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Behavioural Brain Research 247 (2013) 125– 131

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho mepage: www.elsev ier .com/ locate /bbr

esearch report

ole of intra-accumbal cannabinoid CB1 receptors in the potentiation,cquisition and expression of morphine-induced conditionedlace preference

ara Karimia, Pegah Azizib, Ali Shamsizadeha, Abbas Haghparastb,∗

Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, IranNeuroscience Research Center, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, P.O. Box 19615-1178, Tehran, Iran

i g h l i g h t s

ntra-accumbal cannabinoid agonist could dose-dependently induce place preference.Intra-accumbal cannabinoid agonist potentiated morphine reward-ng effect in the rat.Microinjection of AM251 alone in the NAc could induce conditioning place aversion.Blockade of CB1 receptors in the NAc reduced

orphine conditioned place preference.

r t i c l e i n f o

rticle history:eceived 26 January 2013eceived in revised form 10 March 2013ccepted 14 March 2013vailable online xxx

eywords:ewardannabinoid CB1 receptorucleus accumbensorphine

onditioned place preference

a b s t r a c t

Recent studies demonstrate a functional interaction between opioid and endogenous cannabinoid sys-tem. These two systems possess similar pharmacological effects on drug addiction and reward. Thepresent study was designed to investigate the role of intra-accumbal cannabinoid CB1 receptors in theacquisition and expression of morphine-induced conditioned place preference (CPP). Two-hundred fortyeight adult male albino Wistar rats were used in these experiments. Using a 3-day schedule of condition-ing, it was found that subcutaneous administration of morphine (0.2–10 mg/kg) induced CPP at the dosesof 5 and 10 mg/kg. Solely intra-accumbal administration of WIN55,212-2 (1, 2 and 4 mmol/0.5 �l DMSO)as CB1 receptor agonist could induce CPP. Also, our results showed that ineffective dose of WIN55,212-2(1 mmol) when administered before the ineffective dose of morphine (2 mg/kg) could induce the CPPand potentiate the rewarding effect of morphine. On the other hand, intra-accumbal injection of thecannabinoid CB1 receptor antagonist AM251 (90 �mol/0.5 �l DMSO) alone induced a significant condi-

at tioned place aversion. Moreover, intra-NAc injection of AM251 (45 and 90 �mol/0.5 �l DMSO) inhibitedmorphine-induced CPP. Interestingly, injection of WIN55,212-2 (1, 2 and 4 mmol) or AM251 (15, 45 and90 �mol) into the NAc had no effect on the expression of morphine (5 mg/kg)-induced CPP. These obser-vations provide evidence that cannabinoid CB1 receptors in the NAc are involved in development ofreward-related behaviors and they can potentiate the rewarding effects of morphine. It seems that these

eward

receptors can affect the r

. Introduction

The nucleus accumbens (NAc) is a complex forebrain structure1] which plays a key role in addiction to cocaine and morphine [2].t receives massive dopaminergic input from the ventral tegmen-al area (VTA) and glutamatergic input from structures such as theippocampus, prefrontal cortex, and amygdala [3]. Leshner (1997)

otes that “Although each drug that has been studied has some

diosyncratic mechanism of action, virtually all drugs of abuse haveommon effects, either directly or indirectly, on a single pathway in

∗ Corresponding author. Tel.: +98 21 2243 1624; fax: +98 21 2243 1624.E-mail address: Haghparast@yahoo.com (A. Haghparast).

