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Addiction Biology
doi:10.1111/j.1369-1600.2012.00458.x

ORIGINAL ARTICLE

Substituting a long-acting dopamine uptake inhibitor for 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 Universitari d’Investigació en Ciències de la Salut (IUNICS), University of the Balearic Islands, and Redes Temáticas de Investigación Cooperativa en Salud–Red de Trastornos Adictivos (RETICS-RTA), Spain2

ABSTRACT

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The treatment of cocaine addiction remains a challenge. The dopamine replacement approach in cocaine addiction involves the use of a competing dopaminergic agonist that might suppress withdrawal and drug craving in abstinent individuals. Although it has long been postulated that such an approach may be therapeutically successful, preclinical or clinical evidence showing its effectiveness to prevent relapse is scant. We used in rats a procedure that involved substitution of the N-substituted benztropine analog 3a-[bis(4′-fluorophenyl)methoxy]-tropane (AHN-1055), a longacting 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 neurotrophic 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 did saline 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-primed reinstatement. Reductions in drug seeking during reinstatement were matched by downward shifts in the contents of BDNF, c-Fos and FADD proteins in the mPFC, which were elevated in relapsing rats. These data indicate that DUI substitution not only leads to extinction of self-administration behavior but also prevents reinstatement of drug seeking 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 for stimulant 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: juan.canales@canterbury.ac.nz

INTRODUCTION Relapse to drug use is the most significant clinical problem in the treatment of drug addiction. In addition to conditioned drug-related cues and stressors, a major cause of relapse is re-exposure to the drug itself (Kalivas & McFarland 2003; Shaham et al. 2003). In detoxified human addicts, re-experiencing the effects of the drug that was once abused often evokes or intensifies craving and precipitates relapse, even after prolonged periods of drug abstinence (Mahoney et al. 2007). In laboratory animals, priming injection of the drug self-administered previously produces reinstatement of extinguished drug-seeking behavior and, typically, reinstatement can

be elicited after extended (weeks-to-months) drug-free periods (Bossert et al. 2005). The model of reinstatement has been used extensively to evaluate behavioral and pharmacological interventions to prevent relapse (Epstein et al. 2006). The treatment and prevention of cocaine addiction is especially challenging. Strong experimental evidence indicates that withdrawal from several drugs of abuse, including cocaine, decreases the activity of the mesolimbic dopamine system (Rossetti, Hmaidan & Gessa 1992; Weiss et al. 1992). Compromised dopamine function during critical phases of the addiction cycle, such as withdrawal, might fuel anhedonia and dysphoria and decreased motivation for non-drug-related stimuli, in
Addiction Biology

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

Self-administration procedure Rats were trained to lever press in operant chambers (Panlab S. 1987) and to manage nicotine addiction (Cornuz 2006). Newman & Katz 2009). Regions within the prefrontal cortex. Germany) for 7 days. and the prelimbic and anterior cingulate cortices (PrLC and Cg1) within it. For the reinstatement procedure. Madrid. internal diameter 0. Catheters were flushed with heparinized saline (0. However. including brain-derived neurotrophic factor (BDNF). while resetting disrupted dopamine neurotransmission (Rothman et al. Tanda. MO. Garcia-Fuster et al.L. 2003. exiting dorsally between the scapulae.2. have all been implicated in cocaine relapse in humans and animals (Volkow et al. In cocaine addiction.5% tertiary amyl alcohol. Rats were given a maintenance diet of 20 g of rodent chow per day in order to maintain stable body weights during experiments. it remains to be shown that a dopamine-like substitute with low abuse potential affords long-term protection from relapse to cocaine seeking. Cambridge. 2009). as predicted by theory (Gorelick. this cocaine analog exhibits features that are consistent with those of a replacement medication for cocaine addiction. 2010). Leverkusen. Barcelona. rewarding and reinforcing properties (Ferragud et al. MATERIALS AND METHODS Subjects Male Long Evans rats weighing 275–350 g at the beginning of the experiments served as subjects (n = 57).g.17 and 0. 2 ml/100 g of body weight).5 mg/ml. UK) were implanted into the right jugular vein. Water was available ad libitum. as a nodal brain region that regulates reinstatement of cocaine seeking (Shaham et al. 2009) have shown positive outcomes (e. Doses of cocaine and AHN-1055 were based on previous experiments (Ferragud et al. 2009). and catheters (outer diameter 0. Previous data suggest that substitution treatment may be an effective strategy in cocaine addiction.2 Clara Velázquez-Sánchez et al. Cocaine hydrochloride (Alcaliber SL. 2009. in 2. including ability to block cocaine’s effects and weak stimulant. the N-substituted benztropine analog 3a-[bis(4′fluorophenyl)methoxy]-tropane (AHN-1055). 2009). Camcaths. Spain) for cocaine infusions Addiction Biology © 2012 The Authors. Rats were maintained under standard conditions of temperature (21 Ϯ 2°C) and humidity (45–55%) and were kept on a reversed 12-hour light/dark cycle (lights on 21:00 hours). Bayer. To meet these challenges. Ghitza et al. AHN-1055 does not interfere with motoric or motivational processes (e. Shearer et al. Hiranita et al. Rothman et al. Drugs AHN-1055 was synthesized as described previously (Ferragud et al. we focused on the medial prefrontal cortex (mPFC). The replacement approach has been implemented to treat opiate addicts (Bickel et al. facilitated extinction of cocaine self-administration and attenuated cocaine-induced reinstatement in rats (Sorge. In preclinical models. Moreover. Surgery For the intravenous self-administration experiments. 12.50 mg/ kg/infusion. Peters. We tested the ability of AHN-1055 to block cocaine-primed relapse following a self-administration substitution procedure. this hypothesis has inspired efforts to develop long-acting replacement medications that could act as a substitute. rats received injections of cocaine [10 mg/kg intraperitoneal (i. This lack of evidence highlights a gap in our current knowledge. we used a high-affinity.g. Rajabi & Stewart 2005).30 mm. Addiction Biology © 2012 Society for the Study of Addiction . Rats were treated postsurgically with daily injections of antibiotic (Baytril®. All experiments were approved by the Ethical Committee of the University of Valencia or the Animal Ethics Committee of the University of Canterbury.63 mm.9% saline. long-acting dopamine uptake inhibitor (DUI). Gardner & Xi 2004. 2009. including orbitofrontal. All drugs were dispensed in 100-ml boluses following active lever presses. Melis.50 mg/ kg/infusion and d-amphetamine sulfate (AMPH) (SigmaAldrich.p. We analyzed proteins known to be involved in mediating behaviorally relevant effects of cocaine. All drugs available in the self-administration chambers were dissolved in 0. buprenorphine. Experimenter-administered maintenance with the synthetic opioid.)] or vehicle. Kalivas 2008). 10 mg/kg subcutaneous. exceeding 98%. To explore the neural mechanisms underlying the observed effects on behavior. Velazquez-Sanchez et al. improved treatment retention and reduced cocaine use) in cocaine addicts. sucrose reinforcement) at doses that prevent cocaine’s behavioral effects (Ferragud et al. 2009.. 2009).2-tribromoethanol. Kalivas & Quirk 2009). Spiga & Diana 2005). rats were anesthetized with Avertin (2. 2002. 2008). Denehy & Bardo 2008). AHN-1055 at doses of 0. c-Fos and Fas-associated death domain (FADD) (Ciccocioppo. addition to being a major force driving relapse (Koob and Le Moal 1997.10 mg/kg/ infusion (Cain. 2008. Clinical trials using oral administration of dexamphetamine (Grabowski et al. Purity of the product was assessed by magnetic resonance. Saint Louis. USA) at a dose of 0.1 ml. Kalivas & McFarland 2003. 70 IU/ml) before and after each selfadministration session. 2003) and methamphetamine (Mooney et al. 2001. Spain) was administered at a dose of 0. cingulate and prelimbic areas. Sanna & Weiss 2001.

