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Behavioural Brain Research 217 (2011) 408–415

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

LV330, a cannabinoid CB1 receptor antagonist, attenuates ethanol and nicotineeeking and improves inhibitory response control in rats

.M.W.J. de Bruina,∗, J.H.M. Langea, C.G. Krusea, A.H. Herremansa, A.N.M. Schoffelmeerb,. van Drimmelena, T.J. De Vriesb

Abbott Healthcare Products BV, C.J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands1

Neuroscience Campus Amsterdam, Department of Anatomy and Neurosciences, VU University Medical Center, Van der Boechorststaat 7, 1081BT Amsterdam, The Netherlands

r t i c l e i n f o

rticle history:eceived 30 June 2010eceived in revised form 1 November 2010ccepted 5 November 2010vailable online 11 November 2010

eywords:LV330annabinoid CB1 receptor (CB1R)ntagonististar rats

thanolicotineelf-administrationue-induced relapse-choice serial reaction time task (5-CSRTT)

a b s t r a c t

Cannabinoid CB1 receptor (CB1R) signaling has been shown to play a role in the regulation of addictivebehavior. In the present study, our aim was to investigate whether the CB1R antagonist SLV330 couldreduce ethanol and nicotine self-administration and cue-induced reinstatement of ethanol and nicotineseeking behavior in Wistar rats.

In operant chambers, rats were learned to emit a specific response (nose poke) in order to receive anethanol solution or intravenous injections of nicotine. Discrete light and tone cues were presented duringethanol and nicotine delivery. These cues are particularly important for drug self-administration behaviorand, through Pavlovian conditioning, acquire conditioned reinforcing and motivational properties andare therefore able to generate and maintain drug-seeking behavior. Subsequently, the CB1R antagonistSLV330 (doses ranging from 1 to 10 mg/kg, given orally, p.o.) was administered to investigate the effectson drug self-administration. In addition, responding for ethanol and nicotine was extinguished. Then,the animals were tested for cue-induced reinstatement of ethanol and nicotine seeking and treated withvehicle or SLV330. Finally, the effects of SLV330 were studied on the number of anticipatory responsesin the 5-choice serial reaction time task (5-CSRTT) in order to determine whether this compound couldalso increase impulse control in Wistar rats.

The CB1 antagonist SLV330 was effective in reducing ethanol self-administration at a lowest effectivedose (LED) of 10 mg/kg (p.o.) and reinstatement of ethanol seeking at a LED of 3 mg/kg (p.o.). SLV330 was

also effective in reducing nicotine self-administration and reinstatement of nicotine seeking, although ata LED of 10 mg/kg (p.o.). Finally, SLV330 decreased time delay-dependent anticipatory responding (LEDof 3.0 mg/kg, p.o.), indicating an increased inhibitory control.

These findings are in agreement with results reported with other CB1 antagonists. The combined actionof reducing the reinforcing and motivational properties of nicotine and alcohol and the improvement ofimpulse control supports the idea that the cannabinoid system is a promising target for anti-relapse

medication.

. Introduction

The endocannabinoid (eCB) signaling system includes two clas-

ical G-protein-coupled receptors, known as the cannabinoid CB1nd CB2 receptors. Cannabinoid CB1 receptor (CB1R) signaling haseen suggested to play a role in the regulation of addictive behavior,ognition and the control of appetite for food. Preclinical evidence

∗ Corresponding author at: Abbott Healthcare Products BV, PO Box 900, C.J. vanoutenlaan 36, 1381 CP Weesp, The Netherlands, (formerly Solvay Pharmaceuti-als). Tel.: +31 0 294 479665; fax: +31 0 294 415256.

E-mail address: natasja.debruin@gmail.com (N.M.W.J. de Bruin).1 Formerly Solvay Pharmaceuticals.

166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2010.11.013

© 2010 Elsevier B.V. All rights reserved.

suggests that activation of the endogenous cannabinoid systemplays a role in mediating the motivational effects of drugs anddrug-related stimuli and in general brain reward functions and drugabuse [1,2], whereas CB1 antagonists have been reported to coun-teract these effects (reviewed by Beardsley at al. and Fattore et al.[3–5]). For example, rimonabant, a brain-penetrating CB1R antag-onist, attenuates the motivational and dopamine-releasing effectsof nicotine [6], reduces nicotine and ethanol self-administration[6–9] and reverses reinstatement of drug-seeking behavior follow-

ing presentation of drug-associated cues in rats [4,5,10–12]. Also,rimonabant suppresses the appetitive attributes of ethanol in Sar-dinian alcohol-preferring (sP) rats [13]. Furthermore, mice lackingCB1 receptors consume less ethanol than wild type littermates andadministration of a CB1 receptor antagonist reduces consumption

N.M.W.J. de Bruin et al. / Behavioural Brain Research 217 (2011) 408–415 409

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Table 1Pharmacological in vitro and in vivo results for SLV330: CB1 and CB2 receptor binding,CB1 receptor antagonistic activity and two mechanistic pharmacological models.

