INTRODUCTION

Nicotine is considered the major psychoactive component of tobacco leading to smoking addiction, a chronic brain disorder with the specific characteristics of difficulty in stopping smoking, highly frequent relapse after abstinence, and high comorbidity with other mental disorders (Brady and Sinha, 2005; Dalack et al, 1998; DiFranza and Ursprung, 2010; Hitsman et al, 2009; Le Foll and Goldberg, 2006; Morisano et al, 2009; Ziedonis et al, 2008). Nicotine has been reported to support the acquisition, maintenance, and reinstatement of nicotine-seeking behavior induced by the presentation of nicotine-associated cues or by a priming injection of nicotine in rodents, dogs, non-human primates, and humans (Corrigall, 1999; Corrigall and Coen, 1989; Donny et al, 1995; Goldberg et al, 1981; Le Foll et al, 2007; Liu et al, 2006; Risner and Goldberg, 1983; Rose and Corrigall, 1997; Shaham et al, 1997; Sofuoglu et al, 2007). The reinforcing effects of nicotine are primarily due to activation of mesoaccumbal dopamine system (Corrigall and Coen, 1991; Corrigall et al, 1992; Di Chiara, 2000; Laviolette and van der Kooy, 2004; Liu et al, 2010; Mansvelder and McGehee, 2000; Picciotto, 2003; Pidoplichko et al, 1997; Pierce and Kumaresan, 2006; Self, 2004; Wise, 2004), although emerging evidence suggests that other neurotransmitter systems are involved as well. Among the components of the mesoaccumbal dopamine system, dopamine receptors are classified into two super families: D1-like (D1 and D5) and D2-like (D2, D3 and D4), based on their amino-acid sequence homology and modulation of intracellular adenyl cyclase activity. Different subtypes of dopamine receptors have substantially different expression levels in distinct neuronal populations of the brain, thereby having fundamentally different roles in the development of substance dependence/addiction and relapse (Andreoli et al, 2003; Corrigall and Coen, 1991; Di Chiara, 2000; Haile and Kosten, 2001; Khaled et al, 2010; Liu et al, 2010; Ross et al, 2007). Among these dopamine receptors, the role of the dopamine receptor D4 (DRD4) in the development of nicotine dependence and relapse remains unclear (see Le Foll et al, 2009 for review).

It has been reported that chromosome 11p, which includes the DRD4 locus, is associated with increased susceptibility to cigarette smoking (Gelernter et al, 2004). In humans, the DRD4 gene is highly polymorphic with a variable number of tandem repeats (VNTRs) in exon III (in particular, the presence or absence of the 7-repeat (‘long’) allele) and a single-nucleotide polymorphism in the promoter region (C-521T), with both being associated with variation in the expression of DRD4, decreased ligand binding, and attenuation of cyclic adenosine monophosphate (cAMP) formation (Asghari et al, 1994, 1995; Cohen et al, 1992). In depressed smokers, homozygous for the short alleles of the DRD4 gene, smoking practices related to self-medication (ie. smoking to increase arousal or decrease negative affect) were significantly higher when compared with smokers heterozygous or homozygous for the long alleles (Lerman et al, 1998). Recently, these two polymorphisms of the DRD4 gene have been linked to chronic alcoholism, tobacco smoking, opioid dependence, impulsivity, novelty-seeking behavior, and attention-deficit hyperactivity disorder (Chien et al, 2010; Ebstein et al, 1996; George et al, 1993; Ho et al, 2008; Kotler et al, 1997; Li et al, 2000; Luciano et al, 2004). In addition, the VNTR polymorphism of the DRD4 gene has been correlated with smoking and cocaine cue reactivity, as well as alcohol- and heroin-craving behavior (Hutchison et al, 2002a, 2002b, 2003, 2006; McClernon et al, 2007; Munafo and Johnstone, 2008; Shao et al, 2006).

The ability of DRD4 antagonists to reverse the rewarding effects of nicotine that are primarily responsible for tobacco dependence has seldom been explored with animal models. Previously, DRD4 antagonists have been reported to attenuate the discriminative-stimulus effects of cocaine and methamphetamine (Ukai and Mitsunaga, 2005; Yan et al, 2004, 2006), and to attenuate morphine-induced withdrawal signs precipitated by naloxone (Mamiya et al, 2004). Using an intravenous (i.v.) cocaine self-administration model, it was noted that pretreatment with the dopamine D3/D4 receptor antagonist L-745,829 or the selective DRD4 antagonist L-745,870 does not affect cocaine self-administration in rats (Caine et al, 2002). In this study, we examined the effects of the selective DRD4 antagonist (L-745,870) on established nicotine self-administration behavior and on reinstatement of extinguished nicotine-seeking behavior induced by either nicotine-associated cues or by a priming injection of nicotine in rats. We also tested the selective DRD4 agonist (PD 168,077) for its ability to reinstate extinguished nicotine-seeking behavior. Finally, to evaluate whether D4 receptors were affecting reinstatement of nicotine seeking by altering an animal's ability to discriminate the effects of nicotine, we examined the effects of L-745,870 in a classic two-lever nicotine discrimination procedure.