Operant boxes were fitted with two retractable levers serving as active and inactive levers in a counterbalanced fashion. In the reinstatement tests. Rats were trained on a fixed-ratio (FR1) schedule of reinforcement during 12-hour sessions until they obtained 50 reinforcements/session. Addiction Biology © 2012 Society for the Study of Addiction . Garcia-Fuster et al. We added an additional group with no drug experience. The brains Addiction Biology © 2012 The Authors. Brain sample preparation. CO-AMPH (cocaine-AMPH 0. During the substitution procedure. SAL = saline (0. rats received i. These rats underwent cocaine self-administration training and then each rat in this group was paired with a rat in the CO-AHN_0. Rats selfadministered AHN-1055 in 1-hour sessions (Ferragud et al. injections of saline or cocaine (10 mg/kg).Substitution therapy 3 Figure 1 Experimental design to study the effects of DUI substitution therapy on cocaine-primed reinstatement of drugseeking behavior.p.17 (cocaine-AHN-1055 0. which was also divided into two for the reinstatement test.e. the light stimulus and the infusion pumps were disconnected and presses on the active or inactive lever had no programmed consequences. Substitution sessions continued until all groups met this criterion. which showed no evidence of extinction. a yoked group (CO-AHN_yoked) was included. Yoked rats received passively the same infusions of AHN-1055 (0. Inactive lever presses had no programmed consequences. CO = cocaine. we decided to allow 1. When the stability criterion was achieved. Reinstatement test To avoid the possible influence of conditioned cue effects during drug-induced reinstatement. DUI = dopamine uptake inhibitor.p. = intraperitoneal.p. The behavior met criterion for extinction when the last 3 days of substitution differed on average from the last 3 days of cocaine training (Fig.5 mg/kg per infusion in 100 ml/5 seconds).17 mg/kg/infusion) (Fig.50 mg/kg/infusion) or AMPH (0.1 mg/kg/ infusion). including saline. AMPH = d-amphetamine sulfate. After substitution. 1.5 group. some of these groups were split into two in order to test for reinstatement following cocaine or saline challenge. 1). the rats were randomly assigned to one of the four substitution groups: CO-SAL (cocainesaline). Le.5 (cocaine-AHN-1055 0. illumination of a stimulus light for 5 seconds and retraction of the levers for 30 seconds. To be able to dissociate the effects of voluntary drug taking from the pharmacological effects of the treatment with AHN-1055. rats underwent daily 1-hour sessions until they reached a stability criterion (at least 10 1-hour sessions receiving 10 or more infusions per session. Diaz & Sokoloff 2005.5 mg/kg/infusion) that the paired rats self-administered voluntarily. 2009). immunoblotting assays and quantification of target proteins After the reinstatement tests. Active lever presses resulted in infusions of saline or cocaine. i. Priming injections were not given during training. all conditions remained as during training (i. Thereafter.17 or 0. The rats of this group underwent the substitution phase in the same conditions as their associated rats. except the group receiving AMPH substitution. Rats were exposed to 18 substitution sessions before the cocaineprimed reinstatement tests were conducted. Drug-seeking behavior was assessed during 1 hour.1 mg/kg/ infusion). A group of rats was yoked to the group receiving substitution with AHN1055 at the high dose. 2009). CO-AHN_0. but with levers retracted and light stimulus inactivated. 2009.5 hours from the beginning of the reinstatement session. Training and substitution sessions both lasted 1 hour. Cocaine (10 mg/kg) or saline was administered i. rats were left undisturbed in holding cages for 30 minutes and then killed by decapitation under ether anesthesia. levers were retracted and time-out was introduced).50 mg/ kg/infusion) and CO-AHN_0. Rats were trained to selfadminister saline or cocaine (0. thus 30 minutes after it finished (Curran & Morgan 1995. SAL-SAL (saline-saline). VelazquezSanchez et al. the stimulus light was activated during infusions. This allowed the reinstatement test to be conducted after the same period of withdrawal for all groups.5 mg/kg/ infusion) and then received one of the several substitution treatments. 2010b). inset). Based on the kinetics of target proteins. in the home cage 10 minutes prior to placing the animal in the operant chambers. with less than 20% intersession variation in the last three sessions) (Ferragud et al. so that both training and substitution curves could be compared. AHN-1055 (at dose of 0.

