 10; D, n = 10) were killed bydecapitation. Their brains were quickly removed and frozen inisopentane at �30 �C. Twenty micrometer-thick frontal brainsections (anteroposteriority 5.16 mm to 2.52 mm from bregmaaccording to Paxinos and Watson (2005)) were cut at �20 �C,thaw-mounted on Superfrost Plus� slides and stored at �80 �C

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until use. The brain slices were pre-incubated at room temperaturefor 2 � 10 min in 50 mM Tris–HCl buffer (pH 7.4) containing120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. The slideswere then incubated at room temperature for 90 min in the samebuffer containing 3 nM [N-methyl-3H]SCH23390 (2.6–3.2 TBq/mmol, Perkin Elmer). Non-specific binding was assessed in thepresence of 10 lM of SKF 38393 (Tocris). Sections were thenwashed (4 � 5 min) in ice-cold buffer, rapidly rinsed in ice-coldwater, dried and exposed to BAS-TR Fuji Imaging screen for11 days. Standard radioactive microscales were exposed onto eachImaging screen to ensure that labelling densities were in the linearrange. The screens were scanned with a Fuji Bioimaging AnalyserBAS-5000. Densitometry measurements were performed withMCID™ analysis software. Specific labelling of 3–4 sections at thelevel of the mPFC was averaged per rat. The mPFC was sub-dividedin ventral mPFC, including infralimbic (IL) and prelimbic (PrL)cortex, and in dorsal mPFC, including anterior cingulate (ACg)and precentral (PrC) cortex (Dalley, Cardinal, & Robbins, 2004)(Fig. 2A). Furthermore, on the last two most caudal sections, wemeasured D1 receptor labelling in the olfactory tubercle, caudateputamen and nucleus accumbens (core (AccC) and shell (AccS)),three regions containing a high density of the receptor.

2.6. Temporal order memory task and SKF 38393 administration

Rats were 77–84 days old the day of the test. During the fourdays preceding the task, the rats were handled daily and habitu-ated to the test room and to the open-box (100 � 100 � 60 cm;lighting, 50 lux) as previously described (Naudon, Hotte, & Jay,2007). At the fifth day, the task consisted of two consecutivesample phases and one retrieval phase of 4 min each. In the firstsample phase, rats were allowed to explore two identical objects(12–15 cm height) fixed (Patafix�) on the floor of the box in posi-tion 3 cm from the back and 12 cm from the side walls. One hourlater, in the second sample phase, rats were allowed to exploretwo copies of a new object. After an additional delay of 3 h, one‘‘old object’’ from the 1st sample phase and one ‘‘recent object’’from the 2nd sample phase were presented during the retrievalphase. The time exploring each object (i.e. directing the nose tothe object at a distance 62 cm) was scored on videotape. Thediscrimination ratio was calculated as ((time spent exploring theold object) – (time spent exploring the recent object))/(time spentexploring both objects).

To determine the effect of the D1 receptor agonist, each rat(AFR, n = 18; D, n = 20) received an i.p. injection (1 mL/kg) of eithersaline (NaCl 0.9%) or SKF 38393 hydrobromide (5.0 mg/kg, Tocris)10 min prior to the retrieval phase. This dose was chosen basedon the study done by de Lima et al. (2011).

2.7. Statistical analysis

After the SKF 38393 injection, significance between groups wastested by a two-way ANOVA with ‘‘MD’’ and ‘‘Treatment’’ as factors,followed by a post hoc Fisher’s least significance difference test. Allthe other data were compared using an analysis of variance (ANO-VA) and Student’s t-test. Statistical significance was set at p < 0.05.

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Fig. 1. Tissue contents and extracellular levels of DA and its metabolites in themPFC of AFR and D rats. (A) The contents of DA and its metabolites in the mPFCwere similar in AFR and D rats. Results are expressed as mean ± SEM, n = 11–12. (B)Only the extracellular level of DOPAC was significantly decreased in D vs. AFR rats.Mean ± SEM, n = 6–8. � P < 0.05, vs. AFR rats.

