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Behavioural Brain Research 273 (2014) 139–143

Contents lists available at ScienceDirect

Behavioural Brain Research

jou rn al hom epage: www.elsev ier .com/ locate /bbr

hort communication

ex differences in adolescent methylphenidate sensitization:ffects on glial cell-derived neurotrophic factor and brain-derivedeurotrophic factor

oss L. Roeding, Marla K. Perna, Elizabeth D. Cummins, Daniel J. Peterson,atthew I. Palmatier, Russell W. Brown ∗

epartment of Psychology East Tennessee State University, Johnson City, TN 37614, USA

i g h l i g h t s

Adolescent females demonstrated enhanced sensitization to methyphenidate (MPH).Only adolescent females demonstrated MPH conditioned hyperactivity.MPH increased striatal and accumbal glial-cell derived neurotrophic factor (GDNF).MPH increased striatal brain-derived neurotrophic factor (BDNF) in males.Increases in GDNF and BDNF were predicted by behavioral observations.

r t i c l e i n f o

rticle history:eceived 10 April 2014eceived in revised form 3 July 2014ccepted 8 July 2014vailable online 15 July 2014

eywords:dolescenceethylphenidate

ensitizationrain-derived neurotrophic factorlial-cell derived neurotrophic factor

a b s t r a c t

This study analyzed sex differences in methylphenidate (MPH) sensitization and corresponding changesin glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotprhic factor protein (BDNF)in adolescent male and female rats. After habituation to a locomotor arena, animals were sensitized toMPH (5 mg/kg) or saline from postnatal day (P) 33–49, tested every second day. On P50, one group ofanimals were injected with saline and behavior assessed for conditioned hyperactivity. Brain tissue washarvested on P51 and analyzed for GDNF protein. A second group of animals was also sensitized to MPHfrom P33 to 49, and expression of behavioral sensitization was analyzed on a challenge given at P60, andBDNF protein analyzed at P61. Females demonstrated more robust sensitization to MPH than males, butonly females given MPH during sensitization demonstrated conditioned hyperactivity. Interestingly, MPHresulted in a significant increase in striatal and accumbal GDNF with no sex differences revealed. Resultsof the challenge revealed that females sensitized and challenged with MPH demonstrated increasedactivity compared to all other groups. Regarding BDNF, only males given MPH demonstrated an increase in

dorsal striatum, whereas MPH increased accumbal BDNF with no sex differences revealed. A hierarchicalregression analysis revealed that behavioral sensitization and the conditioned hyperactivity test werereliable predictors of striatal and accumbal GDNF, whereas sensitization and activity on the challengewere reliable predictors of accumbal BDNF, but had no relationship to striatal BDNF. These data haveimplications for the role of MPH in addiction and dopamine system plasticity.

Abbreviations: MPH, Methyphenidate; BDNF, Brain-derived neurotrohpic factor;DNF, Glial cell-derived neurotrophic factor.∗ Corresponding author at: Department of Psychology East Tennessee State Uni-ersity 309 Stout Drive, Box 70649, Johnson City, TN 37614, USA.el.: +1 423 439 5863/5694; fax: +1 423 439 5695.

E-mail address: brown1@mail.etsu.edu (R.W. Brown).

ttp://dx.doi.org/10.1016/j.bbr.2014.07.014166-4328/© 2014 Published by Elsevier B.V.

© 2014 Published by Elsevier B.V.

The most commonly prescribed psychostimulant for the treat-ment of ADHD is methylphenidate (MPH) [1]. MPH has potentialfor abuse due to its effects on reward pathways in the brain, whichhas been primarily attributed to MPH inhibition of dopamine (DA)reuptake, similar to cocaine [2]. Reports of MPH misuse in youthrevealed a seven-fold increase in frequency from 1993 to 1999 [3],

and it is been recently estimated that at least 5% of college studentshave used MPH for non-medical use [4].

