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Research Report

Prenatal tactile stimulation attenuates drug-inducedbehavioral sensitization, modifies behavior, and altersbrain architecture

Arif Muhammad⁎, Bryan KolbCanadian Centre for Behavioral Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge AB, Canada, T1K 3M4

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 403 329 2775E-mail addresses: muhammad.arif@uleth

0006-8993/$ – see front matter © 2011 Elsevidoi:10.1016/j.brainres.2011.05.038

A B S T R A C T

Article history:Accepted 16 May 2011Available online 23 May 2011

Based on the findings of postnatal tactile stimulation (TS), a favorable experience in rats, thepresent study examined the influence of prenatal TS on juvenile behavior, adultamphetamine (AMPH) sensitization, and structural alteration in the prefrontal cortex(PFC) and the striatum. Female rats received TS through a baby hair brush throughoutpregnancy, and the pups born were tested for open field locomotion, elevated plus maze(EPM), novel object recognition (NOR), and play fighting behaviors. Development andpersistence of drug-induced behavioral sensitization in adults were tested by repeatedAMPH administration and a challenge, respectively. Structural plasticity in the brain wasassessed from the prefrontal cortical thickness and striatum size from serial coronalsections. The results indicate that TS females showed enhanced exploration in the openfield. TS decreased the frequency of playful attacks whereas the response to face or evade anattack was not affected. Anxiety-like behavior and cognitive performance were notinfluenced by TS. AMPH administration resulted in gradual increase in locomotor activity(i.e., behavioral sensitization) that persisted at least for 2 weeks. However, both male andfemale TS rats exhibited attenuated AMPH sensitization compared to sex-matched controls.Furthermore, the drug-associated alteration in the prefrontal cortical thickness andstriatum size observed in controls were prevented by TS experience. In summary, TSduring prenatal development modified juvenile behavior, attenuated drug-inducedbehavioral sensitization in adulthood, and reorganized brain regions implicated in drugaddiction.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Behavioral sensitizationAddictionPlasticityTactile stimulationMassageRough and tumble play

1. Introduction

Massage therapy, a formof tactile stimulation,duringpregnancyin women is gaining recognition for its anxiolytic and painrelieving properties in addition to mitigating other pregnancy-related problems (Davidson et al., 2000; Field et al., 1999; Hartet al., 2001;Wanget al., 2005).However, despitebeneficial impact

..ca (A. Muhammad), kolb

er B.V. All rights reserved

ofmassage onwomen's health, the long term effects of prenatalstimulation on a child's brain and behavior is understudied andgenerally limited to the first fewmonthsof life.Nevertheless, thefindings of studies related to massage therapy are promising.Massage during pregnancy has a positive outcome for children,alleviating slow sensory-motor development associated withpreterm birth, and enhancing mother–child bonding (Bellieni et

@uleth.ca (B. Kolb).

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Fig. 1 – Mean (SEM) locomotor activity showing open fieldlocomotion for 10 min. Prenatal TS in females compared tosex-matched controls (*, p=0.003) and TS males (*, p=0.001)exhibited increased activity in the open field. TS did not affectexploratory behavior in males.

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al., 2007). Moreover, Cuba's system of maternal health programalso corroborates the benefits of early tactile stimulationintervention on children's learning abilities in addition to apositive health outcome for mother and child (Keon, 2009).

During the last few decades, researchers havemanipulatedthe environment during brain development, by exposinglaboratory animals to a variety of experiences in order tostudy the long term impact of early intervention on brain andbehavior (reviewed by Fone and Porkess, 2008; Weinstock,2008; Zhang and Meaney, 2010). However, most of the earlyinterventions, either favorable (e.g., maternal licking andgrooming) or adverse (e.g., social isolation), are studied duringpostnatal brain development (reviewed by Kaffman andMeaney, 2007; Kikusui and Mori, 2009). On the other hand,the influence of prenatal experiential manipulation on brainand behavior is not well studied and if investigated is mostlyrelated to adverse experiences, such as infectious agents(Watson et al., 1999), malnutrition (Brown et al., 1996), toxins(Tamaru et al., 1988), drugs and alcohol (Eriksson et al., 2000;Noble and Ritchie, 1989; Sobotka, 1989; Streissguth et al., 1989),and stress (Clarke and Schneider, 1997).

Recently, researchers started taking interest in the benefi-cial impact of favorable experiences during prenatal period.For example, prenatal environmental enrichment acceleratedstructuralmaturation of the retina in rat fetus (Sale et al., 2007)and helped in recovery from neonatal brain injuries (R. Gibband B. Kolb, unpublished observations). Similarly, tactilestimulation (TS), a form of sensory stimulation, duringprenatal development improved recovery from neonatalbrain injuries and facilitated motor activity (R. Gibb and B.Kolb, unpublished observations). Additionally, TS altereddendritic morphometry and spine density in various brainregions in rats (Gibb et al., 2010).

We recently reported the favorable long term effect ofpostnatal TS in rats on amphetamine-induced behavioralsensitization, and structural plasticity in brain regions impli-cated in drug addiction (Muhammad et al., 2011). Thepromising results of postnatal TS in rats laid the foundationfor the present study to investigate the long-term effect of TSduring prenatal period on drug-induced behavioral sensitiza-tion. Juvenile exploratory, emotional, cognitive, and socialbehaviors were also studied for the potential modulation byprenatal TS. Previous reports indicated that experience (e.g.,environmental enrichment) interacted with psychomotorstimulants (e.g., nicotine) in structural alteration of brainregions (e.g., nucleus accumbens, prefrontal cortex) (Hamiltonand Kolb, 2005). Prior experience dramatically reduced theresponse to subsequent experience, which was manifested atthe structural level in the brain (Hamilton and Kolb, 2005; Kolbet al., 2003b; Zehle et al., 2007). For example, psychostimulants(e.g., amphetamine), similar to rearing in an enriched envi-ronment, increased the spine density in the nucleus accum-bens (Kolb et al., 2003a; Norrholm et al., 2003; Robinson andKolb, 1997). However, prior drug exposure followed by rearingin the enriched environment blocked the increase in spinedensity associated with the enrichment experience (Hamiltonand Kolb, 2005; Kolb et al., 2003b). In the present study weswitched the order of experiential factors such that anexperience (i.e., TS) was followed by drug exposure, unlikeprevious studies where drug exposure was followed by rearing

experience in complex housing. We examined brain regions,implicated in drug addiction (i.e., medial prefrontal cortex,orbital frontal cortex, and nucleus accumbens) for structuralalterations in cortical thickness and neuronal morphology toinvestigate the interaction of prenatal TS experience andAMPH-induced behavioral sensitization.

