SYNAPSE 69:336–344 (2015)

The Selective Dopamine D3 Receptor Antagonist SB-277011A Attenuates Drug- or Food-Deprivation Reactivation of Expression of Conditioned Place Preference for Cocaine in Male Sprague-Dawley Rats CHARLES R. ASHBY JR.,1* ONARAE V. RICE,2 CHRISTIAN A. HEIDBREDER,3 AND ELIOT L. GARDNER4 1 Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, New York 11439 2 Psychology Department, Furman University, Greenville, South Carolina 29613 3 Indivior PLC, 10710 Midlothian Turnpike, Suite 430, Richmond, VA, U.S.A. 2323 4 Neuropsychopharmacology Section, Molecular Targets and Medication Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224

KEY WORDS

conditioned place preference; dopamine D3 receptors; SB-277011A

ABSTRACT We determined the effect of the selective dopamine D3 receptor antagonist SB-277011A on reactivation of conditioned place preference (CPP) to cocaine elicited by priming injections of cocaine or exposure to food deprivation stress (21 h) in male Sprague–Dawley rats. Animals paired with the cocaine-associated chamber displayed a robust and consistent CPP response. This CPP was extinguished after repeated pairings of the conditioned stimuli (cocaine-paired chamber contextual cues) in the absence of the unconditioned stimulus (cocaine). Twenty-four hours later, the administration of 5 mg kg21 i.p. of cocaine (immediately before the test) or exposure to 21 h of food deprivation reactivated the expression of the cocaine-induced CPP. In contrast, administration of 1 ml kg21 i.p. of vehicle did not reactivate the CPP response. Administration of the selective dopamine D3 receptor antagonist SB-277011A (3–24 mg kg21 i.p.) 30 min before cocaine administration on the test day produced a significant attenuation of CPP reactivation. Reactivation of the CPP response produced by food deprivation was also significantly attenuated by SB-277011A (6 or 12 mg kg21 i.p.) given 30 min before the test session. SB-277011A (12 or 24 mg kg21 i.p.) did not itself produce reactivation of the CPP response. Overall, these results suggest that the reactivation of the incentive value of drug-associated cues by cocaine or food deprivation is attenuated by selective antagonism of D3 receptors. Synapse 69:336–344, 2015. VC 2015 Wiley Periodicals, Inc.

INTRODUCTION Exposure of drug addicts to environmental cues previously associated with their drug-taking behavior (e.g., drug paraphernalia, videos of drug use) (Childress et al., 1986, 1988; Ehrman et al., 1992; Weinstein et al., 1998), stress (Brown et al., 1995; Sinha et al., 2000; Sinha, 2001), or drug (Breiter et al., 1997; de Wit, 1996; Jaffe et al., 1989) can lead to craving and relapse to drug use. This can occur in the absence of the drug and after prolonged periods in a drug free state, suggesting that it is unrelated to the acute effects produced by drug withdrawal. Similarly, it has Ó 2015 WILEY PERIODICALS, INC.

Contract grant sponsor: National Institute of General Medical Sciences; Contract grant sponsor: U.S. National Institutes of Health; Contract grant number: P20-GM103499. *Correspondence to: Dr. Charles R. Ashby, Jr., Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, 8000 Utopia Parkway, Jamaica, NY 11439, USA. E-mail: [email protected] Received 5 November 2014; Revised 26 February 2015; Accepted 12 March 2015 DOI: 10.1002/syn.21820 Published online 24 April 2015 in Wiley Online Library (wileyonlinelibrary. com).

