ORIGINAL ARTICLES

Methylphenidate Treatment during Pre- andPeriadolescence Alters Behavioral Responses toEmotional Stimuli at Adulthood

Carlos A. Bolanos, Michel Barrot, Olivier Berton, Deanna Wallace-Black, andEric J. Nestler

Background: Methylphenidate (MPH) is a psychomotorstimulant medication widely used for the treatment ofattention-deficit/hyperactivity disorder (ADHD). Giventhe extent of prescribed use of MPH, and because MPHinteracts with the same brain pathways activated by drugsof abuse, most research has focused on assessing MPH’spotential to alter an individual’s risk for adult drugaddiction. Data examining other potential long-term be-havioral consequences of early MPH administration arelacking, however.

Methods: We investigated the long-term behavioral con-sequences of chronic administration of MPH (2.0 mg/kg)during pre- and periadolescent development in adult ratsby assessing their behavioral reactivity to a variety ofemotional stimuli.

Results: The MPH-treated animals were significantly lessresponsive to natural rewards such as sucrose, novelty-induced activity, and sex compared with vehicle-treatedcontrol animals. In contrast, MPH-treated animals weresignificantly more sensitive to stressful situations, showedincreased anxiety-like behaviors, and had enhancedplasma levels of corticosterone.

Conclusions: Chronic exposure to MPH during develop-ment leads to decreased sensitivity to rewarding stimuliand results in enhanced responsivity to aversive situations.These results highlight the need for further research toimprove understanding of the effects of stimulants on thedeveloping nervous system and the potential enduringeffects resulting from early-life drug exposure. Biol Psy-chiatry 2003;54:1317–1329 © 2003 Society of BiologicalPsychiatry

Key Words: Attention-deficit/hyperactivity disorder,methylphenidate, development, depression, emotion

Introduction

Attention-deficit/hyperactivity disorder (ADHD) is aneuropsychiatric disorder, commonly diagnosed dur-

ing childhood, characterized by excessive levels of inat-tentiveness, impulsivity, and hyperactivity (Goldman et al1998; Kirby et al 2002; Miller and Castellanos 1998;Swanson et al 1998). Although reported figures varyconsiderably depending on diagnosis criteria, socioeco-nomic status, geographic source of sample, and gender, itis estimated that up to 15%–20% of elementary school–age children meet the criteria for diagnosis of ADHD(Miller and Castellanos 1998; Wilens et al 2002; Zito et al2000), with a reported population prevalence of up to 12%(Brown et al 2001; Goldman et al 1998; LeFever et al1999; Swanson et al 1998). Stimulant medications havebeen effective for the treatment of ADHD (Findling andDogin 1998; Spencer et al 1996; Wolraich et al 1990), andmethylphenidate (MPH) is the most prescribed therapeuticagent (Findling and Dogin 1998; Kirby et al 2002; Millerand Castellanos 1998), accounting for more than 90% ofstimulants used for the management of ADHD in theUnited States (Goldman et al 1998; Zito 2002; Zito et al2000). Treatment duration for the disorder can last foryears (Greenhill et al 2002; Silver 2000; Spencer et al1996), because longitudinal studies report that ADHDsymptoms can continue into adulthood at an estimated rateof persistence of up to 75% of diagnosed children (Bied-erman et al 1996; Faraone et al 2000; Silver 2000; Wilenset al 2002) and because stimulants remain the preferredtreatment for the management of adult ADHD (Taylor andRusso 2001).

Similar to the cellular and behavioral actions of thepsychomotor stimulants cocaine and amphetamine (Koobet al 1998; Wise and Bozarth 1987), basic and clinicalstudies have shown that MPH has psychomotor stimulant–like properties: it blocks the dopamine transporter(Schweri et al 1985; Volkow et al 1998), induces dose-dependent increases in dopamine levels in brain pathwaysactivated by drugs of abuse (Gerasimov et al 2000b;Kuczenski and Segal 1997; Volkow et al 1999, 2001), andproduces heightened motor responses after its repeated

From the Department of Psychiatry and Center for Basic Neuroscience, Universityof Texas Southwestern Medical Center, Dallas, Texas.

Address reprint request to Dr. Eric J. Nestler, Department of Psychiatry, Universityof Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, DallasTX 75390-9070.

Received February 7, 2003; revised May 5, 2003; accepted May 12, 2003.

© 2003 Society of Biological Psychiatry 0006-3223/03/$30.00doi:10.1016/S0006-3223(03)00570-5

administration (Brandon et al 2001; Crawford et al 1998;Kuczenski and Segal 2001; McDougall et al 1999). In thisregard, repeated exposure to psychomotor stimulants re-sults in some of the long-lasting cellular, molecular, andbehavioral adaptations that have been implicated in thetransition from drug use to abuse (Koob et al 1998; Nestler2001; Robinson and Berridge 1993; Self and Nestler1995). Prior exposure to a variety of drugs with abusepotential has been shown to facilitate drug-taking anddrug-seeking behavior (Horger et al 1990, 1992; Pierreand Vezina 1997; Vezina et al 1999), and to enhance theincentive motivational properties of abused drugs (Gaiardiet al 1991; Shippenberg et al 1996). Thus, given the degreeof prescribed used of MPH and its psychostimulant-likeproperties, serious concerns have emerged regarding thepotential addictive properties of MPH (Klein-Schwartz2002; Kollins et al 2001; Meririnne et al 2001; Schenk andDavidson 1998), and consequently most research hasfocused on assessing its potential to predispose individualstoward drug addiction (Andersen et al 2002; Biederman etal 1997; Brandon et al 2001; Schenk and Izenwasser2002).