2. Results

The behavioral data were analyzed using experience (TS orcontrol) and sex as independent factors. However, both sexeswere analyzed independently after the introduction of drug(AMPH or saline) as a factor either because of sex-dependentdifference (e.g., response to AMPH administration) or for theclarity of results description (e.g., in cortical thickness).Furthermore, all ANOVAs were followed by Bonferroni's posthoc test for multiple comparisons.

2.1. Behavior

2.1.1. Open field locomotionPrenatal TS experience led to increased open field exploratorybehavior in females but did not alter the open field locomotionin males. A two-way ANOVA with experience (TS or control)and sex as independent factors revealed a main effect ofexperience [F (1, 68)=4.91, p=0.030], sex [F (1, 68)=7.34,p=0.009], and an interaction between the two [F (1, 68)=4.37,p=0.040]. Pair wise comparison revealed that only prenatal TSfemales exhibited enhanced activity compared to sex-matched controls (p=0.003). Furthermore, TS females weremore active in the open field compared to experience-matched males (p=0.001) (Fig. 1).

2.1.2. Elevated plus mazeThe time spent and the frequency of entry into closed armswere used as indicators of anxiety-like and exploratorybehavior, respectively. The time spent in the closed armswas not affected by early experience but TS rats exhibiteddiminished activity in the maze.

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Fig. 3 – Mean (SEM) of play attacks in 10 min of play fightingbehavior. The playful attackswere sexually dimorphic wherecontrol males attacked more frequently compared toexperience-matched females (†,p=0.039). TS, however, resultedin the feminization of play in males by preventing the sexdifferences.

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A two-way ANOVA (experience×sex) of the time spent inclosedarms revealednomaineffectofexperience [F (1, 67)=0.19,p=0.661], sex [F (1, 67)<0.001, p=0.996], nor an interactionbetween the two [F (1, 67)=0.03, p=0.855]. When subjected to atwo-wayANOVA, the number of entries in closed arms revealeda main effect of experience [F (1, 67)=9.01, p=0.004] and amarginal effect of sex [F (1, 67)=3.79, p=0.057] with nointeraction between the two [F (1, 67)=0.20, p=0.658]. TS ratsentered the closed arms less frequently with a more robusteffect in females (Fig. 2).

2.1.3. Novel object recognitionThe rats tended to spend more time with the old compared torecent familiar object, although not significantly. Prenatal TSdid not influence the object temporal order memory. The ratioof time spent with old compared to recent familiar object inthe third test trial, an index of object temporal order memory,when subjected to a two-way ANOVA (experience×sex)revealed no main effect of experience [F (1, 55)=0.57, p=0.45],sex [F (1, 55)=0.005, p=0.944] nor an interaction between thetwo [F (1, 55)=1.31, p=1.31].

2.1.4. Play fightingEarly TS tended to feminize play fighting in males, but hadlittle effect in females. When subjected to two-way ANOVA(experience×sex), the frequency of play attacks revealed amain effect of experience [F (1, 49)=4.4, p=0.040], no maineffect of sex [F (1, 49)=2.78, p=0.102], nor an interactionbetween the two [F (1, 49)=0.99, p=0.324].Prenatal TS ratscompared to controls showed a reduction in the frequency ofplay attacks. The decrease in playful attacks was morepronounced inmales. Themale rats exhibited enhance playfulattacks in the control group but we did not observe anydifference in the number of attacks in the TS group (Fig. 3).

When subjected to two-way ANOVA (experience×sex), theprobability of complete rotation defense revealed no maineffect of experience [F (1, 49)=0.01, p=0.924], sex [F (1, 49)=0.01,p=0.755], nor an interaction between the two [F (1, 49)=0.63,p=0.431]. The probability of evasion in response to an attackwhen subjected to two-way ANOVA (experience× sex)

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Fig. 2 – Means (SEM) of the total number of entries in closedarms of EPM. Prenatal TS females entered less in the closedarms compared to their sex-matched controls (p=0.017). TSmales appeared to be less active however, the effect wasmarginal (p=0.07).

revealed no main effect of experience [F (1, 49) =0.21,p=0.650], sex [F (1, 49)=0.05, p=0.825], nor an interactionbetween the two [F (1, 49)=0.71, p=0.402].

2.2. Amphetamine sensitization

2.2.1. Acute administrationThe locomotor activity on the first day of a 14-day drugadministration period was analyzed to evaluate the effect ofTS to an acute AMPH injection. Because there are known sexdifferences in both open field activity and the effects ofamphetamine on activity (e.g., Forgie and Stewart, 1993), thetwo sexes were analyzed separately. Amphetamine acutelyincreased activity in both sexes, although the effect was largerin females.

A two-way ANOVA (experience×drug) of activity in themale group revealed a main effect of drug (AMPH or saline) [F(1, 31)=56.83, p<0.001], and nomain effect of experience (TS orcontrol) [F (1, 31)=0.95, p=0.336], nor an interaction betweenthe two [F (1, 31)=0.003, p=0.955]. AMPH-treated groups (bothTS and control) exhibited augmented activity compared tosaline-treated groups (see Fig. 4A for activity on the first day).

A two-way ANOVA of locomotor activity in response toacute AMPH-administration in females showed a main effectof drug [F (1, 33)=107.76, p<0.001], a marginal main effect ofexperience [F (1, 33)=3.90, p=0.057], and no interactionbetween the two [F (1, 33)=0.871, p=0.357]. TS in femalesresponded with diminished locomotor activity to acute AMPHadministration. AMPH administration, in both TS and controlgroups, resulted in increased activity compared to theirrespective saline-treated control groups (see Fig. 4B for activityon the first day).

