ARTICLE Received 1 Aug 2013 | Accepted 7 Oct 2013 | Published 5 Nov 2013 DOI: 10.1038/ncomms3720 Methylphenidate amplifies the potency and reinforcing effects of amphetamines by increasing dopamine transporter expression Erin S. Calipari1, Mark J. Ferris1, Ali Salahpour2, Marc G. Caron3 & Sara R. Jones1 Methylphenidate (MPH) is commonly diverted for recreational use, but the neurobiological consequences of exposure to MPH at high, abused doses are not well defined. Here we show that MPH self-administration in rats increases dopamine transporter (DAT) levels and enhances the potency of MPH and amphetamine on dopamine responses and drug-seeking behaviours, without altering cocaine effects. Genetic overexpression of the DAT in mice mimics these effects, confirming that MPH self-administration-induced increases in DAT levels are sufficient to induce the changes. Further, this work outlines a basic mechanism by which increases in DAT levels, regardless of how they occur, are capable of increasing the rewarding and reinforcing effects of select psychostimulant drugs, and suggests that indivi- duals with elevated DAT levels, such as ADHD sufferers, may be more susceptible to the addictive effects of amphetamine-like drugs. 1 Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. 2 Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada M5S1A8. 3 Department of Cell Biology, Medicine and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA. Correspondence and requests for materials should be addressed to S.R.J. (email: [email protected]). NATURE COMMUNICATIONS | 4:2720 | DOI: 10.1038/ncomms3720 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3720 ecent epidemiological studies show that rates of illicit drug (n ¼ 11). This resulted in escalation of first-hour intake (one-way use have declined in recent years, whereas abuse of analysis of variance (ANOVA); F(4,10) ¼ 20.00, Po0.001) as well Rprescription drugs, including stimulant medications such as overall rate of intake (one-way ANOVA; F(4, 10) ¼ 7.956, as Ritalin, are on the rise1. Methylphenidate (MPH), the active Po0.001; Fig. 1a,b). On an FR1 schedule, it is impossible to compound in Ritalin, is prescribed for attention deficit and differentiate whether increased intake is due to tolerance or hyperactivity disorder (ADHD) and narcolepsy, and is commonly increased motivation to take drug; hence, we measured used off-label, with up to 17% of college students reporting abuse reinforcing efficacy using a progressive ratio (PR) paradigm19. of MPH for its cognitive enhancing or euphoric effects2. MPH is We found an increase in breakpoint for MPH (Student’s t-test; taken orally, intranasally or intravenously, and is one of the t12 ¼ 2.068, Po0.05; Fig. 1c) following MPH self-administration prescribed drugs most diverted onto the illicit market3,4. MPH (n ¼ 7 per group). abuse occurred at rates comparable to cocaine abuse in the 5 United States in 2008 . However, although there are numerous MPH increases MPH potency and MPH-induced dopamine studies outlining the neurochemical consequences of therapeutic overflow. The reinforcing and rewarding effects of many drugs of MPH use, there is a paucity of data outlining the neurobiological abuse are related to their ability to elevate dopamine in the adaptations that occur during MPH abuse. nucleus accumbens (NAc)20. Although the NAc shell is involved When taken via the same route of administration, the in the acute rewarding effects of drugs and acquisition of self- subjective effects of MPH are indistinguishable from cocaine or administration, the NAc core has a critical role in cue-reward amphetamine (AMPH), two commonly abused and highly 6,7 learning as well as continued responding for drugs after repeated addictive drugs . In addition, the behavioural effects of MPH administration as in the current paradigm21,22. We found and other psychostimulants are similar. For example, MPH dopamine uptake in the NAc core to be supersensitive to MPH resembles both cocaine and AMPH in extended access self- inhibition following MPH self-administration (two-way ANOVA; administration experiments, which result in escalation of intake F ¼ 25.11, Po0.05; Fig. 1d,e; n ¼ 5 control, 6 MPH self- over sessions, a change thought to model the switch from abuse to (1, 9) 8–11 administration), which is consistent with our microdialysis addiction . Because of the similar subjective and behavioural data demonstrating increased MPH-induced dopamine overflow profile of MPH compared with highly addictive drugs, it is in the NAc (two-way ANOVA; F(1, 5) ¼ 20.80, Po0.001; critically important to ascertain the neurochemical consequences Supplementary Fig. S1A; n ¼ 4 control, 5 MPH self- of abuse of MPH. administration). The proposed mechanism for augmented MPH MPH, AMPH and cocaine exert their rewarding and reinfor- potency is increased DAT levels, which has been suggested to cing