Abstract
There is an increasing demand for a novel non-nicotinic, nondopaminergic therapeutic approach to nicotine addiction. GABAergic mechanisms have been implicated in drug dependence. Recently, a novel GABAB receptor allosteric-positive modulator, GS39783, was characterized. There are no investigations to date on the effects of GABAB receptor-positive modulators in animal models of nicotine reinforcement. Conditioned place preference (CPP) paradigms are based on the principle that animals, like humans, would learn to seek environmental stimuli that have been previously associated with rewarding events. Here we show that nicotine (0.06 mg/kg s.c.) induced a robust CPP response. Furthermore, GS39783 (30–100 mg/kg p.o.) during the conditioning phase blocked the rewarding effects of nicotine in the CPP paradigm in rats. However, GS39783 did not significantly alter the CPP effects of nicotine when given only immediately before the CPP test. A growing body of evidence suggests that repeated administration of drugs of abuse induced long-term molecular changes in brain plasticity, most notably an accumulation of ΔFosB, in the striatal complex that contribute to the manifestation of dependence. There was a significant accumulation of ΔFosB in the nucleus accumbens, but not in the dorsal striatum, of rats treated daily for 5 days with nicotine (0.06 mg/kg i.p.). GS39783 completely (30–100 mg/kg p.o.) counteracted these nicotine-induced molecular adaptations when given before the CPP acquisition phase but not when administered immediately before the test phase. Taken together, the behavioral and molecular changes induced by nicotine occur in concert and are concomitantly amenable to reversal by GABAB receptor-positive modulators.
Smoking-related illness is a major public health problem in today's society, being the second-leading cause of death in the world (Esson and Leeder, 2004). Therefore, there is great impetus to develop effective therapies that will aid in facilitating smoking cessation. It is largely accepted that nicotine is the active ingredient in tobacco smoke that leads to and maintains tobacco addiction (Goldberg et al., 1989). Therefore, most preclinical research efforts are directed at developing interventions that alter the rewarding components of nicotine.
A growing body of evidence suggests that repeated administration of drugs of abuse induce long-term molecular changes in brain plasticity that contribute to the manifestation of dependence (Kelz and Nestler, 2000). Members of the activator protein-1 family of transcription factors are specifically thought to be involved in the ontogeny of addiction. Indeed, repeated administration of a variety of drugs of abuse, including nicotine, induces an accumulation of ΔFosB, a highly stable truncated form of FosB, in the striatal complex (Kelz and Nestler, 2000). Moreover, mice overexpressing ΔFosB also show enhanced sensitivity to the rewarding effects of cocaine and morphine (Kelz and Nestler, 2000). Thus, one can hypothesize that any potential successful therapy for nicotine dependence should attenuate both the rewarding and the molecular changes induced by nicotine.
A number of preclinical and clinical studies suggest that activation of GABAB receptor may be a useful strategy for cocaine, opiate, and alcohol dependence (Brebner et al., 2002; Cousins et al., 2002). Recently, polymorphisms within the GABAB(2) subunit gene have also been shown to be associated with nicotine dependence in humans (Beuten et al., 2005). Furthermore, GABAB receptor agonists such as baclofen reduce nicotine self-administration in rats (Corrigall, 1999). However, baclofen has many side effects, including sedation, muscle relaxation, and hypothermia, that could limit its widespread use. Positive modulation of metabotropic receptors is a novel principle for enhancing neurotransmission in a use-dependent fashion. Recently, a novel GABAB receptor allosteric-positive modulator, GS39783, was characterized (Urwyler et al., 2003; Cryan et al., 2004). GS39783 increases both the potency and efficacy of endogenous GABA at GABAB receptors without having intrinsic activity (Urwyler et al., 2003; Cryan et al., 2004). Hence, GS39783 lacks many of the behavioral and physiological side effects of full GABAB receptor agonists (Cryan et al., 2004). Of particular interest, GS39783 reduces the behavioral effects of cocaine (Smith et al., 2004; Slattery et al., 2005; Lhuillier et al., 2007). However, to our knowledge, there are no investigations to date on the effects of GS39783 in animal models of nicotine reinforcement.