CB1 rba,b CB2 rb

a,b CB1 functa,b CBBP

c CB1 TEMPd

58 ± 19 3495 ± 968 8.7 ± 0.2 8 10

Source: [24,25].a CB1 rb, Displacement of specific CP-55,940 binding in CHO cells stably trans-

fected with human CB1R, expressed as Ki ± SEM (nM); CB2 rb, Displacement ofspecific CP-55,940 binding in CHO cells stably transfected with human CB2R,expressed as Ki ± SEM (nM); CB1 funct, CB1 functional assay; [3H] arachidonic acidrelease in CHO cells, expressed as pA2 ± SEM values

b All Ki and pA2 values are mean values from at least three independent experi-ments

Fig. 1. Chemical structure of SLV330.

f wild type animals to the same level as receptor null mice [14,15].urprisingly, in another study by Racz et al. [58], CB1 knock-outice had initially a higher preference for ethanol than C57BL/6Jice. Finally, synergistic potentiation of the reducing effect on

thanol drinking and seeking behaviors in rats has been reportedhen administering a combination of rimonabant and the opioid

eceptor antagonist naltrexone (review by Colombo et al. and Carait al. [16,17]).

Animal models of ethanol and nicotine use have been developedith the aim to investigate the effects of compounds on mainte-ance and reinstatement of drug use. In these tasks, animals learno emit a specific response (lever press, nose poke) in order toeceive ethanol or intravenous injections of nicotine [18]. Environ-ental cues are particularly important for drug self-administration

ehavior [10,19–21]. Such stimuli, through Pavlovian conditioning,cquire conditioned reinforcing and motivational properties andre therefore able to generate and maintain drug-seeking behavior.otivational effects of drug-associated stimuli can be measured

sing second-order schedules of drug reinforcement and extinc-ion/reinstatement models.

Whereas the majority of the preclinical studies on CB1 receptorntagonists has focused on the potential impact on obesity-relatedarameters in normal and obese rats, their effects on the treat-ent of drug dependence has received less attention. Hitherto,

nly four CB1 receptor antagonists have been reported in ani-al models of ethanol dependence, viz. the 1,5-diarylpyrazoles

imonabant, surinabant (SR147778) and AM-251 and the triazoleongener LH-21 (see review [22]). In the present study, our aimas to investigate whether the selective CB1R antagonist SLV330

see Fig. 1, [23–25]) could attenuate ethanol and nicotine self-dministration and reinstatement of drug seeking behavior inistar rats.Impulsivity is pathological in several psychiatric disorders

ncluding drug addiction (reviewed by Pattij and Vanderschuren26]). Impulsivity is thought to play a role in drug addictionnd disturbances in inhibition of behavior resulting from pro-onged drug intake have been proposed to maintain drug abuse57]. Furthermore, elevated impulsivity might predispose indi-iduals to initiate or maintain drug seeking and taking in bothumans [27] and laboratory animals [28]. Therefore, compounds

hat increase impulse control might have additional benefits inhe treatment of drug addiction. In the 5-choice serial reac-ion time task (5-CSRTT), rimonabant dose-dependently decreaseshe number of premature responses, potentially reflecting anncrease in inhibitory control [29]. In the present study, we

c Antagonism of CP-55,940 induced hypothension in rat expressed as ED50 (mg/kg,p.o. administration)

d Antagonism of WIN 55,212-2 induced hypothermia in mice expressed as lowesteffective doses (LED, mg/kg, p.o. administration)

investigated whether the pyrazoline derivative SLV330 may havebeneficial effects on impulse control as measured in the 5-CSRTT.

In Table 1, results are described for SLV330 in CB1 and CB2 recep-tor binding studies, in a functional cell assay used to measure CB1antagonistic activity and in mechanistic in vivo pharmacology mod-els (previously reported by [24,25]). SLV330 has been characterizedalso as an inverse agonist. Recently, SLV330 was reported to possesscognitive enhancing effects in rodent models for cognitive impair-ment [23]. SLV330 has been found to have a high brain-to-plasmaratio (8.3) in the rat, suggesting that SLV330 crosses the blood–brainbarrier to affect CB1Rs in the CNS. Therefore, SLV330 is an excellentpharmacological tool compound to investigate the effects on druguse in rats in the present study. SLV330 was tested in doses rangingfrom 1 to 10 mg/kg and given orally (p.o.).

2. Experimental 1: Effects of SLV330 on ethanol self-admininistration andcue-induced reinstatement of ethanol seeking

2.1. Method

2.1.1. SubjectsMale Wistar rats, weighing 280–300 g at arrival (Harlan, the Netherlands) were

used. For the ethanol self-administration study, we started with 64 male Wis-tar rats. For the ethanol cue-induced reinstatement experiment, 28 male Wistarrats were used. The experiment started after one week of acclimatization. Ani-mals were housed individually in a temperature- (Temp = 22 ± 2 ◦C) and humidity(rel% = 60 ± 5%) controlled room on a reverse 12 h light–dark cycle (lights off at7:00 AM) with water available ad libitum. The animals were on a diet of 18 g chow(Teklad, Harlan, the Netherlands) per animal per day. Animals were partially food-deprived to increase motivation, but also to stabilize weight increase of the animalsduring the experiment. Cages were enriched with gnaw woods.

2.1.2. CompoundsEthanol 12% (v/v) was made from ethanol 96% (Antonides-Interchema, Oost-

erzee, Netherlands) and diluted with tap water. SLV330 was supplied and preparedby Solvay Pharmaceuticals by using a crash resistant Cremophor formulation (con-sisting of Polyethylene glycol 200, 8.5% (w/w) Polysorbate 80, 10.0% Propyleneglycol, 41.5% (w/w) Cremophor RH40 and 20.0% (w/w) Vitamin E TPGS). SLV330 wasadministered orally (p.o.) with an injection volume of 2 ml/kg, 60 min before testing.SLV330 was administered in the range 0 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg.

