JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 JPET FastThis articleForward. has not Published been copyedited on and January formatted. 10,The final2007 version as DOI:10.1124/jpet.106.116228 may differ from this version. JPET #116228 1

GABAB Receptor Positive Modulation-Induced Blockade of the Rewarding Properties of is Associated with a Reduction in Nucleus Accumbens ∆FosB Accumulation

Cedric Mombereau, Loic Lhuillier, Klemens Kaupmann and John F. Cryan

Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland (CM, LL, KK, JFC)

Downloaded from

jpet.aspetjournals.org at ASPET Journals on September 25, 2021

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 2

Running Title: GABAB receptor modulator blocks nicotine-induced changes

Corresponding author: John F. Cryan Ph.D School of Pharmacy, Department of Pharmacology & Therapeutics, Cavanagh Pharmacy Building, University College Cork, Cork Ireland Tel: +353 21 4901676 E-Mail: [email protected] Downloaded from

Total number of text pages: 18 Number of figures: 6

Number of references: 37 jpet.aspetjournals.org Number of words in Abstract: 250 Number of words in Introduction: 482 Number of words in Discussion: 1,471 Section: Neuropharmacology

Abbreviations: GABA, γ –aminobutyric acid; at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 3

ABSTRACT

There is an increasing demand for novel non-nicotinic, non-dopaminergic therapeutic approach to nicotine . 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 . Conditioned place preference (CPP) paradigms are

based upon the principle that animals, like humans, would learn to seek environmental stimuli Downloaded from which have been previously associated with rewarding events. Here we demonstrate that nicotine

(0.06 mg/kg s.c.) induced a robust CPP response. Further, GS39783 (30-100 mg/kg. p.o.), during the conditioning phase blocked the rewarding effects of nicotine in the CPP paradigm in rats. jpet.aspetjournals.org

However, GS39783 did not significantly alter the CPP effects of nicotine when given only immediately prior to the CPP test. A growing body of evidence suggests that repeated at ASPET Journals on September 25, 2021 administration of drugs of abuse induced long-term molecular changes in brain plasticity, most notably an accumulation of ∆FosB, in the striatal complex, that contribute to the manifestation of dependence. There was a significant accumulation of ∆FosB in the nucleus accumbens, but not dorsal striatum, of rats treated daily for five days with nicotine (0.06mg/kg i.p). GS39783 completely (30-100 mg/kg, po) counteracted these nicotine-induced molecular adaptations when given prior to the CPP acquisition phase but not when administered immediately prior to the test phase. Taken together, the behavioural and molecular changes induced by nicotine occur in concert and are concomitantly amenable to reversal by GABAB receptor positive modulators. JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 4

Smoking-related illness is a major 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 . It is largely accepted that nicotine is the active ingredient in 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 Downloaded from induce long-term molecular changes in brain plasticity that contribute to the manifestation of dependence (Kelz and Nestler, 2000). Members of the AP-1 family of transcription factors are

specifically thought to be involved in the ontogeny of addiction. Indeed, repeated administration jpet.aspetjournals.org of a variety of drugs of abuse, including nicotine induces an accumulation of ∆FosB, a highly stable truncated form of FosB, in the striatal complex (Kelz and Nestler, 2000). Moreover, mice

∆ overexpressing FosB also show enhanced sensitivity to the rewarding effects of and at ASPET Journals on September 25, 2021 (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 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 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 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 5 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 behavioural 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., 2006). However, to our knowledge there are no investigations to date on the effects of GS39783 in animal models of nicotine reinforcement. Downloaded from Animals, like humans, learn to seek environmental stimuli which 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 jpet.aspetjournals.org a prime test of nicotine reinforcement (Tzschentke, 1998). In the present studies, we investigated the effects of GS39783 on the behavioural (establishment of CPP), and molecular (accumulation

∆ of FosB in the nucleus accumbens) consequences of nicotine exposure. at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 6