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been shown that various stressors (Bossert et al., 2005; Le and Shaham, 2002; Shaham et al., 2003; Shalev et al., 2000; Stewart, 2000), drug-related cues (Bossert et al., 2005; Caggiula et al., 2001; Robinson and Berridge, 1993; Shaham et al., 2003; Stewart et al., 1984; Stewart, 2000) and non-contingent administration of addictive drugs after drug-free periods can reinstate drug-taking behavior in animals (Bossert et al., 2005; de Wit and Stewart, 1987; Le and Shaham, 2002; Shaham et al., 2003; Stewart et al., 1984; Stewart, 1992). Factors that reinstate drug-seeking behavior in laboratory animals, such as drug, stress and drug cues, can elicit relapse to drug use in human addicts (Epstein et al., 2006). Clearly, interventions that significantly attenuate or decrease the likelihood of drug seeking would be useful in the management of addiction. One animal model that has been commonly used in addiction research at the animal level is conditioned place preference (CPP). In this model, drug or nondrug treatment is repeatedly paired with a previously neutral set of environmental stimuli. During the course of this pairing, the environmental stimuli acquire secondary motivational properties and act as conditioned stimuli (CS) to elicit approach or withdrawal (the latter if the primary motivational properties of the treatment were aversive) when the animal is re-exposed to the CS (Bardo and Bevins, 2000; Gardner, 2005; Tzschentke, 1998). CPP may be considered a measure of incentive motivation, defined as processes involving environmental stimuli that predict the perception of an unconditioned stimulus (UCS), which allows the animal on future occasions to seek out and anticipate various rewards in their environment, whether they are drug or non-drug related. One potential target for the pharmacotherapy of drug addiction is the central dopamine (DA) D3 receptor. This receptor, in rodents (Bouthenet et al., 1991; Levant, 1997; Sokoloff et al., 2006) and humans (Gurevich and Joyce, 1999; Landwehrmeyer et al., 1993; Murray et al. 1994; Suzuki et al., 1998), is highly localized to mesolimbic brain areas such as the nucleus accumbens (NAc) and amygdala, which are believed to play a role in mediating the rewarding/reinforcing properties of addictive drugs. Experiments delineating the role of the D3 receptor have been made possible by the synthesis and characterization of highly selective D3 receptor antagonist compounds (for review, see Heidbreder et al., 2005; Joyce and Millan, 2005). One such compound, SB-277011-A, has been shown to have an 80- to 100-fold selectivity for D3 over other DA receptors and 100-fold selectivity over 66 other receptors, enzymes, ion channels, and transporters in the central nervous system (Reavill et al., 2000; Stemp et al., 2000). Accumulating evidence indicates that D3 receptor antagonism

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attenuates the effects of addictive drugs, including cocaine, nicotine, methamphetamine, heroin and ethanol in various animal paradigms such as CPP, brain stimulation reward (BSR), progressive ratio (PR) reinforcement, variable-cost/variable-payoff fixed-ratio reinforcement, second-order reinforcement and drug-, cue- and stress-induced reinstatement (for comprehensive reviews, see Heidbreder and Newman, 2010; Heidbreder et al., 2005). Mueller and Stewart (2000) examined the effect of priming injections of cocaine in a variant of the CPP model. In their study, animals were paired with cocaine and specific environmental cues to establish a CPP. Then, animals were repeatedly given vehicle injections in the cocaine-paired CPP chamber. This produced extinction of the CPP response. On the test day, a priming injection of 5 mg kg21 of cocaine (UCS) was given immediately before the animals were placed in the CPP apparatus. Cocaine reactivated the CPP response, suggesting that relapse to drug use may occur from reactivating or restoring the incentive value of drug-associated cues. Using this model, we examined the effect of the highly selective D3 receptor antagonist SB-277011A on reactivation of the CPP response to cocaine elicited by a priming injection of cocaine or 21-h food deprivation in male Sprague–Dawley rats. MATERIALS AND METHODS Animals Male Sprague–Dawley rats (200 g at the start of CPP pairings, Taconic Farms, Germantown, NY) were used in all experiments. Animals were housed two per cage and there was no more than a 5% difference in body weight between cagemates. Animals were kept on a 12 h lights on/12 h lights off schedule (lights on at 0900 h). Food and water were freely available. Conditioning and testing of all animals was carried out between 1100 and 1800 h. All rats were naive and used only in a single cycle of experiments (i.e., initial CPP, extinction and reactivation). This study was approved by the Institutional Animal Care and Use Committee at St. John’s University. CPP apparatus An automated, two-chambered, Plexiglas CPP apparatus was used as previously described (Horan et al., 2000; Pak et al., 2006; Vorel et al., 2002), with modifications. The two pairing chambers of the apparatus were identical in dimensions (25 3 14 3 36 cm3) and were separated by removable Plexiglas guillotine doors. The pairing chambers had distinctively different visual and tactile cues. The walls of one of the pairing chambers were white with cage bedding (Bed-o-Cob) on the floor; the walls of the second chamber consisted of alternating white and black boxes (1.2 3 1.8 cm2) in a chessboard pattern, and a Synapse