2.2.2. Development of sensitizationLocomotor activity, a standard measure of behavioral sensi-tization in rats, was gradually increased with repeated AMPHadministration. The activities recorded on the first and the lastday of drug administration period were compared to deter-mine the development of sensitization in the 14-day AMPH

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Fig. 4 – Mean (±SEM) locomotor activity (beam crossing×103)recorded for 90 min after saline or AMPH (1 mg/kg)administration over a 14-day period followed by AMPHchallenge in both prior saline- and AMPH-treated rats. PriorAMPH treated rats both males (*, p<0.001) (A) and females(*, p<0.001) (B) compared to their respective saline-treatedgroups exhibited enhanced activity showing persistence ofbehavioral sensitization. TS attenuated AMPH-inducedbehavioral sensitization (all ps<0.05) except for days 1, 2, 5, 7,and 8 in males (A). See the text for the effect of TS on AMPHsensitization in females (B).

56 B R A I N R E S E A R C H 1 4 0 0 ( 2 0 1 1 ) 5 3 – 6 5

administration period. This comparison revealed that AMPHadministration resulted in the development of behavioralsensitization and, in addition, prenatal TS resulted in anattenuated sensitization response compared to control AMPH-administered rats.

MixedANOVAon the first and the last day activities inmalesrevealed amain effect of drug (AMPH or saline) [F (1, 31)=252.10,p<0.001]. AMPH compared to saline administration resulted inaugmentedactivity (i.e., behavioral sensitization)on the last daycompared to the first day in control and TS groups. In contrast,saline administration in both control and TS groups, althoughnot significant, tended to lead to decreased activity on the lastday compared to the first day (see Fig. 4A for the activity on days1 and 14).

Mixed ANOVA of the 14-day activity in males withexperience and drug as independent factors and days asrepeated measure factor revealed a main effect of drug [F (1,

31)=175.86, p<0.001] and experience [F (1, 31)=3.95, p=0.056],with no interaction between the two [F (1,31)=2.87, p=0.10].AMPH compared to saline treatment resulted in enhancedactivity. However, TS compared to control males revealed anattenuated AMPH-induced behavioral sensitization for mostof the days (except days 1, 2, 5, 7, and 8). In contrast, salineadministration did not result in any significant differencesbetween TS and control groups in any of the days (Fig. 4A).

Similar tomales, repeated AMPH administration in femalesresulted in a gradual increase in the locomotor activity (i.e.,behavioral sensitization). The first and the last day activitycomparison revealed a main effect of drug (AMPH or saline) [F(1, 33)=190.07, p<0.001].AMPH administration in femalesresulted in enhanced activity on the last day compared tothe first day (see Fig. 4B for the activity on days 1 and 14).

Mixed ANOVA of 14-day AMPH administration in femalesrevealed a main effect of drug [F (1, 33)=195.65, p<0.001] andno main effect of experience [F (1, 33)=1.16, p=0.289], nor aninteraction between the two [F (1, 33)=0.02, p=0.891]. RepeatedAMPH administration resulted in increased activity in bothcontrol and TS groups. When we looked at the effect of TS onbehavioral sensitization, most of the female rats apparentlyexhibited a blunted sensitization response; however, therewas no significant effect of TS on drug-induced sensitization.Upon close assessment of the data, it was noticed that 3 out of13 rats in the AMPH-treated TS group exhibited hyperactivityeven as juveniles, and which was unrelated to drug sensiti-zation. That is, these 3 rats were outliers throughout thetesting. The 14-day activity data were reanalyzed afterexcluding the hyperactive rats. As anticipated, the analysisrevealed a main effect of experience [F (1, 30)=4.84, p=0.036].TS in females, similar to males, resulted in attenuatedsensitization (Fig. 4B).

2.2.3. Persistence of sensitizationThe persistence of drug-induced sensitization, after a 2-weekwithdrawal period, was determined by challenging both priorsaline- and prior AMPH-treated rats with AMPH administra-tion. Both sexes showed a persistent enhancement in activitywith prior AMPH exposure.

Two-way ANOVA (experience×prior drug treatment) of theactivity recorded on the challenge day in males revealed amain effect of prior drug treatment [F (1, 31)=57.78, p<0.001]and no main effect of experience [F (1, 31)=1.74, p=0.20], noran interaction between the two [F (1, 31)=1.21, p=0.280]. AMPHchallenge resulted in enhanced locomotor activity, exhibitingpersistence of behavioral sensitization in prior AMPH-treatedcompared to prior saline-treated rats. These data appear toshow that prior AMPH-treated TS compared to control ratsexhibited diminished activity in response to a challenge (seeFig. 4A for the activity on the challenge day).

The females rats, in response to AMPH challenge after awithdrawal period, revealed a main effect of prior drugtreatment [F (1, 30)=34.38, p<0.001], and no main effect ofexperience [F (1, 30)=0.01, p=0.938], nor an interactionbetween the two [F (1, 30)=0.12, p=0.730]. Like males, AMPHchallenge in females resulted in augmented locomotor activityexhibiting persistence of behavioral sensitization in priorAMPH-treated compared to prior saline-treated rats (seeFig. 4B for the activity on the challenge day).

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Fig. 6 – Mean (±SEM) of the cortical thickness in (A) Cg 1 and(B) Cg 3 regions of medial PFC and (C) AID region of OFC infemales. TS increased the cortical thickness in the Cg 1(*, p=0.001), Cg 3 (*, p<0.001), and AID regions (*, p=0.009) inAMPH-treated group without any substantial effect onsaline-treated group. In addition, a reduction in thicknesswas observed in AMPH-treated controls in Cg 1 (†, p=0.001),Cg 3 (†, p<0.001), and AID regions (†, p<0.001) however, TSprevented the reduction in all the prefrontal cortical regions.

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2.3. Anatomy

2.3.1. Brain weightTwo-way ANOVA (experience×drug) of brain weights in malesrevealed a main effect of experience (TS or control) [F (1, 31)=10.84, p=0.002] and no main effect of drug (AMPH or saline) [F(1, 31)=0.04, p=0.836], nor an interaction between the two [F (1,31)=0.004, p=0.952]. TS resulted in lighter brainweights inmalerats (Fig. 5).

