Sensitized nucleus accumbens dopamine terminal responses to methylphenidate and dopamine transporter releasers after intermittent-access self-administration

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Neuropharmacology 82 (2014) 1e10

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Sensitized nucleus accumbens dopamine terminal responsesto methylphenidate and dopamine transporter releasers afterintermittent-access self-administration

Erin S. Calipari, Sara R. Jones*

Department of Physiology and Pharmacology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA

a r t i c l e i n f o

Article history:Received 11 December 2013Received in revised form30 January 2014Accepted 11 February 2014Available online 13 March 2014

Keywords:VoltammetrySelf-administrationCocaineMethylphenidateAmphetamineRat

* Corresponding author. Tel.: þ1 336 716 8533; faxE-mail address: [email protected] (S.R. Jon

http://dx.doi.org/10.1016/j.neuropharm.2014.02.0210028-3908/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Long-access methylphenidate (MPH) self-administration has been shown to produce enhancedamphetamine potency at the dopamine transporter and concomitant changes in reinforcing efficacy,suggesting that MPH abuse may change the dopamine system in a way that promotes future drug abuse.While long-access self-administration paradigms have translational validity for cocaine, it may not be asrelevant a model of MPH abuse, as it has been suggested that people often take MPH intermittently.Although previous work outlined the neurochemical and behavioral consequences of long-access MPHself-administration, it was not clear whether intermittent access (6 h session; 5 min access/30 min)would result in similar changes. For cocaine, long-access self-administration resulted in tolerance tococaine’s effects on dopamine and behavior while intermittent-access resulted in sensitization. Here weassessed the neurochemical consequences of intermittent-access MPH self-administration on dopamineterminal function. We found increased maximal rates of uptake, increased stimulated release, andsubsensitive D2-like autoreceptors. Consistent with previous work using extended-access MPH para-digms, the potencies of amphetamine and MPH, but not cocaine, were increased, demonstrating thatunlike cocaine, MPH effects were not altered by the pattern of intake. Although the potency resultssuggest that MPH may share properties with releasers, dopamine release was increased following acuteapplication of MPH, similar to cocaine, and in contrast to the release decreasing effects of amphetamine.Taken together, these data demonstrate that MPH exhibits properties of both blockers and releasers, andthat the compensatory changes produced by MPH self-administration may increase the abuse liability ofamphetamines, independent of the pattern of administration.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Previous work has shown that methylphenidate (MPH) self-administration in rodents enhances the potency and reinforcingefficacy of amphetamine-related drugs, suggesting that MPH abusemay change the dopamine system in a way that promotes futuredrug abuse (Calipari et al., 2013a; Calipari et al., 2014a). MPH, theactive compound in Ritalin, is commonly used off-label orally as a“study drug” (Teter et al., 2006), and in college students it has alsobeen reported to be used to stay awake longer to party onweekends(Hall et al., 2005; Prudhomme-White et al., 2006; Teter et al., 2003).MPH is also taken intranasally or intravenously (IV) in larger doses,to “get high” (Gautschi and Zellweger, 2006; Levine et al., 1986;

: þ1 336 716 8501.es).

Parran and Jasinski, 1991; Sherman et al., 1987; Shaw et al., 2008;Teter et al., 2003); however, currently there is limited informationon these patterns or routes of administration in pre-clinical models(Marusich et al., 2010; Calipari et al., 2013a; Calipari et al., 2014a).When taken via the same route of administration, the subjectiveeffects of MPH are indistinguishable from cocaine or amphetamine,two commonly abused and highly addictive drugs (Rosen et al.,1985; Silverman and Ho, 1980). Although the subjective effects ofMPH and other stimulants are similar, recent work has shown thatthe compensatory changes induced by repeated administration ofMPHaredivergent fromother stimulants of the same type,making itdifficult to predict the neurochemical consequences of MPH abusebased on literature on compounds such as cocaine, the prototypicaldopamine transporter blocker (Calipari et al., 2013a,c; Calipari et al.,2014a,b; Ferris et al., 2011, 2012, 2013a,b). For example, while a five-day history of cocaine self-administration selectively reducedcocaine, but not MPH, potency at the DAT, MPH self-administration

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selectively increased MPH potency (Calipari et al., 2014a). Addi-tionally, although long-accessMPH self-administration did not alterblocker potency (cocaine, nomifensine), it enhanced both the po-tency and reinforcing efficacy of releasers (amphetamine, meth-amphetamine) (Calipari et al., 2013a), suggesting that thesecompounds may have an increased abuse liability followingrepeated MPH exposure.