Behavioral sensitization to psychostimulants is mediated byan increase of dopaminergic activity in the striatum and nucleus

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ccumbens [5]. However, psychostimulant sensitization has alsoeen shown to result in increases of neurotrophic factors in brainreas mediating drug reinforcement, which play a role in mediatingnderlying synaptic changes in these same brain areas. For exam-le, recent work has shown that brain-derived neurotrophic factorBDNF) enhances sensitization or is increased after sensitizationo several different psychostimulants, including methylphenidate6], cocaine [7] and amphetamine [8]. BDNF is in a family of pro-eins known as the neurotrophins that are involved in neuronalurvival, development, and function of neurons [9], and is locatedhroughout the brain. Our laboratory has recently shown that brainissue analyzed 24 h after MPH behavioral sensitization in adoles-ence results in significant increases of striatal BDNF in adolescentale rats, but a significant decrease in striatal BDNF in adoles-

ent female rats 1 day post-drug treatment [6]. Interestingly, thereere no changes in BDNF due to MPH sensitization in the nucleus

ccumbens. Another neurotrophin of interest is glial-cell derivedeurotrophic factor (GDNF), which primarily supports dopamineeurons in the brain and is in relatively high concentrations inopamine terminal areas [10]. Interestingly, there have been notudies to analyze the effects of psychosimulant sensitization onDNF, and the only work on GDNF concerned with psychostimu-

ant addiction has shown long-term increases of GDNF after cocaineelf-administration [11,12].

This study analyzed MPH sensitization (5 mg/kg) and its effectsn BDNF and GDNF in adolescent male and female rats. However,e tested two different groups of animals after induction of sensi-

ization (from postnatal day 33 to 49) on different tasks. One groupf animals was administered a drug-free conditioned hyperactiv-ty test 1 day post-sensitization at postnatal day (P)50, and brainissue analyzed for GDNF at postnatal day P51. A second group ofnimals also sensitized to MPH analyzed expression of MPH behav-oral sensitization on a challenge given at P60 with BDNF analyzedt P61. Neurotrophic factors were analyzed in the dorsal striatumnd nucleus accumbens, two brain areas known to play importantoles in behavioral sensitization [13] as well as addiction [14]. Inddition, sex differences were analyzed in sensitization as well asn the neurotrophic factor response to MPH. The rationale to ana-yze sex differences is based on our past work and others [6,15]hat reported robust sex differences in both MPH sensitization andtriatal BDNF at P50 24 h after the final MPH administration, asell as a number of studies which have shown robust sex differ-

nces in psychostimulant sensitization [for review, see Becker [16])nd sex differences in the development of the dopamine system indolescence [17].

Subjects were male and female Sprague–Dawley rats orderedrom Harlan, Inc. (Indianapolis, IN) that were 21 days of age (P21)pon arrival and socially housed 2–3 per cage. All rats were raisedo P30 when behavioral testing began. A total of 42 males and 40emales were used in this experiment, resulting in 8–9 animals perroup. All animals were housed in an Association for the Assess-ent and Accreditation of Laboratory Animal Care (AALAC) fully

ccredited animal colony with food and water available ad libitum,nd all animals were maintained on a 12-h light:12-h dark on/offycle. All procedures were approved by the East Tennessee Stateniversity Committee on Animal Care which is in compliance with

he NIH Guide on Care and Use of Animals.Animals were behaviorally tested in a locomotor arena that was

square sealed wooden box measuring 30 cm on a side and paintedat black. Behavioral testing was recorded using an automatedomputer program (Any Maze, Stoelting, Wood Dale, IL). The com-uter program superimposes a grid of lines on the arena, and every

nstance the animal crosses each of these lines is counted as a hor-zontal activity count. Animals were habituated to the locomotorrena, 30 min a day for 3 consecutive days from P30 to 32. For habit-ation, animals were administered ip saline 10 min before being

Research 273 (2014) 139–143

placed into the arena. Adolescent drug treatment began on P33,and rats received either saline or a 5 mg/kg dose of MPH (ip) 10 minbefore being placed into the arena, and behavior was recorded fora 30 min testing period. All animals were tested every second dayfor a total nine days of testing from P33 to 49. The age range of dos-ing corresponds to early to mid-adolescence in the rat [18,19]. Therationale for using an every second day drug treatment and testingmethodology follows the paradigm of Pierce and Kalivas [13].

