Substituting a long-acting dopamine uptake inhibitor for cocaine prevents relapse to cocaine seeking

Substituting a long-acting dopamine uptake inhibitorfor cocaine prevents relapse to cocaine seeking

Clara Velázquez-Sánchez1, Antonio Ferragud1, Alfredo Ramos-Miguel2, Jesús A. García-Sevilla2

& Juan J. Canales1

Behavioural Neuroscience, Department of Psychology, University of Canterbury, New Zealand1and Laboratory of Neuropharmacology, Institut Universitarid’Investigació en Ciències de la Salut (IUNICS), University of the Balearic Islands, and Redes Temáticas de Investigación Cooperativa en Salud–Red de TrastornosAdictivos (RETICS-RTA), Spain2

ABSTRACT adb_458 1..11

The treatment of cocaine addiction remains a challenge. The dopamine replacement approach in cocaine addictioninvolves the use of a competing dopaminergic agonist that might suppress withdrawal and drug craving in abstinentindividuals. Although it has long been postulated that such an approach may be therapeutically successful, preclinicalor clinical evidence showing its effectiveness to prevent relapse is scant. We used in rats a procedure that involvedsubstitution of the N-substituted benztropine analog 3a-[bis(4′-fluorophenyl)methoxy]-tropane (AHN-1055), a long-acting dopamine uptake inhibitor (DUI), for cocaine. Maintenance treatment was self-administered. After extinction,reinstatement of drug seeking was induced by cocaine priming. We measured the contents of brain-derived neu-rotrophic factor (BDNF), c-Fos and Fas-associated death domain (FADD) proteins in the medial prefrontal cortex(mPFC) following reinstatement. DUI, but not amphetamine, substitution led to extinction of active lever presses, as didsaline substitution. DUI substitution significantly reduced cocaine-induced reinstatement of drug-seeking behavior,which was strongly elicited after saline substitution. Rats passively yoked to DUI also showed reduced cocaine-primedreinstatement. Reductions in drug seeking during reinstatement were matched by downward shifts in the contents ofBDNF, c-Fos and FADD proteins in the mPFC, which were elevated in relapsing rats. These data indicate that DUIsubstitution not only leads to extinction of self-administration behavior but also prevents reinstatement of drugseeking induced by cocaine re-exposure. Thus, DUI substitution therapy using compounds with low abuse potential,even if received passively in the context previously paired with drug taking, may provide an effective treatment forstimulant addiction.

Keywords Addiction, cocaine, dopamine uptake blocker, relapse, therapy.

Correspondence to: Juan J. Canales, Department of Psychology, The University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand. E-mail:[email protected]

INTRODUCTION

Relapse to drug use is the most significant clinicalproblem in the treatment of drug addiction. In addition toconditioned drug-related cues and stressors, a majorcause of relapse is re-exposure to the drug itself (Kalivas& McFarland 2003; Shaham et al. 2003). In detoxifiedhuman addicts, re-experiencing the effects of the drugthat was once abused often evokes or intensifies cravingand precipitates relapse, even after prolonged periods ofdrug abstinence (Mahoney et al. 2007). In laboratoryanimals, priming injection of the drug self-administeredpreviously produces reinstatement of extinguisheddrug-seeking behavior and, typically, reinstatement can

be elicited after extended (weeks-to-months) drug-freeperiods (Bossert et al. 2005). The model of reinstatementhas been used extensively to evaluate behavioraland pharmacological interventions to prevent relapse(Epstein et al. 2006).

The treatment and prevention of cocaine addiction isespecially challenging. Strong experimental evidenceindicates that withdrawal from several drugs of abuse,including cocaine, decreases the activity of the mesolim-bic dopamine system (Rossetti, Hmaidan & Gessa 1992;Weiss et al. 1992). Compromised dopamine functionduring critical phases of the addiction cycle, such aswithdrawal, might fuel anhedonia and dysphoria anddecreased motivation for non-drug-related stimuli, in

ORIGINAL ARTICLE

bs_bs_bannerAddiction Biologydoi:10.1111/j.1369-1600.2012.00458.x

© 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction Addiction Biology

addition to being a major force driving relapse (Koob andLe Moal 1997; Melis, Spiga & Diana 2005). In cocaineaddiction, this hypothesis has inspired efforts to developlong-acting replacement medications that could act asa substitute, while resetting disrupted dopamine neu-rotransmission (Rothman et al. 2008; Tanda, Newman &Katz 2009). The replacement approach has been imple-mented to treat opiate addicts (Bickel et al. 1987) and tomanage nicotine addiction (Cornuz 2006). Previous datasuggest that substitution treatment may be an effectivestrategy in cocaine addiction. Experimenter-administeredmaintenance with the synthetic opioid, buprenorphine,facilitated extinction of cocaine self-administration andattenuated cocaine-induced reinstatement in rats (Sorge,Rajabi & Stewart 2005). Clinical trials using oral admin-istration of dexamphetamine (Grabowski et al. 2001;Shearer et al. 2003) and methamphetamine (Mooneyet al. 2009) have shown positive outcomes (e.g. improvedtreatment retention and reduced cocaine use) in cocaineaddicts. However, it remains to be shown that adopamine-like substitute with low abuse potential affordslong-term protection from relapse to cocaine seeking,as predicted by theory (Gorelick, Gardner & Xi 2004;Rothman et al. 2008). This lack of evidence highlights agap in our current knowledge.

To meet these challenges, we used a high-affinity,long-acting dopamine uptake inhibitor (DUI), theN-substituted benztropine analog 3a-[bis(4′-fluorophenyl)methoxy]-tropane (AHN-1055). In pre-clinical models, this cocaine analog exhibits features thatare consistent with those of a replacement medication forcocaine addiction, including ability to block cocaine’seffects and weak stimulant, rewarding and reinforcingproperties (Ferragud et al. 2009; Hiranita et al. 2009;Velazquez-Sanchez et al. 2009). Moreover, AHN-1055does not interfere with motoric or motivational processes(e.g. sucrose reinforcement) at doses that preventcocaine’s behavioral effects (Ferragud et al. 2009). Wetested the ability of AHN-1055 to block cocaine-primedrelapse following a self-administration substitution pro-cedure. Regions within the prefrontal cortex, includingorbitofrontal, cingulate and prelimbic areas, have allbeen implicated in cocaine relapse in humans andanimals (Volkow et al. 2002; Kalivas & McFarland 2003;Peters, Kalivas & Quirk 2009). To explore the neuralmechanisms underlying the observed effects on behavior,we focused on the medial prefrontal cortex (mPFC), andthe prelimbic and anterior cingulate cortices (PrLC andCg1) within it, as a nodal brain region that regulatesreinstatement of cocaine seeking (Shaham et al. 2003;Kalivas 2008). We analyzed proteins known to beinvolved in mediating behaviorally relevant effects ofcocaine, including brain-derived neurotrophic factor(BDNF), c-Fos and Fas-associated death domain (FADD)

(Ciccocioppo, Sanna & Weiss 2001; Garcia-Fuster et al.2009; Ghitza et al. 2010).