effects by inhibiting the dopamine transporter (DAT) and 23 12 modulate the potency of dopamine releasers . Indeed, after MPH elevating synaptic dopamine levels . In addition, changes in self-administration (n ¼ 31 per group), the maximal rate of potency at the DAT are predictive of changes in the rewarding dopamine uptake was increased (V ; Student’s t-test; effects of these compounds in place preference paradigms13.To max t60 ¼ 2.434, Po0.01; Fig. 1g,h) as was the peak amplitude of elucidate the consequences of MPH self-administration on the stimulated dopamine release (Fig. 1h; Student’s t-test; t ¼ 2.719, abuse/addiction potential of psychostimulants, we determined 24 Po0.01). We showed previously that the increase in Vmax after the changes in blocker and releaser potency at the DAT as well as MPH was due to increased total DAT levels24. concomitant changes in motivation and drug-seeking behaviours for AMPH, MPH and cocaine. MPH self-administration resulted in increased maximal rates MPH self-administration increases dopamine releaser potency. of dopamine uptake mediated by increased DAT levels. Previous To further understand the impact of MPH self-administration on work has shown that fluctuations in DAT levels can alter the psychostimulant potencies, we used voltammetry and micro- potency of psychostimulants14, although the specific drugs dialysis to examine the ability of a number of DAT blockers and affected and the direction of potency shifts remain equivocal. dopamine releasers to enhance dopamine signals in the NAc core. Here we proposed that MPH self-administration-induced We found that MPH self-administration had no effect on blocker elevations in DAT levels would result in enhanced potency and potency, but enhanced the potency of releasers. Thus, the ability reinforcing efficacy of releasers and MPH. To test this hypothesis, of the blockers cocaine (Fig. 2a,c; n ¼ 5 control, 6 MPH self- we used transgenic DAT overexpressing mice (DAT-tg), which administration) and nomifensine (Fig. 2b,d; n ¼ 5 per group) to have elevated levels of native, non-drug-altered DATs. Using inhibit dopamine uptake was unaffected by MPH self-adminis- these mice, we elucidated a basic mechanism whereby increased tration. In contrast, the potency of AMPH (two-way ANOVA; DAT levels drive enhanced potency and motivation to obtain F(1, 4) ¼ 76.81, Po0.001; Fig. 2e,g; n ¼ 5 per group) and MPH and releaser drugs such as AMPH while leaving the effects methamphetamine (two-way ANOVA; F(1, 10) ¼ 33.51, Po0.001; of blockers such as cocaine unchanged. The robust enhancement Fig. 2f,h; n ¼ 7 control, 5 MPH self-administration) was in the potency of releasers, but not blockers, following increases increased. in DAT levels in DAT-tg mice is particularly important as it The increase in drug potency for dopamine uptake inhibition suggests that individual variations in DAT levels in the human by releasers following MPH self-administration was accompanied population (for example, ADHD15, post-traumatic stress by increases in drug-induced dopamine overflow as measured disorder16, early-life stress17, repeated stressors18) may predict using in vivo microdialysis. AMPH (two-way ANOVA; susceptibility to abuse of compounds such as MPH, AMPH and F(1, 5) ¼ 11.51, Po0.0001; n ¼ 5 control, 6 MPH self-administra- methamphetamine. tion; Supplementary Fig. S1B), but not cocaine (n ¼ 6 per group; Supplementary Fig. S1C), resulted in increased drug-induced dopamine overflow in the NAc. This confirms that shifts in the Results ability of releasers to inhibit dopamine uptake correspond to MPH self-administration increases MPH reinforcement.To shifts in the ability of releaser to augment extracellular dopamine determine the neurochemical consequences of MPH adminis- levels. tration, we allowed rats 6-h access to the most reinforcing dose of MPH (0.56 mg kg À 1 per infusion11) on a fixed ratio one (FR1) MPH increases releaser reinforcement and drug-seeking. Given schedule, with a maximum of 40 injections for 5 consecutive days the enhanced potency of dopamine releasers, it is possible that 2 NATURE COMMUNICATIONS | 4:2720 | DOI: 10.1038/ncomms3720 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3720 ARTICLE a 1 h 1 2 3 Session 4 5 bc 300 CTRL * 12 MPH SA * 250 11 * * 200 10 9 150 Final ratio 8 100 MPH injections per h 7 50 0 0 12345 Session de 10 µM MPH 40 * height CTRL 30 M) MPH SA µ Normalized peak 30 s 20 –0.4 +11.0 * Current (nA) +1.2 +2.0 (app. Km, (app. 10 –0.4 –7.0 –0.4 +21.0 uptake Inhibition of DA 0 vs Ag/AgCl +1.2 +3.0 V –0.4 –14.0 –6.5 –6.0 –5.5 –5.0 –4.5 Log [MPH] (M) 15 s fgh CTRL 4 * 1.5 MPH SA * height 3 M) µ ) 1.0 Normalized peak –1 –0.4 +19.0 M s CTRL Current (nA) µ 2 +1.2 ( –0.4 max 0.5 +2.0 V –0.4 MPH SA 1 per pulse ( [DA] vs Ag/AgCl +1.2 V –0.4 –9.0 0 0.0 15 s CTRL MPH SA CTRL MPH SA Figure 1 | MPH self-administration increases dopamine uptake and MPH potency.