MATERIALS AND METHODS

Subjects and Drugs

Male Long-Evans rats (Charles River, Lachine, PQ, Canada) were used for all nicotine and food self-administration experiments, whereas male Sprague Dawley rats (Charles River, Wilmington, MA, USA) were used for discrimination studies to be consistent with previous studies (Le Foll and Goldberg, 2004, 2005; Le Foll et al, 2008). Animals were initially group housed in a humidity- and temperature-controlled (21–22 °C) vivarium on a 12-h reversed light/dark cycle with access to ad libitum water and food. After a week of habituation to the colony room, animals were singularly housed and diet restricted (∼20g/day) throughout the experiments. All experimental procedures described in this study were carried out in compliance with the guidelines of the Canadian Council on Animal Care and/or the guidelines of the Animal Care and Use Committee of the National Institute on Drug Abuse Intramural Research Program.

(−)Nicotine hydrogen tartrate (Sigma-Aldrich, St Louis, MO, USA) was dissolved in saline and the pH of solution was adjusted to 7.0±0.2. Nicotine solution was freshly prepared and filtered through a 0.22-mm syringe filter (Fisher Scientific, Pittsburgh, PA, USA) to minimize the risk of infection. All nicotine doses are described as free base concentrations. Nicotine was administered i.v. in a volume of 100 μl/kg per infusion or subcutaneously (s.c.) in a volume of 1 ml/kg.

The selective DRD4 antagonist L-745,870 (3-[4-(4-chlorophenyl)piperazin-1-yl]methyl-1H-pyrrolo[2,3-b]pyridine) hydrochloride (Sigma-Aldrich) was reported to have high selectivity for DRD4, >2000-fold compared with other dopamine receptor subtypes and a low-moderate affinity (IC50<300 nM) for non-dopaminergic receptors (Patel et al, 1997). L-745,870 was dissolved in 100% alcohol (final concentration 5% v : v) by a Shaker and/or sonication, and then polyethylene glycol 400 was added and mixed again by a Shaker (final concentration 10% v : v), and, finally, sterile water was added for a final concentration of 1.5 mg/ml L-745,870 (free base). The lower doses of L-745,870 were diluted from the 1.5 mg/ml concentration with vehicle solution (sterile water containing 5% alcohol and 10% polyethylene glycol 400). L-745,870 solution was freshly prepared and administered 30 min before all tests (ie, 30 min before animals were placed in chambers) in a volume of 1 ml/kg. Selection of L-745,870 doses (0.1–3.0 mg/kg), pretreatment time, volume of injection (2 ml/kg), and route of administration were based on previous reports (Caine et al, 2002; Yan et al, 2004, 2006).

The selective DRD4 agonist PD 168,077 (N-([4-(2-cyanophenyl)piperazin-1-yl]methyl)-3-methylbenzamide) maleate (Tocris, Ellisville, MO, USA) was dissolved in saline to a final concentration of 3.0 mg/ml of PD 168,077 (free base) (Glase et al, 1997). Lower doses were prepared by diluting with saline solution. PD 168,077 solution was also freshly prepared and administered 30 min before all tests in a volume of 1 ml/kg.

Apparatus for Nicotine Self-Administration, Food Self-Administration, and Nicotine Discrimination

All experiments were conducted in standard operant conditioning chambers located inside sound-attenuating, ventilated cubicles (Med Associates, St Albans, VT). Each chamber was equipped with two response levers on a panel on one side of the chamber, with a 28-V white cue light above each lever, and with a white chamber light mounted on the ceiling on the opposite side of the chamber. There was a recessed food receptacle between the two levers, which was used for experiments involving food self-administration and reinstatement of food-seeking behavior and nicotine discrimination, and for the initial food training procedure before self-administration. I.v. nicotine infusions were delivered using a syringe pump (PHM-100, Med Associates) located outside each cubicle.

Food Training Procedure before Catheterization

After a 24-h deprivation period, rats were subjected to 1-h daily sessions of lever-press training reinforced by food pellets (45 mg) under a fixed-ratio 1 (FR1) schedule for 5 days. During these sessions, the chamber light was on, but the lever light was unavailable throughout each 1-h session. Assignment of the active lever was counterbalanced across animals, so that in half of the rats, the right lever was active (produced food delivery) and vice versa. Responses on the inactive lever had no programmed consequences. Once assigned, the active lever was fixed for each animal throughout all experiments. These rats were food restricted for the remainder of the experiment but had ad libitum access to water.

Surgical Procedures for Catheterization into the Jugular Vein

After food training, rats were surgically implanted with catheters into the right external jugular vein. Surgery was performed under anesthesia induced by xylazine (10 mg/kg) and ketamine hydrochloride (75 mg/kg). Once anesthetized, marcaine was infused into the incision sites and buprenorphine (0.03 mg/kg) was administered s.c. to provide analgesia. Although surgeries were performed using aseptic techniques, penicillin was administrated s.c. in a single dose of 30 000 Units. The catheter consisted of silastic tubing joined to polyethylene tubing, which terminated in a threaded nylon connector with an attached piece of surgical mesh. The silastic end was inserted into the jugular vein, and the catheter passed s.c. to the dorsal surface between the scapulae where it exited. After surgery, animals recovered from anesthesia on a heating pad, and a s.c. infusion of ∼10 ml/kg sterile isotonic saline was administered for fluid replacement. Animals then had a 1-week recovery period before commencement of experimental procedures. After recovery from surgery, the catheter was flushed daily with saline containing heparin (0.1 ml, 30 Units/ml), and catheter patency was verified by administration of methohexital at the completion of nicotine self-administration experiments. In cases of catheter failure, animals were not re-catheterized but were excluded from the study.