MATERIALS AND METHODS

Subjects

Male Long Evans rats weighing 275–350 g at the begin-ning of the experiments served as subjects (n = 57). Ratswere maintained under standard conditions of tempera-ture (21 � 2°C) and humidity (45–55%) and were kepton a reversed 12-hour light/dark cycle (lights on 21:00hours). Rats were given a maintenance diet of 20 g ofrodent chow per day in order to maintain stable bodyweights during experiments. Water was available adlibitum. All experiments were approved by the EthicalCommittee of the University of Valencia or the AnimalEthics Committee of the University of Canterbury.

Drugs

AHN-1055 was synthesized as described previously (Fer-ragud et al. 2009). Purity of the product was assessed bymagnetic resonance, exceeding 98%. All drugs availablein the self-administration chambers were dissolvedin 0.9% saline. Cocaine hydrochloride (Alcaliber SL,Madrid, Spain) was administered at a dose of 0.50 mg/kg/infusion, AHN-1055 at doses of 0.17 and 0.50 mg/kg/infusion and d-amphetamine sulfate (AMPH) (Sigma-Aldrich, Saint Louis, MO, USA) at a dose of 0.10 mg/kg/infusion (Cain, Denehy & Bardo 2008). All drugs weredispensed in 100-ml boluses following active lever presses.Doses of cocaine and AHN-1055 were based on previousexperiments (Ferragud et al. 2009). For the reinstate-ment procedure, rats received injections of cocaine[10 mg/kg intraperitoneal (i.p.)] or vehicle.

Surgery

For the intravenous self-administration experiments, ratswere anesthetized with Avertin (2,2,2-tribromoethanol,12.5 mg/ml, in 2.5% tertiary amyl alcohol, 2 ml/100 gof body weight), and catheters (outer diameter 0.63 mm,internal diameter 0.30 mm, Camcaths, Cambridge, UK)were implanted into the right jugular vein, exiting dor-sally between the scapulae. Rats were treated post-surgically with daily injections of antibiotic (Baytril®,10 mg/kg subcutaneous, Bayer, Leverkusen, Germany)for 7 days. Catheters were flushed with heparinizedsaline (0.1 ml, 70 IU/ml) before and after each self-administration session.

Self-administration procedure

Rats were trained to lever press in operant chambers(Panlab S.L., Barcelona, Spain) for cocaine infusions

2 Clara Velázquez-Sánchez et al.


(0.5 mg/kg per infusion in 100 ml/5 seconds). Operantboxes were fitted with two retractable levers serving asactive and inactive levers in a counterbalanced fashion.Active lever presses resulted in infusions of saline orcocaine, illumination of a stimulus light for 5 secondsand retraction of the levers for 30 seconds. Inactive leverpresses had no programmed consequences. Rats weretrained on a fixed-ratio (FR1) schedule of reinforce-ment during 12-hour sessions until they obtained 50reinforcements/session. Thereafter, rats underwent daily1-hour sessions until they reached a stability criterion (atleast 10 1-hour sessions receiving 10 or more infusionsper session, with less than 20% intersession variation inthe last three sessions) (Ferragud et al. 2009; Velazquez-Sanchez et al. 2010b). Priming injections were not givenduring training. When the stability criterion wasachieved, the rats were randomly assigned to oneof the four substitution groups: CO-SAL (cocaine-saline), CO-AMPH (cocaine-AMPH 0.1 mg/kg/infusion), CO-AHN_0.5 (cocaine-AHN-1055 0.50 mg/kg/infusion) and CO-AHN_0.17 (cocaine-AHN-10550.17 mg/kg/infusion) (Fig. 1). Training and substitutionsessions both lasted 1 hour, so that both training andsubstitution curves could be compared. Rats self-administered AHN-1055 in 1-hour sessions (Ferragudet al. 2009). After substitution, some of these groupswere split into two in order to test for reinstatementfollowing cocaine or saline challenge. We added anadditional group with no drug experience, SAL-SAL(saline-saline), which was also divided into two for thereinstatement test. During the substitution procedure, allconditions remained as during training (i.e. the stimuluslight was activated during infusions, levers were retractedand time-out was introduced). To be able to dissociate theeffects of voluntary drug taking from the pharmacologi-cal effects of the treatment with AHN-1055, a yokedgroup (CO-AHN_yoked) was included. These ratsunderwent cocaine self-administration training and then

each rat in this group was paired with a rat in theCO-AHN_0.5 group. Yoked rats received passively thesame infusions of AHN-1055 (0.5 mg/kg/infusion) thatthe paired rats self-administered voluntarily. The rats ofthis group underwent the substitution phase in the sameconditions as their associated rats, but with leversretracted and light stimulus inactivated. Rats wereexposed to 18 substitution sessions before the cocaine-primed reinstatement tests were conducted. The behaviormet criterion for extinction when the last 3 days of sub-stitution differed on average from the last 3 days ofcocaine training (Fig. 1, inset). Substitution sessions con-tinued until all groups met this criterion, except thegroup receiving AMPH substitution, which showed noevidence of extinction. This allowed the reinstatementtest to be conducted after the same period of withdrawalfor all groups.

Reinstatement test

To avoid the possible influence of conditioned cue effectsduring drug-induced reinstatement, the light stimulusand the infusion pumps were disconnected and presses onthe active or inactive lever had no programmed conse-quences. Cocaine (10 mg/kg) or saline was administeredi.p. in the home cage 10 minutes prior to placing theanimal in the operant chambers. Drug-seeking behaviorwas assessed during 1 hour.