Long-access self-administration of cocaine in rodents results inescalation of intake over sessions, a phenomenon that has beendocumented in humans and is considered essential to the addictionprocess (Ahmed et al., 2002, 2003; Ahmed and Koob, 2005; Dackisand O’Brien, 2001). However, while self-administration studiesusing long-access paradigms have high translational validity forstimulants such as cocaine and amphetamine, which have beenreported to be taken in multiple day binges (Dackis and O’Brien,2001; Koob and Le Moal, 2001), such paradigms may not modelMPH abuse as accurately. MPH abuse in humans has been sug-gested to occur intermittently, in single large doses many hours ordays apart (Hall et al., 2005; Prudhomme-White et al., 2006; Teteret al., 2003). Thus, it is important to determine if the same neuro-chemical changes occur following amore clinically relevant patternof voluntary MPH intake. Additionally, although previous work hasdetermined the consequences of long-access MPH self-administration on the dopamine system, recent work has shownthat the pattern of self-administration (intermittent versuscontinuous) can play an integral role in determining the neuro-adaptations that occur following stimulant exposure. For example,intermittent versus continuous patterns of cocaine or self-administration resulted in opposite adaptations, characterized bythe development of sensitization and tolerance to cocaine’s effectson the dopamine system, respectively (Calipari et al., 2013b;Calipari et al., 2014c). Hence, we aimed to determine how anintermittent paradigm of MPH administration influences thecompensatory changes that occur within the dopamine system ascompared to previous work following long-access conditions.

2. Methods

2.1. Animals

Male SpragueeDawley rats (375e400 g; Harlan Laboratories, Frederick, Mary-land) were used for all self-administration experiments. Rats were maintained on a12:12 h reverse light/dark cycle (3:00 am lights off; 3:00 pm lights on) with food andwater ad libitum. All animals were maintained according to the National Institutes ofHealth guide for the care and use of Laboratory animals (NIH Publications No. 8023,revised 1978) in Association for Assessment and Accreditation of Laboratory AnimalCare accredited facilities. The experimental protocol was approved by the Institu-tional Animal Care and Use Committee atWake Forest School of Medicine. All effortswere made to minimize suffering, reduce the number of animals, and to utilize al-ternatives to in vivo techniques.

2.2. Self-administration

Rats were anesthetized and implantedwith chronic indwelling jugular cathetersand trained for i.v. self-administration as previously described (Calipari et al.,2013b). Following surgery, animals were singly housed, and all self-administrationsessions took place in the home cage during the active/dark cycle (9:00 ame3:00pm). After a 2-day recovery period, animals underwent a training paradigm withinwhich animals were given access on a fixed ratio one (FR1) schedule to a MPH-paired lever, which, upon responding, initiated an intravenous injection of MPH(0.56 mg/kg, infused over 4 s). After each response/infusion, the lever was retractedand a stimulus light was illuminated for a 20 s timeout period, during which theanimal had no access to the lever, and thus could not respond for drug. Duringtraining, sessions were terminated after a maximum of 20 infusions or 6 h,whichever occurred first. An animal was considered to have acquired uponresponding for 20 injections for two consecutive days and a stable pattern infusionintervals was present. Following training, animals were assigned intermittent accessafter which the dopaminergic alterations that resulted were assessed.

2.3. Controls

Controls were animals housed in the same room, on the same light cycle, withsimilar handling conditions to the animals that performed self-administration. In

previous publications we have confirmed with a number of measures that thesurgery, novelty of being present in the self-administration chambers, and/orlimited drug self-administration, does not influence the results (Calipari et al.,2013b,c).

2.4. Intermittent access group

Animals were given access to MPH on an intermittent schedule of administra-tion described previously (Calipari et al., 2013b). During each 6 h session animalshad access to MPH for 12 five minute trails separated by 25-min timeout periods.Within each five-minute session, there were no timeouts other than during eachinfusion, and the animal could press the lever on an FR1 schedule to receive a 1-sinfusion of MPH (0.140 mg/kg/inf). Upon responding a stimulus light illuminatedconcurrently with the 1-s infusion of drug.

2.5. In vitro voltammetry

Voltammetry experiments were conducted during the dark phase of the lightcycle 18 h after commencement of the final self-administration session. Followingthe completion of the self-administration paradigms animals were deeply anes-thetized with isofluorane and rapidly decapitated. The brain was rapidly removedand the tissue was immediately immersed in ice-cold oxygenated artificial cere-brospinal fluid (aCSF) containing (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2(2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), L-ascorbic acid (0.4) and pH wasadjusted to 7.4. A vibrating tissue slicer was used to prepare 400 mm thick coronalbrain sections containing the NAc. Once sliced, the tissue was transferred to thetesting chambers containing bath aCSF (32 �C), which flowed at 1ml/min. After a 30-min equilibration period, a cylindrical carbon fiber microelectrode (100e200 mMlength, 7 mM radius) and a bipolar stimulating electrode were placed into the core ofthe NAc. The nucleus accumbens (NAc) was selected because of the important role inthe reinforcing and rewarding actions of cocaine. Further, our previous research hasconcentrated on plasticity of dopamine transporters in the core and demonstratedthat these alterations occur due to self-administration history of a number of drugs(Calipari et al., 2013a,b,c; Calipari et al., 2014a,b,c,d; Ferris et al., 2011, 2012, 2013a,b).Fast scan cyclic voltammetry was used to characterize baseline dopamine systemkinetics, D2-like autoreceptor activity, and the ability of psychostimulants to inhibitdopamine uptake. Endogenous dopamine release was evoked by a single electricalpulse (300 mA, 4 ms, monophasic) applied to the tissue every 5 min. Extracellulardopamine was recorded by applying a triangular waveform (�0.4 to þ1.2 to �0.4 Vvs Ag/AgCl, 400 V/s). Once the extracellular dopamine response was stable for threeconsecutive stimulations, cocaine (0.3e30 mmol/L), MPH (0.3e30 mmol/L) and(amphetamine 0.1e10 mmol/L) were applied cumulatively to the brain slice todetermine the effects of cocaine self-administration on drug-induced uptake inhi-bition. In order to determine D2-like autoreceptor sensitivity following these self-administration paradigms, we also ran doseeresponse curves for quinpirole(0.01e1 mmol/L), a D2-like receptor agonist, and reversed the effects with sulpiride(2 mmol/L), a D2-like antagonist.