Animals, like humans, learn to seek environmental stimuli that have been previously associated with rewarding events, a process known as conditioned place preference (CPP). Consequently, most drugs of abuse including nicotine are effective in supporting CPP, making it a prime test of nicotine reinforcement (Tzschentke, 1998). In the present studies, we investigated the effects of GS39783 on the behavioral (establishment of CPP) and molecular [accumulation of ΔFosB in the nucleus accumbens (NAc)] consequences of nicotine exposure.
Materials and Methods
Animals. Wistar rats (Charles River, Lyon, France) weighing 180 to 220 g at the beginning of the experiments were housed four per cage (55 × 33 × 19 cm) in a humidity- and temperature-controlled room under a 12-h light/dark cycle (lights on at 7:00 AM). One week before the beginning of the experiments, all the animals were food-restricted (20 g/day) until the end of the study. This was carried out because food restriction has been reported to enhance the rewarding effects of drugs of abuse (Carr 2002), including nicotine (Donny et al., 1998), and the study on which our protocol is based uses a food-restriction regimen (Forget et al., 2005). Experiments were subject to institutional review and conducted in accordance with the Veterinary Authority of Basel-Stadt (Basel, Switzerland).
CPP. The procedure was carried out essentially as described earlier (Forget et al., 2005). Rats were trained and tested in black wooden open fields (76 × 76 × 50 cm) located in a dimly lit room (red light). The floor of each open field was covered with removable quadrants made from wire mesh or rough Plexiglas. These textures were chosen based on previous studies indicating that naive rats exhibited no unconditioned preference for one of them (Forget et al., 2005). The general procedure consisted of two phases: conditioning and testing. Conditioning consisted of alternating four drug-paired conditioning sessions with four vehicle-paired sessions. During drug-paired sessions, nicotine and/or GS39783 was administered (immediately and 30 min, respectively) before exposure to an open field covered with one floor texture. Conversely, vehicle-paired sessions consisted of pairing vehicle injection with the other floor texture. Initially, half of the rats underwent a vehicle-paired session, whereas the other half underwent a drug-paired session. The day after the last conditioning session the rats were placed for a single 20-min drug-free testing session in the open field whose floor was made up of two quadrants of the vehicle-paired texture and two quadrants of the drug-paired texture. The quadrants of the same texture were positioned diagonally opposite to the other floor texture. During this test session, time spent on each texture was automatically recorded by means of the video system and analyzed.
Drugs. GS39783 was synthesized in-house and made up fresh before use and administered p.o. in a suspension of 0.5% methylcellulose at a volume of 5 ml/kg 30 min before testing. Doses of GS39783 were chosen based on our previous data showing that GS39783 can reverse the behavioral and molecular effects of cocaine in this dose range (Slattery et al., 2005; Lhuillier et al., 2007). Furthermore, they are in line with the pharmacokinetic properties of GS39783 in terms of brain exposure (Smith et al., 2004). (–)Nicotine bitartrate (Sigma Chemicals, St. Louis, MO) was dissolved in saline, and the pH was adjusted to 7.3 to 7.5 with 0.1 M NaOH and administrated immediately before the conditioning session (s.c.). The nicotine dose is expressed as free base and was selected from in-house studies showing robust nicotine-induced place preference at this dose (Chaperon et al., unpublished data) in agreement with other studies (Forget et al., 2005).
Tissue Preparation. All the rats were sacrificed immediately after the final test session. Brains were quickly removed and chilled in ice-cold phosphate-buffered saline. NAc and dorsal striatum were dissected on an ice-chilled glass plate and flash-frozen in dry ice.
Immunoblotting. Western blot analysis was carried out as described previously (Lhuillier et al., 2007). Primary antibodies used were rabbit anti-FosB (sc-48, 1:500; Santa Cruz Biotechnologies, Santa Cruz, CA) and rabbit anti-actin (A2066, 1:5000; Sigma Chemicals). Secondary horseradish peroxidase-linked goat anti-rabbit antibody was obtained from Cell Signaling Technology Inc. (7074, 1:1000–1:5000; Beverly, MA).
Statistical Analysis. In behavioral experiments, the time spent in drug-paired quadrant was analyzed by a one-way analysis of variance (ANOVA) followed by, when appropriate, Fisher's post hoc tests. The level of significance was set at p < 0.05. In molecular studies, significance was assessed by a one-way ANOVA, followed by post hoc Newman-Keuls test. The correlation was performed using a least-square linear regression analysis
Results
Intrinsic Effects of GS39783. There was no unconditioned preference for texture. The one-way ANOVA revealed a significant effect of GS39783 on the time spent in paired quadrant [F(3,47) = 4.771, p = 0.006]. Post hoc analysis indicates rats spent a reduced time in GS39783 (100 mg/kg)-paired quadrants compared with the control group (p < 0.001). In contrast, at 10 and 30 mg/kg, GS39783 was hedonically neutral and failed to affect the time spent in drug-paired quadrants (Fig. 1).