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Plexiglas floor. The two pairing chambers were separated by a third, neutral connecting tunnel, with onehalf of the wall white and the other half consisting of the black and white chessboard pattern. The amount of time spent in each chamber was determined using an automated timing system consisting of infrared microbeams (two per chamber) wired to an automated timer. The beams were arranged such that when an animal entered a chamber, the beam was broken and a timer began recording. Once the animal left the chamber, the timer stopped. The timing circuitry had an internal clock that shut off the microbeams after 15 min. Procedure for determining initial cocaine-induced CPP expression During days 1–3, animals were acclimated to the animal facility. During days 4–6, animals were transported to the laboratory and handled for 5 min each. During days 7–14, animals were exposed to oncedaily cocaine-pairing or vehicle-pairing sessions. For each session, animals were injected with (-)-cocaine HCl (15 mg kg21 i.p. in a volume of 1 ml kg21) or vehicle (1 ml kg21 i.p. of deionized distilled water) and then immediately confined for 30 min in an appropriate cue-specific CPP chamber. Cocaine was always paired with one cue-specific environment, and vehicle was paired with the other; cocaine or vehicle exposure (and appropriate environmental pairing) alternated from day to day over an 8-day period, i.e., animals were given a pairing with cocaine and 24-h later given vehicle and this cycle of pairings was repeated four times. The animals in each group were randomly assigned to a 2 3 2 factorial design with one factor being the pairing chamber and the other factor being the order of pairing. In this counterbalanced procedure, the animals were randomly assigned to one of the two pairing chambers, so that half of the subjects received the drug in one compartment (white walls with bedding on the floor) and the other half in the other compartment (chessboard walls with a smooth floor). This procedure resulted in the animals receiving equal exposure to the two compartments and the random assignment controlled for side preference. One group of animals was paired only with vehicle in both chambers. On the test day (Day 15), animals were randomly divided into groups of 10 and tested in the CPP apparatus. During testing, the guillotine doors were removed and each animal was allowed to move freely within the apparatus for 15 min. Extinction of the initial CPP response to cocaine Following establishment of the initial CPP response to cocaine, animals were given pairings of vehicle (1 ml kg21 i.p.) with each chamber and conSynapse

fined to the appropriate chamber for 30 min every day for 7 consecutive days. The day following the last pairing, animals were placed in the CPP apparatus and the amount of time spent in each chamber was measured for 15 min. Reactivation of the extinguished CPP by a non-contingent injection of cocaine or by food deprivation Twenty-four hours after the last extinction trial, animals (n 5 10 per group) were given vehicle (1 ml kg21 i.p. of 25% w/v solution of 2-hydroxypropylb-cyclodextrin) or 3, 6, 12, or 24 mg kg21 i.p. of SB277011A (dissolved in 25% w/v solution of 2-hydroxypropyl-b-cyclodextrin). Thirty minutes later, a priming injection of 5 mg kg21 i.p. of cocaine was administered and the animals placed in the CPP apparatus and allowed access to all chambers for 15 min. The 5 mg kg21 dose of cocaine was chosen as it has been previously shown that this dose elicits CPP reactivation in male rats that have undergone extinction of cocaine-induced CPP (Mueller and Stewart, 2000). In addition, one set of animals received prior treatment with vehicle or 24 mg kg21 i.p. of SB277011A and then received a priming injection of vehicle and were then tested for CPP. A separate set of animals (n 5 10 per group) were handled, conditioned, and extinguished as before, but were then food deprived for a 21-h period (which reactivates the CPP response). This regimen was used based on previous reports that 21 h of food deprivation reinstates lever-pressing originally associated with cocaine (Shalev et al., 2003) or heroin (Shalev et al., 2002) delivery in rats. On the test day, animals were given vehicle, 3, 6, or 12 mg kg21 i.p. of SB-277011A and 30 min later, were allowed access to all chambers for 15 min. In addition, there were non-food-deprived animals that received vehicle (vehicle 1 no stress) or 12 mg kg21 i.p. of SB-277011A (vehicle 1 12 mg kg21 of SB-277011A 1 no stress) and then tested for CPP. Drugs (-)-Cocaine HCl was purchased from Sigma Chemicals (St. Louis, MO) and 2-hydroxypropyl-bcyclodextrin was purchased from Tocris-Cookson Chemical (St. Louis, MO). SB-277011A was obtained from GlaxoSmithKline (Harlow, Essex, UK). All drug doses are expressed as the salt weight. Statistical analyses The dependent variables were the times spent in the chamber paired with cocaine (paired) or vehicle (unpaired). The data were first analyzed using a twoway (3 3 7) ANOVA (repeated measurements) with main factors of experimental phase (CPP expression, CPP extinction, CPP reactivation) and pairings (Vehicle/Vehicle, Vehicle/Cocaine, SB-277011A 3 mg kg21/

