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Research Report

Corticosterone treatment before puberty sensitizes the effect oforal methylphenidate on locomotor activity in preadolescenceand produces differential effects in adulthood

Jorge Juárez⁎, Cristina Vázquez-CortésLaboratorio de Farmacología y Conducta, Instituto de Neurociencias, Universidad de Guadalajara, Guadalajara, Jalisco, México

A R T I C L E I N F O

⁎ Corresponding author. Laboratorio de FarmQuevedo #180, Col. Arcos Vallarta, CP 44130

E-mail address: jjuarez@cencar.udg.mx (JAbbreviations: MP, methylphenidate; Co, c

adrenal; MP/MP, rats exposed tomethylphenidin preadolescence, and to methylphenidate itreated with corticosterone plusmethylpheniwith saline solution (corticosterone vehicle) plMP, rats treatedwith corticosteronepluswater

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.05.093

A B S T R A C T

Article history:Accepted 30 May 2010Available online 8 June 2010

The first objectiveof this studywas toexaminetheeffects of early exposure tomethylphenidateand then those of re-exposure in adulthood. The second was to analyze the effect ofcorticosterone treatment during pre-puberty on oral methylphenidate consumption and,consequently, the effect of this psychostimulant on locomotor activity in preadolescent andadult rats. Experiment 1: from 31 to 39 days of postnatal age (PA), Wistar rats were exposed toeither oral methylphenidate or water. Experiment 2: from 24 to 39 days PA, the rats receivedeither corticosterone (2.0 mg/kg/day/subject) or a saline solution. From 31 to 39 days PA, ratswere exposed to either methylphenidate or water. During adulthood, all rats in experiments 1and 2 were exposed to either methylphenidate or water, and subsequently exposed to a free-choice condition of the same two substances. Results: Experiment 1. Methylphenidateincreased locomotor activity (LA) regardless of age. In adulthood, higher methylphenidateconsumptionwasobserved in thegroup thathadnotbeenexposed to this substance, comparedto the early methylphenidate-exposed group. Experiment 2. Corticosterone did not affectmethylphenidate consumption during preadolescence or adulthood; however, the LA inducedby methylphenidate was higher in the preadolescents that had been treated withcorticosterone+methylphenidate than in the animals treated only with methylphenidate. Inadulthood, methylphenidate produced higher LA in the animals previously treated withcorticosterone+methylphenidate than in those that had received previous treatmentexclusively with corticosterone. These results suggest that preadolescent corticosteroneexposure produced a sensitizing effect of methylphenidate on LA in preadolescence. Thedifferential effect on LA in adulthood depended on whether the corticosterone wasadministered with or without methylphenidate in preadolescence, which would suggest anenduring effect of the early synergic action between these two substances.

© 2010 Elsevier B.V. All rights reserved.

Keywords:MethylphenidateCorticosteroneSensitizationlocomotor activityPsychostimulants

acología y Conducta, Instituto de Neurociencias, Universidad de Guadalajara, Francisco deGuadalajara, Jalisco, México. Fax: +33 38180740x5851.. Juárez).orticosterone; ADHD, attention deficit hyperactivity disorder; HPA, hypothalamus-pituitary-ate in preadolescence and adulthood;W/MP, rats exposed towater (methylphenidate vehicle)

n adulthood; W/W, rats exposed to water in preadolescence and adulthood; Co+MP/MP, ratsdate in preadolescence and exposed tomethylphenidate in adulthood; Ss+MP/MP, rats treatedusmethylphenidate in preadolescence, and exposed tomethylphenidate in adulthood; Co+W/inpreadolescenceandexposed tomethylphenidate inadulthood;ANOVA,analysis of variance

er B.V. All rights reserved.

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1. Introduction

Methylphenidate (MP) is a psychoactive drug approved forclinical use by the U.S. Food andDrugAdministration (FDA) andindicated in the treatment of attention deficit hyperactivitydisorder (ADHD) and narcolepsy. Methylphenidate acts byblocking the dopamine transporter with an affinity approxi-mately 0.6 times greater than that of cocaine (Ritz et al., 1987).The orally administered therapeutic dose of methylphenidateeffectively blocks more than 50% of the dopamine transporter,thus increasing extracellular levels of DA (Volkow et al., 1998,2001). The neuropharmacological profile of methylphenidate issimilar to that of other stimulants that are commonly used, orabused. Therefore, studies in monkeys have supported thenotion that the reinforcing properties of intravenously admin-istered methylphenidate are similar to those observed forcocaine (Bergman et al., 1989; Johanson and Schuster 1975).Moreover, it has been shown that the psychopharmacologicalproperties of these two substances are very similar in mice(Gatley, et al., 1999).