I.v. Nicotine Self-Administration Procedure

Daily 1-h sessions of nicotine self-administration were conducted Monday through Friday. Acquisition of nicotine self-administration occurred at a dose of 0.03 mg/kg per infusion and an infusion volume of 0.1 ml/kg. Duration of each nicotine infusion was ∼1 s, but was adjusted to accommodate inter-animal or between-session differences in body weight. Initially, each active lever press resulted in an infusion of nicotine (FR1). Nicotine delivery was signaled by illumination of the light above the active lever and turning off of the chamber light. These cues continued for 60 seconds (timeout period), during which time lever presses were recorded but had no programmed consequence. Lever requirements were gradually increased to a final value of 5 (FR1 for 5 days, FR2 for 3 days, and FR5 for at least 12 days). Under an FR5 schedule of reinforcement, rats were considered to have acquired stable nicotine self-administration according to the following criteria: they pressed the active lever more than twice the number of times they pressed the inactive lever, and received a minimum of 10 infusions per 1-h session with <20% variation in the number of infusions earned per session over three consecutive sessions.

Experiment 1: Effects of the DRD4 Antagonist L-745,870 on Nicotine-Taking and Cue-Induced Reinstatement of Nicotine-Seeking Behavior

Animals were first trained to self-administer nicotine at a dose of 0.03 mg/kg per infusion, as described in detail above. Once nicotine self-administration behavior was stable under an FR5 schedule, testing of the effects of L-745,870 on nicotine-taking behavior was carried out according to within-subjects Latin square design. Doses of L-745,870 were: 0 (vehicle), 0.1, 0.3, 1.0, and 3.0 mg/kg and L-745,870 or vehicle was administered intraperitoneally (i.p.) 30 min before animals were placed into the chambers. Test sessions were separated by three or more sessions of nicotine self-administration, which were conducted daily until animals met the above criteria for stable self-administration over two consecutive sessions.

Once this testing with L-745,870 was completed, the same group of animals was again subjected to three or more sessions of nicotine self-administration, which were conducted daily until animals met the criteria for stable self-administration over two consecutive sessions. Daily 1-h sessions of extinction training were then conducted, during which the chamber light was on throughout the whole session, but neither nicotine infusion nor lever lights were available. Responses on either the active or inactive lever had no programmed consequences but were recorded. The criterion for extinction was three consecutive daily sessions with <20 active lever responses or with active lever responses that were <20% of the average for active lever responses that occurred during the last 5 days of nicotine self-administration.

Once animals achieved the criteria for extinction training, testing of effects of L-745,870 on cue-induced reinstatement of nicotine-seeking behavior was conducted according to a within-subjects Latin square design. Pretreatment time and doses of L-745,870 were as the same as described above. Conditions for test sessions involving cue-induced reinstatement were identical to those involving nicotine self-administration, except that (1) a single presentation of the visual cue (active lever light on and chamber light off for 10 s) was non-contingently delivered immediately at the start of the session and (2) responses on the active lever (under an FR5 schedule) resulted in contingent presentation of the cues (active lever light on and chamber light off for 10 s) without nicotine infusion. All responding on the active lever was recorded, including those occurring due to 10-s cue presentations, and served as the measure of reinstatement. Responses on the inactive lever had no programmed consequences but were recorded. Test days were separated by three or more daily 1-h sessions of extinction training until animals met the above criteria for extinction over two consecutive sessions.

Experiment 2: Effects of L-745,870 on Nicotine-Primed Reinstatement of Nicotine-Seeking Behavior

A separate group of naive rats was first trained to self-administer nicotine at 0.03 mg/kg per infusion as described in Experiment 1. Once nicotine self-administration behavior was stable under an FR5 schedule, rats were then exposed to daily 1-h sessions of extinction training until they met the criteria for extinction described above.

Testing for the effects of L-745,870 on nicotine-primed reinstatement of nicotine-seeking behavior was carried out according to a within-subjects Latin square design. The pretreatment time and doses of L-745,870 were the same as in Experiment 1. A priming injection of nicotine (0.15 mg/1 ml per kg) was administered s.c. 10 min before the start of the test session. Nicotine-primed reinstatement tests were conducted under conditions identical to those of extinction training. During test sessions, neither nicotine infusion nor nicotine-associated cues (lever lights) was available. Responses on the active or inactive lever had no programmed consequences but were recorded. Test days were separated by three or more three daily 1-h sessions of extinction training until the above criteria for extinction were met over two consecutive sessions.

Experiment 3: Effects of L-745,870 on Reinstatement of Food-Seeking Behavior Induced by Food Priming or Food-Associated Cues

Another group of naive rats learned to self-administer food (45 mg pellets) under a FR schedule identical to that used for nicotine self-administration. In brief, rats were initially subjected to daily 1-h sessions of lever-press training under an FR1 schedule for 5 days, then FR2 for 3 days, and finally FR5 for 12 days, during which time animals developed stable food-maintained operant self-administration behavior.

Rats were then subjected to extinction training, as described above. Once the criteria for extinction were met, the effects of different doses of L-745,870 on food-primed reinstatement of food-seeking behavior were tested according to a within-subjects Latin square design. Pretreatment time and doses of L-745,870 were the same as in Experiment 1. Priming with food was conducted as follows: three food pellets were non-contingently delivered to the recessed food receptacle between the two levers during the first 3 min of the test sessions. Food-primed reinstatement tests were conducted under conditions identical to those of extinction training, with neither food delivery nor food-associated cues (lever lights) available during the session. Responses on the active or inactive lever had no programmed consequences but were recorded. Test days were separated by three or more daily 1-h sessions of extinction until the above criteria for extinction over two consecutive sessions were met.