3. Results

3.1. Contents of DA and its metabolites in the mPFC

In the mPFC, the contents of DA and its metabolites, DOPAC,HVA and 3MT, were similar in AFR and D rats (Fig. 1A). As a conse-quence, the DA turnover expressed as the ratio metabolites vs. DAwas also not significantly different between both groups (DOPAC/

DA: AFR, 0.10 ± 0.01, D, 0.08 ± 0.01; HVA/DA: AFR, 0.33 ± 0.03, D,0.28 ± 0.02; 3MT/DA: AFR, 0.07 ± 0.01, D, 0.06 ± 0.01). In addition,we observed that the 5HT content and turnover were unaffectedby MD in the mPFC (data not shown).

3.2. Extracellular levels of DA and its metabolites in the mPFC

For each rats the data obtained from the three collected sampleswere averaged. The extracellular levels of DA and HVA, assessed inthe mPFC, were not different in both AFR and D rats; however, wemeasured a significant decrease of the extracellular level of DOPACin D rats in comparison with AFR rats (P < 0.05) (Fig. 1B).

3.3. Labelling of the D1 receptor

In the D rats in comparison with AFR rats, the labelling of the D1receptor was significantly decreased in one sub-region of themPFC, the ACg (P < 0.05), but not in the IL, PrL, and PrC. No changewas noticed in the olfactory tubercle and the caude putamen (notshown), but a significant decrease was observed in the AccC(P < 0.05) and AccS (P < 0.01) of the nucleus accumbens (Fig. 2D)

3.4. Effect of SKF 38393 (5 mg/kg) on the object temporal ordermemory performance

During the sample phases, AFR, AFR-SKF, D and D-SKF rats spentthe same total time exploring both objects (data not shown). Dur-ing the retrieval phase, the total amount of time to explore both‘‘old’’ and ‘‘recent’’ objects was similar in all groups (Fig. 3A). How-ever, statistical analysis of the discrimination ratio showed signif-icant effects MD factor (F(1,34) = 9.0 ; P < 0.01) and Treatmentfactor (F(1,34) = 4.7 ; P < 0.05)). The discrimination ratio was sig-nificantly lower in D rats in comparison with D-SKF (P < 0.01),AFR (P < 0.01) and AFR-SKF rats (P < 0.001), while the discrimina-tion ratio was similar in the three latter groups (Fig. 3B). Indeed,the D rats explored in a comparable manner the two objects

A B C

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Fig. 2. Labelling of the D1 receptor in of the mPFC of AFR and D rats. (A) Schematic delineation of the infralimbic (IL), prelimbic (PrL), anterior cingulate (ACg) and precentral(PrC) subregions of the mPFC. (B and C) Representative autoradiograms of [3H]SCH23390 labelling at the level of the CPFm in AFR rats (3.72 mm from bregma according toPaxinos and Watson (2005), respectively without or in the presence of SKF 38393 (nonspecific labelling). (D) The labelling of the D1 receptor was significantly decreased inthe ACg but not in the IL, PrL, and PrC. On the other hand, a significant decrease was observed in the AccC and AccS. Results are expressed as mean ± SEM, n = 10. P < 0.05,��P < 0.01, vs. AFR rats.

S. Lejeune et al. / Neurobiology of Learning and Memory 106 (2013) 268–273 271

(respectively 7.6 ± 1.1 s and 8.3 ± 1.3 s), while the control AFRgroup spent less time exploring the recent object that the old ob-ject (respectively, 3.4 ± 0.6 and 8.5 ± 1.9, P < 0.05). Interestingly,while the performance of the AFR rats were not modified by SKF38393 (recent object: 3.5 ± 0.7 and old object: 8.2 ± 1.4, P < 0.01),the deficit of D rats was entirely reversed by the D1 receptor ago-nist (recent object: 4.7 ± 0.9 and old object: 9.6 ± 2.1, P < 0.05).Indeed, the discrimination ratio of SKF-treated D rats became nolonger different from the ratio of the two AFR groups.