It has been suggested that early exposure to methylpheni-date may affect the consumption of such drugs as cocaine(Brandon et al., 2001; Thanos et al., 2007); the incentiveproperties of this psychostimulant (Achat-Mendes et al.,2003; Andersen et al., 2002; Augustyniak et al., 2006; Magueet al., 2005); and, the sensitization of the locomotor effects ofcocaine and methamphetamines (Brandon et al., 2001; Guer-riero et al., 2006; Kuczenski and Segal, 2002). However, we arenot aware of any studies concerning the effects of earlyexposure to methylphenidate on sensitization to the effects ofmethylphenidate in preadolescence or adulthood.

In contrast, interaction between the activity of the hypo-thalamus–pituitary–adrenal (HPA) axis and drug abuse hasbeen amply documented. Thus, it is well known that stressplays an important role in the acquisition, maintenance andrecurrence of the self-administration of psychostimulants(Piazza et al., 1991; Piazza and LeMoal, 1998; Goeders, 2002a,b),and that corticosterone treatment also produces an increasein the self-administration of cocaine (Deroche et al., 1997;Goeders 2002a) and amphetamines (Piazza et al., 1991).Similarly, the increase in the sensitization of locomotoractivity produced by amphetamines or cocaine is positivelycorrelated with higher levels of plasma corticosterone, wheth-er induced by stress (Deroche et al., 1992, 1995) or theadministration of exogenous corticosterone (Pauly et al.,1993; Przegaliński et al., 2000). Methylphenidate is notconsidered a drug of abuse as is the case of cocaine andamphetamines, but this psychostimulant is widely used inclinicalmedicine to treat ADHD. There is sufficient evidence toshow that a stressful psychosocial context is more likely topersist in families of children with ADHD than in comparisoncontrol families (Gau, 2007; Whalen et al., 2009). Additionally,important deficits in quality of life related to health issues inseveral psychosocial domains have been described for chil-dren with ADHD (Klassen et al., 2004). If the therapeuticprogram prescribed for children with this disorder includesmethylphenidate, then it is possible that an unfavorablepsychosocial environment may provoke an interaction be-tween the activity of the HPA axis and this psychostimulant;

however, to the best of our knowledge, no studies have yetexplored this issue.

The data available in the literature raise several concernsrelated to the analysis of methylphenidate as a stimulant drugin animal models. First, though various studies haveattempted to elucidate whether early treatment with methyl-phenidate sensitizes the effects of other drugs during adult-hood, there are no reports on the effects of early treatmentwith methylphenidate and the possible sensitizing effects ofmethylphenidate itself in preadolescence and adulthood. Thisquestion is relevant to our understanding of its transient orpermanent effects on the pathways involved in the actionmechanism of this psychostimulant. Second, while there isevidence that corticosterone may propitiate the acquisitionand self-administration of cocaine and amphetamines, andenhances their sensitizing effects on locomotor activity, weknow of no studies of the effects of corticosterone on eitherthe oral consumption of methylphenidate or the sensitizingeffects of this drug. This issue is relevant whether or not onetakes into account the evidence that a stressful psychosocialcontext is more likely to arise in families of children withADHD who are taking methylphenidate treatment. Third, inclinical medicine, methylphenidate is more frequently pre-scribed to patients in the prepuberty age group, but mostexperimental studies have assessed its effects only in adultanimals.

In light of this situation, the first objective of the presentwork was to study the effects of early methylphenidatetreatment on locomotor activity in preadolescence, and thenunder conditions of re-exposure to methylphenidate inadulthood. The second aim was to analyze the effect of earlycorticosterone treatment on the oral consumption of methyl-phenidate and, at the same time, to assess the well-knowneffect of this psychostimulant on locomotor activity in thepreadolescent and adult stages in rats.