2.5.1. Data analysisFor all analysis of FSCV data Demon Voltammetry and Analysis software was

used (Yorgason et al., 2011). To evaluate the effects of MPH self-administration ondopamine system kinetics, evoked levels of dopamine were modeled usingMichaeliseMenten kinetics as described previously (Calipari et al., 2012). Immedi-ately following the completion of each concentrationeresponse curve, recordingelectrodes were calibrated by recording their response (in electrical current; nA) to aknown concentration of dopamine in aCSF (3 mM) using a flow-injection system.This value was then used to convert electrical current to dopamine concentration.For cocaine, MPH, and amphetamine doseeresponse curves, Km, a measure ofapparent affinity for the dopamine transporter, was used to determine changes inability of the psychostimulants to inhibit dopamine uptake in the NAc relative tobaseline. Because quinpirole is a D2-like agonist it concentration-dependently re-duces the peak height of the dopamine signal. As a result we can assess the func-tional sensitivity of D2-like autoreceptors by determining if there is a shift in theconcentrationeresponse curve for quinpirole. All curves for D2-like function areexpressed as percent baseline dopamine release.

2.5.2. Calculating Ki valuesAs described by Jones et al. (1995), inhibition constants (Ki) were determined by

plotting the linear concentrationeeffect profiles and determining the slope of thelinear regression. The Ki was calculated by the equation Km/slope. Ki values are re-ported in mM and are a measure of the drug concentration that is necessary toproduce 50% uptake inhibition.

2.6. Statistics

Graph Pad Prism (version 5, La Jolla, CA, USA) was used to statistically analyzedata sets and create graphs. Pre-drug measures of stimulated dopamine release,dopamine uptake, and Ki values were compared using a two-tailed Student’s t-test.Release data and data obtained after perfusion of cocaine, MPH, amphetamine, orquinpirole were subjected to a two-way analysis of variance (ANOVA) with

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experimental group and concentration of drug as the factors. Behavioral (self-administration) data was analyzed using a one-way ANOVA and regression analysis.When main effects were obtained (p < 0.05), differences between groups weretested using a Bonferroni post hoc test.

3. Results

3.1. Intermittent-access MPH self-administration did not result inescalation

Long-access self-administration of MPH has been previouslybeen shown to result in escalation of intake over sessions (Marusichet al., 2010; Calipari et al., 2013a; Calipari et al., 2014a). One-wayANOVA revealed no main effect of self-administration session onintake, demonstrating that intermittent self-administration of MPHresulted in stable intake over sessions (Fig. 1A). Regression analysisindicated that the slope of intake over sessions is not significantlydifferent from zero indicating that escalation does not occur inthese animals with this model (Fig. 1B).

3.2. Intermittent-access MPH self-administration resulted inincreased stimulated dopamine release and Vmax

Pre-synaptic dopamine system kinetics were assessed usingFSCV in MPH self-administration and control groups. MPH self-administration resulted in an increase in maximal rate of uptake(Fig. 2A) and stimulated dopamine release (Fig. 2B). A two-tailedStudent’s t-test revealed a significant increase in stimulateddopamine release following intermittent MPH self-administration(t36 ¼ 2.191, p < 0.05). In addition, a two-tailed Student’s t-testalso revealed a significant increase in Vmax following MPH self-administration (t33 ¼ 2.919, p < 0.01; Fig. 2C).