Brain sample preparation, immunoblotting assays andquantification of target proteins

After the reinstatement tests, rats were left undisturbed inholding cages for 30 minutes and then killed by decapi-tation under ether anesthesia. Based on the kinetics oftarget proteins, we decided to allow 1.5 hours from thebeginning of the reinstatement session, thus 30 minutesafter it finished (Curran & Morgan 1995; Le, Diaz &Sokoloff 2005; Garcia-Fuster et al. 2009). The brains

Figure 1 Experimental design to studythe effects of DUI substitution therapy oncocaine-primed reinstatement of drug-seeking behavior. Rats were trained to self-administer saline or cocaine (0.5 mg/kg/infusion) and then received one of theseveral substitution treatments, includingsaline, AHN-1055 (at dose of 0.17 or0.50 mg/kg/infusion) or AMPH (0.1 mg/kg/infusion). A group of rats was yoked to thegroup receiving substitution with AHN-1055 at the high dose. In the reinstatementtests, rats received i.p. injections ofsaline or cocaine (10 mg/kg). AMPH =d-amphetamine sulfate; CO = cocaine;DUI = dopamine uptake inhibitor ; i.p. =intraperitoneal; SAL = saline

Substitution therapy 3


were rapidly removed and placed on a brain matrix, and1-mm coronal slices were collected on ice-cold buffer.Bilateral samples from the mPFC were dissected out usinga brain puncher (1 mm in diameter), frozen in liquidnitrogen and finally stored at -80°C. The punches wereaimed at the PrLC and Cg1 areas of the mPFC (Fig. 4a).Before tissue homogenization, brain samples from ratsbelonging to the same experimental group were pooled(n = 5–7). Samples (0.61 � 0.23 mg/sample) from themPFC were then homogenized by gentle sonication (2 ¥ 5seconds, 40 W; Branson Sonifier 250) in 300 ml of ice-cold lysis buffer containing 50 mM Tris HCl (pH 6.8), 2%sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetet-raacetic acid, 1% of a protease inhibitor cocktail (Sigma-Aldrich) and 1% of a phosphatase inhibitor cocktail(Sigma-Aldrich). Total protein concentration in brainsamples was determined by the bicinchoninic acid (BCA)protein assay (Pierce Biotechnology, Rockford, IL, USA).Equal volumes (1:1) of loading buffer, containing100 mM Tris-HCl (pH 6.8), 20% glycerol, 3% SDS, 5%2-mercaptoethanol, 1% bromophenol blue and totalbrain homogenates, were mixed. Finally, samples wereboiled for 4 minutes, divided into working aliquots andstored at -20°C until use. Target proteins were quantifiedby Western blotting following standard procedures(Garcia-Fuster, Miralles & Garcia-Sevilla 2007). Briefly,15-mg protein aliquots from each pool of mPFC wereresolved in 10–12% SDS-PAGE minigels (Bio-Rad Labora-tories, Hercules, CA, USA). Proteins were electrotrans-ferred into 0.45-mm pore-sized nitrocellulosemembranes (Whatman International Ltd, Dassel,Germany). After incubation in a blocking solution, con-taining 5% bovine serum albumin and 1% Tween-20, themembranes were exposed to the primary antibody over-night, at 4°C, in gentle agitation. The primary affinitypurified antibodies used in this study included anti-c-Fos(sc-253, batch D012, 1:2000, Santa Cruz Biotechnology,Santa Cruz, CA, USA), anti-BDNF (sc-546, batch J1110,1:700, Santa Cruz Biotechnology), anti-FADD (sc-5559,batch K1404, 1:5000, Santa Cruz Biotechnology) andanti-p-Ser191 FADD (Ab 2785, batch 1, Cell SignalingTechnology, Beverly, MA, USA). The primary BDNF anti-body detects the immature and mature forms of theprotein (28 and 13 kDa, respectively). Total FADD orp-FADD were detected as dimeric (51 kDa) or oligomeric(116 kDa) species. Membranes were subsequently incu-bated for 1 hour at room temperature in the blockingsolution containing the horseradish peroxidase-linkedantirabbit or antimouse IgG secondary antibody (Cell Sig-naling Technology, Danvers, MA, USA; 1:5000). Immu-noreactivity of target proteins was detected with theenhanced chemiluminiscence (ECL) Western Blot Detec-tion system (Amersham International, Buckingham-shire, UK) and visualized by exposure to Hyperfilm ECL

autoradiograms (Amersham) for 1–60 minutes. As acontrol for sample loading and protein transfer, themembranes were stripped and re-probed with anti-b-actin (clone AC-15, 1:10000, Sigma-Aldrich) antibody(Boronat, Garcia-Fuster & Garcia-Sevilla 2001). Theautoradiograms were quantified by densitometric scan-ning (GS-800 Imaging Densitometer, Bio-Rad Laborato-ries). The immunoreactivities measured by primaryantibody fixation were normalized with those obtained inthe same gels after re-probing with anti-b-actin antibody.The amount of target proteins in mPFC of experimentalrats was compared in the same gel with that of controlanimals (SAL-SAL-SAL group). Experiments were per-formed by using protein amounts known to be within thelinear range of immunolabeling for each target protein(data not shown). The quantification procedure wasrepeated for at least three times, and the mean � thestandard error of the replicates were considered as finalestimates.

Statistical analysis

Behavioral data were analyzed by repeated-measuresanalysis of variance (ANOVA), followed by post hoc com-parisons with the method of Newman–Keuls (N–K) usingthe sampling error from the overall ANOVA as denomi-nator. Statistical significance was set at a = 0.05 perexperiment. Because of the limited availability of braintissue, mPFC samples from rats belonging to the sameexperimental group were pooled. Thus, neurochemicaldata, obtained from repeated measures of the same poolof brain samples, were analyzed by a randomized blockdesign ANOVA (i.e. a two-way ANOVA with experimentand treatment as the two factors) (see Lew 2007). In thiscase, the value of a was set to equal 0.02 (i.e. alpha valueof 0.05 divided by n - 1 replicates).

RESULTS

DUI substitution leads to extinction of active leverpresses in rats with a stable history of cocaineself-administration

Rats were initially trained on an FR1 schedule of rein-forcement until consistent performance was attained. Allfive groups of rats were matched in terms of cocaineintake before introducing the substitution. The meannumber of active lever presses per session was virtuallyidentical in all groups self-administering cocaine, and sowas the number of lever presses on the inactive lever(Fig. 2). After introducing the substitution treatment, thebehavior of the rats changed depending on the substitu-tion treatment received. As expected, when AMPH wassubstituted for cocaine, the rats readily switched from onecompound to the other, showing consistent and stable



responding across all 18 substitution sessions (Fig. 2).However, AHN-1055 did not behave as a classical stimu-lant during the substitution. Initially, responding forlow-dose AHN-1055 was highest. Compared with substi-tution with saline and high-dose AHN-1055, respondingfor low-dose AHN-1055 was significantly increased inday 2 of substitution (Fig. 2). Following several sessionsduring which drug seeking (and drug taking in the caseof substitutions with AHN-1055) remained strong,responding began to steadily decrease until extinctioncriterion was met by all substitution groups, exceptCO-AMPH, whose subjects did not extinguish. Even after18 extinction sessions, substituting saline for cocaine(group CO-SAL) still generated some active leverresponses (mean for the last 3 days was 7.76 � 1.06

active lever presses, with the group SAL-SAL respondinga mean of 1.05 � 0.31 presses, Fig. 2, inset); AHN-1055substitution groups responded similarly to the CO-SALgroup (mean 3.91 � 0.75 for CO-AHN_0.17 and8.42 � 1.93 for CO-AHN_0.5). For the groups CO-SAL,CO-AHN_0.17 and CO-AHN_0.5, but not for theCO-AMPH group, response levels in sessions 16–17-18were significantly reduced relative to stability sessions8–9-10 of cocaine training (Fig. 2, inset).