METHODS

Animals

Wistar rats, (Charles-River, France) weighing 180-220g at the beginning of the experiments were housed four per cage (55x33x19 cm) in a humidity- and temperature-controlled room under a 12 h light/dark cycle (lights on at 0700). One week prior to the beginning of the experiments, all animals were food-restricted (20g/day) until the end of the study. This was carried out as food

restriction has been reported to enhance the rewarding effects of drugs of abuse (Carr 2002) Downloaded from including nicotine (Donny et al. 1998) and the study on which our protocol is based on uses a food restriction regime (Forget et al., 2005). Experiments were subject to institutional review and conducted in accordance with the Veterinary Authority of Basel-Stadt, Switzerland. jpet.aspetjournals.org

Conditioned place preference

The procedure was carried out essentially as described earlier (Forget et al., 2005). Rats were at ASPET Journals on September 25, 2021 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 on the basis of 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 were administered (immediately and 30 minutes, respectively) prior to exposure to an open-field covered with one floor texture. Conversely, vehicle paired session consist in pairing vehicle injection with the other floor texture. Initially, half of rats underwent a vehicle-paired session whereas the other half underwent a drug-paired session. The day following 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 dug-paired texture. The quadrants of the same JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 7 texture were positioned diagonally opposite to each 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 prior to use and administered orally in a suspension of 0.5% methylcellulose at a volume of 5 ml/kg 30 minutes prior to testing. Doses of Downloaded from GS39783 were chosen based on our previous data demonstrating that GS39783 can reverse the behavioral and molecular effects of cocaine in this dose range (Slattery et al., 2005; Lhuillier et

al., 2006). Further, they are in line with the pharmacokinetic properties of GS39783 in terms of jpet.aspetjournals.org brain exposure (Smith et al., 2004). (−)Nicotine bitartrate (Sigma Chemicals, St. Louis, MO,

USA) was dissolved in saline, and the pH was adjusted to 7.3-7.5 with 0.1 M NaOH and

administrated immediately prior conditioning session (s.c.). The nicotine dose is expressed as at ASPET Journals on September 25, 2021 free base and was selected from in-house studies showing robust nicotine-induced place preference at this dose (Chaperon et al., unpublished), in agreement with other studies (Forget et al., 2005).

Tissue Preparation

All rats were sacrificed immediately after the final test session. Brains were quickly removed, chilled in ice-cold phosphate-buffered saline (PBS) . NAc and Dorsal Striatum were dissected on ice-chilled glass plate and flash-frozen in dry ice.

Immunoblotting

Western blot analysis was carried out as previously described (Lhuillier et al., 2006). Primary antibodies used were rabbit rabbit anti-FosB (sc-48, Santa Cruz Biotechnologies, 1:500), rabbit JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 8 anti-actin (A2066, Sigma, 1:5000). Secondary HRP-linked goat anti-rabbit antibody was obtained from Cell Signaling (7074, 1:1000 to 1:5000).

Statistical analysis

In behavioral experiments, the time spent in drug paired quadrant was analyzed by a one-way

ANOVA followed by, where 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 Newmann-Keuls test. The correlation was performed using a least-square linear Downloaded from regression analysis

Results jpet.aspetjournals.org

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 at ASPET Journals on September 25, 2021 analysis indicates rats spent a reduced time in GS39783 (100 mg/kg)-paired quadrants compared to control group (p<0.001). In contrast, at 10 mg/kg and 30 mg/kg, GS39783 was hedonically neutral and failed to affect the time spent in drug-paired quadrants (Figure 1).

The 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 to control animals (p =0.044, Figure 2.) as previously reported (Forget et al.,

2005). Additionally, post-hoc analysis revealed also that GS39783 at 30 mg/kg and 100 mg/kg, when given 30 min prior to each drug-paired session, decreased the amount of time spent in nicotine-paired quadrant compared to animals treated only with nicotine (p =0.005 and p=0.013, respectively; Figure 2). Consequently, these data show that GABAB receptor positive modulation JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 9 during the conditioning phase antagonized the establishment of nicotine-induced place preference.