e. the rats readily switched from one compound to the other. MA. Briefly. divided into working aliquots and stored at -20°C until use. Santa Cruz Biotechnology).1 replicates). 2% sodium dodecyl sulfate (SDS). the value of a was set to equal 0. Beverly. UK) and visualized by exposure to Hyperfilm ECL autoradiograms (Amersham) for 1–60 minutes.8).4 Clara Velázquez-Sánchez et al. at 4°C. CA. mPFC samples from rats belonging to the same experimental group were pooled. neurochemical data. 3% SDS. Experiments were performed by using protein amounts known to be within the linear range of immunolabeling for each target protein (data not shown). batch K1404. anti-FADD (sc-5559. 1:5000). 1% bromophenol blue and total brain homogenates. Proteins were electrotransferred into 0. Miralles & Garcia-Sevilla 2007). 1:5000. brain samples from rats belonging to the same experimental group were pooled (n = 5–7).05 per experiment. The punches were aimed at the PrLC and Cg1 areas of the mPFC (Fig. IL. Equal volumes (1:1) of loading buffer.e. showing consistent and stable Addiction Biology © 2012 The Authors. The mean number of active lever presses per session was virtually identical in all groups self-administering cocaine. followed by post hoc comparisons with the method of Newman–Keuls (N–K) using the sampling error from the overall ANOVA as denominator. in gentle agitation. 1% of a protease inhibitor cocktail (SigmaAldrich) and 1% of a phosphatase inhibitor cocktail (Sigma-Aldrich). Danvers. the membranes were stripped and re-probed with antib-actin (clone AC-15. Santa Cruz. The primary BDNF antibody detects the immature and mature forms of the protein (28 and 13 kDa. obtained from repeated measures of the same pool of brain samples. the behavior of the rats changed depending on the substitution treatment received. Dassel. The immunoreactivities measured by primary antibody fixation were normalized with those obtained in the same gels after re-probing with anti-b-actin antibody. frozen in liquid nitrogen and finally stored at -80°C. 20% glycerol. Statistical analysis Behavioral data were analyzed by repeated-measures analysis of variance (ANOVA). Statistical significance was set at a = 0.8). and 1-mm coronal slices were collected on ice-cold buffer. Addiction Biology © 2012 Society for the Study of Addiction . Target proteins were quantified by Western blotting following standard procedures (Garcia-Fuster. samples were boiled for 4 minutes. USA). 5% 2-mercaptoethanol. USA. batch 1. All five groups of rats were matched in terms of cocaine intake before introducing the substitution. Rockford. Hercules. and so was the number of lever presses on the inactive lever (Fig. 1 mM ethylenediaminetetraacetic acid. 2). Samples (0. USA). Before tissue homogenization. Germany).61 Ϯ 0. containing 5% bovine serum albumin and 1% Tween-20. and the mean Ϯ the standard error of the replicates were considered as final estimates. were rapidly removed and placed on a brain matrix. containing 100 mM Tris-HCl (pH 6. After incubation in a blocking solution. Garcia-Fuster & Garcia-Sevilla 2001). 1:2000. RESULTS DUI substitution leads to extinction of active lever presses in rats with a stable history of cocaine self-administration Rats were initially trained on an FR1 schedule of reinforcement until consistent performance was attained. Bilateral samples from the mPFC were dissected out using a brain puncher (1 mm in diameter). Buckinghamshire. 1:700. Branson Sonifier 250) in 300 ml of icecold lysis buffer containing 50 mM Tris HCl (pH 6. Thus. anti-BDNF (sc-546. The primary affinity purified antibodies used in this study included anti-c-Fos (sc-253.02 (i. Santa Cruz Biotechnology. alpha value of 0. were analyzed by a randomized block design ANOVA (i. batch J1110. 40 W. batch D012. After introducing the substitution treatment. CA. were mixed. Finally. respectively). Immunoreactivity of target proteins was detected with the enhanced chemiluminiscence (ECL) Western Blot Detection system (Amersham International. USA). when AMPH was substituted for cocaine. 4a). Sigma-Aldrich) antibody (Boronat. As a control for sample loading and protein transfer. 1:10000. The quantification procedure was repeated for at least three times. The amount of target proteins in mPFC of experimental rats was compared in the same gel with that of control animals (SAL-SAL-SAL group). Total protein concentration in brain samples was determined by the bicinchoninic acid (BCA) protein assay (Pierce Biotechnology. MA. The autoradiograms were quantified by densitometric scanning (GS-800 Imaging Densitometer. the membranes were exposed to the primary antibody overnight.45-mm pore-sized nitrocellulose membranes (Whatman International Ltd. In this case. Santa Cruz Biotechnology) and anti-p-Ser191 FADD (Ab 2785. Total FADD or p-FADD were detected as dimeric (51 kDa) or oligomeric (116 kDa) species.05 divided by n . Membranes were subsequently incubated for 1 hour at room temperature in the blocking solution containing the horseradish peroxidase-linked antirabbit or antimouse IgG secondary antibody (Cell Signaling Technology. Bio-Rad Laboratories). Cell Signaling Technology. As expected. Because of the limited availability of brain tissue. a two-way ANOVA with experiment and treatment as the two factors) (see Lew 2007).23 mg/sample) from the mPFC were then homogenized by gentle sonication (2 ¥ 5 seconds. 15-mg protein aliquots from each pool of mPFC were resolved in 10–12% SDS-PAGE minigels (Bio-Rad Laboratories. USA).