DUI substitution blocks cocaine-induced reinstatementof drug-seeking behavior

To test the effectiveness of the substitution therapy, a rein-statement test was performed the day after the last session

Figure 2 DUI substitution treatment with AHN-1055 leads to extinction of active lever presses. ANOVA was performed with onebetween-subjects factor, ‘Treatment’, with five levels (four experimental groups and one control group) and one within-subjects variable,‘Session’, with 10 levels (one for each of the sessions of cocaine training).The analysis showed a main effect of the treatment (F4,432 = 60.764,P < 0.0001). During training, the mean number of active lever presses per session was very similar in all groups self-administering cocaine. Ratswere matched in terms of intake before introducing the substitution treatment. During substitution, there were significant differences betweenthe different groups of rats. The ANOVA yielded significant effects of ‘Treatment’ (F4,47 = 25.699, P = 0.0001) and of the interaction‘Treatment’ ¥ ’Sessions’ (F68,799 = 3.325, P = 0.0001).The ANOVA comparing data for the last 3 days of cocaine training with the last 3 days ofsubstitution showed a significant interaction effect (F4,47 = 11.596, P < 0.0001) (extinction panel in inset). The group self-administeringamphetamine maintained similar levels of active lever presses after substitution, whereas the groups that received DUI substitution showedsignificantly decreased response values, which did not differ significantly from those in the SAL-SAL group (N–K tests after ANOVA), meetingcriterion for extinction. The number of subjects per group was as follows: SAL-SAL (n = 13), CO-SAL (n = 15), CO-AHN_0.17 (n = 11),CO-AHN_0.5 (n = 8) and CO-AMPH (n = 5). Dots represent means � standard errors. *P < 0.05, compared with CO-SAL; #P < 0.01,compared with CO-AMPH. AL = active lever ; AMPH = d-amphetamine sulfate; ANOVA = analysis of variance; CO = cocaine; DUI = dopamineuptake inhibitor ; IL = inactive lever ; N–K = Newman–Keuls; SAL = saline



of substitution treatment. Subjects in the CO-AMPHgroup were excluded because they did not extinguishlever pressing. Predictably, cocaine injections (10 mg/kg)produced robust reinstatement of drug-seeking behaviorin the group that received saline substitution (CO-SAL-CO), whereas saline injections (CO-SAL-SAL group), andpresumably the stress associated with the manipulation,induced only a slight increase in responding (Fig. 3).Remarkably, the two DUI substitution groups, CO-AHN_0.17 and CO-AHN_0.5, and the yoked groupexhibited significantly attenuated drug-seeking responses

compared with the CO-SAL-CO condition. Levels of drugseeking in the three DUI substitution groups were notsignificantly different from the cocaine-experiencedgroup, which did not receive cocaine priming (CO-SAL-SAL) (Fig. 3).

Patterns of protein expression in the mPFCparallel changes in reinstatement behavior aftercocaine priming

In drug naïve rats, acute administration of cocaine (SAL-SAL-CO group) significantly increased the immunoreac-tivities of c-Fos (+96%), precursor (+58%) and mature(+115%) BDNF, and total FADD protein (+96%) in themPFC, compared with levels observed in control animals(SAL-SAL-SAL group). In rats pre-exposed to cocaine, theimmunocontents of these proteins did not differ fromthose of drug naïve rats after the extinction of cocaineself-administration (CO-SAL-SAL group) (Fig. 4b–d).

We used c-Fos expression as a marker of neuronalactivation in the mPFC. Following cocaine self-administration, substitution treatment and extinctiontraining, cocaine priming produced robust elevations inc-Fos expression (group CO-SAL-CO). Post hoc tests indi-cated that AHN-1055 substitution treatment preventedthe stimulatory effects of cocaine on c-Fos expression.However, only the high dose of the DUI substitution treat-ment and the yoked treatment blocked cocaine-inducedchanges in c-Fos content (Fig. 4b).

In rats with experience in cocaine self-administration,cocaine challenge before the reinstatement test inducedsignificant elevations in precursor BDNF, over and abovelevels induced by cocaine in drug naïve subjects, and inmature BDNF. DUI substitution dose-dependently and sig-nificantly decreased the over-expression of mature andimmature BDNFs elicited by cocaine challenge (Fig. 4c).

FADD protein was found to be elevated after the chal-lenge of cocaine was given, whether or not animalshad experience in cocaine self-administration. However,cocaine prime treatment failed to increase the levelsof FADD in the mPFC in rats that voluntarily self-administered the DUI substitute or passively received itduring the yoked procedure, as indicated by N–K post hoctests (Fig. 4d). These treatments did not significantly alterthe content of p-Ser191 FADD in the mPFC (data notshown).

DISCUSSION

The data here reported provide a direct demonstrationthat replacement treatment in the drug-taking contextwith a DUI with low abuse liability can lead to extinctionand provide resistance to relapse. The data further indi-cate that this resistance is associated with blunted

Figure 3 DUI substitution therapy prevents cocaine prime-inducedreinstatement of drug seeking. ANOVA was performed with onebetween-subjects factor, ‘Treatment’, with seven levels, which yieldeda significant effect (F6,45 = 9.668, P < 0.0001). Cocaine challenge pro-duced vigorous reinstatement of drug seeking in CO-SAL-CO. Posthoc analysis of the ‘Treatment’ effect showed that responding in theCO-SAL-CO subjects differed significantly from CO-SAL-SAL sub-jects, which did not receive cocaine prime treatment (N–K testfollowing ANOVA). None of the three groups exposed to DUIsubstitution differed significantly from the CO-SAL-SAL group.Moreover, drug-seeking behavior was much reduced in the threeDUI substitution groups compared with the responses of theCO-SAL-CO subjects (N–K test after overall ANOVA). Thenumber of subjects per group was as follows: SAL-SAL-SAL(n = 7), SAL-SAL-CO (n = 6), CO-SAL-SAL (n = 6), CO-SAL-CO(n = 9), CO-AHN_0.17 (n = 11), CO-AHN_0.5 (n = 8) andCO-AHN_yoked (n = 5). Bars represent means � standard errors.**P < 0.01, compared with CO-SAL-SAL; #P < 0.01, comparedwith CO-SAL-CO. ANOVA = analysis of variance; CO = cocaine;DUI = dopamine uptake inhibitor ; i.p. = intraperitoneal; N–K =Newman–Keuls; SAL = saline



responsiveness to cocaine challenge in drug-experiencedsubjects, as measured by decreased cocaine-inducedexpression of c-Fos, BDNF and FADD proteins in themPFC during the reinstatement procedure.