4. Discussion

Consistent with our previous report (Baudin et al. 2012), thepresent study confirmed the inability of adult male D rats, unlikeAFR rats, to succeed in the TMT, a task dependent of the mPFC.We showed that, while the tissue content in the mPFC of DA andof its metabolites were not modified by MD, the extracellular levelof DOPAC was significantly decreased in the mPFC of D rats andthat the density of DA D1 receptors was significantly decreasedin the ACg. Interestingly, we demonstrated that an acute adminis-tration of the dopamine D1 receptor agonist, SKF 38393, was effec-tive to completely correct the cognitive deficit of D rats.

MD did not induce modification of the tissue contents in DA andits metabolites in the mPFC. This result is consistent with previousstudies using similar models of maternal separation (Arborelius &Eklund, 2007; Oreland et al., 2011). However, when the extracellu-lar levels of DA and its metabolites were measured in the mPFC, weobserved lower level of extracellular DOPAC in D rats compared toAFR rats. The effect on the extracellular DOPAC reinforces the pos-sibility of a dysfunction of the DA system in the mPFC of D rats. Thelower level of extracellular DOPAC may reflect a decreased synthe-sis of DA since DOPAC is an index of its intraneuronal metabolism

(Soares-da-Silva & Garrett, 1990; Zetterström, Sharp, Collin, &Ungerstedt, 1988). On the other hand, this lower level in extracel-lular DOPAC and the absence of significant changes in level ofextracellular DA in the mPFC, may indicate that MD induces alter-ations in DA enzymatic catabolism, and more particularly abnor-malities in monoamine-oxidases functioning. It is worthy to note,that promnesic effects of the monoamine-oxidase inhibitors havebeen observed in object recognition tasks (de Lima et al., 2005;Wong et al., 2010).

Since DA neurotransmission plays a central role in cognitiveperformance at the level of the mPFC, principally via its D1 recep-tors (for review, see Hotte et al., 2005) and since DA activity in thiscerebral region was significantly impaired in D rats, we investi-gated whether the density of the D1 receptor was affected byMD in the mPFC. We demonstrated a significant decrease in D1receptor labelling only in the ACg of D rats in comparison withAFR rats. Since the autoradiographic study was performed with aunique concentration of 3H-SCH23390, it do not allow to deter-mine whether the changes in D1 receptors labelling measuredwere due to changes in the number of receptors or to changes inthe affinity of D1 receptors for 3H-SCH23390. The associationbetween the mPFC and the TMT is underlined by lesions or inacti-vations performed more particularly in the PrL (Barker et al., 2007;Hannesson et al., 2004; Mitchell & Laiacona, 1998). However, thedata in some studies suggested that the ACg could be also involvedin temporal order recognition memory (Barker et al., 2007). Thus,the lower density of D1 receptor in the ACg could be partiallyresponsible for the poor performance of D rats in the TMT. Onthe other hand, in the sub-regions of mPFC where MD does notcause a decrease in the density of D1 receptor, it is not excludedthat the activity and/or signalling of D1 receptor was impaired inD rats. For instance, a discrepancy between density and D1 recep-tor desensitization has already been described in other brain

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Fig. 3. Effect of SKF 38393 (5 mg/kg) on performance of AFR and D rats in the objecttemporal order memory task. (A) During the retrieval phase, the total time ofexploration was not different between AFR, AFR-SKF, D and D-SKF rats. (B) Incontrast with AFR, AFR-SKF and D-SKF rats, D rats failed to discriminate betweenthe two objects as indicated by the discrimination ratio. Results are expressed asmean ± SEM. AFR, n = 8; AFR-SKF, n = 10; D, n = 10; D-SKF, n = 10. ��P < 0.01,���P < 0.001, vs. D rats.

272 S. Lejeune et al. / Neurobiology of Learning and Memory 106 (2013) 268–273

regions (Choy, de Visser, & van den Buuse, 2009; Naef et al., 2008).The decreased density of D1 receptor in the ACg constitutes a fur-ther indication of a DA hypoactivity induced in the mPFC by MD.Overall, we observed that MD resulted in an alteration of the func-tioning of the DA system in the mPFC, but that could not be limitedto this region as indicated by the decrease in the density of D1receptors assessed in the core and shell of the nucleus accumbens.