2. Results

2.1. Experiment 1

2.1.1. PreadolescenceIn this experiment, only one group of rats (MP/MP) was exposedto methylphenidate during preadolescence (Fig. 1). It showed adaily consumption of 42.8 mg/kg±3.2 (mean±S.E.M.) during the9 days of methylphenidate exposure. A one-way ANOVA wasused to analyze significant differences among days for thisgroup: consumption on day 1 was higher than on all other days(F8,72=7.89, P<0.0001), but no differences appeared for days 2 to9.After the first dayofmethylphenidate exposure, consumptionof this substance decreased, though it remained stable in thefollowing days. During the 1 h of access to water or water plusmethylphenidate after 12 h of water deprivation, the preado-lescent animals consumed approximately 17% and 14% of thedaily liquid requirements in thewater-only exposure group andin the methylphenidate group, respectively.

The two-way ANOVA (groups×days) showed that thegroup treated with methylphenidate (MP/MP) manifestedhigher locomotor activity compared to the groups (W/W andW/MP) that were not exposed to this psychostimulant during

Fig. 1 – Experimental design. Water (W), methylphenidate (MP), corticosterone (Co), Saline solution (Ss), post-natal days (PnD).Experiment 2: Corticosterone and saline treatment (i.p. daily injections) was begun on post-natal day 24.

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preadolescence (main effect of group: F2,27=19.23, P<0.0001)(Fig. 2).

2.1.2. AdulthoodTo assess the effect of re-exposure to methylphenidate in thisexperiment, twogroups (thoseexposed tomethylphenidate andwater during preadolescence, respectively, [MP/MP and W/MP])

Fig. 2 – Locomotor activity in the open field test inpreadolescence. Data are the mean frequencies (±S.E.M.) ofcrossings in the two groups exposed to water (W/W, W/MP)and in the group exposed to methylphenidate (MP/MP). Ratsfrom the MP/MP group showed significantly (*) higherlocomotor activity than subjects in the W/W and W/MPgroups.

were placed in a condition during adulthood in which methyl-phenidate was the only liquid available during an 8-day period(1 h/day). Methylphenidate consumption was analyzed using atwo-way ANOVA (groups×days), which showed a significantlyhigher consumption of methylphenidate in the group that wasexposed to this psychostimulant for the first time (W/MP) thanin the one that had been pre-exposed to the drug duringpreadolescence (MP/MP) (main effect of group: F1,18=6.48,P=0.02.) (Fig. 3A). In addition, methylphenidate consumptionwas higher on days 1, 4 and 5 than on days 7 and 8, regardless ofthe group (main effect of days: F7,126=4.36, P=0.0002).

In contrast to the differences observed in methylphenidateconsumption, differences in locomotor activity between theW/MP and the MP/MP groups were not significant (Fig. 3B).

2.1.3. Free-choice exposureTo assess free-choice exposure to methylphenidate and waterin adulthood, a two-way ANOVA (groups×days) was per-formed. This analysis showed significantly higher methyl-phenidate consumption in the rats with no previous exposureto methylphenidate (W/W) than in the other two groups thathad been exposed previously to this psychostimulant (Wi/MPand MP/MP) (main effect of group: F2,27=4.09, P=0.02) (Fig. 4).Results for the factor of days showed that methylphenidateconsumption on day 1 was higher than on days 3, 6 and 7,regardless of the group (F6,162=3.71, P=0.001). The interactionbetween the effects of groups and days was also significant, asit revealed that methylphenidate consumption on days 1 and4 was higher in the group with no previous exposure to thisdrug (W/W) than that observed in the other two groups–MP/MP and W/MP–on any day. In addition, methylphenidateconsumption by W/W on days 2 and 3 was higher than thatseen on the same days for MP/MP and on day 3 for the W/MP

Fig. 3 – Methylphenidate (MP) consumption (mg/kg) inadulthood (A) and the frequency of crossings in the open fieldtest (testing days: 1, 3, 5 and 7) in this period of MP treatmentin adulthood (B). Rats had previously been treatedwith eitherwater (W/MP) or methylphenidate (MP/MP) inpreadolescence. The inset bar graphs show the mean total ofMP consumption (A) and the mean total frequency ofcrossings in the open field test (B). The group previouslyexposed to MP in preadolescence (MP/MP) showedsignificantly lower MP consumption than the one that wasexposed to this drug for the first time (W/MP), (inset graph inA). There were no significant differences in the MP-inducedlocomotor activity between these groups in adulthood (B).Data are means (±S.E.M.).