Despite MPH’s potential role in drug abuse liability andthe fact that affected children may be treated with MPHfor years, it is surprising that little is known about thelong-term neuronal and behavioral adaptations resultingfrom MPH exposure during the juvenile period. In thiscontext, it is well-documented that neurobiological sys-tems still undergo substantial neuronal adaptations duringdevelopmental periods before adulthood (Seeman et al1987; Spear 2000; Teicher et al 1995); these age-depen-dent neuroadaptations have been correlated, at least inpart, with changes in cognitive processes and responsivityto emotional stimuli (Hyman 2001; Spear 2000), as well asaltered responsiveness to psychopharmacologic interven-tion (Bolanos et al 1998; Emslie and Mayes 2001; Laviolaet al 1999). In addition, it has been demonstrated thatenvironmental, emotional, and pharmacologic experiencesduring development can dramatically influence neurobe-havioral functioning later in life (Hyman 2001; Ladd et al2000; Spear 2000; Teicher et al 2002).

Given that MPH interacts with brain structures knownto control responses to rewarding as well as aversivestimuli (Kelley and Berridge 2002; Robbins and Everitt1996; Volkow et al 1998), we assessed the reactivity ofvehicle- (VEH-) and MPH-treated animals to a range ofemotional stimuli. This approach was taken given increas-ing evidence that mesolimbic reward pathways are part ofthe neural circuitry that controls mood under normalconditions (Barrot et al 2002; Bolanos et al, in press;Naranjo et al 2001; Nestler et al 2002; Newton et al 2002;Pliakas et al 2001; Yadid et al 2001). Thus, to gain someinsight into the long-term consequences of early-life MPH

treatment, we designed the following study to assesspotential long-term behavioral alterations associated withrepeated exposure to MPH. We treated developing ratsduring the pre- and periadolescence period (postnatal day20–35) with twice-daily injections of a low dose of MPH(2.0 mg/kg), which results in plasma and brain levels ofMPH within the range achieved under clinical conditions(Gerasimov et al 2000a; Volkow et al 1998; Wargin et al1983). After VEH or MPH treatment and early behavioralassessment of play behavior, animals were left undisturbeduntil they became adults (90� days old), at which pointtheir behavioral responses to a battery of emotional stimuliwere assessed.

Methods and Materials

Subjects and Drug TreatmentLitters containing Sprague–Dawley male and female rat pupswith their respective dams were obtained from Harlan (Indianap-olis, Indiana) at postnatal day (PD) 14. After a 5-day acclimationperiod, MPH hydrochloride (2.0 mg/kg, intraperitoneal [IP]; U.S.Pharmacopeia, Rockville, Maryland) or saline vehicle injectionswere given to pups starting on PD 20 twice daily (4 hours apart,first injection at 9 AM) for 16 days (last injections on PD 35). Ratpups were weaned and separated with same-sex littermates ingroups of four animals per cage at PD 23, and double housed inclear polypropyline boxes containing wood shavings at PD 50 inan animal colony maintained at 23°–25°C on a 12-hour light–dark cycle in which lights were on between 7 AM and 7 PM. Onlymale offspring were used for behavioral testing. With theexception of the play behavior experiments that were conductedon PD 40, behavioral testing started 6 weeks after the last MPHinjection. All behaviors, except for locomotor activity to a novelenvironment, were recorded with a video camera located on theceiling of each testing room. Raters unaware of treatmentconditions scored the videotapes. Behavioral observations wererecorded by an observer with no knowledge of the treatmentconditions of each animal. With the exception of the subjectsused for the corticosterone levels experiment (i.e., they were partof the locomotor responses to novel environment experimentonly), animals were tested in the different behavioral paradigmsculminating with the forced swim test. There was a period of atleast 2 weeks before each behavioral test. Animals were providedwith food and water ad libitum. Experiments were conducted incompliance with the National Institute of Health and Universityof Texas Southwestern’s institutional animal review committee.

Play BehaviorWe assessed the short-term effects of MPH treatment by mea-suring behavioral responses to a natural reward, namely, socialplay behavior, during periadolescence (PD 40, 5 days after thelast MPH injection), an ontogenetic period in which social playstill is at its peak (Panksepp et al 1984). Social play is a criticalaspect of rodent development. In addition, play is a powerfulreward to young rodents, and access to “play time” in briefly

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isolated rodents activates brain reward pathways (Gordon et al2002; Panksepp et al 1984; Vanderschuren et al 1997). Animalswere singly housed for 24 hours before testing. All testingoccurred within 2 hours after the start of the dark phase, underred light conditions. Pairs of rats from the same experimentalgroup (i.e., control VEH or MPH), with no previous commonsocial experience, were placed in an arena similar to their homecage, and were allowed to interact freely for 5 min. Interactionswere videotaped and later analyzed using The Observer software,version 4.1 (Noldus Information Technology, Leesburg, Vir-ginia). For each pair, the scored parameters were the number ofpins (scored when one animal has its dorsal surface toward theground with the other animal above) and the number of dorsalcontacts (scored when one animal touches the dorsal surface ofthe other animal with its paws) as previously described byPankseep et al (1984).