There were two different groups of animals tested on differentbehavioral tests after sensitization was complete. For one group ofanimals, on P50, a drug-free conditioned hyperactivity behavioraltest was administered, and all animals were given an ip injection ofsaline 10 min before being placed into the arena. A separate groupof animals was ip administered saline and behaviorally tested fromP33 to 49 and given a challenge to MPH at P60. The rationale for thechallenge being given at P60 was two-fold. First, 60 days of age in arat is considered to be early adulthood, and one focus was to analyzehow adolescent MPH exposure may affect the behavioral responseto a challenge dose of MPH administered to an adult. Secondly, 60days of age is 11 days after the last MPH administration, exceedingthe 7-day period of abstinence as suggested by Pierce and Kalivas[13] as an appropriate test of sensitization expression.

Sex, adolescent drug treatment, and day of testing served asfactors, and horizontal activity counts served as the behavioraldependent measure. GDNF and BDNF protein was analyzed using anELISA kit ordered from Promega, Inc. (Madison, WI). Group codeswere as follows for sensitization and conditioned hyperactivity:Males or females administered saline throughout testing (MS, FS);males or females administered MPH throughout testing but testedon the conditioned hyperactivity test (MM, FM). For the challenge,group codes were as follows: males or females administered MPHduring sensitization and the challenge (MMM, FMM); males orfemales administered saline during sensitization and given MPHon the challenge at P60 (MSM, FSM); males or females given salinethroughout sensitization and the challenge (MSS, FSS). A three-wayANOVA was used as the primary statistic for behavioral sensi-tization and a two-way ANOVA was used to analyze conditionedhyperactivity, the MPH challenge, GDNF protein, and BDNF pro-tein. A Newman–Keuls test was used for all post hoc analyses(p = .05).

For both the GDNF and BDNF ELISA, the nucleus accumbens anddorsal striatum were dissected from the rest of the brain tissue,and analyzed for the appropriate neurotrophin using the Emaximmunoassay system (Promega, Madison, WI). For all samples,250 �l of a RIPA cell lysis buffer (150 mM NaCl, 50 mM Tris–HCl,1.0% NP-40, 0.5% Sodium deoxycholate and 0.1% SDS) plus proteaseand phosphatase inhibitors (P5726, P8340, P0044, Sigma–Aldrich,St. Louis, MO) was added to each tissue sample. Brain regionswere homogenized on ice and centrifuged at 20,000 × g for 25 minat 4 ◦C. The resulting supernatants were removed and stored at−80 ◦C until use. Optical density was measured using a Bio-Tek ELx800 microplate reader (Winooski, VT). For both neurotropic factorassays, the directions provided by the manufacturer were followed.Further, we have previously published specific procedures for ELISAassays in earlier reports [e.g., 6].

Multiple regression was used to determine the relationshipsbetween GDNF and BDNF protein in the dorsal striatum and nucleusaccumbens and three predictor variables which included sex, activ-ity counts on the Conditioned Hyperactivity Test (CHT) or ChallengeTest (CT), as well as a ‘sensitization score’ (SS) which was calcu-lated by subtracting line crosses on Day 1 from line crosses on Day9 for each subject. The goal of this analysis was to investigate the

relationship between behaviors expressed during testing to neu-rotrophin levels in the dorsal striatum and nucleus accumbens.Because MPH significantly increased activity and neurotrophinlevels in the brain regions we investigated, drug treatment was

R.L. Roeding et al. / Behavioural Brain Research 273 (2014) 139–143 141

Fig. 1. Horizontal activity counts as a function of (a) day of treatment and condi-tion for behavioral sensitization. Group FM demonstrated more robust horizontalactivity compared to all other groups (indicated by **). Group MM demonstratedenhanced horizontal activity relative to controls (indicated by *); (b) drug condi-tion and sex for conditioned hyperactivity. Group FM demonstrated conditionedhyperactivity and was significantly greater than all other groups (indicated by **).(c) GDNF as a function of condition for the dorsal striatum and nucleus accumbens.Mt