2.1.3. Apparatus descriptionAll training and testing was conducted in 16 standard operant chambers

(30.5 × 24.1 × 29.2 cm) surrounded by sound attenuating ventilated cubicles (MedAssociates, Georgia, VT). The chambers were fitted with a grid floor, a red dim house-light and opposite the houselight an intelligence panel. In the intelligence panelthere were two nose-poke holes, a red dim stimulus light above both nose-pokeholes, a receptacle between the nosepoke holes in which the ethanol was deliveredand a sonalert tone module located above the receptacle.

One nose-poke hole served as active- and the other as inactive hole. Pokes

made in the active hole resulted in the delivery of an ethanol drop (0.19 ml; 12%)in the receptacle. The ethanol drop was delivered by an infusion pump (PHM-100,MedAssociates, Georgia, VT) in 4.2 s.

During ethanol delivery, the active nose-poke hole was illuminated and a tone(±65 dB) was presented by the tone module for 2 s. The receptacle was illumi-nated for 5 s. The stimulus light switched off for 15 s. In this 15 time-out second

4 ral Brain Research 217 (2011) 408–415

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ment was tested by re-exposing the animals to ethanol-related environment. Wefound that SLV330 with a LED of 3 mg/kg p.o., was able to suppress cue-inducedethanol seeking in a dose-related manner.

10 N.M.W.J. de Bruin et al. / Behaviou

eriod, nose-poking was without consequences. During all sessions, responses inhe inactive hole were monitored, but without consequences.

.1.4. ProceduresAll procedures described were approved by the local Animal Care and Use Com-

ittees of the Free University of Amsterdam and were in accordance with therinciples of Laboratory Animal Care (NIH publication no. 86-23, revised 1985). Allfforts were made to minimize distress and number of animals used.

.1.4.1. Training and testing. Prior to the operant procedure, animals were dailyrained to consume ethanol in a two-bottle free-access and a two-bottle limited-ccess paradigm. In both procedures, two bottles (one with tap water, one withthanol) were placed on the home cage. In the two-bottle free-access procedure,he ethanol percentage was gradually increased from 2% to 12% every ‘ethanol’ day.etween two ‘ethanol’ days was a water day, which means that both bottles werelled with tap water and no ethanol was available. When an ethanol percentagef 12% was reached, ethanol (together with a bottle of water) was available for fiveuccessive days. After this period, the ethanol availability was decreased to 1 h a daytwo bottle limited access procedure), to train the animals in drinking high amountsf ethanol in a short time period.

After this two-bottle limited access procedure, the animals were ready to berained in the operant cage to self-administer ethanol 12% (v/v). The animals wererained on a Fixed Ratio 1 schedule for 10 daily sessions (FR1-schedule; everyctive response resulted in a reward; sessions of 60 min). Subsequently, animalsere trained via an FR2-schedule up to an FR4-schedule (every 2nd and 4th active

esponse, respectively, resulted in a reward; daily sessions of 60 min).

.1.4.2. Test for ethanol self-administration. When response rates were stable,nimals were selected for further testing if they consumed ethanol (in the FR4-chedule) above the in the literature described pharmacological effective dose of.3 g/kg/h [30]. The rate at which a rat metabolizes ethanol is about 0.3 g/kg/h [31],nd thus is likely to produce pharmacological effects. Subsequently, the effects ofhe CB1 antagonist SLV330 on ethanol self-administration was determined using aetween-subject design.

The number of total active and total inactive nose pokes was measured, as wells the number of ethanol deliveries (rewards).

For this study, the total number of rewards and the total number of responseser treatment were subjected to statistical analysis. Sessions were operated and rawata was saved by the MED-PC IV program. For analysis of the self-administrationata, the total number of nose pokes (responses) in the active and inactive hole andhe number of rewards were subjected to a one-way ANOVA, followed by a Dunnettost-hoc test, with the different treatments as between factor.

.1.4.3. Test for reinstatement of ethanol seeking. For the cue-induced reinstatementxperiment, animals were trained as described above until response behavior onhe FR4-schedule was stable. This was followed by a three-week period of absti-ence in the homecage. Subsequently, each animal was tested twice for cue-inducedeinstatement with a 7-day wash-out period in between. Animals were randomlyistributed over the different experimental groups. On the test day, the animals werelaced in the operant cage for 4 h in absence of ethanol and ethanol related stimulio extinguish response behavior. One hour before the end of this extinction session,he animal was removed from the operant cage and treated with SLV330. After drugdministration the animal was placed back into the operant chamber, where theession continued. When the last hour had passed, the cue-induced relapse test ses-ion started (duration: 45 min). During this test, the experimental conditions wereimilar to the conditions during training on an FR4-schedule, with the exceptionshat (1) at the beginning of the session, a 12% ethanol drop of 0.20 ml was availabledelivered in the receptacle by the experimenter at the beginning of the test ses-ion) and (2) after every 4th response, only cues were presented (no ethanol waselivered). Tests were operated and raw data was saved by the MED-PC IV program.or analysis of the cue-induced reinstatement data, the total number of nose pokesresponses) in the active and inactive holes were subjected to a one-way ANOVAollowed by a comparison procedure according to Dunnett, with the different dosesf SLV330 as between factor.

.2. Results

.2.1. Results ethanol self-administration experimentOf 64 animals, 37 animals met the selection criterion (intake of 0.3 g/kg ethanol

uring a 1 h session) and were selected for testing. Normally, the percentage of ani-als excluded is 25%. However, there may be differences between different groups

f animals. All tested animals readily acquired ethanol self-administration behaviornd showed a clear preference for the active nose-poke hole. More particularly, theverage of ethanol consumed in the rats treated with vehicle was 0.77 (±0.12) g/kg

uring the 1 h session, which is well above the criterium of 0.3 g/kg.