Fig. 4 – Voluntary consumption of methylphenidate (MP)during the 7 days of free-choice exposure to water andMP, forthe group previously exposed to MP in preadolescence andadulthood (MP/MP), in the one exposed tomethylphenidate inadulthoodbutnotpreadolescence (W/MP), and in thegroupnotpreviously exposed to methylphenidate (W/W). The inset bargraph shows the mean total of MP consumption. Data aremeans (±S.E.M.). Main effect of group (inset graph): (*) W/W>MP/MP and W/MP. Interaction between effects of groupand days (main graph): (**) Days 1, 4 of the W/W group>days1–7 of the MP/MP andW/MP groups. (*) Days 2, 3 of the W/Wgroup>days 2, 3 and 7 of theMP/MP group, and days 3, 6and 7of the W/MP group.

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group. Finally, intra-group comparisons showed that con-sumption on days 1 and 4 in the W/W group was significantlyhigher than on days 5 and 6. Moreover, on day 1 it was higherthan on day 7 in theW/Wgroup (interaction between effects ofgroup and days: F12,162=2.71, P=0.002).

2.2. Experiment 2

2.2.1. PreadolescenceFor the purpose of assessing the effect of corticosterone onmethylphenidate consumption during preadolescence, a two-wayANOVA (groups×days)wasperformed.This test showedno

significant differences between the group treated with cortico-sterone (Co+MP/MP) and the one that received saline (Ss+MP/MP) both before and during methylphenidate treatment(Fig. 5A). In contrast, the effect of days, regardless of group,showedhighermethylphenidate consumption onday 1 than onany other day, while consumption on days 2, 3, 4 and 5 washigher than that observed on day 8 (F8,144=11.95, P<0.0001).Although methylphenidate consumption was quite similar inthese two groups, MP-elicited locomotor activity was higher inthe corticosterone treated group (Co+MP/MP) during the secondhalf of the treatment period (days 5 and 7) than that observedonall days for the saline-treated group (Ss+MP/MP), (interactionbetween effects of group and days: F3,54=2.95, P=0.04) (Fig. 5B).

2.2.2. AdulthoodAll groups in this experimentwere exposed tomethylphenidateduring adulthood. The ANOVA (groups×days) showed signifi-cant differences only in the factor of days, regardless of group,indicating that methylphenidate consumption was higheron day 1 than on any other day; while day 2 was higher thandays6–7,anddays3and5werehigher thandays4and6–8 (maineffect of days: F7,189=12.05, P<0.0001). Though there were nosignificant differences among groups in methylphenidateconsumption (Fig. 6A), variations in the methylphenidate-elicited locomotor activity were evident in adulthood, as thegroup previously treated with corticosterone plus methylphe-nidate showed higher activity than the one treated withcorticosterone plus saline (in preadolescence) (main effect ofgroup: F2,27=3.79, P=0.03) (Fig. 6B). The group treatedwith saline

Fig. 5 – Methylphenidate (MP) consumption (mg/kg) duringthe eight days of exposure to this psychostimulant in thecorticosterone- (Co+MP/MP) and saline- (Ss+MP/MP) treatedgroups (A) in preadolescence. Frequency of crossings in theopen field test during days 1, 3, 5 and 7 of themethylphenidate or saline treatment in thecorticosterone- (Co+MP/MP) and saline- (Ss+MP/MP) treatedgroups (B) in preadolescence. The inset bar graphs show themean total of MP consumption (A) and the mean totalfrequency of crossings in the open field test (B). Data aremeans (±S.E.M.). Interaction between effects of group anddays: (**) locomotor activity on days 5, 7 of the Co+MP/MPgroup>days 1–7 of the Ss+MP/MP group. (*) locomotoractivity on days 1, 3 of the Co+MP/MP group>day 7 of theSs+MP/MP group.