3.3. D2-like autoreceptors were subsensitive following MPH self-administration

In order to determine if the differential dopamine kinetic effectscould be due to differences in D2-like autoreceptor activity, weassessed the sensitivity of the dopamine system to quinpirole, a D2-like autoreceptor agonist. ANOVA revealed a main effect of self-administration (F1,32 ¼ 5.422, p < 0.05) and a significant self-administration versus dose interaction (F4,32 ¼ 5.437, p < 0.01)(Fig. 3). Bonferroni post-hoc analysis revealed that quinpirole wassignificantly less effective at reducing dopamine release at the10 nM (p < 0.05) and 30 nM (p < 0.01) doses following MPH self-administration, indicating that D2-like autoreceptor activity is

Fig. 1. Animals self-administer methylphenidate (MPH) at high rates when given intermittento induce an intermittent pattern of self-administration within each 6 h session. (A) Represenrepresent earned infusions. (B) Group data plotting total infusions (0.140 mg/kg/inf) in eacindicating that animals do not escalate their intake.

reduced. The subsensitive D2-like autoreceptors that are presentfollowing MPH self-administration could explain, at least in part,the increase in stimulated dopamine release.

3.4. Intermittent-access MPH self-administration resulted inincreased MPH potency

To investigate how intermittent MPH self-administrationaffected MPH potency, we ran cumulative doseeresponse curvesfor MPH. Using FSCV in brain slices, MPH-induced dopamine up-take inhibitionwas recorded over a five-point doseeresponse curvefor the compound. MPH was found to be more effective at inhib-iting dopamine uptake followingMPH self-administration (Fig. 4A).ANOVA revealed a significant main effect of self-administration(F1,40 ¼ 5.316, p < 0.05; Fig. 4B) and a significant interaction ofMPH self-administration on the doseeresponse curve for MPH(F4,40 ¼ 5.387, p < 0.01; Fig. 4B). Bonferroni post hoc analysisrevealed that MPH-induced uptake inhibition was augmented atthe 10 mM (p < 0.05) and 30 mM (p < 0.001) doses following MPHself-administration. A linear doseeresponse curve was plotted tocalculate the Ki of MPH in control and self-administration animals(Fig. 4C). The slopes of the regression lines of the linear doseeresponse curve for MPH-induced uptake inhibition were signifi-cantly different (p < 0.01; Fig. 4D), indicating that MPH was morepotent following MPH self-administration.

The Ki is the dose of drug at which 50% uptake inhibition isachieved, and is calculated from the slope of the linear doseeresponse curve for the compound. A Student’s t-test revealed thatMPH potency as measured by Ki was increased (t10 ¼ 2.698,p < 0.05; Fig 4D), with a lower concentration of MPH resulting in50% uptake inhibition following MPH self-administration ascompared to controls.

3.5. Amphetamine potency was increased following intermittent-access MPH self-administration

Because MPH is structurally similar to the releaser amphet-amine, but functionally similar to the blocker cocaine, weassessed the effects of MPH self-administration on amphetamineand cocaine potencies to determine if the neurochemical areconferred to compounds of a similar structural or functionalclass. If the effects are structure specific amphetamine potencyshould be similar to the effects of MPH self-administration onMPH potency. Amphetamine was found to be more effective atinhibiting dopamine uptake inhibition following MPH self-administration (Fig. 5A), an effect that was similar to that of

t access. Rats were given 5 min unlimited access followed by a 25 min time-out periodtative data showing the pattern of self-administration over the 14 sessions. Tick marksh session over the 14 total sessions. The slope of the regression line is not significant

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Fig. 2. Intermittent methylphenidate (MPH) self-administration leads to increased stimulated dopamine release and uptake. Baseline dopamine system kinetics assessed byfast scan cyclic voltammetry. (A) Representative plot of dopamine release and uptake in control and MPH self-administration groups. Data is normalized to peak height tohighlight differences in uptake between groups. Color plots show dopamine (green) over time as measured in current (z-axis). (B) Group data showing that stimulated release iselevated following intermittent MPH self-administration. (C) Group data demonstrating that maximal rate of dopamine uptake (Vmax) is elevated following MPH self-administration. IntA, intermittent access; *, p < 0.05; **, p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)

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MPH. ANOVA revealed a significant main effect of self-administration (F1,32 ¼ 6.607, p < 0.05; Fig. 5B) and a signifi-cant interaction of MPH self-administration on the doseeresponse curve for amphetamine (F4,32 ¼ 2.744, p < 0.05; Fig. 5B).Bonferroni post hoc analysis revealed that amphetamine-induceduptake inhibition was augmented at the 10 mM dose (p < 0.05)following MPH self-administration. In addition, the linear doseeresponse curve was plotted to calculate the Ki of amphetamine incontrol and self-administration animals (Fig. 5C). The slopes ofthe regression lines of the linear doseeresponse curve foramphetamine-induced uptake inhibition were significantlydifferent (p < 0.05), indicating that amphetamine was morepotent following MPH self-administration.

Fig. 3. Intermittent methylphenidate (MPH) self-administration causes D2-likeautoreceptor subsensitivity. Quinpirole doseeresponse curve represented as percentof baseline dopamine peak height. Intermittent MPH self-administration resulted insubsensitive D2-like autoreceptors as compared to controls. IntA, intermittent access;*, p < 0.05 vs control.