A one-way ANOVA revealed that:

The total number of active nose pokes when treated with SLV330, decreasedcompared with vehicle: F[3,36] = 3.991; p < 0.05, post-hoc 0 mg/kg vs. 3 mg/kgp = 0.067; 0 mg/kg vs. 10 mg/kg p < 0.05 (see Fig. 2; total active nose pokes).

Fig. 2. Effect of SLV330 on ethanol self-administration (means ± SEM): number ofactive and inactive nose pokes and rewards. The difference from the vehicle condi-tion is depicted with *p < 0.05, n = 9–10 per group.

- The number of ethanol deliveries earned during a session was significantly lowerwhen the animals were treated with SLV330: F[3,36] = 3.638; p < 0.05, post hocvehicle vs. 3 mg/kg p = 0.095; vehicle vs. 10 mg/kg p < 0.05 (see Fig. 2; rewards).

- SLV330 showed no effects on the total number of inactive nose pokes(F[3,36] = 0.905; p = 0.449(ns)), see Fig. 2; total inactive nose pokes).

2.2.2. Results cue-induced reinstatement to ethanol seeking experimentIn the operant cage, all 28 selected animals readily acquired ethanol self-

administration behavior and consumed their rewards. They made a clear distinctionbetween the active and inactive hole. Mean ethanol intake amounted to 0.50 g/kg/h.During the test day’s extinction session, responding in the previously active holeextinguished in all of the animals (∼2 active nose pokes in the last hour before thetest session, see Fig. 3).

A one-way ANOVA revealed that during testing for cue-induced ethanol seeking,the amount of active nose-pokes of animals treated with 3 mg/kg and 10 mg/kgSLV330 was significantly lower than animals treated with placebo (F[3,55] = 6.555;p < 0.01). There was no significant difference in inactive nose pokes between placeboand the tested dosages of SLV330 (F[3,55] = 1.134; p = 0.344), see Fig. 4).

2.3. Conclusion

SLV330 was able to suppress responding (nose poking) for ethanol with a LED of10 mg/kg. SLV330 at 3 mg/kg just failed to significantly suppress responding (nosepoking) for ethanol. In a second experiment, animals were withdrawn from ethanolfor three weeks in the homecage. Subsequently, the effect of SLV330 on reinstate-

Fig. 3. Mean amount of responses ±SEM during extinction in the ethanol experi-ment. Following a three-week period of ethanol abstinence in the homecage, eachanimal was tested for cue-induced reinstatement. On the test day, the animals wereplaced in the operant cage for 4 h in absence of ethanol and ethanol related stimulito extinguish response behavior.

N.M.W.J. de Bruin et al. / Behavioural Bra

Fig. 4. Effect of SLV330 on cue induced reinstatement of ethanol seeking(means ± SEM): number of active and inactive nose pokes and rewards. One hourbefore the end of this extinction session, the animal was removed from the operantcbhv

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age and treated with SLV330. After drug administration the animals was placedack into the operant chamber, where the session continued. When the last hourad passed, the cue-induced relapse test session started. The difference from theehicle condition is depicted with *p < 0.05 and **p < 0.01, n = 9–10 per group.

. Experimental 2: Effects of SLV330 on nicotine self-admininistration andue-induced reinstatement of nicotine seeking

.1. Method

.1.1. SubjectsFor the nicotine self-administration study, we started with sixteen male Wistar

ats of 280–300 g (Harlan, the Netherlands). For the nicotine cue-induced rein-tatement experiment, we started with 32 male Wistar rats. Animals were housedndividually in a temperature- (Temp = 22 ± 2 ◦C) and humidity (rel% = 60 ± 5%) con-rolled room on a reverse 12 h light–dark cycle (lights off at 7:00 AM) with foodTeklad, Harlan) and water available ad libitum. Cages were enriched with gnawoods.

.1.2. CompoundsNicotine: (−) nicotine hydrogen tartrate salt (Sigma, St Louis, MO) was dissolved

n sterile saline. The pH of the solution was adjusted to approximately 7.4 withiluted NaOH. Nicotine dose was expressed as free base weight.

SLV330 was supplied and prepared by Solvay Pharmaceuticals*, by using therash resistant Cremophor formulation. SLV330 was administered orally (p.o.) withn injection volume of 2 ml/kg, 60 min before testing. SLV330 was administered inhe range 0 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg.

.1.3. Apparatus descriptionThe apparatus was similar to the one described in Section 2.1.3 with a few

xceptions. During self-administration, animals were connected to an infusionpumpPHM-100, MedAssociates, Georgia, VT) by a swivel to receive intravenous infusionsf nicotine (40 �g/kg/inf). One nose poke hole served as active- and the other as inac-ive hole. Pokes made in the active hole resulted in the delivery of a nicotine infusionor 2 s. During this infusion, the active nose poke hole was illuminated (yellow light)nd an audio stimulus was presented (tone of 2900 Hz and 65 dB for 2 s). From theoment of nicotine delivery, the stimulus light switched off for 15 s. In this time-

ut period, nose poking was without consequences. During all sessions, responsesn the inactive hole were monitored, but without consequences.