Although multiple neurotransmitter systems andreceptors are likely to be implicated in mediating drugreinforcement, transition to compulsive drug taking andrelapse, the dopamine hypothesis remains to this date themost influential account of the behavioral processes thattypify addiction. Changes in dopamine transmission thatoccur during and after drug exposure are believed tocontribute to drug reinforcement (Volkow et al. 1996),withdrawal (Rossetti et al. 1992; Weiss et al. 1992) andrelapse (Wang et al. 2011). Not surprisingly, it has longbeen hypothesized that agents with high affinity for thedopamine transporter (DAT) could be used to suppresswithdrawal and drug craving, thereby mitigating relapsebehavior (Gorelick et al. 2004; Rothman et al. 2008). Thestrategy involves the use of a competing agonist withslower receptor onset and offset and longer half-life thancocaine. In controlled trials with cocaine addicts, drugswith significant abuse liability, including dexamphet-amine (Grabowski et al. 2001; Shearer et al. 2003) andmethamphetamine (Mooney et al. 2009), have shown toreduce cocaine use and craving. However, anotheramphetamine-related compound, methylphenidate, wasineffective at reducing cocaine use (Grabowski et al.1997), a failure that may be linked to its ability to induceself-reports of dysphoria in cocaine addicts (Roache et al.2000). Vanoxerine (GBR-12909), a piperazine derivativewith high affinity for the DAT, underwent phase II clinicaltrials in cocaine addicts but development was halted due

Figure 4 Suppression of cocaine prime-induced c-Fos, BDNF andFADD expression in rat mPFC following substitution treatment withAHN-1055 (0.17 or 0.5 mg/kg). (a) Diagram representing the areatargeted by the punches for mPFC (PrLC and Cg1) dissection andanalysis of protein expression. Coronal sections correspond to dia-grams located 3.2 and 3.7 mm anterior to bregma (Paxinos &Watson 2007). (b–d) Effects of saline or cocaine challenge in ratsreceiving different substitution treatments on the contents ofc-Fos, precursor and mature BDNF and FADD. Columns aremeans � standard errors of three to five replicated samples (pool ofmPFCs, n = 5–7 rats) and expressed as percentage of the controlgroup (SAL-SAL-SAL). Randomized block (two-way) ANOVAshowed significant differences between the groups of treatmentsfor c-Fos (F6,24 = 19.1, P < 0.0001), precursor BDNF (F6,12 = 19.5,P < 0.0001), mature BDNF (F6,12 = 13.8, P < 0.0001) and FADD(F6,24 = 31.9, P < 0.0001). *P < 0.05, **P < 0.01, ***P < 0.001, com-pared with SAL-SAL-SAL group; ##P < 0.01, ###P < 0.001, com-pared with the group CO-SAL-SAL; dP < 0.01, compared withCO-SAL-CO group (N–K post hoc tests after ANOVA). Bottom(b–d): representative immunoblots of c-Fos, BDNF (precursor andmature protein detected in the same gel after different exposuretimes), FADD and b-actin.The molecular masses of target proteinswere estimated from referenced standards. ANOVA = analysis ofvariance; BDNF = brain-derived neurotrophic factor ; Cg1 = anteriorcingulate cortex; CO = cocaine; FADD = Fas-associated protein withdeath domain; mPFC = medial prefrontal cortex; N–K = Newman–Keuls; PrLC = prelimbic cortex; SAL = saline�



to cardiac side effects. Although there is hope that newderivatives and analogs with selective actions at the DATmay remediate these problems, it is important to notethat neither GBR-12909, which reduces cocaine intakein humans (Preti 2000), monkeys (Skjoldager, Winger &Woods 1993) and rats (Tella 1995), nor any other long-acting DUI has been shown to specifically reduce propen-sity to relapse in humans or animals (i.e. animal modelsof reinstatement or context-conditioned relapse). Thisline of research is important in the light of recent evi-dence that reveals differences in DAT structure in cocaineaddicts that are both genetic and cocaine induced (Zhouet al. 2011).

We used AHN-1055, a benztropine derivative thatexhibits strong affinity for the DAT (Ki = 11.8 nM) andmuscarinic M1 receptors (Ki = 11.6 nM) (Agoston et al.1997), and the typical pharmacokinetic/dynamic fea-tures of other N-substituted analogs, including delayedonset and peak of pharmacological action, and longerhalf-life than cocaine (Raje et al. 2005). AHN-1055exhibits desirable behavioral features, including theability to block cocaine- and amphetamine-induced con-ditioned place preference (Velazquez-Sanchez et al. 2009,2010b), as well as cocaine and amphetamine self-administration in rats (Ferragud et al. 2009; Hiranitaet al. 2009; Velazquez-Sanchez et al. 2009). Additionally,AHN-1055 displays weak reinforcing efficacy comparedwith cocaine (Ferragud et al. 2009). To test thehypothesis that DUI substitution may provide long-termprotection from relapse, we took advantage of thesesought-after neurochemical and behavioral features ofAHN-1055. At the outset of the substitution procedure,responding for low-dose AHN-1055 was increased, sug-gesting that the substitute produced some psychoactiveeffects that the rats sought to obtain. However, the rein-forcing efficacy of the DUI treatment was probably weakbecause, as sessions progressed, responding in the twoAHN-1055 substitution groups decreased at the samerate as did in the saline substitution group. The weakreinforcing efficacy observed here is consistent with pre-vious findings (Ferragud et al. 2009). Further indicatingthat the properties of AHN-1055 are dissimilar fromtypical psychostimulants, we confirmed that the groupreceiving amphetamine as the replacement treatmentcontinued to respond consistently throughout thesubstitution procedure, unlike the groups treated withAHN-1055.

We introduced a yoked group to control for therequirement of extinction of lever pressing during substi-tution. Compared with saline treatment, DUI substitutiontherapy produced a striking decrease in the ability ofcocaine to reinstate drug-seeking behavior. Moreover, theresults of the yoked group indicated that voluntary intakeof the substitute and extinction of lever pressing was not

required for the replacement treatment to produce endur-ing resistance to cocaine-primed reinstatement. Thus,passive administration of the substitute in the drug-taking context afforded lasting protection from relapse.One possibility is that substitution treatment producedextinction of conditioned interoceptive cues associatedwith cocaine withdrawal, which are thought to beacquired during drug exposure and cause drug craving(Gray & Critchley 2007). This view is supported by thefinding that cocaine-primed reinstatement of cocaineseeking decreases with repeated cocaine priming (Mihin-dou et al. 2011). Further studies should determinewhether the therapeutic effects of the substitution treat-ment are context specific, thus affording or not resistanceto relapse in a different drug-taking environment. Addi-tionally, it is important to test whether the administrationof the replacement treatment in a given environmentprotects from relapse in the primary drug-taking context.