Sucrose PreferenceThe sucrose preference test consisted of a two-bottle choiceparadigm. In this behavioral test, animals are given the choicebetween consuming water versus sucrose. This paradigm hasbeen used extensively in animal models of depression to assessthe effects of stress-induced anhedonia (Papp et al 1991; Samp-son et al 1992; Willner et al 1987). Additionally, we havesuccessfully used this paradigm to assess changes in appetitivebehavior induced by rewarding stimuli after discrete molecularmanipulations of reward pathways in brain (Barrot et al 2002;Bolanos et al, in press). Animals were habituated to drink waterfrom two bottles for 5 days. At the start of the experiment, ratswere exposed to ascending concentrations of sucrose (.0, .125,.25, .5 and 1%) for 2 days per sucrose concentration. Water andsucrose consumption were measured at 8 AM and 5 PM eachtesting day, at which time the position of the sucrose bottle (leftor right) was balanced between the MPH and VEH groups. Thepreference for sucrose over water was used as a measure foranimals’ sensitivity to reward.

Locomotor Activity Responses to a NovelEnvironmentAnimals were also tested to examine if MPH or VEH treatmentduring development affects general motor activity as indexed bylocomotor responses to a novel environment. Rats were placed inautomated (75 cm diameter � 15 cm wide, four photocell beams)circular activity chambers (Med Associates, St. Albans, Ver-mont; Bolanos et al, in press; Carlezon et al 1997), and theirlocomotor reactivity to the novel environment (i.e., the circularactivity chambers) were assessed for 2 hours divided into 10-minintervals.

Elevated Plus Maze and Self-Grooming BehaviorMPH- and VEH-treated rats were tested for 5 min on the elevatedplus maze, a behavioral model of anxiety-like behavior. Themaze was made of gray plastic and consisted of two perpendic-ular, intersecting runways (12 cm wide � 100 cm long; Barrot etal 2002; Bolanos et al, in press). One runway had tall walls (40

cm high) or “closed arms,” and the other one had no walls or“open arms.” The arms were connected together by a centralarea, and the maze was elevated 1 m from the floor. Testing wasconducted between 9 AM and 1 PM under controlled lightconditions (�90 lux). At the beginning of the 5-min observation,animals were placed in the central area, facing one of the openarms, and the cumulative time spent in the open arms wasvideotaped from a video camera placed on the ceiling of thetesting room. Raters unaware of the treatment conditions scoredthe videotapes. Another behavioral response that rodents exhibitin response to stressful and or anxiogenic stimuli is self-grooming (Gispen et al 1988; Gispen and Isaacson 1981; Spruijtet al 1988). Thus, we also assessed time spent grooming in the“closed arms” during the 5-min test.

Social Interaction in an Aversive EnvironmentIn this test, rats face a conflict between social interacting andtheir innate fear of an aversive environment. In this context, timespent performing social behaviors varies depending on howaversive the testing environment is (File and Hyde 1978; Ramosand Mormede 1998). The MPH- and VEH-treated animals weretested for social interaction in an open-field arena (70 cm square)that was divided into nine virtual squares (23 � 23 cm). Toincrease the aversiveness of the environment, the light intensitywas raised to 250 lux. After social isolation of 1 week, rats wereplaced in pairs (from same drug treatment, but no previouscommon social experience) in two opposite corners of the testarena and were then allowed to interact freely for 5 min.Interactions were videotaped and analyzed using The Observerversion 4.1. For each animal, the scored parameters were thenumber of crossed squares and the total duration of active socialinteractions. The latter dependent variable comprised the follow-ing behavioral classes: sniffing, following, grooming, crawlingover, and crawling under as described by File (1980).

Sexual BehaviorFor the sexual behavior experiments, animals were housed in aseparate room maintained on a 12-hour light–dark cycle (lightson between midnight and 12 noon). Sexual behavior wasassessed under red light conditions between 1:30 PM and 6 PM ina circular arena (60 cm) containing wood chips on the floor. Eachmale was given a 5-min acclimation period to the testing arena.Testing started at the end of the acclimation period by theintroduction of a receptive female to the arena. Testing sessionslasted 90 min unless the male failed to initiate sexual behavior (atleast 1 mount) within 30 min. Behaviors recorded were mounts,intromissions, and ejaculation as previously described (Soderstenet al 1977). More specifically, we measured mount latency (fromthe start of the test to the first mount with or without intromis-sion), intromission latency (from the start of the experiment tothe first intromission), ejaculation latency (calculated from thefirst intromission), and postejaculatory interval (time from ejac-ulation to the first following intromission). For statistical analy-ses, a mount latency of 30 min was attributed to animals thatfailed to initiate the behavior within this time period. Sprague–Dawley ovariectomized female rats (Charles River, Kingston,

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New York) were used in these experiments. Receptivity of thefemales was induced by injection of estradiol benzoate (50 �g,subcutaneous) and progesterone (500 �g, subcutaneous) 48 and4–6 hours before testing, respectively. One week before theexperiment, the females were tested for one intercourse with anexperienced male. Before testing, female receptivity was verifiedby the exhibition of lordosis, in the presence of the experiencedmale, and accepted intromission. Each female was used to testonly one experimental male.