.1.4. ProceduresAll procedures described were approved by the local Animal Care and Use Com-

ittees of the Free University of Amsterdam and were in accordance with therinciples of Laboratory Animal Care (NIH publication no. 86-23, revised 1985). Allfforts were made to minimize distress and number of animals used.

.1.4.1. Surgery. After a week of acclimatization, intravenous silicon catheters weremplanted in the right jugular vein under isoflurane anesthesia as described previ-usly in detail [32].

.1.4.2. Training and testing. Behavioral training started 5–7 days after surgery. Thenimals were trained on a Fixed Ratio 1 schedule (FR1-schedule) for 10 daily sessionsevery active nose poke resulted in a nicotine infusion and associated cues; dailyessions of 60 min). Subsequently, animals were trained on an FR2-schedule for 3ays and then moved up to an FR3-schedule for the remainder of the experiment

in Research 217 (2011) 408–415 411

(respectively, every 2nd and 3rd active nose poke resulted in a reward and associatedcues; daily sessions of 60 min).

3.1.4.3. Test for nicotine self-administration. When response behavior was stable,animals were selected for further testing if they earned at least 5 rewards duringthe last three sessions of the FR3 schedule and the reward delivery was equallydistributed over the session (criterion based on previous experience). Tests wereconducted twice a week, on Tuesdays and Fridays. On intermediary days, rats weretrained to self-administer nicotine without being treated with SLV330. For testingSLV330, a within-subject design was used: every animal received each dose in arandom order. In contrast to the ethanol experiment, a within-subject design wasused in order to reduce the number of animals undergoing the surgery.

During nicotine self-administration, the number of active and inactive nosepokes was measured, as well as the number of nicotine infusions (rewards). Forthis study, the total number of rewards and the total numbers of active and inac-tive responses per treatment were subjected to statistical analysis. Sessions wereoperated and raw data was saved by the MED-PC IV program. For analysis of theself-administration data, the total number of nose pokes (responses) in the activeand inactive hole (time-in + time-out) and the number of rewards were subjectedto a repeated measurement ANOVA with the different treatments as within subjectfactor.

3.1.4.4. Test for reinstatement of nicotine seeking. When response behavior on theFR3-schedule was stable, responding for nicotine was extinguished. In this extinc-tion phase (13 daily sessions of 60 min), the animals were placed in the operantchambers, but the nicotine and nicotine related stimuli (discrete and discriminativecues) were not available. Finally, the animals were tested for cue-induced reinstate-ment. During this test (duration of 30 min), the experimental conditions were similarto the conditions during training on a FR3-schedule, with the exception that nonicotine was available. Tests for cue-induced reinstatement were conducted twice;between these 2 test days, there were 2 days of further extinction training (wash-outperiod). During the relapse tests, effects of SLV330 were studied on output measure-ments: the number of active and inactive nose pokes was measured, as well as thenumber of rewards.

For this study, the total number of active and inactive responses per treatment,made during cue induced reinstatement, was subjected to statistical analysis. Ses-sions were operated and raw data was saved by the MED-PC IV program. A ‘Levene’stest of equality of error variances’ revealed that the error variance of the activeresponses were not homogeneous across groups (even after log-transformation).Since homogeneity is the most important assumption to perform an ANOVA, anon-parametric test was used to analyze this parameter of the cue-induced rein-statement test. The total number of nose pokes (responses) in the active holewas subjected to a non-parametric K-independent samples test (Kruskal–Wallis)followed by a Mann–Whitney test (for post hoc testing), with the different experi-mental groups as between factor. The number of inactive nose pokes was subjectedto a one-way ANOVA followed by a comparison procedure according to Dunnett,with the different experimental groups as between factor.

3.2. Results

3.2.1. Results nicotine self-administration experimentThe 10 animals that met the selection criterion with the best performance dur-

ing nicotine self-administration were selected (out of 16 animals) for further testing.During testing, one animal was excluded, because of blockade of the catheter. All 9tested animals readily acquired nicotine self-administration behavior and showeda clear preference for the active nose poke hole. More particularly, the averagequantity of self-administered nicotine in the rats treated with vehicle was 0.37(±0.02) mg/kg during the test session.

A repeated measurement ANOVA revealed that:

- The total number of active nose pokes when treated with SLV330 decreasedsignificantly (F[3,24] = 17.004; p < 0.001): 0 mg/kg vs. 10 mg/kg (F[1,8] = 32.661;p < 0.001, see Fig. 5, total active nose pokes).

- The total number of infusions of nicotine earned during a session when treatedwith SLV330 also decreased significantly (F[3,24] = 16.829; p < 0.001): 0 mg/kgvs. 3 mg/kg (F[1,8] = 4.267; p = 0.073); 0 mg/kg vs. 10 mg/kg (F[1,8] = 39.130;p < 0.001), see Fig. 5, rewards.

- SLV330 had no effect on the total number of inactive nose pokes (F[3,24] = 1.449;p = 0.253 (ns), see Fig. 5; total inactive nose pokes).

3.2.2. Results cue-induced reinstatement to nicotine seeking experimentOf 32 animals, 25 rats met the criterion for nicotine self-administration and

were selected for further testing. The 25 selected animals readily acquired nico-

poke hole. During extinction sessions, responding in the previously active hole wasextinguished in all of the animals (∼10 active nose pokes in the last session/hour,see Fig. 6). A Kruskal–Wallis test (non-parametric K-independent samples test)followed by a Mann–Whitney test revealed that during testing for cue inducednicotine seeking, the number of active nose pokes when treated with 10 mg/kg

412 N.M.W.J. de Bruin et al. / Behavioural Bra

Fig. 5. Effect of SLV330 on nicotine self-administration (means ± SEM): number ofactive and inactive nose pokes and rewards. The difference from the vehicle condi-tion is depicted with ***p < 0.001, n = 9 per group.