The potential role of the decreased number of D1 receptors inthe impairment of D rats in the TMT may be reinforced by theimprovement of this deficit by D1 receptor agonist. Indeed, wedemonstrated that the acute administration of SKF 38393 (5 mg/kg), 10 min prior the retrieval phase, allowed D rats to discriminatebetween recent and old objects, while SKF 38393 at this dose hadno effect on the performance of AFR rats. Previously, it was shownthat in the TMT, when the delay between the last sample-phaseand the test-phase was increased beyond certain duration the ratscould not distinguish between old and recent objects. This defi-ciency could be corrected by acute systemic injection before thetest-phase of a D1 agonist, suggesting that the activation of D1receptors may optimise the retrieval (Hotte et al., 2005). In ourrecent study, we had chosen a delay of 3 h for which the AFRLong-Evans rats succeeded in the task, but not the D rats (Baudinet al. 2012). Here we show that D1 receptor activation, by SKF38393, was able to improve object temporal order memory in Drats. This result demonstrates the involvement of the D1 receptorin the recovery of the memory performance in the TMT, when ithas been altered following MD. The effect observed on D rats after

SKF 38393 administration could occur through a direct activationof the D1 in the mPFC. Indeed, previous studies in rats, using infu-sions of D1 receptor agonists into the mPFC, have shown that theD1 receptor activity within this region can influence memoryprocesses. Infusions were held either in the PrL (Floresco & Phillips,2001; Runyan & Dash, 2004), or in both PrL and ACg (Rios Valen-tim, Gontijo, Peres, Rodrigues, & Nakamura-Palacios, 2009). Butthe slight change in the density of D1 receptor induced by MD inthe mPFC could also indicate that the amelioration of the retrievalability following D1 receptors stimulation did not originate fromthe mPFC, or at least not only from it. This would not be surprising,since it has been shown that administration of a D1 agonist notonly improves the recall of learned information about objects inthe TMT, but also in the object and place recognition tasks (Hotteet al., 2005). In these last two tasks, the role of the mPFC is muchless obvious. For instance the inactivation or lesion of this cerebralregion does no not prevent the completion of a proper performancein the object recognition task (Barker et al., 2007; Hannesson et al.,2004; Mitchell & Laiacona, 1998). Indeed, the systemic route ofadministration cannot preclude that the action of SKF 38393 in re-gions other than mPFC is responsible for the effects observed. Infact, the mPFC has been shown to form with several brain regions,i.e. the perirhinal cortex, the hippocampus and the mediodorsalthalamic nucleus, functional relationships necessary to process re-cency recognition memory information. Furthermore, these re-gions operate within an integrated neuronal network, whichinvolve different structures such as the orbital PFC and the parietalcortex (Barker & Warburton, 2011; Barker et al., 2007; Cross,Brown, Aggleton, & Warburton, 2013; Hannesson et al., 2004). Toour knowledge the nucleus accumbens, where we have observedlower densities of D1 receptor, has never been involved in tempo-ral order memory. Moreover, several studies demonstrated that theperformance of rat on timing task does not rely to the nucleusaccumbens (Galtress & Kirkpatrick, 2010; Kurti & Matell, 2011;Liu, Strecker, & Brener, 1998; Meck, 2006). However, since a recentstudy indicated that the nucleus accumbens was activated duringretrospective timing tasks (Valencia-Torres et al., 2011), a partici-pation of this region in the TMT performance could not be com-pletely eliminated. Besides D1 receptor, DA D2 and D3 receptorsin the PFC have been recently shown to modulate object recogni-tion in rats (Watson et al., 2012). In the future, it could be interest-ing to study the consequences of MD on the density of thesereceptors in the mPFC and on the effects of D2 and D3 agonistsin the TMT. In a previous systemic pharmacological study, wenoticed that MD led to an alteration of DA neurotransmissionrevealed by a hyposensitivity of the effect of a DA D3 receptoragonist on the rewarding effect of morphine (Vazquez et al., 2007).

In conclusion, the present study showed that D1 receptor stim-ulation improved temporal memory performance in rats subjectedto MD. A pharmacological treatment was able to correct behav-ioural dysfunction induced at adulthood by an early perinatalstress. Whatever the underlying mechanism, we demonstrate thatMD modified the DA system. Additional studies will be necessaryto elucidate how early life adverse events might affect the develop-ment of the DA system and thus potentially increase vulnerabilityfor cognitive and/or mood disorders in later life. This knowledgewill be necessary to design new therapeutic strategies to relievethe patients who have suffered of early life adversity.