After completion of the above testing, rats were exposed to food self-administration once again until they showed stable operant behavior. Once behavior was stable, animals were again subjected to extinction training until criteria for extinction were met. Testing of the effects of L-745,870 on cue-induced reinstatement of food-seeking behavior was then conducted according to a within-subjects Latin square design. Pretreatment time and doses of L-745,870 were as same as described above. Conditions for test sessions studying reinstatement by food-associated cues were identical to those for studying reinstatement by nicotine-associated cues described above. In brief, cue-induced reinstatement tests were conducted under conditions identical to those of food-maintained operant training, except that (1) a single presentation of the visual cue (active lever light on and chamber light off for 10 s) was delivered response independently immediately at the start of the session and (2) responses on the active lever (under an FR5 schedule) resulted in contingent presentation of the cues (active lever light on and chamber light off for 10 s) without food delivery. Responses on the inactive lever had no programmed consequences but were recorded. Each test day was followed by ⩾3 days with 1-h extinction sessions until rats met the criteria for extinction over two consecutive sessions.

Experiment 4: Effects of Selective DRD4 Agonist PD 168,077 on Nicotine- and Food-Seeking Behavior during Extinction

A group of naive rats was first trained to self-administer nicotine at 0.03 mg/kg per infusion and exposed to extinction training as described above. Testing of the effects of PD 168,077 on nicotine-seeking behavior during extinction was carried out according to a within-subjects Latin square design. PD 168,077 (0.03, 0.3, and 3.0/kg) was administered i.p. 30 min before start of the test sessions. Conditions during test sessions were identical to those during extinction training sessions.

A separate group of rats was trained to respond to food and subsequently exposed to extinction training. Once extinction criteria were met, they were tested with PD 168,077 for reinstatement of extinguished food-seeking behavior in a similar manner to that of their nicotine-trained counterparts.

Experiment 5: Effects of Selective DRD4 Antagonist L-745,870 on Nicotine Discrimination

Establishment of nicotine discrimination in male Sprague Dawley rats. Training of the initial food self-administration behavior for these experiments was similar to that reported previously (Caine et al, 2002; Yan et al, 2004, 2006). After this, rats were trained under a discrete-trial schedule of food pellet delivery to respond on one lever when an injection of a training dose of 0.4 mg/kg nicotine was administered s.c. 10 min before the session and on the other lever when an injection of 1 ml/kg of saline vehicle was administered s.c. 10 min before the session. At the start of the session, a white house light was turned on, and in its presence, rats responded to food under a FR10 schedule. The completion of 10 consecutive responses on the appropriate lever delivered a 45 mg food pellet and initiated a 45-s time out during which lever-press responses were recorded but had no programmed consequences and the chamber was dark. Responses on the incorrect lever had no programmed consequences other than to reset the FR requirement on the correct lever. After each time out, the white house light was again turned on and the next trial began. Each session ended after completion of 20 FR trials or after 30 min, whichever occurred first. Discrimination-training sessions were conducted 5 days per week under a double alternation schedule (ie, NNSSNNSS, etc., N=nicotine; S=saline). Training continued until there were eight consecutive sessions during which rats completed at least 90% of their responses during the session on the appropriate lever and no more than four responses occurred on the inappropriate lever during the first trial.

Effects of L-745,870 on nicotine discrimination. After acquisition of nicotine discrimination, test sessions with other doses of nicotine and with the DRD4 antagonist L-745,870 were initiated. Once again, a within-subjects Latin square design was used (ie, the same rats received the various treatments regimen during various test sessions). All doses of L-745,870 (0.1–3.0 mg/kg, i.p., 30 min before the start of the session) were administered alone to examine its ability to produce responding on the nicotine-associated lever (ie, to produce a ‘nicotine-like’ effect). The highest three doses of L-745,870 were administered with various doses of nicotine (0.01, 0.03, 0.1, 0.3, 0.4 mg/kg) and effects on the dose–effect curve for nicotine discrimination were examined. The percentage of nicotine-associated lever responses and the response rate were recorded. Test sessions were identical to training sessions, with the exception of both levers being active and 10 consecutive responses on either 1 of the 2 levers resulting in the delivery of a food pellet. Switching responding from one lever to the other lever reset the ratio requirement. In a test phase, a single alternation schedule was introduced and test sessions were usually conducted on Tuesdays and Fridays. Thus, a 2-week sequence starting on Monday was NTSNTSTNST (T=test). In this manner, test sessions occurred with equal probability after saline and nicotine sessions. Test sessions were conducted only if the criterion of 90% accuracy and not more than four incorrect responses during the first trial was maintained in the two most recent training sessions.

Data Analysis

A two-way repeated-measures analysis of variance (ANOVA), followed by post hoc Bonferroni's test was performed for the difference in the number of active vs inactive lever presses during acquisition of nicotine self-administration, nicotine extinction training, acquisition of food self-administration, food extinction training, and acquisition of nicotine discrimination. Paired Student's t-tests were conducted between baseline and vehicle responding during testing for nicotine-taking, nicotine- and food-seeking behaviors. Subsequently, a one-way repeated-measures ANOVA, followed by post hoc Newman–Keuls multiple comparisons were used to examine the effects of the different doses of L-745,870 on nicotine-taking, cue-induced, or nicotine-primed reinstatement of nicotine-seeking behavior, cue-induced, or food-primed reinstatement of food-seeking behavior and nicotine discrimination.