Fig. 6. Acquisition and extinction curve of tested animals (means ± SEM) in the nico-tine experiment. Rresponding for nicotine was extinguished. In the extinction phase(ta

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13 daily sessions of 60 min), the animals were placed in the operant chambers, buthe nicotine and nicotine related stimuli (discrete and discriminative cues) were notvailable.

LV330, was significantly lower when compared with animals treated with vehiclesee Fig. 7, Kruskal–Wallis: �2 = 19.98; p < 0.001, df = 3). A one-way ANOVA revealedhat SLV330 had no effect on the number of inactive nose pokes, made duringue-induced reinstatement (see Fig. 7, F[3,47] = 0.724; p = 0.543(ns)).

ig. 7. Effect of SLV330 on cue induced reinstatement to nicotine seekingmeans ± SEM): number of active and inactive nose pokes. ***: significant differentrom vehicle, p < 0.001, n = 11–13 per group.

in Research 217 (2011) 408–415

3.3. Conclusion

SLV330 was able to suppress responding (nose poking) for nicotine in a dose-related manner with a LED of 10 mg/kg. In another experiment, the effect of SLV330was studied on cue-induced relapse of nicotine seeking. SLV330 significantly sup-pressed cue-induced reinstatement of nicotine seeking with a LED of 10 mg/kg.

4. Experimental 3: Effects of SLV330 on parameters in the 5-CSRTT

4.1. Method

4.1.1. SubjectsTwelve male Wistar rats were obtained from Harlan (Zeist, The Netherlands)

at approximately 10 weeks of age. They were housed in pairs in Macrolon cages(37 × 25 × 15 cm) under a reversed light–dark cycle (lights on 7.00 pm–7.00 am)with constant room temperature (20–22 ◦C) and humidity (60–70%). One weekafter arrival, the rats were food restricted and fed daily to maintain body weightsat approximately 80% of free-feeding body weights corrected for growth through-out the experiment. Water was always available in the home-cages. Environmentalenrichment was provided with EnviroDri® .

4.1.2. CompoundsSLV330 (0–1–3–10 mg/kg) was suspended in a crash resistant formulation and

injected orally (p.o.) in a volume of 2 ml/kg, 45 min before the session. Doses wereexpressed as free base.

4.1.3. Apparatus descriptionExperiments took place in 12 modular Med-associates rat operant conditioning

chambers (31 × 25 × 33 cm). Each chamber was equipped with a house light and acurved rear wall which contained five 2.5 cm square holes. At the entrance of eachhole a photocell monitored interruptions of a beam of infrared light and at the rearthere was a yellow light-emitting diode. A food dispenser was located in the oppositewall, equidistant from each aperture and delivered one 45 mg Noyes food pelletfor each correct response. The house light was mounted in the middle of this wall6 cm from the ceiling. Each chamber was enclosed in a sound-attenuated ventilatedcabinet. Experimental contingencies and data acquisition were programmed usingMED-PC.

4.1.4. ProceduresAll procedures described were approved by the local Animal Care and Use Com-

mittees of Solvay Pharmaceuticals* and were in accordance with the Principles ofLaboratory Animal Care (NIH publication no. 86-23, revised 1985). All efforts weremade to minimize distress and number of animals used.

4.1.4.1. Training and testing. The training procedure was adapted from Stolermanet al. [33] with a modification to the intertrial interval (ITI) of 1–3–5–7–9 s (variableinstead of stable), randomly presented. In order to earn a food-pellet, an animalhad to detect a short light flash (0.5 s) in one of the five apertures (randomly pre-sented, 20 presentations for each aperture) and respond with a nose poke in thataperture before a fixed time of 5 s had elapsed (correct responses TCOR). If an animalresponded in the wrong aperture (false responses TFT) or did not respond (omis-sion errors TOM), a time-out (TO) of 5 s (all lights turned off) was initiated withoutany reinforcement. Trials were separated by a variable ITI (100 trials per session).Responding during the ITI terminated the trial and was followed by a TO (antic-ipatory responding TANT). The ITI was reset and this cycle was repeated until aresponse after presentation of the stimulus was made. Every response in an aper-ture was recorded, including extra responses after a correct response (perseverativeresponding TPERF) and omission errors. When animals met the criterion (of 70%correct responding over 50% of the trials) and showed stable baseline behavior (nosignificant difference between two blocks of 5 days in mean % correct responding),drug testing was started. Tests were performed during the dark phase of the light-dark cycle. On Mondays and Thursdays, the animals were trained. On Tuesdays andFridays, a dose of SLV330 was administered 45 min prior to the session. On Wednes-days, the animals were not tested or trained. Doses were administered to all subjectsaccording to a semi-random schedule, in such a way that each rat received each doseonce.

Task performance was reflected in the following behavioral parameters thatwere recorded within each session: TANT, TPERF, total responses (TRESP), TCOR,TFT, TOM and percentage of correct responses PERCC = 100 × (TCOR/[TCOR + TFT]).For each test dose, a mean ± SEM for each response-variable was calculated. Theresponse-variables were analyzed by an ANOVA. When a significant compoundeffect was found (p < 0.05), the ANOVA was followed by a comparison procedureaccording to Dunnett. The effect of SLV330 was expressed as LED. Also, analysis wasperformed for each parameter and for each ITI duration.