Fig. 6 – Methylphenidate (MP) consumption (mg/kg) duringthe eight days of exposure to this psychostimulant inadulthood (A), and the frequency of crossings in the openfield test during days 1, 3, 5 and 7 of this methylphenidatetreatment, also in adulthood (B). Rats had previously beentreated with either corticosterone plus methylphenidate(Co+MP/MP), saline plus methylphenidate (Ss+MP/MP), orcorticosterone plus water (Co+W/MP) in preadolescence. Theinset bar graphs show the mean total of MP consumption (A)and the mean total frequency of crossings in the open fieldtest (B). Data are means (±S.E.M.). There were no significantdifferences among groups in methylphenidate consumption(inset graph in A). The locomotor activity of the grouppreviously treated with corticosterone plus methylphenidatewas significantly higher than that observed in thecorticosterone-plus-saline-treated group in preadolescence(inset graph in B).

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plus methylphenidate during preadolescence showed interme-diate values for locomotor activity, butnosignificantdifferenceswhen compared to the other two groups.

2.2.3. Free-choice exposureMethylphenidate consumption during adulthood was similarin the different groups when subjects were allowed to choosefreely between methylphenidate and water (Ss+MP/MP: 10.3;Co+MP/MP: 9.84; Co+W/MP: 10.4 mg/kg). The two-wayANOVA (groups×days) showed higher methylphenidate con-sumption on day 1 than that observed on day 6, regardless ofthe group (main effect of days: F6, 162=2.19, P=0.04).

3. Discussion

3.1. Experiment 1

An increase in locomotor activity in subjects exposed tomethylphenidate is a well-known effect of this substance anda common response to psychostimulant drugs. The relativelyhigh dose of methylphenidate consumed by the rats in thepresent study seems to be due to the following three factors:(1) the previous period of moderate liquid deprivation; (2) the

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availability of the drug during only 1 h per day; and, (3) thetype of consumption, whichwas oral. It is well known that oralconsumption can retard both the absorption of this drug andits subsequent distribution, and thus permit the intake ofhigher doses, such that the effects produced are similar tothose achievedwith lower doses via parenteral administration(Gerasimov et al., 2000). Most studies using water deprivationas an experimental variable perform schedules of 24 to 72 h ofwater deprivation; therefore, the scheme of 12 h/day of liquiddeprivation used in the present study may be considered amild procedure with respect to this parameter. After the firstday of methylphenidate exposure in preadolescent animals,the consumption of this substance decreased, but remainedstable in the following days. This suggests that the decreasemay be more closely related to a regulation of the effects ofmethylphenidate after the high dose that was consumed onthe first day, rather than to a possible development of aversionto methylphenidate under this treatment scheme. On theother hand, the similar liquid consumption during the 1 h ofaccess to either water or water plus methylphenidate in thedifferent groups suggests that the animals exposed tomethylphenidate did not delay satiation of their thirst untilthey were provided with water.

In this experiment, only the MP/MP and W/MP groups(those exposed to methylphenidate and water, respectively,during preadolescence) were later exposed to methylpheni-date in adulthood. The group that was not previously treatedwith methylphenidate showed a significantly higher con-sumption of this substance in adulthood than the one thatreceived it during preadolescence. To the best of our knowl-edge, this is the first study to assess methylphenidateconsumption in adulthood in subjects previously treatedwith this psychostimulant; though several studies haveanalyzed the effects of early methylphenidate consumptionon self-administration, or on the incentive properties of otherpsychostimulants, such as cocaine (Achat-Mendes et al., 2003;Andersen et al., 2002; Augustyniak et al., 2006; Brandon et al.,2001; Mague et al., 2005; Thanos et al., 2007). Some studiesreport that early exposure to methylphenidate increases self-administration of cocaine (Brandon et al., 2001), but mostcoincide in that early methylphenidate exposure decreaseseither the self-administration or the incentive properties ofthat drug (Achat-Mendes et al., 2003; Andersen et al., 2002;Augustyniak et al., 2006; Mague et al., 2005; Thanos et al.,2007). The results of the latter studies agree with thosepresented in this report, as the rats exposed to methylpheni-date during preadolescence consumed lower doses of thisdrug than those that were treated with methylphenidate forthe first time in adulthood. In spite of the differences inmethylphenidate consumption in adulthood, locomotor ac-tivity showed no significant differences among groups. Thissuggests a sensitizing effect on this behavior in the grouppreviously treated with methylphenidate in the preadoles-cence phase; i.e., similar levels of locomotor activity wereobserved in both groups treated with methylphenidate inadulthood, though the group previously exposed to methyl-phenidate during preadolescence (MP/MP) consumed signifi-cantly lower amounts of methylphenidate than the group thatwas exposed to this drug for the first time in adulthood (W/MP). A similar phenomenon has already been documented on

the basis of early methylphenidate treatment and posteriorcocaine challenges (Brandon et al., 2001), however, similarlycontrasting results have been reportedwith cocaine (Guerrieroet al., 2006) andmethamphetamine challenges (Kuczenski andSegal, 2002).