References

Aisa, B., Tordera, R., Lasheras, B., Del Rio, J., & Ramirez, M. J. (2007). Cognitiveimpairment associated to HPA axis hyperactivity after maternal separation inrats. Psychoneuroendocrinology, 32, 256–266.

Aisa, B., Tordera, R., Lasheras, B., Del Rio, J., & Ramirez, M. J. (2008). Effects ofmaternal separation on hypothalamic-pituitary-adrenal responses, cognitionand vulnerability to stress in adult female rats. Neuroscience, 154, 1218–1226.

S. Lejeune et al. / Neurobiology of Learning and Memory 106 (2013) 268–273 273

Arborelius, L., & Eklund, M. B. (2007). Both long and brief maternal separationproduces persistent changes in tissue levels of brain monoamines in middle-aged female rats. Neuroscience, 145, 738–750.

Barker, G. R., Bird, F., Alexander, V., & Warburton, E. C. (2007). Recognition memoryfor objects, place, and temporal order: A disconnection analysis of the role ofthe medial prefrontal cortex and perirhinal cortex. The Journal of Neuroscience,27, 2948–2957.

Barker, G. R., & Warburton, E. C. (2011). When is the hippocampus involved inrecognition memory? The Journal of Neuroscience, 31, 10721–10731.

Baudin, A., Blot, K., Verney, C., Estevez, L., Santamaria, J., Gressens, P., Giros, B., Otani,S., Daugé, V., & Naudon, L. (2012). Maternal deprivation induces deficits intemporal memory and cognitive flexibility and exaggerates synaptic plasticityin the rat medial prefrontal cortex. Neurobiology of Learning and Memory, 98,207–214.

Chiba, A. A., Kesner, R. P., & Reynolds, A. M. (1994). Memory for spatial location as afunction of temporal lag in rats: Role of hippocampus and medial prefrontalcortex. Behavioral and Neural Biology, 61, 123–131.

Choy, K. H., de Visser, Y. P., & van den Buuse, M. (2009). The effect of ‘two hit’neonatal and young–adult stress on dopaminergic modulation of prepulseinhibition and dopamine receptor density. British Journal of Pharmacology, 156,388–396.

Cross, L., Brown, M. W., Aggleton, J. P., & Warburton, E. C. (2013). The medial dorsalthalamic nucleus and the medial prefrontal cortex of the rat function togetherto support associative recognition and recency but not item recognition.Learning & Memory, 20, 41–50.

Dalley, J. W., Cardinal, R. N., & Robbins, T. W. (2004). Prefrontal executive andcognitive functions in rodents: Neural and neurochemical substrates.Neuroscience & Biobehavioral Reviews, 28, 771–784.

de Lima, M. N., Laranja, D. C., Caldana, F., Bromberg, E., Roesler, R., & Schröder, N.(2005). Reversal of age-related deficits in object recognition memory in ratswith l-deprenyl. Experimental Gerontology, 40, 506–511.

de Lima, M. N., Presti-Torres, J., Dornellesn, A., Scalcon, F. S., Roeslern, R., Garcina, V.A., & Schrödern, N. (2011). Modulatory influence of dopamine receptors onconsolidation of object recognition memory. Neurobiology of Learning andMemory, 95, 305–310.

Floresco, S. B., & Phillips, A. G. (2001). Delay-dependent modulation of memoryretrieval by infusion of a dopamine D1 agonist into the rat medial prefrontalcortex. Behavioral Neuroscience, 115, 934–939.

Galtress, T., & Kirkpatrick, K. (2010). The role of the nucleus accumbens core inimpulsive choice, timing, and reward processing. Behavioral Neuroscience, 124,26–43.

Hannesson, D. K., Howland, J. G., & Phillips, A. G. (2004). Interaction betweenperirhinal and medial prefrontal cortex is required for temporal order but notrecognition memory for objects in rats. The Journal of Neuroscience, 24,4596–4604.