4.2. Results

None of the rats was excluded from the experiment, since SLV330 had noinfluence on behavioral performance or general health conditions. It took 60 train-ing sessions before the animals met the criterion of 70% correct responses and

N.M.W.J. de Bruin et al. / Behavioural Brain Research 217 (2011) 408–415 413

Fig. 8. Effect of SLV330 on 5-CSRTT parameters (means ± SEM). *: significant differ-ent from vehicle, p < 0.05; ***: significant different from vehicle, p < 0.001. TANT totalnToo

wi

s

-

-

-

-

a

umber of anticipatory responses; TPERS total number of perseverative responses;RESP total number of responses (correct + false responses); TCOR total numberf correct responses; TFT total number of false responses; TOM total number ofmissions; PERCC percentage of correct responses (accuracy), n = 12 per group.

ere ready for testing. The effect of SLV330 on behavioral parameters is shownn Fig. 8A–C.

In this study (0–1–3–10 mg/kg), three of the response parameters showed aignificant difference compared to vehicle.

SLV330 significantly decreased the anticipatory responses TANT (F[3,41] = 7.42;p < 0.001): at a LED of 3 mg/kg (p < 0.01) and at 10 mg/kg (p < 0.001), see Fig. 8A.SLV330 also significantly decreased the total responses TRESP (F[3,41] = 3.87;p < 0.05): at a LED of 10 mg/kg (p < 0.05), see Fig. 8BIn contrast, SLV330 increased errors of omission TOM (F[3,41] = 3.87; p < 0.05): ata LED of 10 mg/kg (p < 0.05), see Fig. 8B.

No significant effects of SLV330 were found on the accuracy measure (PERCC). seeFig. 8C.

A significant effect of SLV330 was found on the anticipatory responses TANTt the ITI-durations of 7 s (F[3,41] = 3.14; p < 0.05) and 9 s (F[3,41] = 7.51; p < 0.001).

Fig. 9. Effect of SLV330 on anticipatory responses in the 5-CSRTT (means ± SEM).*: significant different from vehicle, p < 0.05; **: significant different from vehicle,p < 0.01; ***: significant different from vehicle, p < 0.001, n = 12 per group.

A comparison procedure (Dunnett) (Fig. 9) revealed that the significant effect ofSLV330 on TANT was due to a significant decrease at the 9 s ITI (LED of 3 mg/kg,p < 0.01 and 10 mg/kg, p < 0.001 vs. vehicle) and at the 7 s ITI (LED 10 mg/kg, p < 0.05vs. vehicle).

For errors of omission TOM, a significant effect of SLV330 was found only at theITI 5 s-duration (F[3,41] = 4.57; p < 0.01): TOM was significantly increased at the 5 sITI at a LED of 10 mg/kg (p < 0.05) compared with vehicle.

4.3. Conclusion

SLV330 (0–1–3 - 10 mg/kg, p.o.) dose-dependently decreased ITI-dependentanticipatory responding (TANT, LED of 3.0 mg/kg), indicating an increased inhibitorycontrol without affecting other behavioral parameters (TRESP, TOM, both affectedat a LED of 10 mg/kg).

5. General discussion

In the present study, the CB1 antagonist SLV330 was effective inreducing ethanol self-administration at a LED of 10 mg/kg (p.o.) andreinstatement of ethanol seeking at a LED of 3 mg/kg (p.o.) in Wistarrats. This coorborates findings with the CB1 antagonist rimonabant,shown to reduce ethanol intake [7] and cue-induced reinstatementto ethanol seeking behavior [34].

To date, only few human surveys testing rimonabant on ethanolabuse and dependence have been conducted (see review [22]). Onetrial was aimed at evaluating the efficacy of rimonabant in the pre-vention of ethanol relapse. A statistically non-significant tendencytoward a protective effect of rimonabant was observed. Anothersurvey was aimed at assessing the effect of rimonabant on ethanolconsumption in heavy ethanol drinkers, in which rimonabant failedto alter daily ethanol consumption or number of ethanol drinksconsumed after the priming ethanol drink.

The results in the present study suggest that SLV330 was effec-tive in reducing nicotine self-administration and reinstatement ofnicotine seeking in Wistar rats, at a LED of 10 mg/kg (p.o.). Thesefindings are in agreement with results reported with other CB1antagonists. Administration of rimonabant (0.3 and 1 mg/kg, i.p.)decreased nicotine self-administration and reinstatement to nico-tine seeking behavior ([6,35,36]). Similarily, AM251, a selective

antagonist at CB1 receptors dose-dependently (1, 3 and 10 mg/kg,i.p.) suppressed intravenous nicotine self-administration and thereinstatement effects produced by nicotine and contingently pre-sented cues [37]. These preclinical findings support the use of CB1antagonists as a smoking cessation aid and highlight the CB1 recep-

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or as a viable target to control intake of nicotine and preventeinstatement of nicotine seeking.

The utility of rimonabant as an anti-smoking aid has been eval-ated in several clinical trials. As concluded by Le Foll et al. [38],it seems that rimonabant is an efficacious treatment for smok-ng cessation (STRATUS clinical trials), although its efficacy doesot exceed that of nicotine-replacement therapy and its use maye limited by emotional side effects (nausea, anxiety and depres-ion, mostly)”. Rimonabant also appears to decrease relapse ratesn smokers. These findings indicate significant, but limited, utilityf rimonabant for smoking cessation [38].