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Cocaine, SB-277011A 6 mg kg21/Cocaine, SB277011A 12 mg kg21/Cocaine, SB-277011A, 24 mg kg21/Cocaine, SB-277011A, 12 or 24 mg kg21/ Vehicle). Subsequently, a one-way ANOVA was performed with a main factor of drug treatment. The post hoc analyses were conducted using Fisher’s least significant difference (LSD) test. RESULTS Cocaine-triggered CPP reactivation Cocaine produces a significant CPP response Statistical analysis indicated a significant effect of experimental phase (F2,54 5 61.4, P < 0.0001), drug

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treatment (F6,54 5 8.18, P < 0.0001), and phase 1 drug treatment (F12,54 5 8.2, P < 0.0001) on the time animals spent in the paired chamber of the CPP apparatus. A subsequent one-way ANOVA revealed an effect of drug treatment on the CPP response (F 5 8.3, P < 0.0001). Post hoc analyses indicated that all groups of animals that were paired with vehicle 1 cocaine spent a significantly greater amount of time (P < 0.0001) in the drug-paired chamber compared to animals that only received vehicle–vehicle pairings (i.e., vehicle in both chambers) (Fig. 1A). These results show that cocaine produced a significant and consistent CPP. The animals that were paired with vehicle in both chambers spent approximately the same amount of time in each chamber, indicating that the CPP in this study was unbiased. There were no significant differences between the initial pairings, extinction and reinstatement values in the vehicle-vehicle paired animals (F2,27 5 0.42, P 5 0.67).

Fig. 1. A. Expression of cocaine-induced CPP. Animals were given four pairings with vehicle (Veh; 1 ml kg21 i.p.) or cocaine HCl (Coc; 15 mg kg21 i.p.) or vehicle only (Veh/Veh) over an 8-day period. Twenty four hours after the last CPP pairing, animals were allowed free access to all chambers of the CPP apparatus and amount of time spent in each compartment was determined over a 15-min test period using an automated timer. Each value represents the mean number of minutes 6 SEM. The 6 Veh/Coc groups are the five different SB-277011A dose test groups (0, 3, 6, 12, 24 mg kg21) plus the Veh 1 24 mg kg21 SB-277011A test group (see C). Ten animals were used for each group. #Significantly greater than noncocaine-paired (vehicle) values, P < 0.0001. *Significantly less than cocaine-paired values, P < 0.0001. (B). Extinction of cocaine-induced CPP. Once a day for 8 consecutive days, animals that had previously acquired CPP expression (A) were put through a CPP extinction regimen. This was achieved by alternately giving each animal vehicle and placing the animal in the CPP chamber that had been previously paired with cocaine, and giving each animal vehicle and placing the animal in the CPP chamber that had been previously paired with vehicle, on alternate days. Twenty four hours after the last extinction pairing, the animals were allowed free access to all chambers of CPP apparatus and the amount of time spent in each compartment was determined over a 15-min test period using an automated timer. Each value represents the mean number of minutes 6 SEM As in (A), the six Veh/Coc groups are the five different SB-277011A dose test groups (0, 3, 6, 12, 24 mg kg21) plus the Veh 1 24 mg kg21 SB-277011A test group (see C). Ten animals were used for each group. As shown in the figure, the CPP extinction regimen successfully extinguished the original cocaine-induced CPP. (C). Reactivation of extinguished cocaine-induced CPP by a single cocaine injection, and effects of the D3 receptor antagonist SB277011A on such reactivation. To assess the effects of D3 receptor antagonism on cocaine-triggered CPP reactivation, animals from the extinction phase were given vehicle (Veh, 1 mL kg21 i.p.) or SB277011A (SB 3, 6, 12, or 24 mg kg21 i.p., dissolved in 25% w/v solution of 2-hydroxypropyl-b-cyclodextrin). Thirty minutes later, animals received 5 mg kg21 i.p. of cocaine (Coc) (five groups) or 24 mg kg21 i.p. of SB-277011A. The amount of time spent in each chamber was measured over a 15-min period using an automated timer. Each value represents the mean number of minutes 6 SEM Ten animals were used for each group. *Significantly greater than the paired values for Veh 1 Veh, SB 3 mg kg21 1 Coc, SB 6 mg kg21 1 Coc, 12 mg kg21 1 Coc and SB 24 mg kg21 1 Coc, P < 0.0001. #Significantly greater than the paired values for SB 6 mg kg21 1 Coc (P 5 0.015), 12 mg kg21 1 Coc (P < 0.0001) and SB 24 mg kg21 1 Coc (P < 0.0001). @Significantly greater than the paired value for SB 24 mg kg21 1 Coc, P < 0.02.