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.03.022

modulatory system at the level of nucleus accumbens in rats.© 2013 Elsevier B.V. All rights reserved.

the brain” [4]. This pathway is the mesolimbic reward system thatgenerates signals from a brain region called the VTA that resultsin the release of chemical dopamine (DA) in the NAc. DA forms acritical link for all reward, including opiates and sedative drugs andit is well documented that release of DA in the NAc and its circuitryis the critical substrate for drug reward [5]. The conditioned placepreference (CPP) is one of the most widespread experimental proto-cols which used for measuring drug reward in laboratory [6]. Basedon Pavlovian conditioning principles, CPP reflects a preference for acontext due to the contiguous association between the context and

a drug-associated stimulus [7]. Although all drug of abuse couldincrease the conditioning time in this protocol but cannabinoidshows the variant effect on conditioning time [7]. Furthermore, it isremarkable that place conditioning is related to the enhancement of

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A release in the NAc by doubling the firing rate of VTA dopaminer-ic neurons [8]. So this is a simple and effective method to assess theewarding properties of drugs [6,9]. Endocannabinoids and theireceptors, mainly the CB1 receptor type, play important roles in dif-erent physiological functions. One function that could be affectedy endocannabinoids is reward. This function is mediated by thepecific brain circuits, which are known to be modulated by thendocannabinoid system [10,11]. They also play a modulatory func-ion on dopamine (DA) transmission, although CB1 receptors do notppear to be located in dopaminergic terminals, at least in the majorrain regions receiving dopaminergic innervation, e.g., the caudate-utamen and the nucleus accumbens/prefrontal cortex [12].

Consequently previous studies showed that the administra-ion of delta-9-tetrahydrocannabinol (�9-THC) as a endogenousannabinoid, and other cannabinoid ligands with agonist actionsCP55940, HU210, and WIN55,212-2) dose-dependently enhancehe firing of dopaminergic neurons in the VTA [13,14]. In addi-ion an increase in DA concentration in the NAc shell [15] in theat also occurs. Previous studies showed that the existence ofoderate density of CB1 receptors in the NAc [16], and Hagh-

arast et al. (2009) also confirmed that CB1 receptors within theAc are involved in the sensitization to morphine in rats [17],nd the attenuation of acquisition and expression in morphine-nduced conditioned place preference (CPP). This is caused bylocking the intra-accumbal CB1 receptors in morphine sensi-ized rats [18]. Furthermore, Braida et al. (2001) showed thatB1 receptors agonist could induced conditioning place prefer-nce like heroin [19]. The existence of parallel neuroadaptionsn both the endogenous opioid and the endogenous cannabi-oid systems that occur during both chronic opiate and chronicannabinoid exposure was confirmed [20]. Recent studies demon-trate a functional interaction between opioids and the endogenousannabinoid system [21,22], they also possess similar pharmaco-ogical effects on drug addiction and reward [23,24]. Endogenouspioids have a crucial role in modulating the addictive proper-ies of cannabinoids [25] and vice versa [26]. The co-localizationf cannabinoid receptors like the CB1 receptor which is involved inhe rewarding effects of cannabinoids [27,28] and �-opioid recep-ors in the presynaptic nerve terminals with the same signalingathway (G-proteins) are important for implying their interac-ions [29]. Behavioral data indicate functional links between CB1annabinoid receptors and the opiate-mediated reward circuitry.revious studies have shown that morphine-induced place con-itioning is attenuated in CB1 knockout mice [3]. Furthermore, itas been elucidated that there is the cross-sensitization [30,31]nd cross tolerance [32] between opioids and cannabinoids. Theresent study was designed to test this hypothesis in animal mod-ls (a) the role of intra-accumbal administration of an agonistnd antagonist of CB1 receptors in the potentiation of morphineewarding properties either in acquisition or expression of con-itioned place preference, and (b) the motivational effects of angonist and antagonist of CB1 receptors by themselves in theats.

. Materials and methods

.1. Animal

Adult male albino Wistar rats weighing 200–250 g were housed in standardlastic cages in groups of three in a controlled colony room (temperature1 ± 3 ◦C). They were maintained on a 12 h light/dark cycle (lights on at 07:00.m.) with food and water ad libitum. The experiments were carried out duringhe light phase of the cycle. Each animal was tested once. Five to eight ani-

als were used per group. All experiments were performed in accordance withhe Guide for the Care and Use of Laboratory Animals (National Institutes ofealth Publication No. 80-23, revised 1996) and were approved by the Researchnd Ethics Committee of Shahid Beheshti and Rafsanjan Universities of Medicalciences.

esearch 247 (2013) 125– 131

2.2. Drugs

In the present study, the following drugs were used: morphine sulfate (Temad,Iran) that was dissolved in sterile saline (0.9%), WIN55,212-2, cannabinoid agonistand AM251 as a CB1 receptor antagonist (Sigma–Aldrich, Germany) which were dis-solved in dimethylsulfoxide, DMSO (Sigma–Aldrich, Germany) as a vehicle. Controlanimals received either saline or DMSO.