Forced SwimmingThe forced swim test is a 2-day procedure in which rats areforced to swim under conditions in which they cannot escape. Onthe first day, rats engage initially in a variety of escape-likebehaviors but eventually adopt a posture of immobility in whichthey make only the movements necessary to maintain their headabove water. When retested 24 hours later, rats become immobilemore quickly; antidepressant treatment between the forced swimexposures can significantly increase their escape-like behaviors,an effect that has been correlated with antidepressant activity inhumans (Cryan et al 2002; Porsolt et al 1977). At the start of theexperiment, rats were placed in plastic cylinders (30 � 45 cm)filled to a depth of 30 cm (so that their paws and tail do not touchthe bottom) with 25°C water and forced to swim for 15 min. Atthe end of this period, rats were removed from the water, driedwith towels, and kept in a warm enclosure for 30 min. Allcylinders were emptied and cleaned between rats. Twenty-fourhours after the forced swim, rats were retested for 5 min underidentical conditions, and sessions were videotaped by a videocamera attached to the ceiling of the testing room. Ratersunaware of the treatment conditions scored the videotapes. In thisstudy, the latency to become immobile was the dependentvariable. Latency to immobility was defined as the time at whichthe rat first initiated a stationary posture that did not reflectattempts to escape from the water (Bolanos et al, in press; Lucki1997; Pliakas et al 2001). To qualify as immobility, this posturehad to be clearly visible and maintained for � 2 sec.

Plasma Corticosterone (CORT) LevelsA separate group of animals (n � 16; VEH � 8, MPH �8) weretreated with MPH (2.0 mg/kg) or VEH during development forthe assessment of CORT levels after restraint stress. Tail bloodsamples were obtained at four time points: 0, 15, 40, and 90 min.Collection of each blood sample was made in about 3 min.Initially we restrained the animals for 20 min, and blood sampleswere taken at time 0 and 15 min. After 20 min of restraint,animals were returned to their home cage and were brieflyrestrained again (twice, for approximately 5 min each time) toobtain blood samples at the 40 and 90 min time points. Bloodwas collected in ice-cold heparin coated tubes and centrifuged(1000 � g, 15 min, 4°C). Aliquots of plasma were stored at–20°C until assayed. Plasma CORT levels were determined bycompetitive enzyme immunoassay (EIA) according to the man-ufacturer’s specifications (ALPCO Diagnostics, Windham, NewHampshire). Data are expressed as ng/mL. The sensitivity of theassay was .23 ng/mL of plasma. Twelve of the animals used for

the assessment of CORT levels (VEH � 6, MPH � 6) hadpreviously been part of the locomotor activity responses tonovelty experiment only.

Statistical AnalysisSignificance was measured using mixed-design (between andwithin variables) repeated analysis of variance (ANOVA) fol-lowed by Scheffe post hoc test. When appropriate, Student’s t,chi-square, and F tests were used to determine statistical signif-icance of preplanned comparisons. Data are expressed as themean � SEM. Statistical significance was defined as p � .05.

Results

Effect of MPH on Weight Gain, Water Intake, andPlay Behavior in the Developing Rat

Rats were weighed once daily (9 AM) during the MPH orVEH treatment (n � 30). Figure 1A shows that a clinicallyrelevant dose of MPH (2.0 mg/kg, IP twice daily [b.i.d.])had no effect on weight gain in pre- and periadolescentrats (p � .05). Water intake (total intake per cage) wasalso monitored during, starting at weaning, and 5 daysfollowing the last MPH injection. No changes in waterintake were observed (data not shown).

To assess short-term influence on behavior by drugtreatment, we measured the ability of MPH treatment toaffect play behavior 5 days after the last MPH injection(PD 40; n � 30). Figure 1B shows the average number ofpins and dorsal contacts, behaviors characteristic of play inyoung rodents, indicating that MPH treatment did notaffect play behaviors in these animals (p � .05).

Effect of MPH on Sucrose Preference in the Adult

Given previous findings that MPH treatment during pre-adolescence can alter the rewarding effects of cocaine inthe adult (Andersen et al 2002), we first assessed thelong-term effects of MPH by measuring animals’ re-sponses to sucrose, a natural reward. We used a sucrosepreference test, which is free of associative memory.Overall repeated-measures ANOVAs indicated thatchronic exposure to MPH did not significantly affect therats’ total fluid intake (water � sucrose; Figure 2 lowerpanel) at any of the sucrose concentrations tested (p �.05); however, sucrose preference varied as a function ofdrug treatment and sucrose concentration (drug treatment� sucrose concentration interaction: F(7,406) � 9.37; p �.0002). Prior MPH treatment decreased sucrose preferenceat .125 (p � .003), .25 (p � .005), and .5% (p � .055)sucrose when compared with the preference exhibited bythe VEH control group (Figure 2 upper panel). Differencesin sucrose preference between groups were not present atthe 1% sucrose concentration (n � 30).