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Extinction of the CPP response to cocaine The animals used in the above CPP expression phase were then subjected to extinction. A one-way ANOVA indicated that following repeated exposure of the animals to vehicle in the chambers previously associated with cocaine, there was no significant CPP in these animals compared to those paired with vehicle in both chambers (F 5 0.13, P 5 0.99) (Fig. 1B). Thus, there was no significant difference in time spent in the paired side between control animals (vehicle–vehicle) and all other groups (vehicle– cocaine), i.e., a CPP response was not observed. These results indicate that the CPP response to cocaine underwent extinction. Reactivation of the CPP response by cocaine is significantly attenuated by SB-277011A Twenty-four hours after CPP extinction, rats were tested for cocaine-triggered CPP reactivation. The vehicle-cocaine paired rats were randomly divided into five groups of 10 animals. On the test day, the animals received one of the following treatments (i.p.) 30 min prior to receiving an injection of 5 mg kg21 i.p. of cocaine and placement in the CPP apparatus: vehicle, 3, 6, 12, or 24 mg kg21 SB-277011A. The vehicle–vehicle paired group was given 24 mg kg21 of SB-277011A followed by an injection of vehicle. A one-way ANOVA (F =13.2, df 5 6, P < 0.0001) indicated a significant effect of treatment on CPP expression. Administration of vehicle did not produce CPP reactivation (Fig. 1C). However, the CPP response was reactivated following administration of 5 mg kg21 i.p. of cocaine (P < 0.0001, Fisher’s LSD test) (Fig. 1C). Post hoc analyses indicated that a single i.p. administration of 3, 6, 12, or 24 mg kg21 of SB-277011A significantly attenuated cocaine-triggered CPP reactivation (P < 0.0001 for all doses, Fisher’s LSD test) (Fig. 1C) compared to animals treated with vehicle. Acute administration of 6, 12, or 24 mg kg21 of SB277011 produced a significantly greater (P < 0.0001) attenuation of CPP reactivation than the 3 mg kg21 dose. In addition, the 24 mg kg21 dose produced a greater reduction in CPP reactivation compared to animals that received 6 mg kg21 (Fig. 1C). The vehicle–vehicle group that received 24 mg kg21 SB-277011A followed by vehicle did not show a significant CPP (Fig. 1C). In fact, there was no difference between their extinction and reactivation CPP responses. This result indicates that SB277011A alone (24 mg kg21) does not reactivate an extinguished cocaine-induced CPP. Food deprivation triggered CPP reactivation Statistical analysis indicated a significant effect of phase (F2,54 5 109.1, P < 0.0001), drug treatment (F6,54 5 7.7, P < 0.0001) and phase 1 drug treatment (F12,54 5 6.2, P < 0.0001) on the time animals spent in Synapse