2.3. Stereotaxic surgery

The animals were anesthetized by Xylazine (10 mg/kg) and Ketamine(100 mg/kg) and placed in the stereotaxic apparatus (Stoelting, USA), with theincisor bar set at approximately 3.3 mm below horizontal zero to achieve a flatskull position. After, an incision was made to expose the rat’s skull. Two pointswere determined and drilled into the skull at the following stereotaxic coordinates:1.4 ± 0.4 mm anterior to bregma, ±1.2 mm lateral to the sagittal suture and 7.8 mmdown from top the skull according to the atlas of rat brain [33]. Two guide cannulae(23-Gauge) with 10 mm length were inserted into the holes aiming at the NAc. Theguide cannulae were anchored with a jeweler’s screw, and the incision was closedwith dental cement. After surgery, dummy inner cannulae that extended 0.5 mmbeyond the guide cannulae were inserted into the guide cannulae and left in theirplace until injections were made. All animals were allowed to recover for one weekbefore behavioral testing began.

2.4. Intra-accumbal injection

The animals were gently restrained by hand and the dummy cannulae wereremoved from the guide cannulae. Drugs were directly injected into the NAc throughthe guide cannulae by using injector cannulae (30-Gauge, 1 mm below the tip of theguide cannulae). Polyethylene tubing (PE-20) was used to attach injector cannulato the 1 �l Hamilton syringe. The injection volume was 0.5 �l/side for all groups.Injections were made bilaterally over a 60 s period, and the injection cannulae wereleft in the guide cannulae for an additional 60 s to facilitate diffusion of the drugs.

2.5. Place conditioning apparatus

A three-compartment conditioned place preference apparatus(30 cm × 30 cm × 40 cm) was used in these experiments. Place conditioningwas conducted using an unbiased protocol, modified slightly from a designpreviously described [34,35]. The apparatus was divided into two equal-sizedcompartments with the third section being the null section which connected thetwo equal-sized sections. Both compartments had white backgrounds with blackstripes in different orientations (vertical vs. horizontal). To provide the tactiledifference between the compartments, one of the compartments had a smoothfloor, while the other compartment had a net-like floor. In this apparatus, ratsshowed no consistent preference for either compartment. In the CPP paradigm,the conditioning score and distance traveled were recorded by a 3CCD camera(Panasonic Inc., Japan) for detecting animal displacement. This camera was placed2 m above the CPP boxes and locomotion tracking was measured by Ethovisionsoftware (version 3.1), a video tracking system for automation of behavioral exper-iments (Noldus Information Technology, the Netherlands). In these experiments,on the pre- and post-conditioning phases, each animal was freely accessed totwo compartments for 10 min. The time spent in each compartment of the CPPapparatus and the total distances traveled were measured during this time. CPPparadigm, took place in 5 continuous days, which consisted of three distinct phases:pre-conditioning, conditioning and post-conditioning (Electronic supplementaryFig. 1). For all phases animals were tested during the same time period each day.

2.5.1. Pre-conditioning phaseOn day 1 (pre-exposure), each rat was separately placed into the apparatus for

10 min, with free access to all compartments. Animal displacement was recordedand analyzed on this day (pre-test day). In the experimental setup used in this study,the animals did not show a conditioned preference for either of the compartments.Animals were then randomly assigned to one of two groups for place conditioningand 5–8 animals were used for each subsequent experiment.