A Student’s t-test revealed that MPH potency as measured by Kiwas increased (t7 ¼ 2.7, p < 0.05; Fig. 5D) as well, with a lowerconcentration of amphetamine resulting in 50% uptake inhibitionfollowing MPH self-administration as compared to controls.Because amphetamine effects are similar to MPH following MPHself-administration it is likely that these effects are specific to thestructure of these compounds rather than their function as blockeror releaser.

3.6. Cocaine potency was unchanged following intermittent-accessMPH self-administration

Because amphetamine and MPH behaved similarly after MPHself-administration, we hypothesized that these effects were due tothe structure of the compound; however, to confirm this, we usedcocaine, a compound structurally dissimilar from MPH, but func-tionally the same. Cocaine was found to be equivalently effective atinhibiting dopamine uptake inhibition following MPH self-administration (Fig. 6A), an effect that was dissimilar to that ofMPH. There was no significant effect of MPH self-administration onthe doseeresponse curve for cocaine (Fig. 6B).

In addition, the linear doseeresponse curve was plotted tocalculate the Ki of amphetamine in control and self-administrationanimals (Fig. 6C). The slopes of the regression lines of the lineardoseeresponse curve for cocaine-induced uptake inhibition werenot significantly different. In addition, cocaine potency asmeasuredby Ki was unchanged (Fig. 6D).

3.7. Stimulated dopamine release profiles for MPH, cocaine, andamphetamine were differentially affected by MPH self-administration

To determine how dopamine release was affected by a history ofMPH self-administration we measured the release effects of MPH,amphetamine, and cocaine across a doseeresponse curve for each

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Fig. 4. Sensitization of the neurochemical effects of methylphenidate (MPH). Fast scan cyclic voltammetry was used to assess changes in the potency of MPH at the dopaminetransporter following MPH self-administration. (A) Representative data showing MPH-induced uptake inhibition at the 10 mM concentration. Traces demonstrate that MPH is moreeffective at inhibiting uptake, as demonstrated by the increased time to return to baseline. Data was normalized to peak height to highlight differences in uptake across groups.Color plots which show dopamine (green) over time as measured in current (z-axis). (B) Group data showing that MPH self-administration results in increased potency of MPH overa doseeresponse curve. (C) Ki values across groups were calculated based off of the linear doseeresponse curve for MPH in each group. (D) Bar graph of Ki values for cocaine incontrol and MPH self-administration groups. Ki values are a measure of the concentration of drug at which 50% inhibition is achieved. IntA, intermittent access; *, p < 0.05 vscontrol; **, p < 0.01 vs control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Enhanced amphetamine potency following methylphenidate (MPH) self-administration. Fast scan cyclic voltammetry was used to assess changes in the potency ofamphetamine following MPH self-administration. (A) Representative data showing amphetamine-induced uptake inhibition at the 1 mM concentration. Amphetamine is moreeffective at inhibiting uptake, as demonstrated by the increased time to return to baseline in the traces. Data was normalized to peak height to highlight differences in uptake acrossgroups. Color plots which show dopamine (green) over time as measured in current (z-axis). (B) Group data showing that MPH self-administration results in increased potency ofamphetamine. (C) Ki values across groups were calculated based off of the linear doseeresponse curve for amphetamine in each group. (D) Bar graph of Ki values for amphetaminein control and MPH self-administration groups. IntA, intermittent access; *, p < 0.05 vs control. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

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Fig. 6. Cocaine potency is unaffected by a history of methylphenidate (MPH) self-administration. Fast scan cyclic voltammetry was used to assess changes in the potency of MPH atthe dopamine transporter following MPH self-administration. (A) Representative data showing cocaine (10 mM)-induced uptake inhibition. Data was normalized to peak height.Color plots which show dopamine (green) over time as measured in current (z-axis). (B) Group data showing no change in cocaine potency following MPH self-administration. (C) Ki

values across groups were calculated based off of the linear doseeresponse curve for cocaine in each group. (D) Bar graph of Ki values for cocaine in control and MPH self-administration groups. Ki values are a measure of the concentration of drug at which 50% inhibition is achieved. IntA, intermittent access; *, p < 0.05 vs control. (For interpre-tation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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of these drugs. First, we assessed the release profile of MPH todetermine how it was affected by a prior MPH self-administrationhistory. MPH exhibits an inverted “U” shaped profile that is indic-ative of a dopamine transporter blocker (Fig. 7A). Curves areexpressed as percent peak height at baseline, and peak height is ameasure of both release and uptake as uptake occurs continuouslyin the presence of dopamine. Because of this, blockers result ininverted-u shaped doseeresponse curves, where they increasepeak height at the early doses, due to their uptake-inhibition af-fects. At the higher doses, the peak height of the signal is reduced.In addition, ANOVA revealed a significant main effect of MPH self-administration (F1,33 ¼ 6.929, p < 0.05), indicating that MPH-induced dopamine release was increased as compared to controlanimals.