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Effect of MPH on Locomotor Reactivity to a NovelEnvironment in the Adult

As can be seen in the inset of Figure 3, animals treatedwith MPH (2.0 mg/kg, b.i.d.) during preadolescence hadsignificantly less spontaneous ambulatory counts in re-sponse to a novel environment (i.e., the locomotor cham-bers) during the 2-hour session than the VEH-treatedcontrol animals (t[40] � 2.9; p � .006; n � 42).Additional analysis indicates that locomotor activity alsovaried as a function of drug-treatment and time spent inthe activity chambers (drug treatment � time interaction:F(11,440) � 2.52; p � .004). Thus, a subsequent Scheffepost hoc test revealed that VEH-treated animals weresignificantly more active during the first hour of testing (p� .05) than the MPH-treated group (Figure 3). In contrast,

given that locomotor activity of MPH- and VEH-treatedanimals did not differ in the second hour of testing, it islikely that baseline locomotor activity is not differentbetween the two groups of animals.

Effect of MPH on Elevated Plus Maze and Self-Grooming Behavior in the Adult

We also studied the effect of MPH on anxiety-like behaviorusing the elevated plus maze (Figure 4A and 4B; n � 30).Drug treatment affected time spent in the open arms of theplus maze (a measure of anxiety-like behavior); animals

Figure 1. Repeated exposure to methylphenidate (MPH) duringpostnatal development (n � 30). (A) There was no change inaverage weight gain during MPH or vehicle (VEH) treatment.(B) Methylphenidate did not affect play behavior in periadoles-cent (PD 40) rats. Data are shown as the average number (mean� SEM) of pins and dorsal contacts.

Figure 2. Methylphenidate (MPH) exposure during developmentregulates adult responses to sucrose reward (n � 30). (A) Develop-mental exposure to MPH significantly decreased sensitivity to therewarding effects of sucrose when compared with vehicle (VEH)-treated rats. Data are presented as percentage preference betweenMPH- and VEH-treated rats. (B) Methylphenidate pretreatment didnot affect total fluid (sucrose � water) intake. *Significantlydifferent at .125% (p � .003) and .25% sucrose (p � .005).†Marginally significant at .5% sucrose (p � .055).

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receiving MPH during preadolescence spent significantlyless time in the open arms than VEH-treated rats (t(28) �2.51; p � .05; Figure 4A). In addition, the MPH-treatedgroup spent significantly more time engaged in self-grooming behavior than the VEH-treated control animals(t(28) � 3.44; p � .001; Figure 4B). These results suggestan increase in anxiety-like behavior in the MPH-exposedanimals.

Effect of MPH on Social Interaction in an AversiveEnvironment in the Adult

We assessed the ability of preadolescence MPH treatmentto affect social interaction in adult rats (n � 30). Figure 4Cand 4D shows the average number of square crosses,duration of interaction, and time noninteracting, all ofwhich show no difference between the MPH and VEHgroups. These findings indicate that MPH did not affectanimals’ behavior in this measure of anxiety (p � .05).

Effect of MPH on Sexual Behavior in the Adult

Animals treated with MPH during adolescence showed adeficit in sexual behavior at adulthood (Table 1; n � 29).Compared with the VEH-treated control animals, a smallerproportion of MPH-treated animals displayed intromission(55.5% vs. 90.9%: 2 � 13.56; p � .0001) and reachedejaculation (27.7% vs. 72.7%: 2 � 7.8; p � .001) within30 min after their first contact with a receptive female.

This deficit appears to be accounted by a delay in latencyto the first mount (p � .015) and to the first intromission(p � .015); however, when considering the subpopulationof MPH-treated animals that successfully completed anintercourse, a tendency for a deficit was still observed asindicated by a larger number of intromissions necessary toreach ejaculation (p � .08; see Table 1). Among thoseanimals that successfully completed the first intercourse,the latency to initiate a second intercourse showed nofurther deficits (p � .38).

Effect of MPH on Forced Swimming Behavior inthe Adult

Given the increasing evidence that the mesolimbic dopa-mine system regulates responses to aversive stimuli as

Figure 3. Methylphenidate (MPH) exposure during developmentregulates adult locomotor responses to a novel environment (n �42). The MPH-treated animals show significantly lower respon-sivity to the locomotor activating effects of exposure to noveltycompared with vehicle (VEH)-treated rats. Data are presented asmean total (mean � SEM) photocell counts during a 120-mintest. *p � .05.

Figure 4. Methylphenidate (MPH) exposure during developmentregulates responses to anxiogenic stimuli (n � 30). (A) TheMPH-treated animals spent significantly less time in the open armsof the elevated plus maze than the vehicle (VEH)-treated controlanimals (*p � .05). (B) The MPH-treated animals spent signifi-cantly more time engaged in self-grooming behavior in the closedarms of the elevated plus maze than the VEH-treated controlanimals (*p � .05). (C, D) There were no significant differences insocial interaction between MPH- and VEH-treated groups.