2.5.2. Conditioning phaseThis phase consisted of a 3-day schedule of conditioning sessions. The condition-

ing training was carried out twice a day, on each of the days 2–4, with six 30 minsessions of saline- and morphine-pairing in an alternate morning-afternoon designwith an interval of 6 h. In this phase, animals received three trials in which theyexperienced the effects of the drugs while confined to one compartment for 30 min,and three trials in which they experienced the effects of saline while confined tothe other compartment by closing the removable gate. Access to the compartmentswas blocked on these days.

2.5.3. Post-conditioning phaseOn the 5th day (test day), the partition was removed and the rats could access

the entire apparatus. The mean time spent for each rat in both compartments dur-ing a 10 min period was recorded. In order to calculate the conditioning score, the

rain Research 247 (2013) 125– 131 127

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S. Karimi et al. / Behavioural B

ifference in time spent for the drug-paired place and saline-paired place was con-idered as the preference criteria. In the acquisition tests, no injection was given onhe post-conditioning day (test day). Total distances traveled for each animal waslso measured on pre and post days for a 10 min period as the locomotor activity inontrol and experimental groups.

.6. Experimental design

The effects of subcutaneous (sc) administrations of morphine on induction oflace conditioning in animals were determined in pervious works [36]. Briefly, in thisxperiment, a dose-response relationship for morphine on conditioned place pref-rence paradigm was established. Each morphine dose (0.2, 0.5, 1, 2, 5 and 10 mg/kg;c) was injected on a 3-day schedule of conditioning as described in the above sec-ion. Then, animals were tested in a morphine-free state to eliminate the influencef morphine induced motor effects on responses [37]. Conditioning score and dis-ance traveled are calculated for each rat [17,38]. Control animals received saline1 ml/kg; sc). After determining the morphine dose, the control and experimentalroups were designed according to Table 1.

.6.1. Effects of intra-accumbal injections of CB1 receptor agonist on thecquisition of morphine-induced conditioned place preference in rats

To test the potentiating effects of CB1 receptor agonist injected into the NAcn morphine rewarding properties, experiments were performed on saline andorphine-treated groups which received the ineffective dose (2 mg/kg; sc) or effec-

ive dose (5 mg/kg; sc) of morphine during the acquisition period. WIN55,212-2 (1, 2nd 4 mmol/0.5 �l DMSO) was bilaterally injected into the NAc, 5 min prior to eitherhe effective or ineffective dose of morphine in the 3-day conditioning phase, theontrol group received vehicle (DMSO). Conditioning score and distance traveledre measured during a 10 min period on the test day.

.6.2. Effects of intra-accumbal injections of CB1 receptor agonist on thexpression of morphine-induced conditioned place preference in rats

In this set of experiments, to evaluate the potentiating effects of single injectionf CB1 receptor agonist on morphine rewarding properties in expression of con-itioned place preference, different doses of WIN55,212-2 (1, 2 and 4 mmol/0.5 �lMSO) were bilaterally injected into the NAc, just 5 min prior to CPP test in saline-nd morphine-treated groups which received ineffective dose (2 mg/kg; sc) or effec-ive dose (5 mg/kg; sc) of morphine during the acquisition period. Conditioning scorend distance traveled are measured during 10 min on the test day.

.6.3. Effects of intra-accumbal injections of CB1 receptor antagonist on thecquisition of morphine-induced conditioned place preference in rats

AM251 (15, 45 and 90 �mol/0.5 �l DMSO) as a CB1 receptor antagonist wasilaterally injected into the NAc, 5 min prior to each morphine (5 mg/kg; sc) injectionuring the acquisition period to investigate its effects on the acquisition of morphine

nduced CPP. In the control group, DMSO (0.5 �l/side), as a vehicle was bilaterallynjected into the NAc instead of AM251 during the acquisition period. Conditioningcore and distance traveled are measured during a 10 min period on the test day.