the cocaine-paired CPP chamber. A subsequent one-way ANOVA revealed a significant effect of drug treatment on CPP response (F 5 8.7, P < 0.0001). Post hoc analyses indicated that all groups of animals that were paired with vehicle 1 cocaine spent a significantly greater amount of time (P < 0.0001) in the cocaine-paired chamber, as compared to animals that only received vehiclevehicle pairings (Fig. 2A). Thus, cocaine produced a significant and consistent CPP response. Animals receiving only vehicle did not show a CPP. Extinction of the cocaine-induced CPP The above animals were subsequently paired with vehicle for four pairings in the chamber in which they had previously received cocaine. A one-way ANOVA indicated that, after these repeated vehicle pairings, there was no significant difference in time spent in the previously cocaine-paired chambers as compared to the previously vehicle-paired chambers between groups of animals (F 5 0.4, df 5 6, P 5 0.88) (Fig. 2B). In addition, the CPP response in these animals was significantly lower than their original CPP expression values, and not significantly different from vehicle–vehicle paired animals. Thus, the CPP response to cocaine had been extinguished. Reactivation of the CPP response by fooddeprivation is significantly attenuated by SB277011A A two-way repeated-measures ANOVA on fooddeprivation-induced CPP reactivation indicated a significant effect of phase (extinction vs. reactivation; F1,54 5 287.5, P < 0.0001) but not group (F5,54 5 0.23, P 5 0.95) or group 3 phase (F5,54 5 0.37, P 5 0.87). Administration of vehicle did not produce a CPP reactivation. However, food deprivation reactivated expression of the CPP response (P < 0.0001) (Fig. 2C). One-way repeated-measures ANOVA indicated a significant effect of SB-277011A treatment on food deprived-induced reactivation of cocaine-induced CPP (F 5 10.5, P < 0.0001). Subsequent post hoc analysis indicated that that food deprivation produced CPP reactivation (P < 0.0001, Fig. 2A). The 3 mg kg21 i.p. dose of SB-277011A did not significantly (P 5 0.12) alter food deprived-induced CPP (Fig. 2C). However, a single i.p. administration of 6 (P 5 0.0015) or 12 (P < 0.0001) mg/kg i.p. of SB-277011A significantly attenuated food deprived-induced reactivation of the CPP response. In addition, the 12 mg kg21 dose of SB-277011A produced significantly greater attenuation of the reactivated CPP response compared to 3 mg kg21 (P < 0.0001) or 6 mg kg21 (P 5 0.0016) of SB277011A. Administration of 12 mg kg21 SB-277011A to animals that were not food deprived (Veh 1 SB 12 mg kg21/Not Food Deprived, Group 2F) did not

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reactivate the CPP response (not significantly different from Veh/Veh/Not Food Deprived (Group 2A), P 5 0.54). DISCUSSION As previously reported (for review, see Aguilar et al., 2009; Tzschentke, 2007), the pairing of adult male rats with cocaine and specific visual/tactile cues produces a robust and consistent CPP. In the present work, we show that this cocaine-induced CPP can undergo extinction (i.e., the cocaine-paired environmental cues in the CPP chamber lose their incentive motivational value, and no longer elicit drug-seeking behavior). This is congruent with previous reports

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that expression of cocaine-induced CPP can be extinguished by repeated pairing of the animals with vehicle injections in the original drug-paired chamber (e.g., Lu et al., 2002; Mueller and Stewart, 2000; Wang et al., 2000). We also here show that a noncontingent injection of cocaine reactivates the extinguished CPP response, which is congruent with previous reports (for review, see Aguilar et al., 2009). It has been posited that CPP reactivation is attributable to the discriminative stimulus characteristics of the drug—acting in a manner so as to produce statedependent learning. This is unlikely because (as pointed out by Aguilar et al., 2009): (1) the drug administration is not paired with any specific instrumental response; (2) reactivation can be elicited by various non-drug stimuli; (3) the expression of CPP to one drug can be reactivated by administration of drugs from other pharmacological classes, and does not appear to be due to generalization to the drug used in initial CPP pairing. It could be argued that reactivation is related to the phenomenon of sensitization, as the animals are challenged with a nonFig. 2. A. Expression of cocaine-induced CPP. Animals were given four pairings with vehicle (Veh; 1 ml kg21 i.p.) or cocaine HCl (Coc; 15 mg kg21 i.p.) or vehicle only (Veh/Veh) over an 8-day period. Twenty four hours after the last CPP pairing, animals were allowed free access to all chambers of the CPP apparatus and amount of time spent in each compartment was determined over a 15-min test period using an automated timer. Each value represents the mean number of minutes 6 SEM The six Veh/Coc groups are the five different SB277011A dose test groups (0, 3, 6, 12, 24 mg kg21) plus the Veh 1 24 mg kg21 SB-277011A test group (see C). Ten animals were used for each group. *Significantly greater than non-paired values, P < 0.0001. #Significantly less than Veh/Coc paired values, P < 0.0001. (B). Extinction of cocaine-induced CPP. Once a day for 8 consecutive days, animals that had previously acquired CPP expression (Fig. 1A) were put through a CPP extinction regimen. This was achieved by alternately giving each animal vehicle and placing the animal in the CPP chamber that had been previously paired with cocaine, and giving each animal vehicle and placing the animal in the CPP chamber that had been previously paired with vehicle, on alternate days. Twenty four hours after the last extinction pairing, the animals were allowed free access to all chambers of CPP apparatus and the amount of time spent in each compartment was determined over a 15-min test period using an automated timer. Each value represents the mean number of minutes 6 SEM. As in (A), the 6 Veh/Coc groups are the five different SB-277011A dose test groups (0, 3, 6, 12, 24 mg kg21) plus the Veh 1 24 mg kg21 SB-277011A test group (see C). Ten animals were used for each group. As shown in the figure, the CPP extinction regimen successfully extinguished the original cocaine-induced CPP. (C). Reactivation of extinguished cocaine-induced CPP by food deprivation, and effects of the D3 receptor antagonist SB-277011A on such reactivation. To assess the effects of D3 receptor antagonism on food deprivation -triggered CPP reactivation, animals from the extinction phase were subjected to food deprivation for 21 h. Then, on the test day, the food deprived animals were given vehicle (Veh, 1 ml kg21 i.p.) or SB-277011A (SB 3, 6, or 12 mg kg21 i.p., dissolved in 25% w/v solution of 2-hydroxypropyl-bcyclodextrin). One group of non-food deprived animals was given vehicle plus vehicle (Group 2A) and another non-food deprived group was given vehicle plus 12 mg kg21 i.p. of SB-277011A (Group 2F). The amount of time spent in each chamber was measured over a 15min period using an automated timer. Each value represents the mean number of minutes 6 SEM Ten animals were used for each group. *Significantly greater than Veh 1 Food Deprived Stress, P < 0.0001. #Significantly less than Veh 1 Food Deprived, P < 0.0001. & Significantly less than Veh 1 Food Deprived, P < 0.0001.