Hotte, M., Naudon, L., & Jay, T. M. (2005). Modulation of recognition and temporalorder memory retrieval by dopamine D1 receptor in rats. Neurobiology ofLearning and Memory, 84, 85–92.

Hui, J. J., Zhang, Z. J., Liu, S. S., Xi, G. J., Zhang, X. R., Teng, G. J., Chan, K. C., Wu, E. X.,Nie, B. B., Shan, B. C., Li, L. J., & Reynolds, G. P. (2011). Hippocampalneurochemistry is involved in the behavioural effects of neonatal maternalseparation and their reversal by post-weaning environmental enrichment: Amagnetic resonance study. Behavioural Brain Research, 217, 122–127.

Hulshof, H. J., Novati, A., Sgoifo, A., Luiten, P. G., den Boer, J. A., & Meerlo, P. (2011).Maternal separation decreases adult hippocampal cell proliferation and impairscognitive performance but has little effect on stress sensitivity and anxiety inadult Wistar rats. Behavioural Brain Research, 216, 552–560.

Huot, R. L., Plotsky, P. M., Lenox, R. H., & McNamara, R. K. (2002). Neonatal maternalseparation reduces hippocampal mossy fiber density in adult Long Evans rats.Brain Research, 950, 52–63.

Kesner, R. P., & Holbrook, T. (1987). Dissociation of item and order spatial memoryin rats following medial prefrontal cortex lesions. Neuropsychologia, 25,653–664.

Kurti, A. N., & Matell, M. S. (2011). Nucleus accumbens dopamine modulatesresponse rate but not response timing in an interval timing task. BehavioralNeuroscience, 125, 215–225.

Liu, X., Strecker, R. E., & Brener, J. (1998). Dopamine depletion in nucleus accumbensinfluences locomotion but not force and timing of operant responding.Pharmacology Biochemistry and Behavior, 59, 737–745.

Meck, W. H. (2006). Neuroanatomical localization of an internal clock: A functionallink between mesolimbic, nigrostriatal, and mesocortical dopaminergicsystems. Brain Research, 1109, 93–107.

Mello, P. B., Benetti, F., Cammarota, M., & Izquierdo, I. (2009). Physical exercise canreverse the deficit in fear memory induced by maternal deprivation.Neurobiology of Learning and Memory, 92, 364–369.

Mitchell, J. B., & Laiacona, J. (1998). The medial frontal cortex and temporalmemory: Tests using spontaneous exploratory behaviour in the rat. BehaviouralBrain Research, 97, 107–113.

Mourlon, V., Baudin, A., Blanc, O., Lauber, A., Giros, B., Naudon, L., & Daugé, V. (2010).Maternal deprivation induces depressive-like behaviours only in female rats.Behavioural Brain Research, 213, 278–287.

Mourlon, V., Naudon, L., Giros, B., Crumeyrolle-Arias, M., & Daugé, V. (2011). Earlystress leads to effects on estrous cycle and differential responses to stress.Physiology & Behavior, 102, 304–310.

Naef, L., Srivastava, L., Gratton, A., Hendrickson, H., Owens, S. M., & Walker, C. D.(2008). Maternal high fat diet during the perinatal period altersmesocorticolimbic dopamine in the adult rat offspring: Reduction in thebehavioral responses to repeated amphetamine administration.Psychopharmacology (Berl), 197, 83–94.

Naudon, L., Hotte, M., & Jay, T. M. (2007). Effects of acute and chronic antidepressanttreatments on memory performance: A comparison between paroxetine andimipramine. Psychopharmacology (Berl), 191, 353–364.

Oreland, S., Raudkivi, K., Oreland, L., Harro, J., Arborelius, L., & Nylander, I. (2011).Ethanol-induced effects on the dopamine and serotonin systems in adult Wistarrats are dependent on early-life experiences. Brain Research, 1405, 57–68.

Paxinos, G., & Watson, C. (2005). The rat brain in stereotaxic coordinates (5th ed.). SanDiego: Academic Press.

Pryce, C. R., & Feldon, J. (2003). Long-term neurobehavioural impact of the postnatalenvironment in rats: Manipulations, effects and mediating mechanisms.Neuroscience and Biobehavioral Reviews, 27, 57–71.