The 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 demonstrated, nicotine supported conditioned place preference (p = 0.03, compared to control group). GS39783 at 30 mg/kg and 100 mg/kg, Downloaded from when given 45 min prior to testing session, failed to decrease significantly the amount of time spent in the nicotine-paired quadrant compared to 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 jpet.aspetjournals.org texture by rats given GS39783 (100 mg/kg) did not differ from the control level (p =0.408).

Consequently, although GS39783 affects the acquisition of nicotine-induced place preference, it

appears to be without effect on its expression (Figure 3). at ASPET Journals on September 25, 2021

Effects of GS39783 on nicotine-induced increases in the expression of ∆FosB.

We studied the influence of both repeated and single administration of GS39783 on ∆FosB expression in NAc and dorsal striatum by semi-quantitative western blot analysis immediately after completion of the last test. Chronic nicotine stimulated a robust increase in ∆FosB expression in NAc (Figure 4A; p < 0.001 versus saline). This upregulation was completely blocked by GS39783 (p < 0.001 versus nicotine) at both doses used, when daily injected during the acquisition phase. GS39783 did not have any intrinsic effect on basal ∆FosB levels. In NAc, a single administration of GS39783 before the final CPP testing failed to inhibit ∆FosB induction at both doses (Figure 5A). One concern of the experimental procedure we used is that the ∆FosB signal observed might be a direct consequence of the last CPP test, as opposed to a slow build up of the protein during chronic nicotine exposure. This hypothesis can however be ruled out since JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 10

1) only chronic treatment with GS39783 resulted in inhibition of ∆FosB induction and 2) in experiment 2, the last GS39783 administration was performed 48 hours before CPP testing. In the dorsal striatum, ∆FosB levels were not altered by chronic nicotine or by chronic or acute

GS39783 (Figure 4B, 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 ∆FosB expression (p < 0.001, R2= 0.38; Figure 6). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 11

Discussion

The present report demonstrates 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 long lasting molecular adaptation, accumulation of accumbal ∆FosB, induced by repeated

administration of nicotine. Downloaded from

It appears that GS39783, at the highest dose tested (100 mg/kg), elicited a place aversion in the present study which can be interpreted as an aversive or anhedonic property of the compound at jpet.aspetjournals.org 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 at ASPET Journals on September 25, 2021 protocol employed 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 demonstrated that GS39783, when administrated during conditioning phase, blocked conditioned place preference 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 drugs intrinsic effects. Nonetheless, the fact that

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 prior to each drug pairing session attenuates the rewarding properties of nicotine.

GABAergic mechanisms have long been implicated in drug dependence largely due to known direct interactions of the GABA and the transmitter systems (Kalivas et al., JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 12

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, decreases also nicotine self-administration (Corrigall et al., 2000; Paterson et al., 2005). Downloaded from Interestingly, we also observed that a single administration of GS39783 prior to 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 jpet.aspetjournals.org blocked the establishment but not the expression of behavioural sensitization to cocaine

(Lhuillier et al., 2006). 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 at ASPET Journals on September 25, 2021 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, 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 might be postulated that these response 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 in order to confirm this hypothesis. However, caution needs to be taken regarding the use of baclofen in animal models which are based primarily on either, or both, motor performance and cognitive function as baclofen markedly impairs both parameters (Cryan et al.,