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contingent injection of cocaine 8 days after sub-chronic cocaine pairings. However, a recent study in mice using the CPP reactivation paradigm found no significant correlation between reactivation and sensitization to a challenge dose of cocaine (Brown et al., 2010). The present study is the first to show that a single administration of 6, 12, or 24 mg kg21 of SB-277011A significantly attenuates cocaine-induced reactivation of the CPP response following CPP extinction in rats. In fact, the 12 and 24 mg kg21 doses of SB-277011A completely reversed the cocaine-induced reactivation. Our results are in line with other studies that have examined the effect of highly selective D3 receptor antagonists on cocaine’s action in other paradigms. For example, Vorel et al. (2002), using the reinstatement paradigm, reported that SB-277011A produced a dosedependent decrease in cocaine-induced reinstatement of i.v. cocaine self-administration. In addition, administration of the highly selective D3 receptor antagonists YQA-14 (Song et al., 2012, 2014) and NGB2904 (Xi et al., 2006; Gilbert et al., 2005) and the preferential D3/D2 antagonist S33138 (Peng et al., 2009) significantly decreased cocaine-induced reinstatement of i.v. cocaine self-administration in rodents. Our current findings suggest that the D3 receptor is instrumental in mediating cocaine-induced reactivation of the CPP response. It might be argued that antagonism of D3 receptors interferes with the detection of the environmental stimuli normally associated with the drugpaired chambers. This is unlikely because SB-277011A does not alter learning or memory in rodents. In fact, it enhances social recognition (Loiseau and Millan, 2009; Millan et al., 2007) and reduces the amnesic effects of scopolamine in rodent models that measure memory deficits (Laszy et al., 2005) at the doses used in the present study. In addition, compared to vehicle-treated rats, acute administration of SB-277011A (9.2, 20.1 or 42.2 mg/kg) does not significantly alter the startle response or pre-pulse inhibition (Reavill et al., 2000). Another significant finding of the present study is that exposure of animals to 21 h of food deprivation significantly reactivates the expression of cocaineinduced CPP in animals that have undergone CPP extinction. In the present study, a single i.p. administration of 6 or 12 mg kg21 of SB-277011A significantly attenuated food-deprivation-induced reactivation of the CPP response to cocaine. The mechanisms underlying such food-deprivation-induced CPP reactivation are not known. It has been reported that food deprivation activates the hypothalamic-pituitary axis in rats and mice (Dallman et al., 1995; Kiss et al., 1994; Mantella et al., 2005), producing an increase in corticosterone, a hormone involved in modulating responses to stressful stimuli. However, studies using the selfadministration/reinstatement model indicate that stress-induced increases in corticosterone release are not involved in reinstatement (Erb et al., 1998; Shalev Synapse