01.91 Ϯ 0.0001) and of the interaction ‘Treatment’ ¥ ’Sessions’ (F68.432 = 60. responding for low-dose AHN-1055 was highest. Rats were matched in terms of intake before introducing the substitution treatment. The group self-administering amphetamine maintained similar levels of active lever presses after substitution. 2). whose subjects did not extinguish. Initially. Dots represent means Ϯ standard errors.5 (n = 8) and CO-AMPH (n = 5). *P < 0.31 presses.17 and 8. the mean number of active lever presses per session was very similar in all groups self-administering cocaine. P = 0. inset). CO = cocaine. CO-AHN_0.93 for CO-AHN_0.The analysis showed a main effect of the treatment (F4. 2).05.06 active lever presses. with 10 levels (one for each of the sessions of cocaine training). Addiction Biology © 2012 Society for the Study of Addiction . P = 0.42 Ϯ 1. Following several sessions during which drug seeking (and drug taking in the case of substitutions with AHN-1055) remained strong. ‘Treatment’. 2. DUI = dopamine uptake inhibitor. inset). meeting criterion for extinction.76 Ϯ 1. AHN-1055 did not behave as a classical stimulant during the substitution.764. ‘Session’.325. CO-SAL (n = 15).75 for CO-AHN_0. but not for the CO-AMPH group. Even after 18 extinction sessions. For the groups CO-SAL.699.5). P < 0. N–K = Newman–Keuls.47 = 25. Compared with substitution with saline and high-dose AHN-1055. #P < 0. AHN-1055 substitution groups responded similarly to the CO-SAL group (mean 3. CO-AHN_0. The ANOVA yielded significant effects of ‘Treatment’ (F4. responding for low-dose AHN-1055 was significantly increased in day 2 of substitution (Fig. AL = active lever. ANOVA was performed with one between-subjects factor. there were significant differences between the different groups of rats. The number of subjects per group was as follows: SAL-SAL (n = 13). P < 0.5. which did not differ significantly from those in the SAL-SAL group (N–K tests after ANOVA). However. DUI substitution blocks cocaine-induced reinstatement of drug-seeking behavior To test the effectiveness of the substitution therapy.17 and CO-AHN_0. whereas the groups that received DUI substitution showed significantly decreased response values. CO-AHN_0. Fig. except CO-AMPH. AMPH = d-amphetamine sulfate. During substitution. IL = inactive lever.17 (n = 11). compared with CO-SAL.05 Ϯ 0. SAL = saline responding across all 18 substitution sessions (Fig.0001).596. with the group SAL-SAL responding a mean of 1.Substitution therapy 5 Figure 2 DUI substitution treatment with AHN-1055 leads to extinction of active lever presses.The ANOVA comparing data for the last 3 days of cocaine training with the last 3 days of substitution showed a significant interaction effect (F4. 2. with five levels (four experimental groups and one control group) and one within-subjects variable. compared with CO-AMPH. ANOVA = analysis of variance.47 = 11.799 = 3.0001). substituting saline for cocaine (group CO-SAL) still generated some active lever responses (mean for the last 3 days was 7. response levels in sessions 16–17-18 were significantly reduced relative to stability sessions 8–9-10 of cocaine training (Fig. During training. a reinstatement test was performed the day after the last session Addiction Biology © 2012 The Authors. responding began to steadily decrease until extinction criterion was met by all substitution groups.0001) (extinction panel in inset).

P < 0. The number of subjects per group was as follows: SAL-SAL-SAL (n = 7). 3). as indicated by N–K post hoc tests (Fig. which yielded a significant effect (F6.45 = 9. with seven levels. In rats with experience in cocaine self-administration. Remarkably. ANOVA = analysis of variance. DUI = dopamine uptake inhibitor. Moreover. 4b). whereas saline injections (CO-SAL-SAL group). Post hoc analysis of the ‘Treatment’ effect showed that responding in the CO-SAL-CO subjects differed significantly from CO-SAL-SAL subjects. cocaine challenge before the reinstatement test induced significant elevations in precursor BDNF. **P < 0. and in mature BDNF. However.0001). N–K = Newman–Keuls. precursor (+58%) and mature (+115%) BDNF. CO-SAL-CO (n = 9).01. None of the three groups exposed to DUI substitution differed significantly from the CO-SAL-SAL group. cocaine prime treatment failed to increase the levels of FADD in the mPFC in rats that voluntarily selfadministered the DUI substitute or passively received it during the yoked procedure. CO = cocaine. which did not receive cocaine prime treatment (N–K test following ANOVA). the two DUI substitution groups. CO-AHN_0. induced only a slight increase in responding (Fig. = intraperitoneal. and the yoked group exhibited significantly attenuated drug-seeking responses DISCUSSION The data here reported provide a direct demonstration that replacement treatment in the drug-taking context with a DUI with low abuse liability can lead to extinction and provide resistance to relapse.17 (n = 11). substitution treatment and extinction training.6 Clara Velázquez-Sánchez et al. 4b–d). cocaine priming produced robust elevations in c-Fos expression (group CO-SAL-CO). ‘Treatment’.5 (n = 8) and CO-AHN_yoked (n = 5). However. which did not receive cocaine priming (CO-SALSAL) (Fig. compared with the CO-SAL-CO condition. only the high dose of the DUI substitution treatment and the yoked treatment blocked cocaine-induced changes in c-Fos content (Fig.5. 3). In rats pre-exposed to cocaine. acute administration of cocaine (SALSAL-CO group) significantly increased the immunoreactivities of c-Fos (+96%). compared with levels observed in control animals (SAL-SAL-SAL group). Addiction Biology © 2012 Society for the Study of Addiction . Patterns of protein expression in the mPFC parallel changes in reinstatement behavior after cocaine priming In drug naïve rats. CO-SAL-SAL (n = 6). 4c). DUI substitution dose-dependently and significantly decreased the over-expression of mature and immature BDNFs elicited by cocaine challenge (Fig. compared with CO-SAL-SAL. FADD protein was found to be elevated after the challenge of cocaine was given. and total FADD protein (+96%) in the mPFC. The data further indicate that this resistance is associated with blunted Addiction Biology © 2012 The Authors. Levels of drug seeking in the three DUI substitution groups were not significantly different from the cocaine-experienced group.668. drug-seeking behavior was much reduced in the three DUI substitution groups compared with the responses of the CO-SAL-CO subjects (N–K test after overall ANOVA).p. ANOVA was performed with one between-subjects factor.17 and CO-AHN_0. the immunocontents of these proteins did not differ from those of drug naïve rats after the extinction of cocaine self-administration (CO-SAL-SAL group) (Fig. 4d). compared with CO-SAL-CO. over and above levels induced by cocaine in drug naïve subjects. CO-AHN_0.01. SAL = saline of substitution treatment. Post hoc tests indicated that AHN-1055 substitution treatment prevented the stimulatory effects of cocaine on c-Fos expression. cocaine injections (10 mg/kg) produced robust reinstatement of drug-seeking behavior in the group that received saline substitution (CO-SALCO). COAHN_0. SAL-SAL-CO (n = 6). #P < 0. Figure 3 DUI substitution therapy prevents cocaine prime-induced reinstatement of drug seeking. Cocaine challenge produced vigorous reinstatement of drug seeking in CO-SAL-CO. Following cocaine selfadministration. Bars represent means Ϯ standard errors. We used c-Fos expression as a marker of neuronal activation in the mPFC. These treatments did not significantly alter the content of p-Ser191 FADD in the mPFC (data not shown). Predictably. and presumably the stress associated with the manipulation. Subjects in the CO-AMPH group were excluded because they did not extinguish lever pressing. whether or not animals had experience in cocaine self-administration. i.