Voluntary consumption of methylphenidate was signifi-cantly higher in the group that had not been exposed to thispsychostimulant either inpreadolescenceor adulthood,when itwas theonly liquidavailable in the cage.Again, it ispossible thata sensitization phenomenon in the groups with early exposureto methylphenidate may account for these results. However,methylphenidate consumption by the group not previouslyexposed tomethylphenidate (W/W) decreasedonday 5 to levelssimilar to those seen in the other two groups (Fig. 4). Thissuggests that sensitization to methylphenidate in the W/Wgroupmight have occurred during the period of the few days ofdaily exposure to this drug, but only became evident when theanimals were exposed to methylphenidate in the free-choicecondition. It is noteworthy that subjects in all groups consumedmethylphenidatewhen they had a free choice betweenwater orthe MP solution, and were not food- or liquid-deprived in anyway; therefore, it appears that a possible development ofaversion to methylphenidate can be discarded, at least in thisexperimental condition. As far aswe know, there are no studiesdescribing this free-choice paradigm between water and asolution of water plus methylphenidate; therefore we do notknow if rats recognize the taste of methylphenidate. Nonethe-less,we candiscarda randomchoiceof liquids, sincedailywaterconsumption was several times higher than that of themethylphenidate solution. Considering that consumption didnot decline at the end of the days of exposure, it is possible thatthe effects of methylphenidate were sufficiently rewarding tomaintain a stable consumption over time.

3.2. Experiment 2

Corticosterone treatment did not affect methylphenidate con-sumption when compared to saline treatment in the preadoles-cent stage. However, the corticosterone-treated group showedsignificantly higher locomotor activity elicited by methylpheni-date thandid the salinegroup,especiallyat theendof treatment.While locomotor activity in the saline group had a tendency todecrease, an upwardsmovementwas observed in the activity ofthe corticosterone group throughout the testingdays. Therefore,corticosterone treatment seems to sensitize the effects ofmethylphenidate on locomotor activity, though without affect-ing consumption of thisdrug. The present results donot supportthe facilitation of corticosterone in methylphenidate consump-tion, as has been described for the consumption of other drugs,such as cocaine ((Deroche et al., 1997; Goeders 2002a), amphe-tamines (Piazza et al., 1991) and alcohol (Fahlke and Hansen,1999; Prasad and Prasad, 1995). They do, however, support thedescribed effect of corticosterone as enhancing the sensitizationof the locomotor response elicited by other drugs, such asmorphine (Deroche et al., 1993, 1994), cocaine (Marinelli et al.,1996; Przegaliński et al., 2000; Stamp et al., 2008) and ampheta-mines (Deroche et al., 1993; Pauly et al., 1993). The results of thisexperiment thus extend this effect of corticosterone to includemethylphenidate-induced locomotor activity, anaspect thathasnot been reported up to the present time.

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When the subjects in the three groups were exposed tomethylphenidate in adulthood, there were no significantdifferences in the consumption of this substance. However,the group treatedwith corticosteronewithoutmethylphenidateduring preadolescence, showed a tendency towards higherconsumption than the other two groups that had beenpreviously exposed to this drug. It should be noted that thisgroup–the one treated in preadolescence with corticosteronebutnotmethylphenidate–showedsignificantly lower locomotoractivity elicited by this psychostimulant in adulthood than thegroup previously treated with corticosterone plus methylphe-nidate in preadolescence. This result, whichwould appear to bedependent on earlier treatment in preadolescence, suggests anenduring plastic effect in the brain that manifests itself whensubjects are re-exposed to methylphenidate in adulthood. Thiseffect suggests an apparent facilitation of locomotor activitywhencorticosterone is combinedwithmethylphenidate, andanapparent attenuation ofmethylphenidate's effect on locomotoractivity when corticosterone is administered alone (with water)in preadolescence.