To determine if the release effects were common to all drugs orspecific to a certain class/structure, cocaine was also run. As withMPH, cocaine exhibited an inverted “U” shaped release profileindicative of blockers (Fig. 7B). Although the release profile wassimilar to MPH, the release effects of cocaine were unaffected byMPH self-administration. This gives further support to the hy-pothesis that the effects of MPH self-administration on subsequentpsychostimulant effects are driven by the structure of the com-pounds and are not specific to function as blocker or releaser.

To further characterize the release effects of MPH self-administration, the release profile over a doseeresponse curve foramphetamine was assessed. Amphetamine has a different profiledue to its mechanism of action as a dopamine releaser. Becauseamphetamine is releasing dopamine at all times, independent ofstimulated release, it concentration-dependently depletes dopa-mine releasable pools leading to decreased dopamine release overthe concentrationeresponse curve (Fig. 7C). ANOVA revealed asignificantmain effect ofMPH self-administration on amphetamine

release (F1,28 ¼ 13.96, p < 0.01). Bonferroni post hoc analysisrevealed that amphetamine releasewas significantly reduced at the300 nM dose (p < 0.001). The increase in amphetamine-induceddopamine reduction indicates an increase in the potency ofamphetamine to release dopamine in these animals. In addition,the fact that both MPH and amphetamine are more potent in theirdopamine releasing effects, although it is through different releaseprofiles, gives further support to the idea that the effects of MPHself-administration on subsequent psychostimulant effects aredriven by structure.

4. Discussion

The present study found that intermittent MPH self-administration resulted in dopamine system changes that may in-crease the abuse liability of some psychostimulants. With regard todopamine signaling in the absence of drug, we found increasedmaximal rates of uptake and increased stimulated dopaminerelease as well as subsensitive D2-like autoreceptors. Further, thepotencies of amphetamine and MPH were increased, as indicatedby decreased Ki values for uptake inhibition, while cocaine effectswere unchanged. Amphetamine and MPH effects on the peakheight of evoked dopamine signals were also augmented, althoughin opposite directions, wherein MPH-induced elevations andamphetamine-induced reductions in peak height were enhancedafter intermittent MPH self-administration. Additionally, MPH didnot lead to reductions in release due to depletion of dopaminevesicles, confirming that MPH is not a classical releaser, eventhough it is similar to releasers with regard to potency changes atthe dopamine transporter. The enhanced potency of MPH andreleasers is consistent with previous work showing enhancedamphetamine- and MPH-seeking behaviors following extended-

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Fig. 7. Stimulated dopamine release profile of cocaine, methylphenidate (MPH), andamphetamine following intermittent MPH self-administration. Stimulated release wasdetermined by the peak height of the signal over the doseeresponse curve for eachdrug. Data was then reported as percent pre-drug dopamine peak height. (A) MPHdoseeresponse curve that indicates that dopamine release in the presence of MPH isaugmented following MPH self-administration. The release profile of MPH indicatesthat it is a dopamine transporter blocker. The profile of dopamine transporter blockersis characterized by an inverted “U” shaped curve with increased evoked dopaminerelease at lower concentrations and reduced evoked dopamine release at higherconcentrations. (B) Cocaine doseeresponse curve that indicates that dopamine releaseis unchanged following MPH self-administration. (C) Amphetamine doseeresponsecurve demonstrating that evoked release in the presence of amphetamine is reducedfollowing MPH self-administration. The profile of releasers is characterized bydecreased exocytotic dopamine release, due to vesicular depletion, as doses areincreased. IntA, intermittent access; *, p < 0.05 vs control; ***, p < 0.001 vs control.

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access MPH administration (Calipari et al., 2014a). The similaritiesbetween dopamine system adaptations induced by extended-access protocols and the current intermittent study further high-light the unique properties of MPH, as cocaine exhibited oppositeeffects when self-administered intermittently versus continuously(Calipari et al., 2013a,c; Calipari et al., 2014a). Taken together, theseresults suggest that MPH abuse, regardless of pattern of intake, mayresult in increased abuse potential for amphetamine-like drugs.

The NAc core region was examined because of its strongdopamine innervation as well as its critical role in aspects of drug