Effect of GS39783 on the Establishment of Nicotine-Induced Place Preference. ANOVA revealed an overall effect of treatment on CPP [F(3,40) = 3.524, p = 0.023]. Rats who previously received nicotine spent significantly more time in quadrants previously paired with nicotine compared with control animals (p = 0.044) (Fig. 2) as previously reported (Forget et al., 2005). In addition, post hoc analysis revealed also that GS39783 at 30 and 100 mg/kg, when given 30 min before each drug-paired session, decreased the amount of time spent in nicotine-paired quadrant compared with animals treated only with nicotine (p = 0.005 and p = 0.013, respectively) (Fig. 2). Consequently, these data show that GABAB receptor-positive modulation during the conditioning phase antagonized the establishment of nicotine-induced place preference.
Effects of GS39783 on the Expression of Nicotine-Induced Place Preference. ANOVA revealed an overall effect of treatments on the time spent in drug-associated quadrants [F(3,40) = 3.352, p = 0.028]. As we previously showed, nicotine supported CPP (p = 0.03, compared with control group). GS39783 at 30 and 100 mg/kg, when given 45 min before testing session, failed to decrease significantly the amount of time spent in the nicotine-paired quadrant compared with animals treated only with nicotine (p = 0.558 and p = 0.167, respectively). However, it is noteworthy that the time spent on the nicotine-paired texture by rats given GS39783 (100 mg/kg) did not differ from the control level (p = 0.408). As a result, although GS39783 affects the acquisition of nicotine-induced place preference, it seems to be without effect on its expression (Fig. 3).
Effects of GS39783 on Nicotine-Induced Increases in the Expression of ΔFosB. We studied the influence of both repeated and single administration of GS39783 on ΔFosB expression in NAc and dorsal striatum by semiquantitative Western blot analysis immediately after completion of the last test. Chronic nicotine stimulated a robust increase in ΔFosB expression in NAc (Fig. 4A) (p < 0.001 versus saline). This up-regulation was completely blocked by GS39783 (p < 0.001 versus nicotine) at both doses used when injected daily during the acquisition phase. GS39783 did not have any intrinsic effect on basal ΔFosB levels. In NAc, a single administration of GS39783 before the final CPP testing failed to inhibit ΔFosB induction at both doses (Fig. 5A). One concern of the experimental procedure we used is that the ΔFosB signal observed might be a direct consequence of the last CPP test, as opposed to a slow buildup of the protein during chronic nicotine exposure. However, this hypothesis can be ruled out because 1) only chronic treatment with GS39783 resulted in inhibition of ΔFosB induction, and 2) in experiment 2, the last GS39783 administration was performed 48 h before CPP testing. In the dorsal striatum, ΔFosB levels were not altered by chronic nicotine or by chronic or acute GS39783 (Figs. 4B and 5B) (p > 0.05 versus saline). Finally, a plot of the data obtained from individually paired behavioral and biochemical data reveals a significant positive correlation between CPP scores and levels of ΔFosB expression (p < 0.001, R2 = 0.38) (Fig. 6).
Discussion
The present report shows that GABAB receptor activation, via administration of GABAB receptor-positive modulator, blocked the acquisition of nicotine-induced place preference without affecting its expression. In tandem, we report that GABAB receptor-positive modulation during the conditioning phase of place preference counteracts the correlated longlasting molecular adaptation, accumulation of accumbal ΔFosB, induced by repeated administration of nicotine.