In contrast, while the group treated with corticosterone plusmethylphenidate in preadolescence showed the highest valuesof methylphenidate-induced locomotor activity in adulthood,this effect was not significantly different from that observed inthe group that received early methylphenidate treatmentwithout corticosterone. This suggests that the apparent sensi-tization of the locomotor activity produced by the combinationof corticosterone plusmethylphenidate in preadolescence doesnot persist–or, at least, not significantly–in adulthood whensubjects are re-exposed to methylphenidate.

These results support the well-known effect of methylphe-nidate on locomotor activity as much in preadolescence as inadulthood. Methylphenidate consumption seems to sensitizethe central nervous system in such a way that subsequentexposure to it is characterized by lower consumption com-pared to that of subjects that are exposed to methylphenidatefor the first time. Corticosterone treatment did not affectmethylphenidate consumption, but results support a sensiti-zation of its effects on locomotor activity in the preadolescentstage. During adulthood, corticosterone had no effect onmethylphenidate consumption, but methylphenidate in-duced-locomotor activity was affected differentially depend-ing on the combination of corticosterone with, or without,methylphenidate during preadolescence. Methylphenidateconsumption was observed in the absence of food or waterdeprivation and in the completely free-choice conditionbetween water and this psychostimulant, suggesting thatthe oral consumption of methylphenidate may have reward-ing effects in rats.

4. Experimental procedures

4.1. Subjects

Male Wistar rats were obtained from a colony bred at theInstitute of Neurosciences at the University of Guadalajara.Subjects were maintained on a 12-h light–dark cycle (lights onat 8:00 a.m.), and food pellets (Ralston Rations, Purina) wereavailable ad libitum. The temperature, feeding and light-dark

cycle conditions were maintained constant throughout thecourse of the study.

Two experiments were performed. The experimentaltreatments in preadolescence were different in each case,but the experimental conditions were basically the same forthe different groups in adulthood in both experiments. Hence,themethodswill be described separately for the preadolescentstage, but conjointly for adulthood.

4.2. Experiment 1

4.2.1. Preadolescent treatmentAt weaning (21 days of age), thirty males from different litterswere assigned to one of three treatment groups and placed incollective cages in subgroups of five rats each. At 31 to 39 days ofpostnatal age, 10 rats were individually exposed during 1 h/dayto a bottle containing methylphenidate (Ritalin™, methylphe-nidate hydrochloride) dissolved in distillated water at aconcentration of 1 mg/ml (MP/MP group). Two other groups often males each (W/MP and W/W), were individually exposedduring 1 h/day to a bottle containing tap water (Fig. 1). Thoughthe rats in the second and third groups received the same liquidtreatment during prepuberty, we decided to separate them intotwo groups for this phase of the experiment because themotoractivity recorded in this preadolescent phase might be consid-ered as a reference for that whichwould be recorded during thedifferent pharmacological treatments in adulthood. Subjects inall three groups were deprived of liquids during the 12 hprevious to the daily exposure to methylphenidate or water.The amount of methylphenidate or water consumed dependedon self-administration by subjects during the 1 hof daily access.

4.2.2. Motor activityImmediately after the 1 h of daily individual exposure tomethylphenidate or water, the motor activity of males wasrecorded every other day using the open field test (post-nataldays 31, 33, 35 and 37). Thus, motor activity was recorded on4 days during the treatment period, between 31 and 39 days ofage. Open field activity was measured by an apparatusconsisting of a 60×60 cm, square black floor with a 25-cm-highperipheral wall. The floor was marked with thin intersectinggray lines to form 10 cm squares. At the beginning of the test, aratwas placed in the center of this apparatus and thenumber ofintersecting lines crossed in 10minwas recorded. The ratswerereturned to their collectivehomecagesaftereachopen field test.Water was replaced in those cages about 1.5 h after the openfield test (post-natal days 31, 33, 35 and 37) or after each sessionof exposure tomethylphenidate orwater (post-natal days32, 34,36, 38 and 39). This meant that tap water was availablecontinuously for at least 9 h per day before the next 12 h-periodof liquid deprivation. From 40 to 70 days of age, the rats of allthree groups remained undisturbed in their home cages withfood and water ad libitum.