2004; Jacobson and Cryan 2005). Therefore, because of these confounds we did not test baclofen JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 13

in nicotine CPP in the current series of studies. While 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 demonstrate 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 Downloaded from 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 jpet.aspetjournals.org ∆FosB in the NAc, but not in dorsal striatum (Pich et al., 1997). Here we confirm these results in our model of nicotine reinforcement (Figure 4A and 5A). Further, GS39783 was effective in inhibiting this accumulation when injected chronically during the acquisition phase of place at ASPET Journals on September 25, 2021 preference. In contrast to this result, a single administration of GS39783 before the final test failed to block nicotine-induced ∆FosB accumulation. This result is in agreement with the observation that ∆FosB is an extremely stable variant of FosB which 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. ∆FosB 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 fine an increased excitability of VTA neurons, through direct activation of DA VTA neurons (Pidoplichko et al., 1997), cholinergic activation of these neurons by the pediculopontine 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 ∆FosB observed here (Imperato et al., 1986; Nisell et al., 1994). Additionally, JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 14

GABAB receptors tonically modulate the excitability of VTA DA neurons and GABAB antagonism enhances nicotine-induced increase of firing frequency (Erhardt et al., 2002). It is therefore possible that chronic GS39783 strengthens GABAergic tonic inhibition of VTA neurons, which would in turn decrease DA output in the NAc and the ensuing build up of ∆FosB, 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 Downloaded from correlation between ∆FosB expression and preference for nicotine. Genetic overexpression of

∆FosB in striatal tissues enhances cocaine place preference at low doses (Kelz et al., 1999) jpet.aspetjournals.org while, mice carrying an inactivating mutation of FosB show reduced preference for cocaine

(Hiroi et al., 1997). Our data therefore strengthen this existing body of literature suggesting strong associations between the accumulation of ∆FosB and the manifestation of rewarding at ASPET Journals on September 25, 2021 properties of drugs of abuse.

There is an increasing demand for a non-nicotinic, non-dopaminergic therapeutic approach to addiction (Cryan et al., 2003). In line with this, several clinical studies demonstrated the efficacy of GABAB receptor agonists for the treatment of , 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 demonstrate 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 obtain 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. Further, it is clear that there are JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 15 many more aspects of the addiction process that need to be countered in order to develop successful therapeutic strategies for smoking (Cryan et al., 2003). Among those, counteracting the impact of drug-induced withdrawal and are essential. Therefore, interventions should not be limited to only inhibiting the rewarding effects of a drug per se, but should also 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) Downloaded from suggests that they may have additional benefits as smoking cessation aids. Nonetheless, the examination of the behavioral effects of GS39783 in animal models of and

relapse is now warranted. Furthermore, given that the molecular effects of GS39783 in the jpet.aspetjournals.org current study parallels its behavioural effect, studies investigating the molecular mechanisms underlying how GABAB receptor positive modulators can modify NAc ∆FosB may provide

novel therapeutic targets for nicotine dependence. at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 16

References. Bechtholt AJ and Cunningham CL (2005) Ethanol-induced conditioned place preference is

expressed through a ventral tegmental area dependent mechanism. Behav Neurosci

119:213-223.

Beuten J, Ma JZ, Payne TJ, Dupont RT, Crews KM, Somes G, Williams NJ, Elston RC and Li

MD (2005) Single- and multilocus allelic variants within the GABA(B) receptor subunit

2 (GABAB2) gene are significantly associated with nicotine dependence. Am J Hum

Genet 76:859-864. Downloaded from

Brebner K, Childress AR and Roberts DC (2002) A potential role for GABA(B) agonists in the

treatment of psychostimulant addiction. Alcohol Alcohol 37:478-484. jpet.aspetjournals.org Brunzell DH, Russell DS and Picciotto MR (2003) In vivo nicotine treatment regulates

mesocorticolimbic CREB and ERK signaling in C57Bl/6J mice. J Neurochem 84:1431-

1441. at ASPET Journals on September 25, 2021 Carr KD (2002) Augmentation of drug reward by chronic food restriction: behavioral evidence

and underlying mechanisms. Physiol Behav 76:353–364

Chester JA, Cunningham CL. (1999) Baclofen alters ethanol-stimulated activity but not

conditioned place preference or taste aversion in mice. Pharmacol Biochem Behav.