RESULTS

No Effect of the Selective DRD4 Antagonist L-745,870 on Nicotine Self-Administration

After animals had acquired stable self-administration, pretreatment with L-745,870 doses ranging from 0.1 to 3.0 mg/kg (i.p.) did not affect the number of nicotine infusions self-administered, compared with vehicle treatment (Figure 1a, ANOVA, p>0.05). L-745,870 pretreatment at the doses tested also did not alter either active lever presses or inactive lever presses (Figure 1b, ANOVA, p>0.05). These results suggest that the role of D4 receptors in modulating the reinforcing effects of nicotine is minimal at best.

Figure 1
figure 1

Effect of DRD4 antagonist L-745,870 on nicotine self-administration behavior under a fixed ratio (FR5) schedule in rats. (a) No effect of L-745,870 at the dose range of 0.1–3.0 mg/kg on the number of nicotine infusions. (b) No effect of L-745,870 at the dose range of 0.1–3.0 mg/kg on the number of active and inactive lever presses. N=13. Data are expressed as means (±SEM) of the number of injections per session during baseline conditions (baseline) and during sessions with vehicle (0 mg/kg) or L-745,870 pretreatment. The antagonist did not alter nicotine-taking behavior.

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Attenuation of Cue-Induced Reinstatement of Nicotine-Seeking Behavior by L-745,870

After extinction of their nicotine-seeking behavior, animals showed clear cue-induced reinstatement of nicotine-seeking behavior after vehicle pretreatment compared with baseline extinction (paired Student's t-test of baseline vs vehicle, p<0.001). In contrast, L-745,870 pretreatment, at doses ranging from 0.1 to 3.0 mg/kg, significantly decreased the number of active lever presses induced by nicotine-associated cues (Figure 2a, upper panel, ANOVA with post hoc Newman–Keuls comparison, p<0.01, 0.05, 0.05, 0.01, respectively), although there was no difference in the number of inactive lever presses during these tests (Figure 2a, lower panel, ANOVA, p>0.05). These findings suggest that D4 receptors are important for cue-induced relapse to nicotine-seeking behavior.

Figure 2
figure 2

Effect of DRD4 antagonist L-745,870 on the reinstatement of nicotine-seeking behavior in rats. (a) Pretreatment of L-745,870 significantly reduced the number of active (upper) but not inactive (lower) lever presses triggered by nicotine-associated cues. N=13. (b) Pretreatment of L-745,870 significantly attenuated the number of active (upper) but not inactive (lower) lever presses induced by priming injection of nicotine (0.15 mg/kg, s.c.). N=23. *p<0.05, **p<0.01, ***p<0.001 vs the baseline (without nicotine-associated cues); Student's paired t-test. ##p<0.01, ###p<0.001 vs the vehicle pretreatment; Newman–Keuls multiple comparison test after significant ANOVA for repeated measures. Data are expressed as means (±SEM) of the number of lever presses during extinction training, during baseline conditions (baseline), and during sessions with vehicle (0 mg/kg) or L-745,870 pretreatment.

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Attenuation of Nicotine-Primed Reinstatement of Nicotine-Seeking Behavior by L-745,870

In the group of rats trained to self-administer nicotine, which subsequently underwent extinction of their nicotine-seeking behavior, a priming injection of nicotine (0.15 mg/kg, s.c.) reliably induced reinstatement of nicotine-seeking behavior (paired Student's t-test of baseline vs vehicle, p<0.001). Pretreatment with L-745,870 over the dose range of 0.1–3.0 mg/kg (i.p.) significantly reduced the number of active lever presses induced by a priming injection of nicotine (Figure 2b, upper panel, ANOVA with post hoc Newman–Keuls comparison, p<0.01, 0.01, 0.01, 0.001, respectively), whereas there was no change in the number of inactive lever presses during these tests (Figure 2b, lower panel, ANOVA, p>0.05) across different doses of L-745,870. These observations indicate that D4 receptors have an important role in nicotine-primed reinstatement of nicotine-seeking behavior and in cue-induced reinstatement of nicotine-seeking behavior.

No Effect of L-745,870 on Reinstatement of Food-Seeking Behavior Induced by either Food Priming or Food-Associated Cues

In the group of rats trained to self-administer food, which subsequently underwent extinction of their food-seeking behavior, priming with food pellets reliably induced reinstatement of food-seeking behavior (paired Student's t-test of baseline vs vehicle, p<0.001). However, L-745,870 doses ranging from 0.1 to 3.0 mg/kg (i.p.) did not affect reinstatement of food-seeking behavior (Figure 3a, upper panel, ANOVA, p>0.05). Similarly, food-associated cues also reliably induced reinstatement of food-seeking behavior (paired Student's t-test of baseline vs vehicle, p<0.01) and L-745,870, at all doses tested, failed to alter cue-induced reinstatement of food-seeking behavior (Figure 3b, upper panel, ANOVA, p>0.05).