4.3. Experiment 2

4.3.1. Preadolescent treatmentAt weaning (21 days of age), males from different litters wereassigned to one of 3 treatment groups, each one with 10 rats.The animals were placed in collective cages in subgroups of

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five. From 24 to 39 days of postnatal age, twenty malesreceived i.p. injections of corticosterone (Co), (2.0 mg/kg/day/subject), (Sigma-Aldrich 46148), suspended in a saline solution(NaCl, 0.9 %), while the other ten received i.p. injections ofsaline solution (NaCl, 0.9 %), (Fig. 1).

From 31 to 39 days of postnatal age, ten of the twenty ratstreated with corticosterone (Co+MP/MP group) and the ten ratstreated with saline (Ss+MP/MP) were individually exposed for1 h/day to a bottle containing methylphenidate (Ritalin®,methylphenidate hydrochloride) dissolved in distillated waterat a concentration of 1 mg/ml. The remaining ten males, thosetreated with corticosterone, were individually exposed to abottle containing tap water for 1 h/day (Co+W/MP group). Asdescribed for experiment 1, subjects were deprived of liquidsduring the 12 h previous to the daily exposure to methylpheni-date or water (Fig. 1).

4.3.2. Motor activityAs described for experiment 1, motor activity was recordedusing the open field test every other day immediately after the1 h of exposure to MP or water (i.e., days 1, 3, 5 and 7).Therefore, motor activity was recorded on 4 days during thistreatment period from 31 to 39 days of age. Water wasreplaced in the home cages about 1.5 h after the open fieldtest (post-natal days 31, 33, 35 and 37) or after each individualsession of exposure to methylphenidate or water (post-nataldays 32, 34, 36, 38 and 39). From 40 to 70 days of age, the rats inall three groups remained undisturbed in their home cageswith food and water ad libitum.

4.4. Treatment in adulthood (experiments 1 and 2)

From 71 to 78 days of age, all rats in all groups, with theexception of W/W from experiment 1, were exposed to a bottlecontaining 30ml of methylphenidate dissolved in 30ml ofdistillated water at a concentration of 1 mg/ml for 1 h/day. TheW/W group, in contrast, was exposed to a bottle containing tapwater for 1 h/day in the same period of 71–78 days of age. Asdescribed above for the preadolescent stage, subjects of allgroups were deprived of liquids during the 12 h/day previous tothe daily individual exposure to methylphenidate or water(Fig. 1). Motor activity was recorded using the open field testevery other day immediately after the 1 h of exposure tomethylphenidate or water (post-natal days 71, 73, 75 and 77);i.e., on 4 daysduring this treatment period that lasted from71 to78 days of age, using the same procedure as that described forthe pre-puberty period. Water was replaced in the home cagesabout 1.5 h after the open field test (days 71, 73, 75 and 77) orafter each individual session of exposure tomethylphenidate orwater (post-natal days 72, 74, 76 and 78). In this way, tap waterwas continuously available for at least 9 h per day before thenext 12 h-period of liquid deprivation. From79 to 85 days of age,the rats in all six groups remained undisturbed in their homecages with food and water available ad libitum.

4.5. Free choice of methylphenidate and water duringadulthood

At 86 days of age, all subjects from all groups in the twoexperiments, including those from the W/W group, were

individually exposed to a free-choice condition of tap waterand a solution of methylphenidate at 1 mg/ml for 12 h/day for7 days,withno foodorwaterdeprivation (Fig. 1).After each12 h-period of this free-choice condition, subjects were returned totheir home cages, where they had food andwater ad libitum forthe remaining 12 h of each day. Motor activity was not recordedin this period of the liquid free-choice condition becausemethylphenidate consumption could have occurred at anytime during the 12 h period of exposure to the liquids. In suchconditions, the values for motor activity obtained at anymoment of this period would be related to highly variabledoses of methylphenidate among subjects.

4.6. Statistical analyses

In experiment 1, only one group of rats (MP/MP) was exposed tomethylphenidate for 9 days during preadolescence; therefore, aone-way analysis of variance (ANOVA) was used to analyzesignificant differences amongdays in this group. In all the otheranalyses involving methylphenidate intake or locomotor activ-ity, two-way ANOVAs [groups (kind of treatment)×days (oftreatment or activity assessment)] were used. When a maineffect (three or more levels) or interaction between effects wasseen to be significant, a Tukey's test was used to conduct post-hoc comparisons, with the level of significance set at P≤0.05.

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