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Fig. 2. GDNF from the dorsal striatum (a) and nucleus accumbens (b) as a function ofthe standardized predictors in the multiple regression analyses. Males are plotted

PH increased GDNF protein in both dorsal striatum and nucleus accumbens, andhere were no sex differences. All asterisks indicate p < .05.

ollinear with the activity predictors and was not included in theodel. Separate analyses were performed for each brain region.For behavioral sensitization, horizontal activity counts are pre-

ented as a function of day and drug condition in Fig. 1(a). Annitial analysis of the 3 days of habituation (expressed as one

ean) revealed no significant main effects or interactions. Ahree-way ANOVA of behavioral sensitization revealed significant

ain effects of sex F(1,30) = 56.26, p = .001, adolescent drug treat-ent F(1,30) = 579.76, p = .001, day of testing F(4,30) = 6.93, p = .001

nd significant two-way interactions of sex × adolescent drugreatment F(1,30) = 69.8, p = .001, adolescent drug treatment × day

(4,30) = 3.44, p = .017, and a significant three-way interaction ofex × adolescent drug treatment × day of testing F(4,30) = 2.86,

= .027. Post hoc analyses revealed that Group FM demonstrated

as square symbols and females are plotted as triangles. Filled symbols representgroups sensitized to MPH, open symbols represent saline controls.

significantly greater activity counts throughout testing than allother groups. Group MM males demonstrated significantly greateractivity counts than saline-treated groups throughout testing, andthere were no other group differences. Conditioned hyperactivityhorizontal activity counts are presented as a function of day andgroup in Fig. 1(b). A two-way ANOVA (sex, drug) revealed a sig-nificant main effect of sex F(1,31) = 5.36, p = .028, adolescent drugtreatment F(1,31) = 17.18, p = .001 and a significant interaction ofsex × adolescent drug treatment F(1,31) = 6.21, p = .019. Group FMdemonstrated significantly higher horizontal activity counts thanall other groups, Group MM was equivalent to saline controls, andthere were no other group differences. Therefore, only femalesgiven MPH demonstrated conditioned hyperactivity. GDNF protein(pg/mg tissue) is presented as a function of group for the dor-sal striatum and nucleus accumbens in Fig. 1(c). For the dorsalstriatum, a two-way ANOVA revealed a significant main effect ofadolescent drug treatment F(1,20) = 12.0, p = .003. Animals givenMPH demonstrated a significant 52% increase of striatal GDNFcompared to saline controls. For the nucleus accumbens, a two-way ANOVA also revealed a significant main effect of adolescentdrug treatment F(1,20) = 8.5, p = .011. Similar to effects in the dor-sal striatum, MPH significantly increased GDNF protein, but in theaccumbens the increase was less robust at 38%.

In the dorsal striatum, sex, CHT, and sensitization score werestrong predictors of GDNF (see Fig. 2(a)) accounting for almost 70%of the variance in levels of this neurotrophin. The original model

(sex and CT) was a significant predictor of striatal GDNF [adjustedR2 = 0.45, F(2,19) = 9.74, p = 0.01]. Further, addition of sensitiza-tion score did significantly improve the model [R2 Change = 0.2, F

142 R.L. Roeding et al. / Behavioural Brain Research 273 (2014) 139–143

Fig. 3. (a) Horizontal activity counts are presented as a function of condition for theMPH challenge administered on P60. Group FMM demonstrated significantly higheractivity as compared to all other groups (indicated by **). Groups FSM and MMMwere equivalent, but demonstrated higher horizontal activity counts than salinecontrols (FSS, MSS) as well as Group MSM (indicated by *). (b) BDNF as a function ofcondition for the dorsal striatum and nucleus accumbens. For the dorsal striatum,Group MMM demonstrated significantly higher levels of BDNF compared to all othergroups (indicated by **). For the nucleus accumbens, animals given MPH demon-strated an increase in BDNF compared to saline controls, regardless of whether thegi