It seems that GS39783, at the highest dose tested (100 mg/kg), elicited a place aversion in the present study that can be interpreted as an aversive or anhedonic property of the compound at this relatively high dose. Nonetheless, it should be noted that the same dose of GS39783 has no effect on baseline intracranial self-stimulation (ICSS) thresholds (Slattery et al., 2005) and spontaneous locomotor activity (Cryan et al., 2004). Nevertheless, the place-conditioning protocol used here requires four administrations of GS39783 versus one for the ICSS and locomotor activity. Although these dissimilarities in the regimen of GS39783 could explain the differences between the studies, the processes involved in the aversive effect of GABAB positive modulator in the present paradigm remain elusive. Regardless of these considerations, we clearly showed that GS39783, when administrated during conditioning phase, blocked CPP elicited by nicotine at both doses used. However, given that GS39783 (100 mg/kg) induces an aversion in its own right, it is difficult to dissociate the reduction in time spent in the nicotine-associated quadrant from the drug's intrinsic effects. Nonetheless, GS39783, even at a dose that was devoid of intrinsic activity (30 mg/kg), completely reversed nicotine-induced CPP, and we can deduce that administration of GS39783 before each drug-pairing session attenuates the rewarding properties of nicotine.
GABAergic mechanisms have long been implicated in drug dependence largely because of known direct interactions of the GABA and the dopamine transmitter systems (Kalivas et al., 1990). It is probable that GS39783 attenuates the increase of dopamine release via its action on dopaminergic neurons of the VTA, resulting in a reduction of the salience of rewarding stimulus. Thus, this reduction might limit the establishment of the association between nicotine and paired cues. These data are in line with the ability of GS39783 to decrease rewarding properties of cocaine in both self-administration (Smith et al., 2004) and ICSS paradigms (Slattery et al., 2005). Correspondingly, GABAB receptor activation, via agonist administration, also decreases nicotine self-administration (Corrigall et al., 2000; Paterson et al., 2005).
Interestingly, we also observed that a single administration of GS39783 before the test failed to significantly affect the expression of nicotine-induced place preference. These present results are consistent with previous data showing that GABAB receptor-positive modulator blocked the establishment but not the expression of behavioral sensitization to cocaine (Lhuillier et al., 2007). This absence of effects of GS39783 on the expression of CPP might be attributed to the inability of GABAB receptor-positive modulator to affect conditioned motivational properties of nicotine-paired cues. Nevertheless, it has been shown that CGP44532, a GABAB agonist, blocks cue-induced reinstatement of nicotine-seeking in rats (Paterson et al., 2005). Moreover, a number of studies have reported that baclofen affects the expression of place preference elicited by ethanol, methamphetamine, or morphine [Li et al., 2001; Bechtholt and Cunningham, 2005; but see Chester and Cunningham, 1999, who have demonstrated that baclofen failed to alter ethanol CPP]. Thus, it is postulated that these responses require agonistic activity and that enhancement of GABAB tone induced by GS39783 is not sufficient to affect the expression of place preference elicited by nicotine. Nevertheless, further studies are required to confirm this hypothesis. However, caution needs to be taken regarding the use of baclofen in animal models that are based primarily on either, or both, motor performance and cognitive function because baclofen markedly impairs both parameters (Cryan et al., 2004; Jacobson and Cryan, 2005). Therefore, because of these confounds we did not test baclofen in nicotine CPP in the current series of studies. Whereas GABAB receptor agonists impair memory in several paradigms, there is no evidence for amnesic-like effects of GS39783 (Cryan et al., 2004). Overall, our data show that GABAB receptor-positive modulation markedly and selectively attenuates the acquisition but not the expression of nicotine-induced place preference.
We next assessed whether the behavioral changes observed translated into alterations at the molecular level. Although molecular adaptations to chronic nicotine have been hypothesized to play a major role in the manifestation of nicotine dependence (Brunzell et al., 2003; Walters et al., 2005), very few studies to date have investigated the ability of potential therapeutic agents to modulate such responses. Repeated nicotine self-administration induces a strong accumulation of ΔFosB in the NAc but not in the dorsal striatum (Pich et al., 1997). Here we confirm these results in our model of nicotine reinforcement (Figs. 4A and 5A). Furthermore, GS39783 was effective in inhibiting this accumulation when injected chronically during the acquisition phase of place preference. In contrast to this result, a single administration of GS39783 before the final test failed to block nicotine-induced ΔFosB accumulation. This result is in agreement with the observation that ΔFosB is an extremely stable variant of FosB that slowly accumulates during chronic drug exposure (Kelz and Nestler, 2000). Taken together, these results show that chronic GABAB-positive modulation is sufficient to counteract the effects of chronic nicotine administration. ΔFosB is accumulated in the mesolimbic system in response to various chronic stimuli, and this phenomenon is dependent on dopamine signaling (Zhang et al., 2002). Several lines of evidence have shown that nicotine administration causes, in short, an increased excitability of VTA neurons through direct activation of dopamine VTA neurons (Pidoplichko et al., 1997), cholinergic activation of these neurons by the pedunculopontine nucleus (Forster and Blaha, 2003), or inhibition of VTA GABA interneurons (Mansvelder et al., 2002). All these effects result in an increased dopamine release in the NAc, which could therefore be the neurochemical basis for the accumulation of ΔFosB observed here (Imperato et al., 1986; Nisell et al., 1994). In addition, GABAB receptors tonically modulate the excitability of VTA dopamine neurons, and GABAB antagonism enhances nicotine-induced increase of firing frequency (Erhardt et al., 2002). Therefore, it is possible that chronic GS39783 strengthens GABAergic tonic inhibition of VTA neurons, which would in turn decrease dopamine output in the NAc and the ensuing buildup of ΔFosB, although this hypothesis deserves further clarification.