addiction, including learning of drug-cue associations and drugseeking behaviors (Willuhn et al., 2010). Alterations in dopaminesignaling in this region have also been implicated in the learningand execution of goal-oriented behaviors in the absence of drugs(Berridge, 2007; Yawata et al., 2012; Wassum et al., 2013). Weshow an increase in evoked dopamine release following MPH self-administration, which could lead to increased associations be-tween drugs and their rewarding/reinforcing effects (Flagel et al.,2011; Wanat et al., 2009). This increase in evoked dopaminerelease is consistent with the decrease in D2-like autoreceptoractivity found in the current study. It has been suggested thatthere is no dopamine “tone”, or unstimulated extracellular dopa-mine levels, in slice preparations; therefore, during single pulsestimulations it is unlikely that D2-like autoreceptors activelyregulate dopamine release (Kennedy et al., 1992). However,because D2-like autoreceptors function to reduce dopamine syn-thesis as well as release, it is possible that their reduced activityleads to increased dopamine synthesis via increased tyrosine hy-droxylase activity (Håkansson et al., 2004). Indeed, following MPHself-administration tyrosine hydroxylase expression is increased(Calipari et al., 2014a). It is important to note that, although thereare increases in evoked dopamine release following intermittentMPH self-administration, uptake rates are also increased, whichcould be a compensatory response to the increased dopaminerelease. The increase in uptake rates is most likely caused by anincrease in dopamine transporter levels (Salahpour et al., 2008;Ferris et al., 2013b), which has been shown previously followingMPH self-administration (Calipari et al., 2013a). Increases indopamine transporter levels are particularly important in this case,as increasing dopamine transporter levels by transgenic over-expression has been shown to increase the behavioral andneurochemical potency of releasers such as amphetamine(Salahpour et al., 2008; Calipari et al., 2013a), highlighting the ideathat these changes may increase the susceptibility of individuals toabuse/addiction of psychostimulants.

In addition to changes in evoked release in the absence of drugs,stimulant-enhanced release was also altered following MPH self-administration. MPH self-administration enhanced the effects ofMPH and amphetamine on dopamine release, but not cocaine.However, unlike amphetamine, MPH did not cause reductions inevoked release due to depletion of vesicles, suggesting that MPH isnot a classical dopamine releaser. At high concentrations,amphetamine leads to depletion of vesicles in dopamine terminals,which is experimentally observed, using voltammetric techniques,as reduced stimulated dopamine release (Jones et al., 1998), aneffect that is enhanced by intermittent MPH self-administration.Although the effects of MPH self-administration on MPH potencyat the dopamine transporter are similar to those found withdopamine releasers, MPH exhibits the release profile of a blocker,which is characterized by and inverted “U” shaped release curvewith increased stimulated dopamine release at low concentrations(above pre-drug) which return to pre-drug levels at higher doses.This demonstrates that MPH is not a pure dopamine releaser, butrather exhibits properties of both releasers and blockers. This is inline with previous work showing that although MPH is not trans-ported into cells, and thus cannot release dopamine fromvesicles, itbinds to the dopamine transporter in a way that is similar toreleasers (Sonders et al., 1997; Dar et al., 2005; Wayment et al.,1999).

Following methylphenidate self-administration, cross-sensiti-zation occurs only for amphetamine-like compounds, and notcocaine, a stimulant of the same pharmacological class. Although itis not at novel concept that exposure to one stimulant may increasethe abuse potential for other stimulants (Shuman et al., 2012;Rosine et al., 2009; Valvassori et al., 2007; Tronci et al., 2006;

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Yang et al., 2003), this paper is particularly relevant when consid-ering previous data on cross sensitization of stimulants. Whilemany groups have previously shown that stimulants do, indeed,produce cross sensitization and cross-tolerance (Shuman et al.,2012; Rosine et al., 2009; Valvassori et al., 2007; Tronci et al.,2006; Yang et al., 2003; Ferris et al., 2012, 2013a; Calipari et al.,2013b), here we expand and clarify this idea. First, until recently,sensitization and cross-sensitization have been phenomena pri-marily attributed to experimenter-delivered stimulant adminis-tration protocols, while self-administration of stimulants hasrepeatedly been shown to result in tolerance (Hurd et al., 1989;Calipari et al., 2013b; Calipari et al., 2014a,b; Ferris et al., 2011,2012, 2013a,b). Therefore, this is one of the first reports of stimu-lant self-administration resulting in the sensitization of theneurochemical effects of a drug. Second, we show that not onlydoes cross-sensitization not occur for all stimulants, but it may noteven occur for stimulants of the same blocker or releaser class.Therefore the idea of cross-sensitization as a general process needsto be refined and made more specific for individual drugs.

A potential mechanism for the increased potency and enhancedeffects on dopamine release in the presence of amphetamine andMPH is via increased dopamine transporter levels. Previous workdemonstrated that similar changes following extended access MPHself-administration were due to elevated dopamine transporterlevels, and here we observe the same neurochemical effects, sug-gesting that the current changes may occur via the same mecha-nism (Calipari et al., 2013a; Calipari et al., 2014a). The current studyshowed that intermittent-access MPH self-administration resultedin increased uptake rates, suggesting that dopamine transporterlevels are likely to be elevated as well (Ferris et al., 2013b). Further,it has been shown that elevations in dopamine transporter levelsalone are capable of enhancing both the behavioral and neuro-chemical effects of amphetamine and MPH, but not cocaine(Calipari et al., 2013a; Salahpour et al., 2008). Although this is thefirst study to show enhanced MPH-induced dopamine releasefollowing MPH self-administration, it is possible that dopaminetransporter levels influence these effects as well. MPH has beenshown to redistribute vesicles from storage pools into the readilyreleasable pool (Chadchankar et al., 2012; Volz et al., 2008). Thiscould be a potential mechanism for its release effects and it ispossible that these effects could be dependent on MPH binding tothe dopamine transporter. Additionally, the increases in dopaminetransporter levels may also explain the enhanced effects ofamphetamine on evoked dopamine release following MPH self-administration. Increased uptake/dopamine transporter levelsmay function to allow increased concentrations of amphetamineinto the cell, resulting in enhanced vesicular dopamine depletion.