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Fig. 4. BDNF signal from the dorsal striatum (a) and nucleus accumbens (b) as a

BDNF. Regarding the regression analysis, the negative �-weight

roup was administered saline or MPH on the challenge (indicated by *). All asterisksndicate p < .05.

hange (1,17) = 12.9, p = 0.01]. Activity on the conditioned hyper-ctivity test and sensitization scores were robust predictors oftriatal GDNF, with more activity and greater increases in activ-ty over testing associated with higher levels of striatal GDNF. Sex

as negatively associated with GDNF levels, indicating that malesemonstrated increased striatal GDNF compared to females. In theucleus accumbens, sex and activity on the conditioned hyperactiv-

ty test (CHT) were significant predictors of GDNF and accounted forpproximately 34% of the variance in GDNF [R2 = 0.34, F(2,21) = 6.4,

= 0.01]. Fig. 2(b) plots GDNF over the standardized predictorsnd the line of best fit for the accepted model. Although sex wasncluded in the model it did not robustly predict GDNF levels inhe nucleus accumbens and most of the variance in GDNF wasccounted for by activity on the conditioned hyperactivity test.

For the MPH challenge at P60, horizontal activity counts as aunction of adolescent drug treatment are presented in Fig. 3(a).

two-way ANOVA revealed a significant main effect of sex(1,49) = 45.78, p = .001, adolescent drug treatment F(2,49) = 65.69,

= .001 and a significant interaction of sex × adolescent drug treat-ent F(2,49) = 11.86, p = .001. Post hoc analyses revealed that group

M demonstrated significantly higher activity counts than allther groups. Group MM demonstrated significantly higher activ-ty counts than Group MS and male Group MSM, but interestingly,

as equal to Group FSM. In addition, female Group FSM demon-trated significantly higher activity counts than their counterpartroup MSM on the challenge.

function of the standardized predictors in the multiple regression analyses. Malesare plotted as square symbols and females are plotted as triangles. Filled symbolsrepresent groups sensitized to MPH, open symbols represent saline controls.

BDNF is presented as a function of condition for the dor-sal striatum and nucleus accumbens in Fig. 3(b). For the dorsalstriatum, a two-way ANOVA revealed a significant main effect ofadolescent drug treatment F(2,39) = 6.80, p = .003 and a significantsex × adolescent drug treatment interaction F(2,39) = 3.43, p = .044.Group MM demonstrated significantly higher levels of BDNF pro-tein compared to all other groups (74.2%) but there were no othersignificant differences between groups. For the nucleus accumbens,a two-way ANOVA revealed a significant main effect of adolescentdrug treatment F(2,39) = 6.13, p = .005. Animals given MPH demon-strated a significant 63% increase in BDNF compared to salinecontrols and a 78% increase compared to animals administeredsaline during sensitization and MPH on the challenge.

Sex, CT and SS were not significant predictors of BDNF sig-nal in the dorsal striatum, presented in Fig. 4(a). The best modelincluded only sex and CT as factors and only accounted for 6%of the variance in BDNF signal [adjusted R2 = 0.061, F(2,26) = 1.84,p = 0.18]. In the nucleus accumbens, sex and activity on the chal-lenge test (CT) were statistically significant predictors of BDNFlevels and the adjusted R2 of Model 1 was 0.16 [F(2,25) = 3.39,p = 0.05]. However, the model was significantly improved by theaddition of sensitization score (SS) and all three predictors com-bined accounted for approximately 40% of the variance in striatalBDNF [R2 Change = 0.24, F Change(1,22) = 9.72, p = 0.005]. The scat-terplot of standardized predictors and accumbal BDNF signal withthe line of best fit are presented in Fig. 4(b). As illustrated in thefigure, these predictors were strongly associated with accumbal

and partial correlation for sex indicates higher accumbal BDNF ofmale rats. Also, whereas activity on the challenge test positivelypredicted accumbal BDNF, the sensitization score was a negative

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redictor–indicating that rats with larger increases in activity fromay 1 to Day 9 of testing had lower accumbal BDNF.