63:325-31

Corrigall WA (1999) Nicotine self-administration in animals as a dependence model. Nicotine

Tob Res 1:11-20.

Corrigall WA, Coen KM, Adamson KL, Chow BL and Zhang J (2000) Response of nicotine

self-administration in the rat to manipulations of mu- and gamma-aminobutyric

acid receptors in the ventral tegmental area. Psychopharmacology (Berl) 149:107-114.

Cousins MS, Roberts DC, de Wit H. (2002) GABA(B) receptor agonists for the treatment of

drug addiction: a review of recent findings. Drug Alcohol Depend. 65:209-20. JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 17

Cryan JF, Gasparini F, van Heeke G and Markou A (2003) Non-nicotinic neuropharmacological

strategies for nicotine dependence: beyond . Drug Discov Today 8:1025-1034.

Cryan JF, Kelly PH, Chaperon F, Gentsch C, Mombereau C, Lingenhoehl K, Froestl W, Bettler

B, Kaupmann K and Spooren WP (2004) Behavioral characterization of the novel

GABAB receptor-positive modulator GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-

nitro-pyrimidine-4,6-diamine): anxiolytic-like activity without side effects associated

with baclofen or . J Pharmacol Exp Ther 310:952-963. Downloaded from Donny EC, Caggiula AR, Mielke MM, Jacobs KS, Rose C, Sved AF. (1998) Acquisition of

nicotine self-administration in rats: the effects of dose, feeding schedule, and drug

contingency. Psychopharmacology 136:83-90. jpet.aspetjournals.org

Erhardt S, Mathe JM, Chergui K, Engberg G and Svensson TH (2002) GABA(B) receptor-

mediated modulation of the firing pattern of ventral tegmental area dopamine neurons in

vivo. Naunyn Schmiedebergs Arch Pharmacol 365:173-180. at ASPET Journals on September 25, 2021

Esson L and Leeder SR (2004) The millennium development goals and tobacco control: an

opportunity for global partnership., in, World Health Organization (WHO), Geneva.

Forget B, Hamon M and Thiebot MH (2005) Cannabinoid CB1 receptors are involved in

motivational effects of nicotine in rats. Psychopharmacology (Berl): 181:722-34.

Forster GL and Blaha CD (2003) Pedunculopontine tegmental stimulation evokes striatal

dopamine efflux by activation of and glutamate receptors in the midbrain

and pons of the rat. Eur J Neurosci 17:751-762.

Goldberg SR, Risner ME, Stolerman IP, Reavill C and Garcha HS (1989) Nicotine and some

related compounds: effects on schedule-controlled behaviour and discriminative

properties in rats. Psychopharmacology (Berl) 97:295-302. JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 18

Hiroi N, Brown JR, Haile CN, Ye H, Greenberg ME and Nestler EJ (1997) FosB mutant mice:

loss of chronic cocaine induction of Fos-related proteins and heightened sensitivity to

cocaine's psychomotor and rewarding effects. Proc Natl Acad Sci U S A 94:10397-10402.

Imperato A, Mulas A and Di Chiara G (1986) Nicotine preferentially stimulates dopamine

release in the limbic system of freely moving rats. Eur J Pharmacol 132:337-338.

Kalivas PW, Duffy P and Eberhardt H (1990) Modulation of A10 dopamine neurons by gamma-

aminobutyric acid agonists. J Pharmacol Exp Ther 253:858-866. Downloaded from Kelz MB, Chen J, Carlezon WA, Jr., Whisler K, Gilden L, Beckmann AM, Steffen C, Zhang YJ,

Marotti L, Self DW, Tkatch T, Baranauskas G, Surmeier DJ, Neve RL, Duman RS,

Picciotto MR and Nestler EJ (1999) Expression of the deltaFosB in jpet.aspetjournals.org

the brain controls sensitivity to cocaine. Nature 401:272-276.