1. dP < 0. The molecular masses of target proteins were estimated from referenced standards. Vanoxerine (GBR-12909).24 = 31. have shown to reduce cocaine use and craving. ###P < 0. However. mPFC = medial prefrontal cortex. Columns are means Ϯ standard errors of three to five replicated samples (pool of mPFCs. including dexamphetamine (Grabowski et al. Cg1 = anterior cingulate cortex. n = 5–7 rats) and expressed as percentage of the control group (SAL-SAL-SAL). PrLC = prelimbic cortex. Not surprisingly. a failure that may be linked to its ability to induce self-reports of dysphoria in cocaine addicts (Roache et al. CO = cocaine.2 and 3. **P < 0. Rothman et al.01. the dopamine hypothesis remains to this date the most influential account of the behavioral processes that typify addiction.05.5 mg/kg).5. 2009).001. 2011). N–K = Newman– Keuls. ANOVA = analysis of variance. Coronal sections correspond to diagrams located 3. Addiction Biology © 2012 Society for the Study of Addiction . compared with the group CO-SAL-SAL. P < 0. it has long been hypothesized that agents with high affinity for the dopamine transporter (DAT) could be used to suppress withdrawal and drug craving. 1992) and relapse (Wang et al. Randomized block (two-way) ANOVA showed significant differences between the groups of treatments for c-Fos (F6. 2003) and methamphetamine (Mooney et al. transition to compulsive drug taking and relapse. a piperazine derivative with high affinity for the DAT. thereby mitigating relapse behavior (Gorelick et al.9.8.17 or 0. BDNF and FADD expression in rat mPFC following substitution treatment with AHN-1055 (0.0001).0001). 2004. precursor BDNF (F6.001. In controlled trials with cocaine addicts. compared with SAL-SAL-SAL group. P < 0. SAL = saline ᭣ responsiveness to cocaine challenge in drug-experienced subjects. (a) Diagram representing the area targeted by the punches for mPFC (PrLC and Cg1) dissection and analysis of protein expression. (b–d) Effects of saline or cocaine challenge in rats receiving different substitution treatments on the contents of c-Fos.24 = 19. Changes in dopamine transmission that occur during and after drug exposure are believed to contribute to drug reinforcement (Volkow et al. *P < 0. precursor and mature BDNF and FADD. Although multiple neurotransmitter systems and receptors are likely to be implicated in mediating drug reinforcement.12 = 13.12 = 19. P < 0. Bottom (b–d): representative immunoblots of c-Fos. BDNF = brain-derived neurotrophic factor.Substitution therapy 7 Figure 4 Suppression of cocaine prime-induced c-Fos. Weiss et al. 1997). FADD and b-actin. 2001. 2008). 1992. 2000). compared with CO-SAL-CO group (N–K post hoc tests after ANOVA).0001). methylphenidate. Shearer et al.01. was ineffective at reducing cocaine use (Grabowski et al. ##P < 0. P < 0. The strategy involves the use of a competing agonist with slower receptor onset and offset and longer half-life than cocaine. another amphetamine-related compound.0001) and FADD (F6. underwent phase II clinical trials in cocaine addicts but development was halted due Addiction Biology © 2012 The Authors. FADD = Fas-associated protein with death domain. mature BDNF (F6.7 mm anterior to bregma (Paxinos & Watson 2007).01. withdrawal (Rossetti et al. drugs with significant abuse liability. ***P < 0. BDNF (precursor and mature protein detected in the same gel after different exposure times). 1996). as measured by decreased cocaine-induced expression of c-Fos. BDNF and FADD proteins in the mPFC during the reinstatement procedure.

animal models of reinstatement or context-conditioned relapse). This finding suggests that the induction of c-Fos. it is important to note that neither GBR-12909. including the ability to block cocaine. the reinforcing efficacy of the DUI treatment was probably weak because. Additionally. important evidence showed that following cocaine withdrawal. Compared with saline treatment. The current data are consistent with these findings. Whitfield & Berglind 2010).and amphetamine-induced conditioned place preference (Velazquez-Sanchez et al. We used AHN-1055. Moreover. AHN-1055 displays weak reinforcing efficacy compared with cocaine (Ferragud et al. DUI substitution therapy produced a striking decrease in the ability of cocaine to reinstate drug-seeking behavior. suggesting that the substitute produced some psychoactive effects that the rats sought to obtain. however. which are thought to be acquired during drug exposure and cause drug craving (Gray & Critchley 2007). including delayed onset and peak of pharmacological action. Furthermore. we took advantage of these sought-after neurochemical and behavioral features of AHN-1055. 1997). Additionally. BDNF and FADD proteins in the PrLC/Cg1 area in rats that received saline substitution and showed strong reinstatement. Cocaine priming produced enhanced expression of c-Fos. and the typical pharmacokinetic/dynamic features of other N-substituted analogs. as well as cocaine and amphetamine selfadministration in rats (Ferragud et al. which reduces cocaine intake in humans (Preti 2000). and longer half-life than cocaine (Raje et al. Addiction Biology © 2012 Society for the Study of Addiction . to cardiac side effects. 2005). responding for low-dose AHN-1055 was increased.8 Clara Velázquez-Sánchez et al. 2009).e. including the mPFC. At the outset of the substitution procedure. 2011). Hiranita et al.8 nM) and muscarinic M1 receptors (Ki = 11. Winger & Woods 1993) and rats (Tella 1995). 2008). Ziolkowska et al. 2009). AHN-1055 exhibits desirable behavioral features. We used the analysis of several proteins as a read-out of activity and adaptive change in the mPFC. 2011). One possibility is that substitution treatment produced extinction of conditioned interoceptive cues associated with cocaine withdrawal. To test the hypothesis that DUI substitution may provide long-term protection from relapse. We introduced a yoked group to control for the requirement of extinction of lever pressing during substitution. thus affording or not resistance to relapse in a different drug-taking environment. Peters et al. 2009. which may indicate functional engagement of the mPFC during reinstatement. This line of research is important in the light of recent evidence that reveals differences in DAT structure in cocaine addicts that are both genetic and cocaine induced (Zhou et al. However. 2009. 2001. This view is supported by the finding that cocaine-primed reinstatement of cocaine seeking decreases with repeated cocaine priming (Mihindou et al. we confirmed that the group receiving amphetamine as the replacement treatment continued to respond consistently throughout the substitution procedure. 2010b). nor any other longacting DUI has been shown to specifically reduce propensity to relapse in humans or animals (i. Thus. Although there is hope that new derivatives and analogs with selective actions at the DAT may remediate these problems. monkeys (Skjoldager. this increase also occurred in cocaine naïve rats. 2003). responding in the two AHN-1055 substitution groups decreased at the same rate as did in the saline substitution group. the results of the yoked group indicated that voluntary intake of the substitute and extinction of lever pressing was not required for the replacement treatment to produce enduring resistance to cocaine-primed reinstatement. 2011). BDNF and FADD proteins in the mPFC may be associated not only with cocaine-primed reinstatement but also with general sensitivity to cocaine. Pathways recruited during cocaine relapse may be segregated in the mPFC. Re-exposure to the cocaine taking context and relapse produced induction of BDNF mRNA in the mPFC (Hearing et al. 2009). with infralimbic cortex mediating suppressive effects after extinction and PrLC and Cg1 subserving renewed drug seeking by way Cg1 projections to the nucleus accumbens (Kalivas & McFarland 2003. and BDNF has been postulated to be a gatekeeper of synaptic efficacy and plasticity controlling addictive-like behavior (McGinty. as sessions progressed.6 nM) (Agoston et al. passive administration of the substitute in the drugtaking context afforded lasting protection from relapse. Further studies should determine whether the therapeutic effects of the substitution treatment are context specific. a benztropine derivative that exhibits strong affinity for the DAT (Ki = 11. 2009). Velazquez-Sanchez et al.and cocaine-induced reinstatement of drug seeking (Ciccocioppo et al. 2009. unlike the groups treated with AHN-1055. increases in Addiction Biology © 2012 The Authors. Cocaine treatment elevates BDNF expression at multiple brain sites. Activation of c-Fos and early response genes in the mPFC occurs following cue. Both PrLC and Cg1 are central elements of the circuits mediating craving and drug-seeking behavior. We searched for neural mechanisms associated with the therapeutic effects of the substitution treatment. Evidence has shown that tetrototoxin-induced inactivation of the PrLC blocks cocaine-primed reinstatement of drug seeking in selfadministration assays (Capriles et al. it is important to test whether the administration of the replacement treatment in a given environment protects from relapse in the primary drug-taking context. The weak reinforcing efficacy observed here is consistent with previous findings (Ferragud et al. Further indicating that the properties of AHN-1055 are dissimilar from typical psychostimulants.