This study reported several important findings relative to theffects of MPH on behavioral sensitization, its associative effects,s well as the effects of MPH on neurotrophic factor proteins.he observation of behavioral sensitization to MPH in adoles-ent females, and a lack of sensitization in adolescent males, isonsistent with past work from our laboratory [6] and others15]. Interestingly, these sex differences remained on the drugree conditioned hyperactivity test 1 day post-sensitization, withdolescent females given MPH demonstrating conditioned hyper-ctivity, whereas adolescent males administered MPH failed toxpress conditioned hyperactivity. This result would appear tondicate a stronger contextual association with MPH in adolescentemales, consistent with findings from Wooters et al. [20].

One day after the conditioned hyperactivity test, MPH produced significant increase in striatal and accumbal GDNF and there wereo sex differences in this response. This is the first report to ana-

yze subchronic MPH on GDNF in drug reward areas of the brain,nd would indicate that MPH is likely producing robust effects onopaminergic plasticity which has implications towards the effectsf MPH on the brain’s reward system, especially if administered at

dose higher than prescribed levels. In a much different light, theffects of MPH on GDNF may have therapeutic significance towardsarkinson’s disease. Experimental studies in animal models suggesthat the GDNF family of ligands (GFLs) have the potent ability torotect degenerating dopamine neurons as well as promote regen-ration of the nigrostriatal dopamine system [for review, see 21].ne shortcoming is that we did not analyze long-term effects ofPH on GDNF in this particular study, as it was previously unknownhether MPH would produce any change in GDNF. Thus, we ana-

yzed the effects of MPH on GDNF at a time point at which weypothesized a change would be more likely observed. Future workan investigate whether MPH produces long-term effects on GDNF.

Regarding the challenge, MPH resulted in an increased expres-ion of sensitization in females compared to all other groups,lthough males also demonstrated increased expression comparedo saline controls. Interestingly, animals given an acute dose of MPHn the challenge demonstrated an increase in activity compared toontrols, with females demonstrating a more robust increase rela-ive to males, further establishing a sex difference in the behavioralesponse to MPH. BDNF analyses 1 day after the challenge revealed

sex difference in the striatum, with males given MPH demon-trating a robust increase compared to all other groups, somewhatonsistent with our past work [6]. In the nucleus accumbens, MPHroduced an overall increase compared to saline controls with noex differences revealed. An acute dose of MPH given on the chal-enge day was not sufficient to produce an increase in BDNF inroups administered saline during sensitization.

A hierarchical regression analysis was used to analyze whetheranipulated factors played a role in predicting changes in striatal

nd accumbal GDNF and BDNF protein. Regarding GDNF, resultsndicated a behavioral test-dependent association, with accum-al GDNF as the best predictor of strength of association on theonditioned hyperactivity test, and striatal GDNF as the best pre-ictor of overall activity regardless of the test. This result wouldeem to indicate striatal and accumbal GDNF play somewhat dif-erent roles in MPH sensitization and the associative effects of

PH. Concerning BDNF, there was a strong relationship discov-

red between accumbal BDNF with sex, sensitization score andevels of activity on the challenge test, but there was no relation-hip between any manipulated variable with striatal BDNF. Thus,ensitization to MPH and increases of activity on the challenge are

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Research 273 (2014) 139–143 143

most related to accumbal BDNF. This finding is consistent with workthat has shown psychostimulant sensitization results in an increaseof accumbal dopaminergic activity that ultimately results in anincrease of activity-dependent BDNF [8,22], and suggest accumbalBDNF plays the more important role in MPH sensitization and itsassociative effects.

In conclusion, this study revealed robust sex differences in thebehavioral and neurotrophic factor response to MPH. In combina-tion with our past study [6], MPH results in robust changes in boththe behavioral and neural plasticity response in brain areas knownbe important in drug reward and addiction, and these data haveimportant implications towards recreational use of MPH during acritical period of development.

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