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well as rewarding ones (Barrot et al 2002; Bolanos et al, inpress), it was of interest to study the effect of MPHtreatment during development on behavioral tests of aver-sion (n � 30). We used the forced swim test to study ananimal’s responses to stressful conditions (Cryan et al2002; Pliakas et al 2001). In this test, animals initiallystruggle, apparently trying to escape, but within a minuteor two they become immobile. The amount of time ratsengaged in escape-directed behaviors (i.e., latency toimmobility) in the forced swim test was dependent on drugtreatment; animals treated with MPH during developmenthad significantly shorter times to become immobile (treat-ment main effect: F(1,28) � 5.2; p � .0009) than theVEH-treated control group (Figure 5A).

Effect of MPH on CORT Levels during RestraintStress in the Adult

Given our findings of increased anxiety-like behavioralresponsiveness in MPH-treated animals, we assessedplasma CORT levels in a separate group of VEH- andMPH-pretreated adult rats (n � 16). Repeated-measureANOVA indicated that MPH treatment had a significanteffect on CORT levels in response to restraint stress(treatment � time interaction: F(3,42) � 40.227; p � .0001;Figure 5B). More specifically, subsequent post hoc testsrevealed that MPH-treated animals had significantlyhigher CORT levels at 15 min (p � .04) and wererelatively higher than the VEH-treated control animals at90 min (p � .09; Figure 5B).

Discussion

Psychostimulants have proven to be an effective pharma-cotherapy for ADHD, and MPH is the preferred medica-tion for the treatment and management of this neuropsy-chiatric disorder. Given the prevalence of prescribed useof MPH in pediatric populations, it is surprising that fewstudies have used developmental animal models to analyzethe long-term neurobiological and behavioral effects ofMPH treatment. The purpose of this study was to assesspotential enduring behavioral alterations in adult animalsas a consequence of repeated MPH exposure during theirjuvenile period. We show that repeated exposure to atherapeutic dose of MPH (2.0 mg/kg b.i.d.) during thejuvenile period results in altered behavioral responses to avariety of rewarding and aversive stimuli during adult-hood.

More specifically, our findings show that adult animalspretreated with MPH during the juvenile period are lesssensitive to the rewarding effects of natural rewards suchas sucrose, are less responsive with respect to the motoractivation exhibited by animals when first exposed to a

novel environment, and show deficits in the initiation andperformance of sexual behavior. In contrast, these animalsshow greater sensitivity to several types of aversivestimuli, including swim stress and anxiogenic challenges.Together, these data suggest that MPH treatment duringdevelopment (i.e., pre- and periadolescence periods) re-sults in decreased responsiveness to rewarding stimuliwhile enhancing a negative emotional state characterizedby increased sensitivity to aversive stimuli.

The findings that adult animals treated with MPHduring development are less sensitive to sucrose (i.e.,concentrations lower than 1%) are likely due to MPH’sability to alter an animal’s responsiveness to the rewardingeffects of sucrose, because the overall liquid intake duringsucrose testing did not differ between the treatmentgroups. Moreover, no alterations in liquid intake wereobserved during or after MPH treatment. Our findingsfurther show that MPH-treated animals have deficits insexual behavior, although in this study it was difficult todifferentiate between “interest” versus “efficiency.” It iswell-established that the brain’s reward pathways, such asthe nucleus accumbens (NAc) and its dopaminergic inputfrom the ventral tegmental area, are involved in regulatingmotivated behavior and responses to drugs of abuse andnatural rewards (Di Chiara and North 1992; Kelley andBerridge 2002; Robbins and Everitt 1996; Wise andBozarth 1987). Experimental evidence indicates that ex-posure to sweet solutions such as sucrose (Datla et al2002; Hajnal and Norgren 2001) and to the opposite sex(Becker et al 2001; Damsma et al 1992; Fiorino et al 1997)are rewarding; they activate the mesolimbic dopaminesystem and cause increases in dopamine release in theNAc, whereas lesions to these reward pathways blocksucrose preference (Shimura et al 2002) and impair sexualbehavior (Everitt 1990; Robbins et al 1989). Together, ourresults are in agreement with recent findings indicatingthat early exposure to MPH leads to long-lasting alter-ations in brain dopamine systems that result in decreasedsensitivity to cocaine reward and diminished cocaine-seeking behavior (Andersen et al 2002), and we nowextend these findings to natural rewards.

We also assessed the short-term effects of MPH treat-ment by measuring behavioral responses to a naturalreward, namely, social play behavior, during periadoles-cence (PD 40, 5 days after the last MPH injection), anontogenetic period in which social play is still at its peak(Panksepp et al 1984). Our results indicate that MPHpretreatment did not affect play behavior in these animals.Social play is a critical aspect of rodent development. Ithas been shown that play is a powerful reward to youngrodents and that access to “play time” in briefly isolatedrodents activates brain reward pathways (Gordon et al2002; Panksepp et al 1984; Vanderschuren et al 1997).