et al., 2003). Food deprivation has also been reported to enhance levels of a number of endogenous molecules that activate mesocorticolimbic DA neurons—including (but not limited to) ghrelin and orexins (Aston-Jones et al., 2009; Bingham et al., 2006; Jerlhag et al., 2006; Quarta et al., 2009). It is unlikely that SB-277011A directly antagonizes receptors of these or other peptidergic molecules involved in mediating homeostatic response(s) to food deprivation. However, it is possible that SB-277011A’s D3 receptor antagonism attenuates the enhanced DA produced by such molecules. Xi et al. (2004), using the reinstatement paradigm, reported that SB-277011A significantly attenuated foot-shock-induced reinstatement of i.v. selfadministration of cocaine in rats. Although the CPP reactivation paradigm is different from that used by Xi et al. (2004), the pharmacological effect produced by SB-277011A was similar in each—decreased drugseeking behavior elicited by stressful stimuli. Such findings are consistent with reports that stress alters the function/activity of mesocorticolimbic DA neurons (for reviews, see Koob, 2008; Ungless et al., 2010). D3 receptors are present in brain areas innervated by these neurons (Sokoloff et al., 2006). Also, the hypothalamus, a brain region involved in the stress response, has a high concentration of D3 receptor mRNA (Reavill et al., 2000). Thus, by antagonizing D3 receptors, SB-277011A may inhibit effects produced by stress-induced release of DA. It is also possible that SB-277011A produces an anti-stress/ anxiolytic action, and that this attenuates fooddeprivation-induced reactivation of cocaine-induced CPP. Currently, there are no published reports on the effect of systemic SB-277011A in animal models of anxiety. However, Diaz et al. (2010) reported that injection of the selective D3 antagonist GR103691 (D3/D2 in vitro 5 60; Audinot et al., 1998) into the basolateral amygdala produces anxiolytic action in the elevated plus maze test in rats. There are further data suggesting that SB-277011A may have antiaversive actions. For example, SB-277011A (6, 12, or 24 mg kg21 i.p.) significantly decreases the conditioned place aversion produced by naloxoneprecipitated morphine withdrawal in rats (Rice et al., 2012). SB-277011A also dose-dependently blocks the expression of conditioned fear in rats produced by stimulus-shock pairings (Swain et al., 2008). Also, SB-277011A’s action(s) could result from its interaction with DA D2 or other receptors. This is unlikely as, in vitro, SB-277011A has 100-fold selectivity for D3 receptors compared to (1) D2 receptors; (2) >66 other receptors (including D1, D4 and D5), enzymes, ion channels and transporters in brain tissue (Reavill et al., 2000; Stemp et al., 2000). Furthermore, the doses of SB-277011A used in the present study do not produce behavioral effects typically elicited by D2 antagonists (Reavill et al., 2000; for review, see

D3 RECEPTOR ANTAGONISM BLOCKS RELAPSE TO COCAINE SEEKING

Heidbreder et al., 2005). SB-277011A’s attenuation of stress- and drug-induced reactivation of CPP could also conceivably be due to interference with memory retrieval. However, acute administration of SB277011A actually enhances social memory (Millan et al., 2007) and attenuates the deleterious effects of scopolamine in rodent models that assess memory deficits (Laszy et al., 2005; Millan et al., 2007). In addition, SB-277011A (3–24 mg/kg) has been shown to not produce adverse motoric effects (Reavill et al., 2000). Conceivably, the present findings could have been due to SB-277011A producing either appetitive or aversive actions by itself. However, this possibility is unlikely, as acute administration of SB-277011A alone has been shown to not be rewarding or aversive (Vorel et al., 2002; Xi et al., 2005). Also, acute administration of SB-277011A has no significant effect on oral self-administration of sucrose pellets in rats (Song et al., 2012), suggesting that it does not alter the appetitive effects of natural rewards. In conclusion, acute i.p. administration of SB277011A significantly decreases reactivation of the expression of cocaine-induced CPP produced by cocaine or by food deprivation. These findings are concordant with data from studies using operant paradigms indicating that highly selective D3 receptor antagonists significantly attenuate reinstatement of cocaine i.v. selfadministration produced by cocaine priming or food deprivation. Human relapse to drug-seeking behavior is often triggered by environmental cues (e.g., people, places, things) and contexts previously paired with drug-taking behavior. The present results suggest that antagonism of D3 receptors could decrease the association of cues originally paired with the appetitive effects of cocaine and therefore have therapeutic value. Specifically, highly selective D3 antagonists could decrease the incentive motivational value of conditions or situations that previously led to drug-taking behavior. Ultimately, it is important to determine if the preclinical findings with selective D3 receptor antagonists can be translated into treatment for drug dependence and addiction in humans (Newman et al., 2012).

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