We searched for neural mechanisms associated withthe therapeutic effects of the substitution treatment. Weused the analysis of several proteins as a read-out ofactivity and adaptive change in the mPFC. Both PrLC andCg1 are central elements of the circuits mediatingcraving and drug-seeking behavior. Evidence has shownthat tetrototoxin-induced inactivation of the PrLC blockscocaine-primed reinstatement of drug seeking in self-administration assays (Capriles et al. 2003). Pathwaysrecruited during cocaine relapse may be segregated in themPFC, with infralimbic cortex mediating suppressiveeffects after extinction and PrLC and Cg1 subservingrenewed drug seeking by way Cg1 projections to thenucleus accumbens (Kalivas & McFarland 2003; Peterset al. 2009). The current data are consistent with thesefindings. Cocaine priming produced enhanced expressionof c-Fos, BDNF and FADD proteins in the PrLC/Cg1 areain rats that received saline substitution and showedstrong reinstatement; however, this increase alsooccurred in cocaine naïve rats. This finding suggests thatthe induction of c-Fos, BDNF and FADD proteins in themPFC may be associated not only with cocaine-primedreinstatement but also with general sensitivity to cocaine.Activation of c-Fos and early response genes in the mPFCoccurs following cue- and cocaine-induced reinstatementof drug seeking (Ciccocioppo et al. 2001; Ziolkowskaet al. 2011), which may indicate functional engagementof the mPFC during reinstatement. Cocaine treatmentelevates BDNF expression at multiple brain sites, includ-ing the mPFC, and BDNF has been postulated to be agatekeeper of synaptic efficacy and plasticity controllingaddictive-like behavior (McGinty, Whitfield & Berglind2010). Re-exposure to the cocaine taking context andrelapse produced induction of BDNF mRNA in the mPFC(Hearing et al. 2008). Furthermore, important evidenceshowed that following cocaine withdrawal, increases in



BDNF in the mPFC enhanced activity-induced long-termpotentiation, effectively sensitizing excitatory synapses inthe mPFC and contributing to behavioral sensitizationand drug reactivity (Lu et al. 2010). In agreement withthese findings, our data suggest that DUI substitutionafter cocaine taking reduces cocaine-induced BDNFexpression in the mPFC of rats with diminished drugreactivity and susceptibility to drug-induced relapse.Finally, the induction of FADD by cocaine challenge wasinhibited in rats that received DUI substitution treatment.Although the main role of FADD adaptor is pro-apoptotic,recent evidence suggests additional roles in cocaine-induced synaptic plasticity (Garcia-Fuster et al. 2009,2011; Alvaro-Bartolome et al. 2011). The present datafurther indicate that cocaine promotes the expression ofFADD protein in rat cortex, with an apparent lack of con-version to the phosphorylated FADD species (Garcia-Fuster et al. 2007; Alvaro-Bartolome et al. 2011).Interestingly, elevated levels of basal FADD have beenfound in rats that display enhanced cocaine sensitizationand have greater propensity to self-administer cocaine(Garcia-Fuster et al. 2009). In line with these observa-tions, our data now show that DUI substitution treatmentreduces cocaine-induced increases in FADD content inthe mPFC, which in turn associates with reduced suscep-tibility to cocaine prime-induced relapse.

We acknowledge that the molecular data presentedshould be interpreted with caution due to the absence ofadditional groups of rats that could have been treatedwith saline after substitution (Fig. 3). These groups werenot included as rats exposed to such condition would notbe expected to show reinstatement of cocaine seeking(similarly to rats in the CO-SAL-SAL condition). Takingthese data as a whole, while we recognize that the neuro-chemical evidence presented is only correlational, thestriking changes in cocaine-induced protein expressionduring the reinstatement test strongly suggest that DUItherapy decreased reactivity to cocaine at the level of themPFC, thereby reducing craving and vulnerability torelapse. While the substitution therapy may have pre-vented some of the molecular and neurochemical adap-tations that associate with withdrawal from stimulantexposure, which involve changes in dopamine transmis-sion (Rossetti et al. 1992; Weiss et al. 1992), the specificmechanisms responsible for its effectiveness in this modelof relapse remain to be determined.

Another aspect that requires further investigation isthe relative contribution of DAT inhibition and M1 recep-tor blockade to the therapeutic-like effects of AHN-1055.Although previous evidence indicates that DAT inhibi-tion with benztropine derivatives is sufficient to blockcocaine’s locomotor, rewarding and reinforcing effects(Desai et al. 2005; Velazquez-Sanchez et al. 2010a), rein-statement of drug-seeking behavior constitutes a distinct

behavior potentially modulated by multiple transmittersystems. Moreover, muscarinic receptors undergo down-regulation in rat brain following discontinuation of sub-chronic cocaine exposure (Macedo et al. 2004). Thus,replications with high-affinity DAT blockers without sig-nificant activity at M1 receptors are warranted.

Regarding the clinical implications of our findings,it is critical to emphasize that we investigated thetherapeutic-like effects of DUI substitution in one modelof reinstatement only (i.e. drug-induced reinstatement)and at one timepoint after withdrawal (i.e. after 18 daysof substitution). Stimulant addicts are more prone tosuffer craving and relapse during the first few days andweeks of discontinuation, and therefore our data areclinically relevant. However, future studies should incor-porate protracted relapse tests and other forms of rein-statement (e.g. stress- and cue-induced reinstatement). Insummary, the present findings provide a straightforwarddemonstration that an atypical, long-acting DUI effec-tively reduced relapse to cocaine seeking following substi-tution therapy in the drug-taking context, thus stronglysupporting the rationale for developing medications thattarget the DAT and exhibit low abuse liability as treat-ment for cocaine addiction.

Acknowledgements

This work was supported by grants from the Departmentof Psychology of the University of Canterbury (NewZealand), Instituto de Salud Carlos III (grant PI10/00297, Spanish Ministry of Science and Innovation),Plan Nacional Sobre Drogas (grant PNSD2008-057,Spanish Ministry of Health) and Red de Trastornos Adic-tivos. We thank Agueda Ferrer for technical assistance.

Disclosure/Conflict of Interest

All authors report no biomedical financial interests orpotential conflicts of interest.