Figure 3
figure 3

Effect of DRD4 antagonist L-745,870 on reinstatement of food-seeking behavior induced by either food-priming or food-associated cues in rats. (a) Pretreatment of L-745,870 did not affect food-primed reinstatement of food-seeking behavior. ##p<0.01 vs the baseline; Student's paired t-test. (b) Pretreatment of L-745,870 did not influence food cue-induced reinstatement of food-seeking behavior. #p<0.05 vs the baseline; Student's paired t-test. N=6–10. Data are expressed as means (±SEM) of the number of active lever presses during baseline conditions (baseline) and during sessions with vehicle (0 mg/kg) or L-745,870 pretreatment.

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No Effect of the Selective DRD4 Agonist PD 168,077 on Nicotine- or Food-Seeking Behavior during Extinction

In the group of rats trained to self-administer nicotine, which subsequently underwent extinction of their nicotine-seeking behavior, PD 168,077 (dose range 0.3–3.0 mg/kg; i.p.), did not affect active lever responding during nicotine extinction sessions (ANOVA, p>0.05).

Another group of naive rats was first trained to self-administer food pellets and then subjected to extinction training as described above. Once criteria for extinction were met, tests for effects of PD 168,077 on food-seeking behavior began. PD 168,077 (dose range 0.3–3.0 mg/kg; i.p.) did not affect active lever responding during food extinction sessions (ANOVA, p>0.05).

No Effect of the Selective DRD4 Antagonist L-745,870 on Nicotine Discrimination

A two-way ANOVA on the percentage of nicotine-associated lever responses for the dose–response curve for nicotine discrimination (0.01–0.4 mg/kg), with or without L-745,870 pretreatment (0.3–3 mg/kg), indicated a significant nicotine effect (F(5, 241)=323, p<0.0001), and post hoc analysis showed a dose-dependent increase in nicotine-lever selection with maximal nicotine-lever selection at the 0.4 mg/kg training dose of nicotine (Figure 4a). L-745,870 did not significantly affect the percentage of nicotine-associated lever responses for any dose of nicotine (Figure 4a, ANOVA, L-745,870 effect: F(3, 241)=1.225, p>0.05), indicating that DRD4 blockade did not alter the discriminative effects of nicotine in rats. A second ANOVA was performed on the rate of lever responding, which indicated a significant effect of both nicotine (F(5, 241)=2.86, p<0.05) and L-745,870 (F(3, 241)=2.88, p<0.05), but no significant interaction (Figure 4b, F(15, 241)=0.46, p>0.05). However, post hoc analysis revealed that only the 3.0 mg/kg dose of L-745,870 significantly decreased the rate of lever responding when compared with vehicle, but only at the 0.3 mg/kg dose of nicotine (p<0.05). Finally, L-745,870 did not produce any nicotine-like discriminative effects (<5% of responses emitted on nicotine-associated lever) at any of the three doses examined (Figure 4c, ANOVA, L-745,870 effect: F(3, 79)=0.675, p>0.05), nor did it produce any effects on rate of lever-press responding (Figure 4d ANOVA, L-745,870 effect: F(3, 79)=0.63, p>0.05).

Figure 4
figure 4

(a) Dose-effect functions for the discriminative-stimulus effects of nicotine in rats trained to discriminate 0.4 mg/kg nicotine from saline. The percentage of responses on the lever associated with nicotine administration is shown as a function of dose (mg/kg) during tests with various doses of nicotine. The DRD4 antagonist L-745,870, administered acutely 30 min before the sessions, did not modify discriminative-stimulus effects of nicotine (no curve shift). (b) L-745,870 also did not significantly affect rates of lever-press responding when administered together with different doses of nicotine or with vehicle. (c) Effects of different doses of L-745,870 on discriminative-stimulus effects of the training dose of nicotine when L-745,870 was administered with vehicle. (d) Effects of different doses of L-745,870 on rates of lever-press responding when administered together with the training dose of nicotine or with vehicle. N=9–12. Data are expressed as means (±SEM) of the percentage of responses on the nicotine lever.

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DISCUSSION

In this study, we demonstrated that the selective DRD4 antagonist, L-745,870, significantly attenuates both nicotine- and cue-induced reinstatement of nicotine-seeking behavior in rats, but does not alter the ability of rats to discriminate nicotine from saline. Yet, over the same dose range, L-745,870 has no effect on ongoing nicotine self-administration behavior and does not alter food- or cue-induced reinstatement of food-seeking behavior. In clinical studies, polymorphisms of the DRD4 gene have been linked to chronic alcoholism, tobacco smoking, opioid dependence, impulsivity, and novelty-seeking behavior, which are considered potential predictors of vulnerability to drugs of abuse (Chien et al, 2010; Ebstein et al, 1996; George et al, 1993; Ho et al, 2008; Kotler et al, 1997; Li et al, 2000; Luciano et al, 2004). Previously, polymorphisms of DRD4 were reported to modulate DRD4 expression, decrease ligand binding to DRD4, and attenuate the resulting cAMP formation (Asghari et al, 1994, 1995; Cohen et al, 1992; Le Foll et al, 2009 for review). More recently, the VNTR polymorphism of the DRD4 gene has been correlated with smoking cue reactivity, cocaine cue reactivity, and alcohol- and heroin-craving behavior (Hutchison et al, 2002a, 2002b, 2003, 2006; McClernon et al, 2007; Munafo and Johnstone, 2008; Shao et al, 2006). In addition, previous animal studies have demonstrated that DRD4 blockade with L-745,870 blocks the discriminative-stimulus effects of methamphetamine in rats (Yan et al, 2004, 2006) and attenuates the morphine-induced withdrawal syndrome precipitated by naloxone (Mamiya et al, 2004). The data presented here suggest that DRD4 is very likely involved in relapse to tobacco smoking behavior. Along with the above-mentioned evidence, our data also suggest DRD4 as a potential target for development of therapeutic agents against certain aspects of addictive behavior, particularly tobacco smoking relapse.