Kelz MB and Nestler EJ (2000) deltaFosB: a molecular switch underlying long-term neural

plasticity. Curr Opin Neurol 13:715-720. at ASPET Journals on September 25, 2021

Lhuillier L, Mombereau C, Cryan JF and Kaupmann K (2006) GABA(B) Receptor-Positive

Modulation Decreases Selective Molecular and Behavioral Effects of Cocaine.

Neuropsychopharmacology. In Press

Li SM, Yin LL, Ren YH, Pan LS and Zheng JW (2001) GABA(B) receptor agonist baclofen

attenuates the development and expression of d-methamphetamine-induced place

preference in rats. Life Sci 70:349-356.

Mansvelder HD, Keath JR and McGehee DS (2002) Synaptic mechanisms underlie nicotine-

induced excitability of brain reward areas. Neuron 33:905-919.

Mombereau C, Kaupmann K, Froestl W, Sansig G, van der Putten H and Cryan JF (2004)

Genetic and pharmacological evidence of a role for GABA(B) receptors in the

modulation of anxiety- and antidepressant-like behavior. Neuropsychopharmacology

29:1050-1062. JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 19

Nisell M, Nomikos GG and Svensson TH (1994) Infusion of nicotine in the ventral tegmental

area or the nucleus accumbens of the rat differentially affects accumbal dopamine

release. Pharmacol Toxicol 75:348-352.

Paterson NE, Froestl W and Markou A (2005) Repeated administration of the GABAB receptor

agonist CGP44532 decreased nicotine self-administration, and acute administration

decreased cue-induced reinstatement of nicotine-seeking in rats.

Neuropsychopharmacology 30:119-128. Downloaded from Pich EM, Pagliusi SR, Tessari M, Talabot-Ayer D, Hooft van Huijsduijnen R and Chiamulera C

(1997) Common neural substrates for the addictive properties of nicotine and cocaine.

Science 275:83-86. jpet.aspetjournals.org

Pidoplichko VI, DeBiasi M, Williams JT and Dani JA (1997) Nicotine activates and desensitizes

midbrain dopamine neurons. Nature 390:401-404.

Slattery DA, Markou A, Froestl W and Cryan JF (2005) The GABAB receptor-positive at ASPET Journals on September 25, 2021

modulator GS39783 and the GABAB receptor agonist baclofen attenuate the reward-

facilitating effects of cocaine: intracranial self-stimulation studies in the rat.

Neuropsychopharmacology 30:2065-2072.

Smith MA, Yancey DL, Morgan D, Liu Y, Froestl W and Roberts DC (2004) Effects of positive

allosteric modulators of the GABAB receptor on cocaine self-administration in rats.

Psychopharmacology (Berl) 173:105-111.

Tzschentke TM (1998) Measuring reward with the conditioned place preference paradigm: a

comprehensive review of drug effects, recent progress and new issues. Prog Neurobiol

56:613-672.

Urwyler S, Pozza MF, Lingenhoehl K, Mosbacher J, Lampert C, Froestl W, Koller M and

Kaupmann K (2003) N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6- JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 20

diamine (GS39783) and Structurally Related Compounds: Novel Allosteric Enhancers of

{gamma}-Aminobutyric AcidB Receptor Function. J Pharmacol Exp Ther 307:322-330.

Volkow ND (2005) What do we know about drug addiction? Am J Psychiatry 162:1401-1402.

Walters CL, Cleck JN, Kuo YC and Blendy JA (2005) Mu-opioid receptor and CREB activation

are required for nicotine reward. Neuron 46:933-943.

Zhang D, Zhang L, Lou DW, Nakabeppu Y, Zhang J and Xu M (2002) The dopamine D1

receptor is a critical mediator for cocaine-induced gene expression. J Neurochem Downloaded from 82:1453-1464. jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 21

FOOTNOTES CM and LL contributed equally to this work.