We took advantage of the fact that both behavioral and biochemical tests in this study were performed on the same group of animals to compare both data. We found a strong positive correlation between ΔFosB expression and preference for nicotine. Genetic overexpression of ΔFosB in striatal tissues enhances cocaine place preference at low doses (Kelz et al., 1999), whereas mice carrying an inactivating mutation of FosB show reduced preference for cocaine (Hiroi et al., 1997). Therefore, our data strengthen this existing body of literature, suggesting strong associations between the accumulation of ΔFosB and the manifestation of rewarding properties of drugs of abuse.
There is an increasing demand for a non-nicotinic, nondopaminergic therapeutic approach to addiction (Cryan et al., 2003). In line with this, several clinical studies showed the efficacy of GABAB receptor agonists for the treatment of alcoholism, cocaine, or heroin dependence (Brebner et al., 2002). However, the long-term side effects of baclofen may affect its compliance in the smoking population. Together, our data show that GABAB receptor-positive modulation during the conditioning phase of place preference counteracts the rewarding and the long-lasting molecular adaptation induced by repeated administration of nicotine. However, the fact that we only obtained a significant blockade of the acquisition of CPP and not on its expression may limit the therapeutic potential, given that most smokers are already dependent when they attempt smoking cessation therapies. However, future studies must examine the effects of GS39783 in other models of nicotine dependence such as in the acquisition, maintenance, and reinstatement of nicotine self-administration. Furthermore, it is clear that there are many more aspects of the addiction process that need to be countered to develop successful therapeutic strategies for smoking (Cryan et al., 2003). Among those, counteracting the impact of drug-induced withdrawal and craving is essential. Therefore, interventions should not only be limited to inhibiting the rewarding effects of a drug per se but also should be aimed at reducing the manifestation of withdrawal and craving and reducing withdrawal-induced deficits in mood and anxiety (Volkow, 2005). It should be noted that GABAB receptor-positive modulators reduce anxiety in preclinical paradigms (Cryan et al., 2004; Mombereau et al., 2004), suggesting that they may have additional benefits as smoking cessation aids. Nonetheless, the examination of the behavioral effects of GS39783 in animal models of drug withdrawal and relapse is now warranted. Furthermore, given that the molecular effects of GS39783 in the current study parallel its behavioral effect, studies investigating the molecular mechanisms underlying how GABAB receptor-positive modulators can modify NAc ΔFosB may provide novel therapeutic targets for nicotine dependence.
Footnotes
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This work was supported by National Institute on Drug Abuse/National Institute of Mental Health Grant U01MH60962.
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C.M. and L.L. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.116228.
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ABBREVIATIONS: CPP, conditioned place preference; NAc, nucleus accumbens; ANOVA, analysis of variance; ICSS, intracranial self-stimulation; GS39783; N,N′-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine; VTA, ventral tegmental mass; CGP44532, (3-amino-2(S)-hydroxypropyl) methylphosphinic acid.
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↵1 Current affiliation: Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania.
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↵2 Current affiliation: Lectus Therapeutics, Babraham, United Kingdom.
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↵3 Current affiliation: Department of Pharmacology and Therapeutics, School of Pharmacy, University College Cork, Cork, Ireland.
- Received October 26, 2006.
- Accepted January 8, 2007.
- The American Society for Pharmacology and Experimental Therapeutics