The mechanisms that underlie the modulatory role of cannabi-oids on drug seeking remain to be elucidated (Review [39]).oodman Goodman (2008 review) has proposed a possible mech-nism through which the endocannabinoids (eCBs) may influencehe reinforcing properties of drugs. The proposal is that CB1Rsocated on the presynaptic terminals of GABAergic neurons inhe ventral tegmental area (VTA) modulate the activity of VTAopaminergic neurons. The function of dopamine (DA) is the signal-

ng of the incentive salience of events (including rewarding stimuli),n driving motivated behavior, in predicting reward or non-reward.nder ordinary circumstances, eCBs activate the presynaptic CB1Rs

hat mediate the inhibition of GABA release, thereby decreasinghe GABA-mediated inhibition of DA release. Inactivation or elim-nation of the CB1 receptors, for example by CB1R antagonists,nterrupts this process and thus attenuates the DA responses toehaviors (e.g. drug and food intake) that are associated with DAelease in the nucleus accumbens. Also, activation of endocannabi-oid systems at postsynaptic sites from the DA synapse in thetriatum and prefrontal cortex might contribute to drug seekingreview [11]).

It can be argued whether the effects of SLV330 effects areeally specific for drug reinforcement and not due to a decrease ofesponding at high doses. We believe that the effects of SLV330 areue to effects on drug reinforcement, since SLV330 was also ableo significantly attenuate cue-induced reinstated cocaine seekingat the 3 and 10 mg/kg doses, data not shown). In this study, Wistarats were trained to self-administer cocaine (500 �g/kg/infusion)n an operant conditioning chamber.

As stated in the introduction, impulsivity is thought to play aole in drug addiction and disturbances in inhibition of behav-or resulting from prolonged drug intake have been proposedo maintain drug abuse [41,57]. In addition, elevated impulsivity

ight predispose individuals to initiate or maintain drug seek-ng and taking in both humans [27] and laboratory animals [28].herefore, compounds that increase impulse control might havedditional benefits in the treatment of drug addiction. So, inhe present study, the effects of SLV330 on 5-CSRTT parame-ers were tested in order to determine whether this compoundould also increase impulse control. SLV330 (p.o.) indeed dose-ependently decreased ITI-dependent anticipatory (premature)esponding (LED of 3.0 mg/kg), indicating an increased inhibitoryontrol without affecting other behavioral parameters at this effec-ive dose. As mentioned earlier, SLV330 was also effective ineducing reinstatement of ethanol seeking at a LED of 3 mg/kg (p.o.).his finding is particularly important, since this underlines the con-ept proposed by Jentsch [42] that impulse control may be a criticalarget for pharmacological intervention in the treatment of addic-ion and his suggestion that further developments in animal modelsor impulsivity may be useful in expanding the understanding andreatment of drug abuse. He finally concludes that new pharma-

ological treatments selected according to their ability to enhancempulse control in animal models should be increasingly the focusf future drug treatment trials in humans. As shown here, SLV330eems to both reduce impulsivity and reinstatement of drug seek-ng in rodents.

in Research 217 (2011) 408–415

SLV330 at 10 mg/kg also increased omission responses, whichcould be secondary to a possible anorectic effect of SLV330. Indeed,this could be underlined by the finding [43] that SLV330 (two weektreatment, 10 mg/kg, i.p.) significantly reduced bodyweight andfood intake of male Wistar rats compared to vehicle-treated ani-mals. Hagn [43] concluded that SLV330 could have potential in thetreatment of obesity.

Although CB1 receptor antagonists may be effective in reduc-ing drug use and preventing relapse in drug abuse, it shouldbe noted that the risk of psychiatric adverse effects has termi-nated many development programs for CB1R antagonists [44].As a result, the future therapeutic perspectives of CB1R antag-onists are under debate [45,46]. Whether this risk also holdstrue for SLV330 is not known, since we have not tested thisaspect in human subjects. It has been suggested that the cannabi-noid system can be modulated differently, producing blockadeof reinstatement without anxiogenic/depressive side-effects. Forexample, URB597 is a relatively selective inhibitor of the enzymefatty acid amide hydrolase (FAAH). FAAH is the primary degrada-tory enzyme for the endocannabinoid anandamide and, as such,inhibition of FAAH leads to an accumulation of anandamide(Narachidonoyl-ethanolamide) in the CNS and periphery where itactivates cannabinoid receptors. Anandamide is an endocannabi-noid, as defined by its ability to bind to and activate cannabinoid CB1and CB2 receptors [47]. Scherma et al. [48] have shown that URB597,at a dose (0.3 mg/kg) that had no behavioral effects by itself,prevented development of nicotine-induced conditioned placepreference (CPP) and acquisition of nicotine self-administration.URB597 also reduced nicotine-induced reinstatement in both CPPand self-administration models of relapse. Furthermore, URB597increased the production of endocannabinoids resulting in anantidepressant and anxiolytic-like profile in rodents [12,49,50].Therefore, it has been suggested that such compounds may possi-bly be better tolerated in humans than rimonabant, which inducesadverse psychiatric side effects [12]. However, other effects ofanandamine besides activation of cannabinoid receptors should beconsidered here. For example, anandamide directly binds to Perox-isome Proliferator-Activated Receptor Gamma (PPAR�), activatesPPAR� transcriptional activity [51,52] and activates the transientreceptor potential V1 (TRPV1) receptor [53]. Thus, drugs actingon anandamide signaling may produce a variety of side effectsthat would limit their utility and these alternate pathways forthe actions of anandamide warrant further careful examination[54–56].

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