Current addresses:

Dept Pharmacology & Therapeutics, School of Pharmacy, University College Cork, Cork,

Ireland (JFC); Lectus Therapeutics, Babraham, UK (LL); Dept Pharmacology, University of

Pennsylvania, Philadelphia, USA (CM).

Acknowledgements: Downloaded from

Supported by NIDA/NIMH grant U01MH60962. jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 22

LEGENDS FOR FIGURES

Figure 1. Intrinsic properties of GS39783 in the place conditioning paradigm. GS30783 (10-

30 mg/kg) was hedonically neutral in CPP. However. at 100 mg/kg, GS39783 elicited a significant place aversion. Each bar represents the mean (n = 12 per group) ± S.E.M. of the time spent in drug associated quadrants. *, **, groups that differed significantly from vehicle treated animals (p < 0.05 and p < 0.01, respectively).

Downloaded from Figure 2. The effects of GS39783 on the acquisition of the nicotine-induced place preference.

Nicotine, (0.06 mg/kg s.c.), elicited significant place preference (black bar) which was blocked

by administration of GS39783, at both 30 and 100 mg/kg, during the conditioning phase (grey jpet.aspetjournals.org

Bars). Each bar represents the mean (n = 11 per group) ± S.E.M. of the time spent in the drug associated quadrants. * and + represent differences to saline or nicotine groups, respectively; **,

++, p < 0.01; +, p < 0.05. at ASPET Journals on September 25, 2021

Figure 3. The effects of GS39783 on the expression of nicotine-induced place preference.

Nicotine (0.06 mg/kg s.c.), induced a significant place preference (black bar). A single administration of GS39783 (grey bars) prior to the test phase failed to block the nicotine-induced conditioned place preference. Each bar represents the mean (n = 11 per group) ± S.E.M. of the time spent in drug associated quadrants. *, **, groups that differed significantly from vehicle treated animals (P < 0.05 and P < 0.01, respectively)

Figure 4. Effects of chronic GS39783 administration on ∆FosB accumulation. Rats (n = 5-8 animals/experimental group) were treated daily with saline (white bars) or nicotine, (0.06mg/ kg i.p; black bars) during the 5 days of pre-exposure and during the acquisition phase of place preference. During conditioning, GS39783 was injected (at indicated concentrations, p.o.) 30 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. JPET #116228 23 minutes prior to nicotine/saline administration. Animals were sacrificed immediately after the final test. NAc and Dorsal striatum dissected and processed for immunoblot analysis.

Representative immunoblots (top panels) and averaged densitometry values (bottom panels) are shown. Nicotine induces ∆FosB upregulation in NAc (A) but not in the dorsal striatum (B).

GS39783 completely inhibits ∆FosB induction in NAc at both doses used (A).

Figure 5. Lack of effect of a single administration of GS39783 on ∆FosB accumulation. Rats Downloaded from (n=10 animals/group) underwent nicotine pre-conditioning and conditioning without exposure to

GS39783. The modulator was administered 30 min prior to final test at the doses indicated.

∆

FosB protein levels are upregulated by nicotine pre-treatment in NAc (A) and not in dorsal jpet.aspetjournals.org striatum (B). Acute GS39783 treatment fails to inhibit this upregulation in NAc. * and + represent differences to saline or nicotine groups, respectively; **, p < 0.05; ***, +++ p < 0.001.

at ASPET Journals on September 25, 2021

Figure 6. ∆FosB induction is correlated to nicotine reinforcement. Data in both behavioral and biochemical analyses are included (n = 39 animals). Accumbal ∆FosB relative abundance is plotted against the subject’s CPP score. The red line represents the best fit with a least-square method. Dashed lines indicate chance level (x-axis) and ∆FosB basal level (y-axis).

JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 10, 2007 as DOI: 10.1124/jpet.106.116228 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021