Rios Valentim, S. J., Jr., Gontijo, A. V., Peres, M. D., Rodrigues, L. C., & Nakamura-Palacios, E. M. (2009). D1 dopamine and NMDA receptors interactions in themedial prefrontal cortex: Modulation of spatial working memory in rats.Behavioural Brain Research, 204, 124–128.

Runyan, J. D., & Dash, P. K. (2004). Intra-medial prefrontal administration of SCH-23390 attenuates ERK phosphorylation and long-term memory for trace fearconditioning in rats. Neurobiology of Learning and Memory, 82, 65–70.

Sandstrom, N. J., & Hart, S. R. (2005). Isolation stress during the third postnatal weekalters radial arm maze performance and corticosterone levels in adulthood.Behavioural Brain Research, 156, 289–296.

Soares-da-Silva, P., & Garrett, M. C. (1990). A kinetic study of the rate of formation ofdopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid(HVA) in the brain of the rat: Implications for the origin of DOPAC.Neuropharmacology, 2, 869–874.

Teicher, M. H., Andersen, S. L., Polcari, A., Anderson, C. M., Navalta, C. P., & Kim, D. M.(2003). The neurobiological consequences of early stress and childhoodmaltreatment. Neuroscience and Biobehavioral Reviews, 27, 33–44.

Uysal, N., Ozdemir, D., Dayi, A., Yalaz, G., Baltaci, A. K., & Bediz, C. S. (2005). Effects ofmaternal deprivation on melatonin production and cognition in adolescentmale and female rats. Neuro Endocrinology Letters, 26, 555–560.

Valencia-Torres, L., Olarte-Sánchez, C. M., Body, S., Fone, K. C., Bradshaw, C. M., &Szabadi, E. (2011). Fos expression in the prefrontal cortex and nucleusaccumbens following exposure to retrospective timing tasks. BehavioralNeuroscience, 125, 202–214.

Vazquez, V., Farley, S., Giros, B., & Daugé, V. (2005a). Maternal deprivation increasesbehavioural reactivity to stressful situations in adulthood: Suppression by theCCK2 antagonist L365,260. Psychopharmacology (Berl), 181, 706–713.

Vazquez, V., Giros, B., & Daugé, V. (2006). Maternal deprivation specificallyenhances vulnerability to opiate dependence. Behavioural Pharmacology, 17,715–724.

Vazquez, V., Penit-Soria, J., Durand, C., Besson, M. J., Giros, B., & Daugé, V. (2005b).Maternal deprivation increases vulnerability to morphine dependence anddisturbs the enkephalinergic system in adulthood. Journal of Neuroscience, 25,4453–4462.

Vazquez, V., Weiss, S., Giros, B., Martres, M. P., & Daugé, V. (2007). Maternaldeprivation and handling modify the effect of the dopamine D3 receptoragonist, BP 897 on morphine-conditioned place preference in rats.Psychopharmacology (Berl), 193(4), 475–486.

Watson, D. J., Loiseau, F., Ingallinesi, M., Millan, M. J., Marsden, C. A., & Fone, K. C.(2012). Selective blockade of dopamine D3 receptors enhances while D2receptor antagonism impairs social novelty discrimination and novel objectrecognition in rats: A key role for the prefrontal cortex.Neuropsychopharmacology, 37, 770–786.

Wong, F. K., Lee, S. H., Atcha, Z., Ong, A. B., Pemberton, D. J., & Chen, W. S. (2010).Rasagiline improves learning and memory in young healthy rats. BehaviouralPharmacology, 21, 278–282.

Zetterström, T., Sharp, T., Collin, A. K., & Ungerstedt, U. (1988). In vivo measurementof extracellular dopamine and DOPAC in rat striatum after various doparnine-releasing drugs; implications for the origin of extracellular DOPAC. EuropeanJournal of Pharmacology, 148, 327–334.

Zhu, X., Li, T., Peng, S., Ma, X., Chen, X., & Zhang, X. (2010). Maternal deprivation-caused behavioral abnormalities in adult rats relate to a non-methylation-regulated D2 receptor levels in the nucleus accumbens. Behavioural BrainResearch, 209, 281–288.