It has been reported that, in the nicotine self-administration paradigm, nicotine itself has two actions: a primary reinforcing effect and a non-associative enhancement of responding for weakly reinforcing visual stimuli, or ‘reinforcement-enhancing’ effect (Chaudhri et al, 2006a, 2006b; Palmatier et al, 2006, 2007a, 2007b). In this study, we observed that blockade of DRD4 with L-745,870 failed to affect nicotine self-administration. It is difficult to determine whether this failure resulted from an inability of DRD4 blockade to alter the primary reinforcing effect of nicotine or the reinforcement-enhancing effect of nicotine; however, future study should further explore this aspect.

It has been previously reported that blockade of DRD4 with L-745,870 (dose range 0.1–10.0 mg/kg) does not affect cocaine self-administration in rats (Caine et al, 2002). Moreover, deletion of the DRD4 gene in mice does not affect food or cocaine self-administration behavior (Thanos et al, 2010). These findings are in agreement with our observation in this study that L-745,870 (0.1–3.0 mg/kg) did not alter nicotine self-administration behavior. It seems unlikely that this lack of effect of L-745,870 is due to insufficient dosage, as estimates of D4 receptor occupancy in vivo suggest that 1.0 mg/kg of L-745,870 occupies up to 90% of D4 receptors in the mouse brain (Patel et al, 1997). In addition, it is interesting to note that the blockade of another member of the D2-like receptor family, DRD3, also fails to affect nicotine-taking behavior, yet attenuates the reinstatement of nicotine-seeking behavior (Khaled et al, 2010; Andreoli et al, 2003). As L-745,870 had no effect on nicotine discrimination, it is unlikely that its effect on nicotine-seeking behavior was due to a reduced ability of rats to discriminate the effects of nicotine. Moreover, it is unlikely that the dissociable effects of L-745,870 on nicotine-taking behavior vs nicotine relapse resulted from food training before nicotine self-administration, as there was no effect of L-745,870 on food-seeking behavior induced by presentation of food pellets or food-associated cues. In contrast, the fact that blockade of DRD4 with L-745,870 did not affect nicotine-taking behavior, but disrupted reinstatement of nicotine-seeking behavior, may be due to scarce distribution of D4 receptors in reinforcement-related brain subregions.

The expression of DRD4 is most abundant in the retina, followed by the prefrontal cortex, hippocampus, amygdala, and hypothalamus, but the level of DRD4 is sparse in the basal ganglia, including the nucleus accumbens (Cohen et al, 1992; Khan et al, 1998; Oak et al, 2000). The relatively low level of DRD4 expression that does exist in the shell of nucleus accumbens appears to be mainly localized on presynaptic structures (Svingos et al, 2000). The DRD4 is also found on both pyramidal and non-pyramidal neurons of the cerebral cortex, particularly layer V, and in the hippocampus, where they are known to regulate both GABAergic signaling and NMDA receptor surface expression (Mrzljak et al, 1996; Wang et al, 2002, 2003). Overall, the expression pattern of DRD4 in the brain suggests that DRD4 activity participates only marginally in the reinforcing effects of abused drugs such as nicotine and cocaine. The lack of effect of intra-accumbal administration of L-750,667, another selective DRD4 antagonist, on cocaine-seeking behavior (Anderson et al, 2006) supports the view that expression of DRD4 is too low in the nucleus accumbens (Khan et al, 1998; Oak et al, 2000) to modulate the reinforcing effects of abused drugs. In contrast, brain areas such as the insular cortex or the frontal cortex, which more abundantly express DRD4 are more likely to mediate the inhibitory effects of L-745,870 on nicotine-seeking behavior induced either by a nicotine prime or by nicotine-associated cues (Forget et al, 2010; Hai-Bin et al, 2005). This hypothesis seems to be in agreement with recent clinical and preclinical animal studies showing that the insular cortex is critical for tobacco smoking behavior (Forget et al, 2010; Janes et al, 2010; McClernon et al, 2007; Naqvi et al, 2007).

In this study, the DRD4 antagonist L-745,870 at all doses tested, significantly attenuated reinstatement of nicotine-seeking behavior induced either by nicotine-associated cues or by a priming injection of nicotine. Interestingly, across the doses studied (0.1–3.0 mg/kg, i.p.), L-745,870 did not show a typical pharmacological dose–effect function for blockade of reinstatement of nicotine-seeking behavior. In the paradigm of methamphetamine discrimination, it was also found that L-745,870, at all doses tested (0.025–0.5 mg/kg, i.p.), significantly attenuated the discriminative-stimulus effects of methamphetamine in rats, yet it also did not show a typical pharmacological dose–effect function (Yan et al, 2006). A previous in vivo study confirmed that a dose of 0.035 mg/kg of L-745,870 is sufficient to occupy 50% DRD4 in the mouse brain (Patel et al, 1997), whereas, in our current study, the lowest dose of 0.1 mg/kg is about three times greater. One parsimonious explanation is that occupancy of DRD4 by L-745,870, at all of doses tested in this study, was sufficient to fully block the bioactivity of DRD4 in the rat brain. With regard to the inability of L-745,870 to affect nicotine discrimination, one possible explanation is that the DRD4 is specifically involved in relapse to nicotine-seeking behavior, but not in the discriminative-stimulus effects of nicotine in rats. However, it is noteworthy that different rat strains have been used in this study for nicotine discrimination and nicotine self-administration, and that differences in rat strains can be an important factor in the development of drug dependence and relapse (Hamilton et al, 2010; Kacew and Festing, 1996).