replications with high-affinity DAT blockers without significant activity at M1 receptors are warranted. 2010). Addiction Biology © 2012 The Authors. elevated levels of basal FADD have been found in rats that display enhanced cocaine sensitization and have greater propensity to self-administer cocaine (Garcia-Fuster et al. the present findings provide a straightforward demonstration that an atypical. Disclosure/Conflict of Interest All authors report no biomedical financial interests or potential conflicts of interest.Substitution therapy 9 BDNF in the mPFC enhanced activity-induced long-term potentiation. Another aspect that requires further investigation is the relative contribution of DAT inhibition and M1 receptor blockade to the therapeutic-like effects of AHN-1055. In summary. These groups were not included as rats exposed to such condition would not be expected to show reinstatement of cocaine seeking (similarly to rats in the CO-SAL-SAL condition). In line with these observations. 2005. 1992. future studies should incorporate protracted relapse tests and other forms of reinstatement (e.and cue-induced reinstatement). our data now show that DUI substitution treatment reduces cocaine-induced increases in FADD content in the mPFC. 2009). 3). Instituto de Salud Carlos III (grant PI10/ 00297. George C. Acknowledgements This work was supported by grants from the Department of Psychology of the University of Canterbury (New Zealand). Kline RH. and JJC supervised the experiments. it is critical to emphasize that we investigated the therapeutic-like effects of DUI substitution in one model of reinstatement only (i.e. the specific mechanisms responsible for its effectiveness in this model of relapse remain to be determined. Although previous evidence indicates that DAT inhibition with benztropine derivatives is sufficient to block cocaine’s locomotor. Izenwasser S. which in turn associates with reduced susceptibility to cocaine prime-induced relapse. thereby reducing craving and vulnerability to relapse. Alvaro-Bartolome et al. 2011). rewarding and reinforcing effects (Desai et al. Thus. recent evidence suggests additional roles in cocaineinduced synaptic plasticity (Garcia-Fuster et al. Although the main role of FADD adaptor is pro-apoptotic. analyzed the data and wrote the manuscript. Wu JH. In agreement with these findings. 2007. We thank Agueda Ferrer for technical assistance. the induction of FADD by cocaine challenge was inhibited in rats that received DUI substitution treatment. Weiss et al. While the substitution therapy may have prevented some of the molecular and neurochemical adaptations that associate with withdrawal from stimulant exposure. and therefore our data are clinically relevant. Plan Nacional Sobre Drogas (grant PNSD2008-057. Spanish Ministry of Health) and Red de Trastornos Adictivos. Velazquez-Sanchez et al. muscarinic receptors undergo downregulation in rat brain following discontinuation of subchronic cocaine exposure (Macedo et al. The present data further indicate that cocaine promotes the expression of FADD protein in rat cortex. References Agoston GE. 2010a). Moreover.g. AF performed the behavioral assays. 2011. Spanish Ministry of Science and Innovation). drug-induced reinstatement) and at one timepoint after withdrawal (i. 2004). Regarding the clinical implications of our findings. However. 1992). Stimulant addicts are more prone to suffer craving and relapse during the first few days and weeks of discontinuation. effectively sensitizing excitatory synapses in the mPFC and contributing to behavioral sensitization and drug reactivity (Lu et al.e. Newman AH (1997) Novel N-substituted 3 alpha-[bis(4′fluorophenyl)methoxy]tropane analogues: selective ligands for the dopamine transporter. reinstatement of drug-seeking behavior constitutes a distinct behavior potentially modulated by multiple transmitter systems. J Med Chem 40:4329–4339. long-acting DUI effectively reduced relapse to cocaine seeking following substitution therapy in the drug-taking context. thus strongly supporting the rationale for developing medications that target the DAT and exhibit low abuse liability as treatment for cocaine addiction. Alvaro-Bartolome et al. 2009. 2011). with an apparent lack of conversion to the phosphorylated FADD species (GarciaFuster et al. stress. Taking these data as a whole. AR-M and JAG-S carried out the neurochemical experiments and analyzed the protein content data. while we recognize that the neurochemical evidence presented is only correlational. which involve changes in dopamine transmission (Rossetti et al. Authors Contribution CV-S performed the behavioral and neurochemical assays. Interestingly. Katz J. our data suggest that DUI substitution after cocaine taking reduces cocaine-induced BDNF expression in the mPFC of rats with diminished drug reactivity and susceptibility to drug-induced relapse. Addiction Biology © 2012 Society for the Study of Addiction . the striking changes in cocaine-induced protein expression during the reinstatement test strongly suggest that DUI therapy decreased reactivity to cocaine at the level of the mPFC. Finally. after 18 days of substitution). We acknowledge that the molecular data presented should be interpreted with caution due to the absence of additional groups of rats that could have been treated with saline after substitution (Fig.