In contrast to males, neither TS nor AMPH administrationaffected brainweights in females. A two-way ANOVA revealedno main effect of experience [F (1, 33)=1.88, p=0.179], drug [F(1, 33)=0.93, p=0.343] nor an interaction between the two [F (1,33)=0.036, p=0.851].

2.3.2. Cortical thickness in Cg 1AMPH administration in females, but not males, reduced theCg 1 thickness in the control group and this effect was blockedby TS. In addition, TS interacted with AMPH administrationresulting in increased Cg 1 thickness in both sexes whereas nosuch effect was observed in the saline-treated group.

A two-way ANOVA (experience×drug) on the Cg 1 thicknessin females showed no main effect of experience [F (1, 70)=3.12,p=0.082] but a main effect of drug [F (1, 70)=3.93, p=0.051] withan interaction between the two [F (1, 70)=9.64, p=0.003]. Theinteraction reflected the decreased cortical thickness in thecontrol group but not TS group (Fig. 6A). A two-way ANOVA(experience×drug) inmales revealednomaineffect of experience[F (1, 66)=1.51, p=0.224], drug [F (1, 66)=0.44, p=0.507], nor aninteraction between the two [F (1, 66)=2.95, p=0.091]. Inspectionof the data did suggest that AMPH increased cortical thickness inthe TS group.

2.3.3. Cortical thickness in Cg 3As in Cg 1, prenatal TS in females interacted with AMPHadministration: AMPH reduced Cg 3 thickness and this wasblocked by TS. Unlike females, neither early experience nordrug influenced Cg 3 thickness in males.

A two-way ANOVA (experience×drug) of Cg 3 thickness infemales showed a main effect of experience [F (1, 70)=5.03,p=0.028], drug [F (1, 70)=16.58, p<0.001], and an interactionbetween the two [F (1, 70)=7.74, p=0.007]. The interactionreflected the AMPH-induced decrease in thickness that wasblocked by the prenatal TS (Fig. 6B). A two-way ANOVA(experience×drug) in males revealed no main effect of

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Fig. 5 – Mean (±SEM) brain weight (in grams). Prenatal TSresulted in lighter brain weights in both saline- (*, p=0.033)and AMPH-treated male groups (*, p=0.020).

experience [F (1, 66)=1.19, p=0.280], drug [F (1, 66)=1.61,p=0.209], nor an interaction between the two [F (1, 66)=0.711,p=0.402].

2.3.4. Cortical thickness in AIDAs in themedial regions, AID thickness was reduced in femalesby AMPH, and this effect was blocked by TS. In contrast tofemales, males did not show a substantial alteration in the AIDthickness associated with early experience or drug.

The AID thickness in females, when subjected to a two-wayANOVA (experience×drug), revealed no main effect of expe-rience [F (1, 70)=0.18, p=0.675] but there was a main effect ofdrug [F (1, 70)=6.72, p=0.012] with an interaction between the

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two [F (1, 70)=12.69, p=0.001]. Once again, the interactionreflected the blockade of the AMPH-related drop in thickness(Fig. 6C). Dorsal agranular insular cortical thickness data inmales,whensubjected toa two-wayANOVA (experience×drug),revealed no main effect of experience [F (1, 66)=0.02, p=0.875],drug [F (1, 66)=1.50, p=0.225], nor an interaction between thetwo [F (1, 66)=0.47, p=0.497].

2.3.5. Striatum sizePrenatal TS resulted in a larger anterior striatum in bothsaline- and AMPH-treated male and female groups. TSincreased posterior striatal size in saline-treated males andAMPH-treated females. Repeated AMPH administration en-larged the posterior striatum in male controls, a result thatwas prevented by TS.

A two-way ANOVA (experience×drug) of the anteriorstriatum in males revealed a main effect of experience [F (1,66)=24.61, p<0.001] with no main effect of drug [F (1, 66)=0.76,p=0.385], nor an interaction between the two [F (1, 66)=0.005,p=0.944]. Prenatal TS resulted in larger anterior striatum inmales (Fig. 7A). When subjected to a two-way ANOVA (experi-ence×drug), the posterior striatum in males revealed a maineffect of experience [F (1, 66)=4.05, p=0.048], and nomain effectof drug [F (1, 66)=1.45, p=0.233], nor an interaction between thetwo [F (1, 66)=3.43, p=0.068]. TS in saline-treated rats resulted inlarger posterior striatum (p=0.012) but AMPH treatment pre-

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Fig. 7 – Mean (±SEM) of anterior (Bregma,~1.7 mm) andposterior striatum (Bregma,~0.2 mm). Prenatal TS resultedin larger anterior striatum in the saline- and AMPH-treatedTS groups in males (*, p=0. 043 and 0.015, respectively) andfemales (*, p=0. 009 and *, p<0.001, respectively). In contrast,the posterior striatum was enlarged by TS in saline-treatedmales (*, p=0.012). TS in females increased the posteriorstriatum size in AMPH-treated females (*, p=0.035).Furthermore, AMPH compared to saline administration incontrols resulted in enlarged posterior striatum (†, p=0.039)but TS prevented the AMPH-induced striatal enlargement.

vented the TS-dependent increase in size. Furthermore, AMPHcompared to saline administration in controls resulted inenlarged striatum (p=0.039) and TS alleviated the AMPH-induced striatal enlargement effect in controls (Fig. 7B).

A two-way ANOVA (experience×drug) of the anteriorstriatum in females revealed a main effect of experience [F (1,70)=19.99, p<0.001] with no main effect of drug [F (1, 70)=3.13,p=0.081], nor an interaction between the two [F (1, 70)=0.25,p=0.616]. Prenatal TS compared to the respective control groupresulted in an enlarged striatum in females (Fig. 7A).Whensubjected to a two-way ANOVA (experience×drug), the poste-rior striatum in females revealednomain effect of experience [F(1, 68)=0.43, p=0.511] nor drug [F (1, 68)=0.32, p=0.570] but therewas an interaction between the two [F (1, 68)=5.00, p=0.029]. TScompared to controls resulted in larger posterior striatum inAMPH-treated group (p=0.035) without affecting the saline-treated females. Furthermore, AMPH compared to salineadministration in TS group resulted in larger posterior striatum(p=0.041), with no effect on the control group (Fig. 7B).