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Our findings are in agreement with previous researchshowing that exposure to relatively low doses of MPHdoes not affect play or social interaction in young adultrats (Sproson et al 2001). It is interesting to note, however,that although in this previous study MPH pretreatment didnot affect social interaction, it did affect mesolimbicneuronal functioning because presynaptic dopamine re-lease was significantly attenuated by repeated MPH ad-ministration during development (Sproson et al 2001). Inaddition, research has shown that social interaction andplay behavior are not entirely dopamine-dependent be-cause nondopaminergic mechanisms are also involved inmediating this behavior (Panksepp et al 1980; Vander-schuren et al 1995). Thus, it is conceivable that whereasrepeated MPH exposure affects mesolimbic dopaminesystems, it does not affect social play in periadolescentanimals.

Our findings also indicate that MPH treatment duringdevelopment alters an animal’s sensitivity to aversivestimuli. Given that MPH’s primary sites of action arelocated within limbic structures, these findings lend fur-ther support to the notion that the mesolimbic rewardpathway may play a role in the symptoms of depressionand other stress-related syndromes (Bolanos et al, in press;Kapur and Mann 1992; Naranjo et al 2001; Nestler et al2002; Pliakas et al 2001; Yadid et al 2001). Thus, animalspretreated with MPH during development exhibitedshorter latency to immobility in the forced swim test, aneffect opposite to that of antidepressant treatments (Cryanet al 2002; Porsolt et al 1977). Further studies with theelevated plus maze found that MPH exposure increasedresponses to anxiogenic stimuli, because these animalsspend significantly less time in the “open arms” and

significantly more time engaged in grooming behavior, abehavioral response to stressful or anxiogenic stimuli(Gispen et al 1988; Gispen and Isaacson 1981; Spruijt et al1988). Another behavioral measure of anxiety, namely,adult social interaction under increased light conditions,did not reveal deficits, because there were no differencesin social interaction responding between the treatmentgroups. In this test, rats face a conflict between socialinteraction, a behavioral measure of natural reward, andtheir innate fear of an aversive environment. Thus, thetime spent performing social behaviors varies as a functionof how aversive the testing environment is or is perceivedby the animal (File 1980; File and Hyde 1978; Ramos andMormede 1998). In this context, it is conceivable that theopportunity to “interact” is an incentive stimulus withpositive motivational valence that is powerful enough tomask potential MPH-induced alterations. Additionally, itis likely that the lack of an effect of MPH on socialinteraction may be due to repeated handling (i.e., samegroup of animals used for the locomotor activity and theelevated plus maze test) because it is well-established thatprior handling has anxiolytic effects in several testingsituations (Britton and Britton 1981; Ferre et al 1995;Hogg 1996). Conversely, and similarly to our findingsduring periadolescence, it is possible that MPH pretreat-ment does not affect the positive motivational aspects ofplay and social interaction. Despite the lack of effect onthis measure of anxiety, our results of increased CORTlevels in response to restraint stress further suggest thatMPH treatment during development results in enhancedreactivity to stressful situations at adulthood.

In our attempt to develop a general profile of behavioralresponsivity to emotional stimuli after early-life MPHtreatment, we also assessed the motor activity induced byexposure to a novel environment (Hooks and Kalivas1995; Kabbaj et al 2000). In this test, MPH-treatedanimals showed significantly lower behavioral reactivityto the novel test environment compared with the VEH-treated control animals. Novelty has positive incentivevalence (Bardo et al 1996; Pierce et al 1990), and exposureto novel environments increases dopamine release in theNAc (Rebec et al 1997). Accordingly, it is conceivablethat the hypoactivity exhibited by the MPH group was dueto decreased sensitivity to the rewarding aspects associ-ated with exploring a novel environment. On the otherhand, novelty also has some aversive-stressful components(Bardo et al 1996; Kabbaj et al 2000); thus, the lowerreactivity to the novel environment exhibited by the MPHgroup could be an indication of lesser sensitivity to stress,not to reward. Nevertheless, it has been established thatnovelty-elicited behavioral activation is largely dependenton the integrity of the mesolimbic dopamine system(Bardo et al 1990; Fink and Smith 1980; Koob et al 1981)

Table 1. Measures of Copulatory Behavior

Control(n � 11)

Methylphenidate(n � 18)

Mount Latency (sec) 526 � 155 1131 � 152a

Intromission Latency (sec) 654 � 191 1269 � 145a

Ejaculation Latency (sec) 653 � 110 (n � 10) 865 � 150 (n � 11)Number of Mounts 6.3 � 1.2 13.1 � 4.4Number of Intromissions 14.9 � .9 20.4 � 2.7b

Mounts � Intromissions 21.2 � 1.8 33.4 � 6.0b

Intromission Ratio 72% � 4.1 68% � 5.3PEI (sec) 338 � 12 325 � 9% I 30 90.9 (10/11) 55.5 (10/18)c

% E 30 72.7 (8/11) 27.7 (5/18)d

Methylphenidate treatment during development affects sexual behavior duringadulthood. Data are presented as mean � SEM. The ejaculation latency wascalculated from the first intromission. PEI, postejaculatory interval; % I 30,proportion of animals (10/18) displaying intromission within 30 min; % E 30,proportion of animals (5/18) reaching ejaculation within 30 min.

ap � .015 (t test).bp � .08 (t test).cp � .0001 (2 test).dp � .001 (2 test).