Authors Contribution

CV-S performed the behavioral and neurochemicalassays, AF performed the behavioral assays, AR-M andJAG-S carried out the neurochemical experiments andanalyzed the protein content data, and JJC supervisedthe experiments, analyzed the data and wrote themanuscript.

References

Agoston GE, Wu JH, Izenwasser S, George C, Katz J, Kline RH,Newman AH (1997) Novel N-substituted 3 alpha-[bis(4′-fluorophenyl)methoxy]tropane analogues: selective ligandsfor the dopamine transporter. J Med Chem 40:4329–4339.



Alvaro-Bartolome M, La HR, Callado LF, Meana JJ, Garcia-Sevilla JA (2011) Molecular adaptations of apoptotic path-ways and signaling partners in the cerebral cortex of humancocaine addicts and cocaine-treated rats. Neuroscience196:1–15.

Bickel WK, Johnson RE, Stitzer ML, Bigelow GE, Liebson IA,Jasinski DR (1987) A clinical trial of buprenorphine: I.Comparison with methadone in the detoxification of heroinaddicts. II. Examination of its opioid blocking properties. NIDARes Monogr 76:182–188.

Boronat MA, Garcia-Fuster MJ, Garcia-Sevilla JA (2001)Chronic morphine induces up-regulation of the pro-apoptoticFas receptor and down-regulation of the anti-apoptotic Bcl-2oncoprotein in rat brain. Br J Pharmacol 134:1263–1270.

Bossert JM, Ghitza UE, Lu L, Epstein DH, Shaham Y (2005) Neu-robiology of relapse to heroin and cocaine seeking: an updateand clinical implications. Eur J Pharmacol 526:36–50.

Cain ME, Denehy ED, Bardo MT (2008) Individual differences inamphetamine self-administration: the role of the centralnucleus of the amygdala. Neuropsychopharmacology 33:1149–1161.

Capriles N, Rodaros D, Sorge RE, Stewart J (2003) A role for theprefrontal cortex in stress- and cocaine-induced reinstatementof cocaine seeking in rats. Psychopharmacology (Berl) 168:66–74.

Ciccocioppo R, Sanna PP, Weiss F (2001) Cocaine-predictivestimulus induces drug-seeking behavior and neural activationin limbic brain regions after multiple months of abstinence:reversal by D(1) antagonists. Proc Natl Acad Sci U S A98:1976–1981.

Cornuz J (2006) Treating tobacco use and dependence in clinicalpractice. Expert Opin Pharmacother 7:783–792.

Curran T, Morgan JI (1995) Fos: an immediate-early transcrip-tion factor in neurons. J Neurobiol 26:403–412.

Desai RI, Kopajtic TA, Koffarnus M, Newman AH, Katz JL (2005)Identification of a dopamine transporter ligand that blocks thestimulant effects of cocaine. J Neurosci 25:1889–1893.

Epstein DH, Preston KL, Stewart J, Shaham Y (2006) Toward amodel of drug relapse: an assessment of the validity of thereinstatement procedure. Psychopharmacology (Berl) 189:1–16.

Ferragud A, Velazquez-Sanchez C, Hernandez-Rabaza V, NacherA, Merino V, Carda M, Murga J, Canales JJ (2009) A dopaminetransport inhibitor with markedly low abuse liability sup-presses cocaine self-administration in the rat. Psychopharma-cology (Berl) 207:281–289.

Garcia-Fuster MJ, Clinton SM, Watson SJ, Akil H (2009) Effect ofcocaine on Fas-associated protein with death domain in therat brain: individual differences in a model of differentialvulnerability to drug abuse. Neuropsychopharmacology 34:1123–1134.

Garcia-Fuster MJ, Flagel SB, Mahmood ST, Mayo LM, ThompsonRC, Watson SJ, Akil H (2011) Decreased proliferation of adulthippocampal stem cells during cocaine withdrawal: possiblerole of the cell fate regulator FADD. Neuropsychopharmacol-ogy 36:2303–2317.

Garcia-Fuster MJ, Miralles A, Garcia-Sevilla JA (2007) Effects ofopiate drugs on Fas-associated protein with death domain(FADD) and effector caspases in the rat brain: regulation by theERK1/2 MAP kinase pathway. Neuropsychopharmacology32:399–411.

Ghitza UE, Zhai H, Wu P, Airavaara M, Shaham Y, Lu L (2010)Role of BDNF and GDNF in drug reward and relapse: a review.Neurosci Biobehav Rev 35:157–171.

Gorelick DA, Gardner EL, Xi ZX (2004) Agents in developmentfor the management of cocaine abuse. Drugs 64:1547–1573.

Grabowski J, Rhoades H, Schmitz J, Stotts A, Daruzska LA,Creson D, Moeller FG (2001) Dextroamphetamine for cocaine-dependence treatment: a double-blind randomized clinicaltrial. J Clin Psychopharmacol 21:522–526.

Grabowski J, Roache JD, Schmitz JM, Rhoades H, Creson D,Korszun A (1997) Replacement medication for cocaine depen-dence: methylphenidate. J Clin Psychopharmacol 17:485–488.

Gray MA, Critchley HD (2007) Interoceptive basis to craving.Neuron 54:183–186.

Hearing MC, Miller SW, See RE, McGinty JF (2008) Relapse tococaine seeking increases activity-regulated gene expressiondifferentially in the prefrontal cortex of abstinent rats. Psy-chopharmacology (Berl) 198:77–91.

Hiranita T, Soto PL, Newman AH, Katz JL (2009) Assessment ofreinforcing effects of benztropine analogs and their effectson cocaine self-administration in rats: comparisons withmonoamine uptake inhibitors. J Pharmacol Exp Ther 329:677–686.

Kalivas PW (2008) Addiction as a pathology in prefrontal corti-cal regulation of corticostriatal habit circuitry. Neurotox Res14:185–189.

Kalivas PW, McFarland K (2003) Brain circuitry and the rein-statement of cocaine-seeking behavior. Psychopharmacology(Berl) 168:44–56.

Koob GF, Le Moal M (1997) Drug abuse: hedonic homeostaticdysregulation. Science 278:52–58.

Le FB, Diaz J, Sokoloff P (2005) A single cocaine exposureincreases BDNF and D3 receptor expression: implications fordrug-conditioning. Neuroreport 16:175–178.

Lew M (2007) Good statistical practice in pharmacology.Problem 2. Br J Pharmacol 152:299–303.

Lu H, Cheng PL, Lim BK, Khoshnevisrad N, Poo MM (2010)Elevated BDNF after cocaine withdrawal facilitates LTP inmedial prefrontal cortex by suppressing GABA inhibition.Neuron 67:821–833.