In this study, the DRD4 agonist PD 168,077, at all doses tested, failed to reinstate nicotine-seeking behavior in rats, although the selective DRD4 antagonist L-745,870 significantly reduced reinstatement of nicotine-seeking behavior. It seems unlikely that the failure of PD 168,077 to induce nicotine-seeking behavior resulted from a low selectivity for DRD4, because the agonist has been demonstrated to be highly selective for DRD4, showing low binding affinities for other dopaminergic, adrenergic, or serotoninergic receptors (eg, with >100-fold selectivity over DRD1, >400-fold selectivity over DRD2, and >300-fold selectivity vs DRD3) (Glase et al, 1997). It also seems unlikely that the failure of PD 168,077 to induce nicotine-seeking behavior resulted from an insufficient dosage as a similar range of doses has been used to elucidate the specific function of DRD4 in in vivo studies (Bernaerts and Tirelli, 2003; Gago et al, 2007; Nayak and Cassaday, 2003). Previously, it was reported that the role of both DRD1 antagonists and agonists in the reinstatement of cocaine-seeking behavior is dissociable in rats: a selective DRD1 antagonist, SCH23390, attenuates, whereas a selective DRD1 agonist has no affect on, the reinstatement of cocaine-seeking behavior in rats (Self, 2004). In addition, it was also reported that co-activation of DRD1 and DRD2 is required for long-term synaptic depression in the striatum (Calabresi et al, 1992) and that long-term synaptic depression in the basal ganglia is critical for drug-seeking behavior (Kasanetz et al, 2010; Thomas et al, 2008). These findings suggest that co-activation of dopamine D1-like and D2-like receptors is essential for the reinstatement of drug-seeking behavior (Ikemoto et al, 1997; Schmidt and Pierce, 2006). The hypothesis of co-activation of D1-and D2-like receptors may lay a framework for the dissociable role of both dopamine receptor antagonists and agonists in the reinstatement of drug-seeking behavior. Accordingly, it seems reasonable to postulate that DRD4 is a key component in the reinstatement of nicotine-seeking behavior, but its activation alone is insufficient for inducing reinstatement of nicotine-taking behavior. In addition, using the drug-conditioned place-preference paradigm, it was also reported that another selective DRD4 agonist (A-412997) does not produce conditioned place preference, a measure of reward in rats (Woolley et al, 2008), although there no information is available for the effect of DRD4 antagonists on reward-related behavior with the place-preference paradigm. Future studies should examine the potential interaction between DRD4 and DRD1-like or other DRD2-like subtypes during reinstatement of nicotine-seeking behavior.

Previously, it was reported that DRD4 knockout mice are supersensitive to the psychomotor stimulant effects of ethanol, cocaine, methamphetamine, and amphetamine (Katz et al, 2003; Kruzich et al, 2004; Rubinstein et al, 1997). Cocaine has also been reported to be more potent in stimulating locomotor activity and in producing discriminative-stimulus effects in DRD4 knockout mice compared with wild-type littermates (Katz et al, 2003). These findings suggest that DRD4 functions as an inhibitory modulator of locomotor stimulation induced by abused drugs. On the basis of these findings, it would be expected that pharmacological blockade of DRD4 should produce changes in the behavioral effects of abused drugs similar to those observed in DRD4 knockout mice. More recently, it has been reported that DRD4 knockouts do not show any difference from wild-type mice in food-reinforced operant behavior or in cocaine self-administration behavior (Thanos et al, 2010). These findings seem to be in discrepancy with our results in this report. One possible explanation for contrasting observations is that differences between the effects of DRD4 gene deletion and pharmacological blockade of DRD4 on the behavioral effects of abused drugs results from compensatory adjustments to DRD4 gene deletion during development. For example, expression of DRD1 and glutamate NMDA receptors is enhanced in the forebrain of DRD4 knockout mice (Gan et al, 2004), and DRD4 knockout mice exhibit neurochemical changes consistent with decreased dopamine release (Thomas et al, 2007).

In conclusion, our new results suggest that DRD4 is critical for nicotine relapse behavior, but are not involved in nicotine-taking behavior, natural reward-seeking behavior, or in the discriminative-stimulus effects of nicotine. Recently, there has been a marked interest in DRD4 involvement in psychiatric diseases (Hoenicka et al, 2007), and these findings suggest that the DRD4 is critically involved in the pressing problem of tobacco dependence. High relapse rates after smoking cessation constitute, perhaps, the major impediment to successful treatment of tobacco dependence (Hughes et al, 2004), and our findings may point to pharmacological blockade of DRD4 as a novel and potentially effective treatment against relapse to tobacco smoking in clinical situations. Future identification of the precise subregion(s) and molecular mechanism(s) underlying the pharmacological effects of the selective DRD4 antagonist L-745,870 on relapse to nicotine-seeking behavior that we observed in the present experiments would help to elucidate the in vivo role of this receptor subtype in nicotine abuse and tobacco dependence.