3. Discussion

Tactile stimulation during prenatal brain development alteredbrain and behavioral development in rats. TS effectivelymodulated juvenile exploratory and social behaviors, andattenuated adult AMPH-induced behavioral sensitization. Inaddition, TS in interaction with AMPH altered prefrontalcortical thickness and striatum size, brain regions implicatedin drug addiction. The structural alterations in the brainhowever, were sex dependent.

3.1. Behavior

Juvenile rats that received prenatal TSwere tested in a numberof behavioral tasks to investigate the effect of early TSexperience on exploratory, emotional, cognitive, and socialbehaviors. The tasks included open field locomotion, elevatedplus maze, novel object recognition, and play fightingbehavior. Prenatal TS enhanced the exploratory behavior,tested as open field locomotion, which was more robust infemales (Maruoka et al., 2009). However, reduced explorationwas observed in the EPM, tested as the number of entries inclosed arms, in bothmale and female. The contrary results forexploratory behavior in open field and EPM points to theimportance of context of testing paradigm (e.g., EPM iscomparatively more anxiogenic) that may have an adaptiverole in exploratory behavior. The time spent in closed arms ofthe EPM, used as a measure of anxiety-like behavior, was notaffected by TS in both male and female groups (Cirulli et al.,2010). However, the findings for females are not consistentwith that of Maruoka et al. (2009) who reported reducedanxiety-like behavior in mice raised in enriched environmentand tested in open field. The difference could be due toprocedural differences such as the experience paradigm,animal species, and age at testing.

Play fighting behavior, comprised of a series of attacks anddefense generated in response to an attack, was modifieddifferentially by prenatal TS. Prenatal TS modulated juvenile

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play differently in male and female rats. Male rats showedenhanced playful attacks in the control group (Pellis and Pellis,1990). However, prenatal TS in males resulted in female-likeplay behavior with reduced number of playful attacks.Previous studies related to early experience manipulations(e.g., prenatal stress or maternal licking and grooming)reported sex-dependent alterations in social behavior withpronounced influence on males (Parent and Meaney, 2008;Ward and Stehm, 1991). The strategy to face (i.e., completerotation defense) or evade a play attack was not altered byearly TS experience. Arnold and Siviy (2002) also reportednegligible influence of early handling experience on defensestrategy.

The effects of prenatal TS or enrichment experience onbrain development are little studied to date. Consequently, itis hard to speculate about possible mechanisms involved inmodulating the exploratory, cognitive, and social behaviorsexamined in the current study. But previous reports related toprenatal experience in general associate the alteration in brainand behavior with the modulation of neurotrophic factors,crucial in early brain development (Riva and Mocchetti, 1991),such as BDNF (Maier et al., 1999) and NGF (Fiore et al., 2009). Inaddition, the PFC is altered structurally and functionally (Luet al., 2009) by early experiences and is a key player inmodulating behaviors such as play behavior (Bell et al., 2010).

There is an obvious dissociation between male and femalegroups in juvenile behavior, which points to the likelihood ofsome sort of early experience-dependent differences. It ishypothesized that experience during development has differ-ential sex-specific influence in rodents that could possibly belinked to gonadal hormones and associated receptors. Forexample, previous reports described that only prenatallystressed females exhibited enhanced anxiety-like behaviorwhereas only prenatally stressed males were impaired inspatial learning (Zagron and Weinstock, 2006). Similarly, thesex-specific differences in behavior associated with earlyexperience have been correlated with epigenetic alteration inthe brain (e.g., estrogen receptors methylation) (McCarthyet al., 2009).

3.2. Amphetamine sensitization

Repeated amphetamine administration in rats produces agradual increase in locomotor activity (i.e., behavioral sensi-tization) in experimental animals (Kalivas and Stewart, 1991).In contrast, saline administration over time in rats reduces theactivity that could be the result of habituation to the testingenvironment. In addition, the behavioral sensitization alsopersists after months and even years of withdrawal period inrodents (Kolb et al., 2003b) and monkeys (Castner and Gold-man-Rakic, 1999), respectively. We manipulated prenatalexperience by exposing rats to TS, which resulted in attenu-ated sensitization response to acute as well as chronic AMPHadministration. The AMPH-induced attenuated sensitizationin males was very conspicuous; however, TS females did notexhibit a marked attenuation because of three very hyperac-tive rats. The hyperactivity was unrelated to drug-inducedsensitization because these rats were hyperactive from thefirst day of drug administration period and even as juveniles(Fig. 1). When the hyperactive rats were excluded from the

analysis, as anticipated the females showed an attenuatedAMPH-induced behavioral sensitization.

There is no report to our knowledge that has studied theinfluence of prenatal stimulation or enrichment on drug-induced behavioral sensitization. However, we previouslyreported comparable findings, where postnatal TS resultedin attenuated AMPH-induced sensitization in both male andfemale rats (Muhammad et al., 2011). The attenuation of drug-induced behavioral sensitization exhibited by pre- andpostnatal TS rats demonstrates the enrichment effect ofearly stimulation experience.

Repeated stimulant administration results in drug-inducedbehavioral sensitization but the degree of sensitization isexperience dependent. For example, stress during develop-ment has been correlated with augmented psychomotorstimulant sensitization later in life (Deminiere et al., 1992).Although there are published studies on the influence ofprenatal adverse experience on drug sensitization, we areunaware of any published reports that have investigated therelationship of prenatal enrichment or stimulation and drug-induced behavioral sensitization. Based upon earlier ‘enrich-ment’ studies of postnatal brain development such as earlyenvironmental enrichment (Bardo et al., 1995) or maternallicking and grooming (Francis and Kuhar, 2008), whichresulted in blunted drug-induced sensitization, we mayconjecture that the effects of prenatal TS are similar in nature.