.6.4. Effects of intra-accumbal injections of CB1 receptor antagonist on thexpression of morphine-induced conditioned place preference in rats

In this set of experiments, morphine (5 mg/kg; sc) was injected during condi-ioning phase while AM251 (15, 45 and 90 �mol/0.5 �l DMSO) as a CB1 receptorntagonist was bilaterally injected into the NAc just before the CPP test on theost-conditioning phase. In the control group, DMSO (0.5 �l/side), as a vehicle wasilaterally injected into the NAc instead of AM251 on the test day. Conditioningcore and distance traveled are measured during a 10 min period on the test day.

.7. Conditioning score measurement

Conditioning scores (CPP score) during 10 min (600 s) were calculated on pre-nd post-conditioning phases. Conditioning scores represents the time spent inrug-paired compartment minus the time spent in the saline-paired compartments39]. This is used for evaluating the effect of drugs on morphine-induced conditionedlace preference in rats.

.8. Locomotor activity measurement

To evaluate the effect of drugs on locomotor activity in animals, total distanceraveled (cm) during the 10 min test period, on the pre- and post-conditioninghases was measured by Ethovision software.

.9. Histology

After completion of behavioral testing, the animals were deeply anesthetized

ith ketamine and xylazine. Then, they were transcardially perfused with 0.9%

aline and 10% formalin solution. The brains were removed, blocked and cut coro-ally in 50 �m sections through the cannulae placements. The neuroanatomical

ocation of cannulae tips placements, were confirmed using rat brain atlas. Onlyhe animals with correct cannulae placements were included in the data analysis.

approximate location of the NAc injection sites in the experiments. The numbersindicate anterioposterior coordinates relative to bregma. Atlas plates are adaptedfrom Paxinos and Watson [33].

Fig. 1 shows the approximate point of the drug injections in the NAc. The histologicalresults were plotted on the representative section taken from the rat brain atlas ofPaxinos and Watson [33].

2.10. Statistics

Conditioning score and distance traveled are expressed as mean ± SEM(standard error of mean). Data were processed by commercially available softwareGraphPad Prism® 5.0. In order to compare the conditioning scores and distance trav-eled obtained in all groups (vehicle and experimental groups), one- and two-wayanalysis of variance (ANOVA) and randomized blocks design model followed by posthoc analysis (Newman–Keuls multiple comparison test and Bonferroni test, respec-tively) were used, as appropriate. In this study, CPP scores and motor activity canbe evaluated with a two-way ANOVA with two between factors: Cannabinoid drugsand Morphine treatments. P-values less than 0.05 (P < 0.05) were considered to bestatistically significant.

3. Results

Dose-response effects of morphine in the CPP paradigm hasbeen determined in all previous studies; however in this study, sixgroups of animals also received different doses of morphine sulfate(0.2, 0.5, 1, 2, 5, and 10 mg/kg; sc). One-way ANOVA followed byNewman–Keuls test [F(6.41) = 7.785, P < 0.0001] showed that thedose of 5 mg/kg is caused a significant increase in time spent in thedrug-paired compartment compared to the saline-paired compart-ment (Electronic supplementary Fig. 2). Subcutaneous injection ofsaline instead of morphine to the animals (saline control group) inthe conditioning compartments did not produce any preference oraversion for either place. Based on these data, we selected the dosesof 2 and 5 mg/kg as the ineffective and effective doses of morphinefor the next experiments, respectively.

3.1. Effect of intra-accumbal cannabinoid agonist on theacquisition of conditioned place preference in the absence or

presence of morphine

Two-way ANOVA followed by Bonferroni’s test indicates sig-nificant differences between response to different doses of

128 S. Karimi et al. / Behavioural Brain Research 247 (2013) 125– 131

Table 1The control and experimental groups used in the present study and the treatment received during the acquisition or expression of conditioned place preference (CPP)paradigm.