Stotts A. Melis M. Schmitz J. Bossert JM. Correia EE. See RE. CA: Academic Press. Neurosci Biobehav Rev 35:157–171. II. Shaham Y (2006) Toward a model of drug relapse: an assessment of the validity of the reinstatement procedure. Capriles N. La HR. Diaz J. Vasconcelos SM. Bardo MT (2008) Individual differences in amphetamine self-administration: the role of the central nucleus of the amygdala. Xi ZX (2004) Agents in development for the management of cocaine abuse. Katz JL (2005) Identification of a dopamine transporter ligand that blocks the stimulant effects of cocaine. Watson CR (2007) The Rat Brain in Stereotaxic Coordinates. Critchley HD (2007) Interoceptive basis to craving. Morgan JI (1995) Fos: an immediate-early transcription factor in neurons. Cain ME. Diana M (2005) The dopamine hypothesis of drug addiction: hypodopaminergic state. Denehy ED.and cocaine-induced reinstatement of cocaine seeking in rats. Flagel SB. Newman AH. Le FB. J Clin Psychopharmacol 21:522–526. Brain Res 1314: 183–193. Watson SJ. Vouillac C. Carda M. Addiction Biology © 2012 Society for the Study of Addiction . Nacher A. Akil H (2009) Effect of cocaine on Fas-associated protein with death domain in the rat brain: individual differences in a model of differential vulnerability to drug abuse. Garcia-Fuster MJ. Problem 2. Quirk GJ (2009) Extinction circuits for fear and addiction overlap in prefrontal cortex. Lu L. Koob GF. Green CE. Johnson RE. McGinty JF. Katz JL (2009) Assessment of reinforcing effects of benztropine analogs and their effects on cocaine self-administration in rats: comparisons with monoamine uptake inhibitors. Garcia-Fuster MJ. Zhai H. Mahoney JJ. Cornuz J (2006) Treating tobacco use and dependence in clinical practice. Stewart J. NIDA Res Monogr 76:182–188. Learn Mem 16:279–288. Clinton SM. Cheng PL. Psychopharmacology (Berl) 168: 66–74. Newman AH. Canales JJ (2009) A dopamine transport inhibitor with markedly low abuse liability suppresses cocaine self-administration in the rat. Biol Psychiatry 70:593–598. Neuropsychopharmacology 32:399–411. Ghitza UE. Kalechstein AD. Mihindou C. Br J Pharmacol 134:1263–1270. Berglind WJ (2010) Brain-derived neurotrophic factor and cocaine addiction. Curran T. Neuroreport 16:175–178. Garcia-Fuster MJ. Psychopharmacology (Berl) 198:77–91. Ciccocioppo R. Koffarnus M. Hernandez-Rabaza V. Grabowski J. Drugs 64:1547–1573. Merino V. J Neurobiol 26:403–412. Murga J. Aguiar LM. Sokoloff P (2005) A single cocaine exposure increases BDNF and D3 receptor expression: implications for drug-conditioning. Lew M (2007) Good statistical practice in pharmacology. Weiss F (2001) Cocaine-predictive stimulus induces drug-seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: reversal by D(1) antagonists. Neuroscience 196:1–15. double-blind. Psychopharmacology (Berl) 207:281–289. J Neurosci 25:1889–1893. Preston KL. Neurotox Res 14:185–189. Neuropsychopharmacology 34: 1123–1134. Lu L (2010) Role of BDNF and GDNF in drug reward and relapse: a review. Prog Neuropsychopharmacol Biol Psychiatry 31:593–599. Airavaara M. Schmitz JM. Macedo DS. Creson D. Thompson RC. Garcia-Sevilla JA (2007) Effects of opiate drugs on Fas-associated protein with death domain (FADD) and effector caspases in the rat brain: regulation by the ERK1/2 MAP kinase pathway. Science 278:52–58. Gardner EL.10 Clara Velázquez-Sánchez et al. Daruzska LA. Grabowski J (2009) Effects of oral methamphetamine on cocaine use: a randomized. Eur J Pharmacol 526:36–50. Viana GS. McGinty JF (2008) Relapse to cocaine seeking increases activity-regulated gene expression differentially in the prefrontal cortex of abstinent rats. J Clin Psychopharmacol 17:485– 488. Rhoades H. J Pharmacol Exp Ther 329: 677–686. Callado LF. Herin DV. Psychopharmacology (Berl) 189:1– 16. Mayo LM. Korszun A (1997) Replacement medication for cocaine dependence: methylphenidate. Comparison with methadone in the detoxification of heroin addicts. Miller SW. Kalivas PW. De La Garza R. Mahmood ST. Neuron 67:821–833. Desai RI. Liebson IA. Shaham Y (2005) Neurobiology of relapse to heroin and cocaine seeking: an update and clinical implications. Shaham Y. Rhoades H. GarciaSevilla JA (2011) Molecular adaptations of apoptotic pathways and signaling partners in the cerebral cortex of human cocaine addicts and cocaine-treated rats. Ferragud A. Drug Alcohol Depend 101:34–41. Newton TF (2007) A qualitative and quantitative review of cocaine-induced craving: the phenomenon of priming. Grabowski J. Neuropsychopharmacology 33: 1149–1161. Akil H (2011) Decreased proliferation of adult hippocampal stem cells during cocaine withdrawal: possible role of the cell fate regulator FADD. Neuron 54:183–186. Khoshnevisrad N. Velazquez-Sanchez C. Garcia-Sevilla JA (2001) Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain. Examination of its opioid blocking properties. Jasinski DR (1987) A clinical trial of buprenorphine: I. Ghitza UE. Moeller FG (2001) Dextroamphetamine for cocainedependence treatment: a double-blind randomized clinical trial. Bickel WK. Int Rev Neurobiol 63:101–154. Peters J. Proc Natl Acad Sci U S A 98:1976–1981. Gray MA. Watson SJ. Le Moal M (1997) Drug abuse: hedonic homeostatic dysregulation. Gorelick DA. Koob GF. Paxinos G. Schmitz JM. Rodaros D. San Diego. McFarland K (2003) Brain circuitry and the reinstatement of cocaine-seeking behavior. Creson D. Moukaddam N. Lu H. Garcia-Fuster MJ. Soto PL. Br J Pharmacol 152:299–303. Ahmed SH (2011) Preclinical validation of a novel cocaine exposure therapy for relapse prevention. Stitzer ML. Epstein DH. Stewart J (2003) A role for the prefrontal cortex in stress. Boronat MA. Addiction Biology © 2012 The Authors. Kalivas PW (2008) Addiction as a pathology in prefrontal cortical regulation of corticostriatal habit circuitry. Bigelow GE. Poo MM (2010) Elevated BDNF after cocaine withdrawal facilitates LTP in medial prefrontal cortex by suppressing GABA inhibition. Kopajtic TA. Meana JJ. Neuropsychopharmacology 36:2303–2317. Kalivas PW. Sousa FC (2004) Cocaine treatment causes early and longlasting changes in muscarinic and dopaminergic receptors. Expert Opin Pharmacother 7:783–792. Epstein DH. Hiranita T. Sanna PP. Whitfield TW Jr. Cell Mol Neurobiol 24:129–136. Alvaro-Bartolome M. Lim BK. Mooney ME. Roache JD. Wu P. Spiga S. Miralles A. placebo-controlled trial. Hearing MC. Psychopharmacology (Berl) 168:44–56. Sorge RE.