Brain during prenatal period goes through different stages ofdevelopment such as neurogenesis, proliferation, synaptogen-esis, and apoptosis. Exposure to any experience would poten-tially alter the rate or degreeof braindevelopment,whichwill bereflected at the behavioral level later in life. The possiblemechanisms of how experience might alter brain and behaviorcould be via modulation of neurotrophic factors and neuro-transmitters. For example, maternal stimulation through exer-cise during pregnancy was associated with enhancedhippocampal neurogenesis and increased BDNF production inrat pups (Lee et al., 2006). Similarly, prenatal stress modifiedcatecholamine transmission in the PFC, whichmight serve as acontributing factor in vulnerability to drug addiction (Carboni etal., 2010). The attenuated drug-induced sensitization resultingfrom prenatal TSmight be the result of epigenetic alterations inbrain regions associated with drug sensitization. For instance,TS (Bear et al., 2006) up regulates glucocorticoid receptors in thehippocampus and the PFC, key regions in theHPA axis feedbackmechanism (Sapolsky et al., 1984). Intracerebroventricularadministration of the corticotropin releasing factor in therodents on the other hand are positively correlated withincreased sensitivity to the effects of amphetamine (Cadoret al., 1993). Nevertheless, themodulation of behavior anddrug-induced behavioral sensitization need further investigation topin point the possible mechanism(s).

3.3. Anatomy

3.3.1. Prefrontal cortexWe investigated the interaction of experience (i.e., TS) anddrug (i.e., AMPH) on the structural plasticity of the PFC. Kolband associates previously reported that psychostimulantadministration followed by rearing experience in an enrichedenvironment blocked the structural alterations associated

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with environmental enrichment (Hamilton and Kolb, 2005;Kolb et al., 2003b). We used cortical thickness measurement toinvestigate structural alteration in the PFC subregions (i.e.,mPFC and OFC) associated with early TS experience and laterAMPH exposure.

Our findings suggest that prenatal TS experience interactedwith later AMPH exposure to modulate cortical thickness inthe PFC subregions. However, the structural alteration wasmore pronounced in females. Whereas AMPH reduced theprefrontal cortical thickness in control females, prenatal TSprevented the drug-induced reduction in the cortical thick-ness. We did not observe any substantial alteration inprefrontal cortical thickness in control males, which isconsistent with the findings of Bartzokis' et al. (2000) studyof frontal lobe volume in human male addicts and controls.

The prefrontal cortex of the rat is divided in various regionsbased on cytoarchitectonic features. Following the terminologydescribed by Zilles (1985), Cg 1 and Cg 3 regions of anteriorcingulate cortex, form part of the medial PFC whereas AID, aregion of the insular cortex, forms part of the OFC in rats. Boththe PFC subregions (i.e., medial and OFC) play a vital role in thedevelopment of drug addiction in experimental animals(reviewed by Porrino and Lyons, 2000) and humans (reviewedby Volkow et al., 2003). In addition to drug-induced impairmentof PFC functions (Baron et al., 1998), altered structural plasticity(e.g., spine density) has also been reported in experimentalanimals (Robinson and Kolb, 2004; Selemon et al., 2007).Although there are no, or at best limited, work related to theprefrontal cortical thickness inexperimentalanimals, structuralalterations have been extensively studied in human patientsthrough the application of imaging technology (Bartzokis et al.,2000; Kim et al., 2006; Lawyer et al., 2010).

We observed that repeated AMPH administration reducedthe cortical thickness in themPFC and the OFC regions but onlyin control females. Structural alteration in the cortical regions inhuman studied through imaging techniques supports ourfindings. A decrease in the cortical gray matter density orvolume in the anterior prefrontal cortex (Kim et al., 2006;Schwartz et al., 2010) and medial orbital cortex (Tanabe et al.,2009) was reported in chronic AMPH or methamphetamineabusers. Other PFC-related neurological disorders, such asschizophrenia, similar to drug addiction, also reduce the frontalcortical thickness (Kuperberg et al., 2003; Rimol et al., 2010).

The drug-induced reduction in the prefrontal corticalthickness observed in control rats, however, was preventedby prenatal TS experience: AMPH compared to saline admin-istration did not alter the cortical thickness. In addition, themedial prefrontal cortical thickness was not affected in thesaline-treated TS rats but AMPH administration increased thethickness in this region. We observed interesting findings inthe AID region, where a reduction in the cortical thicknesswasobserved in the saline-treated TS females. In contrast, AMPHadministration in the same group resulted in increased AIDthickness. Sensory stimulation (e.g., environmental enrich-ment) appears to prevent the decrease in cortical thicknessassociated with an insult, for instance, neonatal brain injury(Comeau et al., 2008) or early social isolation stress (Hellemanset al., 2004). We believe that the prevention of drug-inducedreduction in the prefrontal cortical thickness by TS could bethe protective mechanism that resulted in attenuated AMPH-

induced behavioral sensitization observed in our study.Further studies are needed to expand the mechanistic (e.g.,epigenetic or molecular) details of the enrichment experienceand drug associated structural alterations in the PFC.

3.3.2. StriatumThe striatum has been associated with drug addiction inlaboratory animals and human patients (Gerdeman et al.,2003; Ito et al., 2002; Li et al., 2003; Volkow et al., 2003). Similarto the prefrontal cortical alteration, we observed sex-depen-dent modulation of the striatum size. But unlike the PFC,where the reduction in the cortical thickness was observedonly in control females, AMPH administration increased thesize of the posterior striatum but only in control males. Thechange in the striatum size has also been reported in humandrug addicts. For example, MRI studies related to the alterationin the striatal morphology in chronic cocaine abusers showedan increase in the striatal volume (Jacobsen et al., 2001). Thedrug-induced enlargement in striatum size could be attributedto the augmented dopamine release in the striatum (Robinsonand Becker, 1982).

Interestingly, TS prevented the drug-induced increase inthe posterior striatum observed in control rats. In addition, TSincreased the size of anterior striatum regardless of sex ordrug exposure. In contrast, the increase in the posteriorstriatum was sex and drug dependent. An increase wasobserved in saline-treated male rats with no effect in theAMPH-treated TS group, whereas AMPH-administration in-creased the posterior striatum size in TS females withoutaffecting the saline-treated females. Similar to our investiga-tion of experience- and drug-associated metaplasticity atstructural levels, researchers also studied the same phenom-enon at molecular levels. Levels of the basic fibroblast factor(FGF-2), a protein associated with neuronal plasticity, havebeen increased in stress (Riva et al., 1995) and psychostimu-lants administration (Fumagalli et al., 2006) in various brainregions. However, chronic stress exposure interacted withcocaine administration in region-dependent modulation ofFGF-2 in the PFC and the striatum (Fumagalli et al., 2008).