Macedo DS, Correia EE, Vasconcelos SM, Aguiar LM, Viana GS,Sousa FC (2004) Cocaine treatment causes early and long-lasting changes in muscarinic and dopaminergic receptors.Cell Mol Neurobiol 24:129–136.

Mahoney JJ, Kalechstein AD, De La Garza R, Newton TF (2007)A qualitative and quantitative review of cocaine-inducedcraving: the phenomenon of priming. Prog Neuropsychop-harmacol Biol Psychiatry 31:593–599.

McGinty JF, Whitfield TW Jr, Berglind WJ (2010) Brain-derivedneurotrophic factor and cocaine addiction. Brain Res 1314:183–193.

Melis M, Spiga S, Diana M (2005) The dopamine hypothesis ofdrug addiction: hypodopaminergic state. Int Rev Neurobiol63:101–154.

Mihindou C, Vouillac C, Koob GF, Ahmed SH (2011) Preclinicalvalidation of a novel cocaine exposure therapy for relapse pre-vention. Biol Psychiatry 70:593–598.

Mooney ME, Herin DV, Schmitz JM, Moukaddam N, Green CE,Grabowski J (2009) Effects of oral methamphetamine oncocaine use: a randomized, double-blind, placebo-controlledtrial. Drug Alcohol Depend 101:34–41.

Paxinos G, Watson CR (2007) The Rat Brain in Stereotaxic Coor-dinates. San Diego, CA: Academic Press.

Peters J, Kalivas PW, Quirk GJ (2009) Extinction circuits forfear and addiction overlap in prefrontal cortex. Learn Mem16:279–288.



Preti A (2000) Vanoxerine National Institute on Drug Abuse.Curr Opin Investig Drugs 1:241–251.

Raje S, Cornish J, Newman AH, Cao J, Katz JL, Eddington ND(2005) Pharmacodynamic assessment of the benztropineanalogues AHN-1055 and AHN-2005 using intracerebralmicrodialysis to evaluate brain dopamine levels andpharmacokinetic/pharmacodynamic modeling. Pharm Res22:603–612.

Roache JD, Grabowski J, Schmitz JM, Creson DL, Rhoades HM(2000) Laboratory measures of methylphenidate effects incocaine-dependent patients receiving treatment. J Clin Psy-chopharmacol 20:61–68.

Rossetti ZL, Hmaidan Y, Gessa GL (1992) Marked inhibition ofmesolimbic dopamine release: a common feature of ethanol,morphine, cocaine and amphetamine abstinence in rats. Eur JPharmacol 221:227–234.

Rothman RB, Baumann MH, Prisinzano TE, Newman AH(2008) Dopamine transport inhibitors based on GBR12909and benztropine as potential medications to treat cocaineaddiction. Biochem Pharmacol 75:2–16.

Shaham Y, Shalev U, Lu L, De WH, Stewart J (2003) Thereinstatement model of drug relapse: history, methodo-logy and major findings. Psychopharmacology (Berl) 168:3–20.

Shearer J, Wodak A, van Beek I, Mattick RP, Lewis J (2003) Pilotrandomized double blind placebo-controlled study of dexam-phetamine for cocaine dependence. Addiction 98:1137–1141.

Skjoldager P, Winger G, Woods JH (1993) Effects of GBR 12909and cocaine on cocaine-maintained behavior in rhesusmonkeys. Drug Alcohol Depend 33:31–39.

Sorge RE, Rajabi H, Stewart J (2005) Rats maintained chroni-cally on buprenorphine show reduced heroin and cocaineseeking in tests of extinction and drug-induced reinstatement.Neuropsychopharmacology 30:1681–1692.

Tanda G, Newman AH, Katz JL (2009) Discovery of drugs totreat cocaine dependence: behavioral and neurochemicaleffects of atypical dopamine transport inhibitors. Adv Phar-macol 57:253–289.

Tella SR (1995) Effects of monoamine reuptake inhibitors oncocaine self-administration in rats. Pharmacol BiochemBehav 51:687–692.

Velazquez-Sanchez C, Ferragud A, Hernandez-Rabaza V,Nacher A, Merino V, Carda M, Murga J, Canales JJ(2009) The dopamine uptake inhibitor 3 alpha-[bis(4′-fluorophenyl)metoxy]-tropane reduces cocaine-inducedearly-gene expression, locomotor activity, and conditionedreward. Neuropsychopharmacology 34:2497–2507.

Velazquez-Sanchez C, Ferragud A, Murga J, Carda M, Canales JJ(2010a) The high affinity dopamine uptake inhibitor, JHW007, blocks cocaine-induced reward, locomotor stimulationand sensitization. Eur Neuropsychopharmacol 20:501–508.

Velazquez-Sanchez C, Ferragud A, Renau-Piqueras J, Canales JJ(2010b) Therapeutic-like properties of a dopamine uptakeinhibitor in animal models of amphetamine addiction. Int JNeuropsychopharmacol 24:1–11.

Volkow ND, Fowler JS, Wang GJ, Goldstein RZ (2002) Role ofdopamine, the frontal cortex and memory circuits in drugaddiction: insight from imaging studies. Neurobiol Learn Mem78:610–624.

Volkow ND, Wang GJ, Fowler JS, Gatley SJ, Ding YS, Logan J,Dewey SL, Hitzemann R, Lieberman J (1996) Relationshipbetween psychostimulant-induced ‘high’ and dopaminetransporter occupancy. Proc Natl Acad Sci U S A 93:10388–10392.

Wang GJ, Smith L, Volkow ND, Telang F, Logan J, Tomasi D,Wong CT, Hoffman W, Jayne M, Alia-Klein N, Thanos P,Fowler JS (2011) Decreased dopamine activity predicts relapsein methamphetamine abusers. Mol Psychiatry doi: 10.1038/mp.2011.86. [Epub ahead of print].

Weiss F, Markou A, Lorang MT, Koob GF (1992) Basal extracel-lular dopamine levels in the nucleus accumbens are decreasedduring cocaine withdrawal after unlimited-access self-administration. Brain Res 593:314–318.

Zhou Y, Michelhaugh SK, Schmidt CJ, Liu JS, Bannon MJ, Lin Z(2011) Ventral midbrain correlation between genetic varia-tion and expression of the dopamine transporter gene incocaine-abusing versus non-abusing subjects. Addict Bioldoi: 10.1111/j.1369-1600.2011.00391.x. [Epub ahead ofprint].

Ziolkowska B, Kielbinski M, Gieryk A, Soria G, Maldonado R,Przewlocki R (2011) Regulation of the immediate-early genesarc and zif268 in a mouse operant model of cocaine seekingreinstatement. J Neural Transm 118:877–887.



Documents

Substituting a long-acting dopamine uptake inhibitor for cocaine prevents relapse to cocaine seeking