Shalev U. Newman AH (2008) Dopamine transport inhibitors based on GBR12909 and benztropine as potential medications to treat cocaine addiction. Shearer J. Markou A. Velazquez-Sanchez C. Rothman RB. locomotor activity. [Epub ahead of print]. Schmitz JM. Brain Res 593:314–318. Wang GJ. Lin Z (2011) Ventral midbrain correlation between genetic variation and expression of the dopamine transporter gene in cocaine-abusing versus non-abusing subjects. Wang GJ. Katz JL. Dewey SL. Volkow ND. Rossetti ZL. Fowler JS. Eddington ND (2005) Pharmacodynamic assessment of the benztropine analogues AHN-1055 and AHN-2005 using intracerebral microdialysis to evaluate brain dopamine levels and pharmacokinetic/pharmacodynamic modeling. Hmaidan Y. the frontal cortex and memory circuits in drug addiction: insight from imaging studies. Woods JH (1993) Effects of GBR 12909 and cocaine on cocaine-maintained behavior in rhesus monkeys. Michelhaugh SK.2011. Shaham Y. Cornish J. J Clin Psychopharmacol 20:61–68. Winger G. Curr Opin Investig Drugs 1:241–251. Katz JL (2009) Discovery of drugs to treat cocaine dependence: behavioral and neurochemical effects of atypical dopamine transport inhibitors. Eur Neuropsychopharmacol 20:501–508. Fowler JS. Lieberman J (1996) Relationship between psychostimulant-induced ‘high’ and dopamine transporter occupancy. Volkow ND. Merino V. Carda M. Ziolkowska B. Gieryk A. Proc Natl Acad Sci U S A 93:10388– 10392. Rhoades HM (2000) Laboratory measures of methylphenidate effects in cocaine-dependent patients receiving treatment. Gatley SJ. Skjoldager P. Velazquez-Sanchez C. Neuropsychopharmacology 34:2497–2507. Zhou Y. Liu JS. Eur J Pharmacol 221:227–234. Pharm Res 22:603–612.1038/ mp. Velazquez-Sanchez C. Pharmacol Biochem Behav 51:687–692. Canales JJ (2010b) Therapeutic-like properties of a dopamine uptake inhibitor in animal models of amphetamine addiction. Logan J.86. Carda M. Cao J. Grabowski J. De WH. Soria G. Wong CT. Neurobiol Learn Mem 78:610–624.Substitution therapy 11 Preti A (2000) Vanoxerine National Institute on Drug Abuse. Smith L. Schmidt CJ. Int J Neuropsychopharmacol 24:1–11. J Neural Transm 118:877–887. Hernandez-Rabaza V. © 2012 The Authors. Sorge RE. Renau-Piqueras J. Hoffman W. Neuropsychopharmacology 30:1681–1692.1111/j. Lu L. Stewart J (2005) Rats maintained chronically on buprenorphine show reduced heroin and cocaine seeking in tests of extinction and drug-induced reinstatement. Alia-Klein N. Ferragud A. Przewlocki R (2011) Regulation of the immediate-early genes arc and zif268 in a mouse operant model of cocaine seeking reinstatement. Koob GF (1992) Basal extracellular dopamine levels in the nucleus accumbens are decreased during cocaine withdrawal after unlimited-access selfadministration. Logan J. Kielbinski M. Biochem Pharmacol 75:2–16. Tomasi D. Maldonado R. Ding YS. Ferragud A. Mol Psychiatry doi: 10. Lewis J (2003) Pilot randomized double blind placebo-controlled study of dexamphetamine for cocaine dependence. Addiction 98:1137– 1141. Raje S. Tella SR (1995) Effects of monoamine reuptake inhibitors on cocaine self-administration in rats. Canales JJ (2009) The dopamine uptake inhibitor 3 alpha-[bis(4′fluorophenyl)metoxy]-tropane reduces cocaine-induced early-gene expression. Jayne M. Lorang MT. and conditioned reward. Murga J. Nacher A. Creson DL. locomotor stimulation and sensitization. blocks cocaine-induced reward. Tanda G. van Beek I. Thanos P. methodology and major findings. Gessa GL (1992) Marked inhibition of mesolimbic dopamine release: a common feature of ethanol. Wodak A. Adv Pharmacol 57:253–289.1369-1600.2011. Telang F. Canales JJ (2010a) The high affinity dopamine uptake inhibitor. [Epub ahead of print]. Stewart J (2003) The reinstatement model of drug relapse: history. Drug Alcohol Depend 33:31–39. Psychopharmacology (Berl) 168:3– 20. Baumann MH. Goldstein RZ (2002) Role of dopamine. Hitzemann R. Weiss F. morphine. Bannon MJ. Newman AH. Addiction Biology © 2012 Society for the Study of Addiction Addiction Biology . Mattick RP. JHW 007. Ferragud A. Addict Biol doi: 10. Murga J. Rajabi H.x. Fowler JS (2011) Decreased dopamine activity predicts relapse in methamphetamine abusers. Prisinzano TE. Newman AH. Volkow ND. cocaine and amphetamine abstinence in rats. Roache JD. Wang GJ.00391.