In summary, prenatal tactile stimulation in rats modulatedsocial behavior with diminished playful attacks resulting in thefeminization of male play fighting behavior. Exploration in theopen field, anxiety-like behaviors, and cognitive performancewere not affected. Repeated amphetamine administrationgradually increased the locomotor activity resulting in thedevelopment of behavioral sensitization that persisted at leastfor 2 weeks. Prenatal tactile stimulation, however, blunted thedrug-induced behavioral sensitization. Furthermore, prenatalTS modulated the cortical thickness and striatum size in asexually dimorphicmanner. TS increased the cortical thicknessin the subregions of the prefrontal cortex in the AMPH-treatedfemaleswithout any substantial effect onmales. In addition, anoverall reduction in the prefrontal cortical thickness wasobserved in AMPH-treated controls but TS prevented thereduction in cortical thickness. Prenatal TS resulted in a largeranterior striatum regardless of drug exposure and sex. But theposterior striatum showed enlargement depending on theexperience, drug, and sex. TS increased the posterior striatalsize only in saline-treated males but, in contrast, an increasewas observed only in AMPH-treated females. Furthermore,

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repeated AMPH administration in control males resulted inenlarged posterior striatum but TS prevented the AMPH-induced striatal enlargement.

The present study highlighted the role of a favorableexperience during prenatal brain development in modulatingthe subsequent response to drug-induced behavioral sensiti-zation and associated anatomical changes in the brain. Theattenuated behavioral sensitization as a result of earlyfavorable experience (i.e., TS) might be related to theprevention of drug-induced structural reorganization of thePFC and the striatum and therefore, may play a protective roleagainst stimulant-induced behavioral sensitization. The pre-sent findings have practical implication for child developmentpractices as it underpins the importance of early interventionin brain development that starts with a successful conception(or even earlier than that). Massage in women duringpregnancy, therefore, could potentially play a protective rolein the prevention of drug abuse, which is hard to treat later inlife because of limited plasticity in the brain.

4. Experimental procedures

4.1. Animals

Long Evans dams received tactile stimulation while pregnant,the pups born were randomly selected with not more than twopups of each sex from the same litter. Male (control: 16; TS: 19)and female (control: 16; TS: 21) ratswereused in theexperiment.The number of rats run in the behavioral tasks varies owing totechnical problems, such as, video filming or inadvertentmoving of one of the objects from its original position in theNORtask.The control ratswerealsoused inanotherexperiment(Muhammad et al., 2011). The pupswere housed in the breedingcolony with their respective dams at the Canadian Centre forBehavioral Neuroscience (CCBN), University of Lethbridge,Alberta, Canada. At weaning, prenatal TS rats were housed instandard shoe-box cages with same sex in a group of two in atemperature- andhumidity-controlled room.Rat chowfoodandwater were provided ad lib. The rats were left undisturbedexcept for regular cage cleaning, running behavioral tasks, andamphetamine (AMPH) administration.

4.2. Tactile stimulation

Long Evans female rats, raised in the breeding colony at ourfacility, received tactile stimulation (TS) throughout pregnancystarting a week before being paired with males. Briefly, femalerats were transported in a box with new bedding to a separateroom. Individual rats were placed in the experimenter's lap andgently stroked with a baby hairbrush by stroking from the neckto the lower back. The procedure was carried out for 15minthree times a day, approximately the same time and by thesame experimenter. The control dams were transported alongthe TS dam to the separate room but were not subjected to TSprocedure. Once born, the pups and its mother were leftundisturbed (aside from animal care maintenance) until thecommencement of behavioral testing.

4.3. Behavior

The effect of early TS on juvenile behavior was investigated bytesting the rats between postnatal (P) 30–40 in a battery ofbehavioral tasks. The tests included open field locomotion,elevated plus maze (EPM), novel object recognition (NOR)-recency discrimination version, and play fighting behavior.

4.3.1. Open field locomotionExploratory behavior of the rats was evaluated as open fieldlocomotion, recorded for 10 min using Accuscan activitymonitoring Plexiglas boxes (L 42 cm, W 42 cm, H 30 cm). Theactivitywas recorded as the number of sensors beam breaks inthe boxes attached to a computer. The horizontal beambreaks, used as an index of locomotor activity, were recordedon the computer with VersaMax™ program and converted tospreadsheet using VersaDat™ software (AccuScan Instru-ments, Inc., Columbus, OH).

4.3.2. Elevated plus mazeThe EPM, a ‘+’ shapemazewith two closed and two open arms,was used to test anxiety-like behavior exhibited by the rats.The length of each arm of the maze measured 113 cm with awidth of 10 cm while the maze was elevated 88 cm above theground. Rats were placed in the center of the maze facing aclosed arm and were allowed to explore the maze for 5 min.Exploration behavior was videotaped with a camera installedin such a way as to spot both open arms. The time spent inclosed arms and the number of entries in each open andclosed arm were scored and analyzed to assess anxiety-likeand exploratory behaviors, respectively.

4.3.3. Novel object recognitionNovel object recognition was carried out to evaluate explora-tion of novel object as well as exploration of objects intemporal order. Rats were habituated to a Plexiglas box for15–20 min for 4 days prior to the commencement of the testingsessions. The NOR taskwas comprised of three trials followingthe procedure with minor modification described elsewhere(Hannesson et al., 2004; Mitchell and Laiacona, 1998). Briefly,rats were allowed to explore objects and taped with a videocamera. Glass candle holders, with similar sizes but differentshapes and colors, were used as objects. During the firsttraining trial, a rat was exposed to 2 novel but similar objectsfor a 4-minute period. Followed by a 60 min delay, the rat wasagain exposed to 2 new objects, again similar but differentobjects from the first trial. After an additional 60-minutedelay, the rat was exposed to 1 object each from the first andsecond trial, termed as ‘old’ and ‘recent’ familiar objects,respectively. The objects and testing area were cleaned with30% alcohol between each trial for disinfection and odorremoval. Rats were transported back to their home cages inthe 60-minute delay between the trials.