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and that the level of behavioral responding is correlatedwith the activity of ventral tegmental area dopamineneurons (Marinelli and White 2000). In this context,regardless of whether the reduced behavioral reactivitydisplayed by the MPH group is an indication of alterationsin their ability to respond to a rewarding or stressfulstimulus, it is likely that the attenuated reactivity tonovelty is an indication of deficits in the mesolimbicdopamine system induced by early-life MPH exposure.

The mechanisms underlying these enduring behavioraleffects induced by repeated MPH exposure during devel-opment are unknown. Based on previous research showingthat changes in the transcription factor cAMP responseelement-binding protein (CREB) within the mesolimbicdopamine system regulates responsiveness to rewardingand aversive stimuli, we hypothesize that changes in thistranscription factor may underlie these MPH-induced be-havioral adaptations. For example, activation of CREB orits overexpression in the NAc or ventral tegmental areadecreases the rewarding effects of drugs of abuse (Carle-zon et al 1997, 1998; Self et al 1998) and natural rewards(Barrot et al 2002), while enhancing an animal’s sensitiv-ity to aversive stimuli (Pliakas et al 2001). In addition,recent findings by Andersen et al (2002), indicating that

treatment with a similar dose of MPH (2.0 mg/kg) duringthe same developmental period as in our study causes asustained increase in CREB levels in the NAc and asubsequent decrease in cocaine-induced reward, lendsfurther support to this hypothesis.

Although our findings are in general agreement withthose suggestive of MPH-induced dysregulation of rewardsubstrates leading to decreased cocaine reward (Andersenet al 2002), our results must also be viewed in light ofthose obtained by Brandon et al (2001), which showincreased sensitivity to cocaine reward after MPH treat-ment. In the latter study, the authors administered MPH(2.0 mg/kg) for a relatively short period (7 days, oncedaily), whereas in this study we used a longer treatmentregimen (15 days) that included twice-daily injections ofthe drug. In addition, we treated our animals between 20 to35 days postnatal, whereas in the Brandon et al studyanimals were exposed to the drug between postnatal days35 to 42. Accordingly, it is conceivable that whereasshorter MPH treatment during adolescence causes sensi-tized responses to rewards (Brandon et al 2001), othertreatment regimens, involving longer treatment durationand earlier developmental stages (Andersen et al 2002;this study) may yield different results. In this ontogeneticcontext, it is not surprising to encounter different and, attimes, conflicting results given that qualitative and quan-titative differences frequently emerge when manipulatingthe nervous system during a particular maturational stage(Bolanos et al 1996; McDougall and Bolanos 1995;McDougall et al 1992; Spear 2000; Spear and Brake 1983;Zavala et al 2002).

To summarize, results of our study indicate that treat-ment with MPH during development can significantlyalter responsiveness to natural rewards and aversive stim-uli in adulthood. These findings provide further impetusfor the development of effective, nonstimulant treatmentsfor ADHD; however, it is imperative to note that theMPH-induced long-term behavioral effects described inthis study were derived from “normal” animals, andsimilar MPH treatment using established animal modelsfor ADHD might yield different, even contrary, results.Indeed, the results of this study should not be overinter-preted, because MPH remains a safe and effective treat-ment for ADHD. Still, basic and human developmentalpsychopharmacology is an area of study that has receivedrelatively little attention. Thus, little is known about howpsychotropic drugs may influence the developmental pro-cesses of the central nervous system or the enduringeffects during adulthood. Our findings show that develop-mental administration of MPH results in aberrant behav-ioral adaptations during adulthood, and these results un-derscore the need for further developmental researchgeared toward a better understanding of the mechanisms

Figure 5. Methylphenidate (MPH) exposure during developmentregulates responses to forced swim stress. (A) Latencies tobecome immobile varied as a function of drug treatment.Latencies were significantly decreased in rats treated with MPH(2.0 mg/kg, twice daily) during development compared with thevehicle (VEH)-treated control animals. Data are presented aslatencies (mean � SEM, in sec) to become immobile (*p �.0009; n � 30). (B) Corticosterone levels at several time pointsduring restraint stress. Corticosterone levels were generallyhigher in adult rats treated with MPH during development. Ittook less than 3 min to collect each blood sample. Initially,animals were retrained for 20 min, and blood samples were takenat times 0 and 15 min. After the first restraint period, animalswere returned to their home cage and were briefly restrainedagain (twice, approximately 5 min each time) to take bloodsamples at the 40 and 90 min time points. Data are presented asng/mL. *p � .04; †p � .09; n � 16.

Methylphenidate Modulates Mood-Related Behaviors 1325BIOL PSYCHIATRY2003;54:1317–1329

underlying MPH-induced behavioral plasticity. Researchexamining the effects of psychotropic drugs during devel-opment and their potential enduring effects will lead to abetter understanding of the neural and molecular basis forbetter psychopharmacologic therapies aimed at pediatricpopulations.

This work was supported by grants from the National Institute on DrugAbuse (EJN) and the National Institute of Mental Health (EJN), as wellas a National Research Service Award (CAB) from the National Instituteon Drug Abuse.

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