It is also possible that the augmented uptake inhibition effectsthat occur at the DAT for MPH following MPH self-administrationare driving the increased peak height of evoked dopamine sig-nals. The peak height of evoked dopamine release is dictated by theopposing processes of release and uptake (Wightman andZimmerman, 1990; Yorgason et al., 2011), which are occurringsimultaneously; therefore, when uptake inhibition occurs it canmanifest as increases in the peak height of evoked dopamine,especially at lower concentrations. Indeed, we do observe increasesin the peak height of evoked dopamine release at the lower con-centrations of MPH (0.3, 1 mM). Thus it is possible that the increaseduptake inhibition effect of MPH is contributing to the enhancedevoked release effects of the compound, in addition to potentialdirect effects of MPH on dopamine release. It should be noted thatboth MPH and amphetamine exhibit enhanced uptake inhibitionfollowing MPH self-administration, but amphetamine’s increasedpotency leads to greater decreases in the evoked dopamine peakheight, presumably through an increased rate of entry into the cells

due to the higher uptake rate and subsequent interactions withexocytotic vesicles.

These neurochemical changes have important behavioral im-plications, because stimulant effects on behavior, such as rewardand reinforcement, are primarily mediated by the ability of thedrug to inhibit the dopamine transporter and elevate dopaminelevels (Ritz and Kuhar, 1989; Roberts et al., 1977). Further, changesin the NAc core have been shown to be directly associated withchanges in reinforcement-related behaviors, where increasedaccumbal dopamine responses to cues or drugs result in increasedin reinforcing efficacy/drug-seeking behaviors (Calipari et al.,2014a; Saunders et al., 2013; Graf et al., 2013; Holmes and Fam,2013). This suggests that intermittent administration of MPH maylead to an increase in reward and reinforcement-related behaviorsfor amphetamines and other releasers, which could increase theabuse liability/addiction potential of these compounds. Although inthe current study, we did not measure postsynaptic changes, whichcould compensate for the enhanced potency of MPH and releasers,previous work on long-access MPH self-administration wouldsuggest that the changes specifically at the DAT are predictive ofself-administration behavior and reinforcing efficacy as measuredby both progressive ratio and behavioral economic approaches(Calipari et al., 2013a). Calipari et al. (2013a) demonstrated thatlong-access MPH self-administration resulted in the same changesin DAT function and psychostimulant potency and showed thatthese changes were positively correlated with reinforcing efficacy.This is of particular importance, given the high and increasinglevels of MPH abuse in the adult and adolescent populations inrecent years (Imbert et al., 2013; Frauger et al., 2013; Semboweret al., 2013; Zosel et al., 2013), as well as the co-abuse of MPHwith other drugs such as amphetamines (Teter et al., 2006).

A broader implication of findings such as these can help toelucidate the mechanisms by which stimulants alter the effects ofother stimulant effects to promote addictive-like behaviors.Knowingwhat components of a compound (structure, function) areresponsible for specific neurochemical adaptations that occurfollowing repeated use of a stimulant is important because it allowsfor the determination of the consequences/abuse potential ofdifferent treatment compounds or new drugs that are developedfor either treatment or abuse. Additionally, knowing the structure/function relationship of the neurochemical changes that occur afterthe administration of a compound could allow for the developmentof novel and specialized treatment options. Thus, understandingthe factors that are responsible for the expression of changes instimulant potency at the DAT can allow for the prediction of theneurochemical effects of other compounds, or the development ofnovel therapeutics that could be used to treat addiction.

5. Conclusions

Taken together, this work highlights the idea that MPH is aunique compound, both in its acute effects as well as thecompensatory effects that occur following self-administration.Acutely, MPH exhibits the uptake-inhibition profile of a releaserbut the release profile of a blocker, suggesting that MPH is part of aunique class of drugs that does not fit into either the releaser orblocker category. Further, the compensatory effects that occurfollowing MPH self-administration are not dependent on thetemporal profile of MPH intake, an effect that is different fromprevious work with cocaine self-administration. Thus MPH, whentaken in larger amounts than prescribed and via any pattern ofadministration, could result in an increased susceptibility to abuseof/addiction to dopamine releaser compounds such as amphet-amine, methamphetamine, and designer amphetamines such a“bath salts”.

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Acknowledgments

We would like to thank Dr. Mark J. Ferris for his input and forediting the manuscript. This work was funded by NIH grants R01DA021325, R01 DA030161, R01 DA014030 (SRJ) and T32 DA007246and F31 DA031533 (ESC).

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