Exploration of each novel object in the first two trials and‘old’ and ‘recent’ familiar objects in the third trial was scored.The ratio of time spent with ‘old’ familiar object wascalculated as the difference between times spent with ‘old’and ‘recent’ familiar object divided by the total time exploringboth objects [i.e., (old−recent)/(old+recent)] (Hannesson et al.,2004).

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4.3.4. Play fightingJuvenile rats were allowed to play in pairs to assess the effectof TS on social behavior. TS and control rats were housed inpairs as playmates for a period of about 2 weeks. Theplaymates, as juveniles, were habituated to a play box(50 cm×50 cm×50 cm) for about 30 min for 3 days. For playdeprivation before testing, rats were housed individually in anisolation room for 24 h at the end of habituation period. Theplay behavior was recorded for 10 min with a night shotcamera. On testing day both playmates were color marked onthe tail with two separate paints to make them identifiable inthe video recording, which was shot in the dark. Rats weretransported to their home cage after the play session and pair-housed for the rest of the experiment. The video recording,analyzed frame by frame, was scored for attacks, andcomplete rotation defense or evasion generated in responseto an attack (Pellis and Pellis, 1990). The defense was scored as‘complete rotation’ when a rat had both fore- and hind limbsin the air while lying in a supine position. If the rat turnedaway from the attacker instead of facing an attack, it wasscored as evasion. The probability of complete rotation orevasion was calculated as the number of complete rotation orevasion divided by the total number of attacks carried out bythe playmate (Pellis and Pellis, 1990).

4.4. Amphetamine sensitization

4.4.1. Amphetamine administrationTo see the effects of prenatal TS on psychomotor stimulantbehavioral sensitization, adult rats (P80) were administeredwith D-amphetamine sulfate (Sigma Aldrich, St. Louis, MO,USA) dissolved in sterile 0.9% saline. Locomotor activity, usedas an index of behavioral sensitization (Wise and Bozarth,1987), was recorded using Accuscan activity monitoringsystem, comprised of Plexiglas boxes (L 42 cm, W 42 cm, H30 cm) connected to a computer. The rats were habituated tothe activity boxes for 30 min followed by AMPH (1 mg/kg bodyweight, i.p.) or 0.9% saline administration, both at a volume of1 ml/kg. Rats were immediately placed back in the activityboxes post injections and the activity was recorded for 90 min.The drugwas administered once a day for 14 consecutive days,approximately the same time every day. Locomotor activityrecorded on a computer with VersaMax™ program wasconverted to spreadsheet using VersaDat™ software (AccuS-can Instruments, Inc., Columbus, OH). The rats were returnedback to their home cages each day after the end of AMPH thetesting session. The development of sensitization was deter-mined by analyzing activity recorded over a 14-day periodusing mixed ANOVA with experience (TS or control) and drug(AMPH or saline) as independent factors, and day (1–14 days)

AMPH administration

Behavioral tasks (P30-40)

TS (-7 to 21 ED)

Fig. 8 – A timeline of the experiment. Rats received tactile stimbefore conception till birth. As juvenile, rats were run in a batteryadministration. The rats were challenged with AMPH after a wit

as a repeated measure factor, followed by Bonferroni's posthoc test for multiple comparisons.

4.4.2. ChallengeThe rats were given a withdrawal period of 2 weeks after AMPHadministration period, followed by a challenge with AMPH(1mg/kg, i.p.) given to both prior AMPH- andprior saline-treatedrats. Locomotor activity was recorded in activity monitoringboxes similar to the development of AMPH sensitizationprocedure described above. All rats were challenged withAMPH to see the persistence of behavioral sensitization inprior AMPH-treated rats compared to prior saline-treated rats.Please refer to Fig. 8 for the timeline of the experiment.

4.5. Anatomy

4.5.1. Perfusion and stainingApproximately 24 h post AMPH challenge, the rats were givenan overdose of sodiumpentobarbital solution i.p. and perfusedwith 0.9% saline solution intracardially. The brains removedfrom the skull were trimmed by cutting the olfactory bulb,optic nerves and spinal cord. The brains were then weighedand preserved in Golgi–Cox solution for 14 days followed bytransfer to a 30% sucrose solution at least for 3 days. Thebrainswere sliced at a thickness of 200 μmon a Vibratome andfixed on gelatinized slides. The slides mounted with brainsections were processed for Golgi–Cox staining, following theprotocol described by Gibb and Kolb (1998). Unfortunately,owing to unforeseen technical problems, the Golgi stainingproved unreliable so the tissue was used for a grossermeasureof cortical change, namely cortical thickness and striatalcross-sectional area.

4.5.2. Prefrontal cortical thicknessGolgi–Cox stained coronal sections were used to measureprefrontal cortical thickness following the procedure, withminor modification, described by Stewart and Kolb (1988).Briefly, the coronal sections containing slides were mountedon a magnifying projector at a magnification of 17.5×. Themeasurement was carried out in three regions; Cg 1 and Cg 3regions of anterior cingulate of the medial prefrontal cortex,and dorsal agranular insular cortex (AID) region of the orbitalfrontal cortex described by Zilles (1985). The cortical thickness,measured with a metric ruler on a petrographic projector, wasanalyzed for hemispheric difference and the data werecollapsed in the absence of any difference.

4.5.3. Striatum sizeThe striatal area was measured following the proceduredescribed elsewhere (Kolb et al., 1983) withminor modification.

(P80-93) Perfusion (P109)

AMPH challenge (P108)

ulation during embryonic development (ED) starting a weekof behavioral tasks followed by chronic amphetamine (AMPH)hdrawal period of two weeks and perfused the next day.

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Briefly, digital images were taken from Golgi–Cox stainedcoronal brain sections from the anterior (Bregma, ~1.7 mm)and posterior (Bregma, ~0.2 mm) of the striatum. The totalstriatal area was measured using NIH Image software in bothanterior and posterior planes.

Acknowledgments

This researchwas supported by NSERC of Canada grants to BK.The authors thank Dawn Danka, Shakhawat Hossain, IvyZuidhof, and Barbara Medland for their help in running theexperiments.

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