Acquisition expe rimen ts Expre ssion expe rimen ts

(Intra -ac cumbal a dministrat ion * duri ng th e 3 -day condit ioning phase ) (Intra -ac cumbal a dminis trat ion on the tes t day)

DMSO WIN55,212 -2 AM251 DMSO WIN55,212-2 AM251

1 mmol (n= 8) 15 mmol (n= 8) 1 mmol (n=7 ) 15 mmol (n= 7)

Saline (1 ml/kg) 0.5 μl (n= 8) 2 mmol (n= 6) 45 mmol (n=8) 0.5 μl (n=6) 2 mmol (n=6 ) 45 mmol (n= 6)

4 mmol (n=7 ) 90 mmol (n=8) 4 mmol (n=7 ) 90 mmol (n= 7)

1 mmol (n= 6) 1 mmol (n=6 )

Morphine (2 mg/kg) 0.5 μl (n= 7) 2 mmol (n=8 ) 0.5 μl (n=5) 2 mmol (n =7)

4 mmol (n= 6) 4 mmol (n=6 )

1 mmol (n= 6) 15 mmol (n= 8) 1 mmol (n=6 ) 15 mmol (n= 7)

Morphine (5 mg/kg) 0.5 μl (n= 8) 2 mmol (n= 7) 45 mmol (n= 8) 0.5 μl (n=8) 2 mmol (n= 7) 45 mmol (n= 7)

4 mmol (n= 6) 90 mmol (n= 8) 4 mmol (n= 6) 90 mmol (n= 6)Su

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IN 55,212-2 as a cannabinoid receptor agonist and morphinereatments compared to those of saline-treated group [factorannabinoid drugs: F(3.67) = 7.263, P = 0.0003; factor morphinereatments: F(2.67) = 16.03, P < 0.0001; interaction: F(6.67) = 1.201,

= 0.3165]. Data revealed that intra NAc administration of WIN5,212-2 could induce CPP in saline treated animals. Two higheroses of WIN 55,212-2 (2 and 4 mmol/0.5 �l DMSO) alone induced

significant place preference (Fig. 2A; left panel). Furthermore,oncurrent administration of intra-NAc injection of different dosesf WIN 55,212-2 and the ineffective dose of morphine, duringhe 3-day conditioning phase, could induce CPP (Fig. 2A; middleanel). Additionally, statistical analysis showed that there were noignificant differences in conditioning scores among all doses of

IN55,212-2 compared to the respective vehicle (DMSO) group innimals that received the effective dose of morphine (Fig. 2A; rightanel). However, one-way ANOVA followed by Newman–Keulsest [F(7.53) = 6.885, P < 0.0001] showed that ineffective dose of

IN55,212-2 (1 mmol) when administered before the ineffectiveose of morphine (2 mg/kg) can induce the CPP and potentiate theewarding effects of this dose of morphine in the rats (see Fig. 2A;eft and middle panels). On the other hand, Fig. 2B indicated that allifferent doses of drugs in saline and morphine-treated groups didot change the locomotor activity during the 10 min test periodpost-conditioning phase; day 5) in comparison with that of thealine control group.

.2. Effects of intra-accumbal administration of WIN55,212-2 onhe expression of conditioned place preference in the absence or

resence of morphine

Data was analyzed with the two-way ANOVA followed byonferroni’s test [factor cannabinoid drugs: F(3.64) = 0.3985,

trol and experimental groups.

P = 0.7545; factor morphine treatments: F(2.64) = 25.33, P < 0.0001;interaction: F(6.64) = 0.2301, P = 0.9654]. As shown in Fig. 3A, ourdata did not show any significant difference in conditioning scoresin any drug injected groups in saline (right panel) and morphine(ineffective dose; 2 mg/kg) treated animals (middle panel). Nev-ertheless, there were significant increases in conditioning scoresin all morphine (effective dose; 5 mg/kg) treated animals com-paring to the respective control group in saline-treated animals[F(7.51) = 6.472, P < 0.0001] but no changes were observed in theconditioning scores in comparison with the respective vehiclegroup (Fig. 3A; right panel). In this set of experiments, data alsoindicated that all different doses of drugs in saline- and morphine-treated groups did not change the locomotor activity during the10 min test period (Electronic supplementary Fig. 3).

3.3. Effects of CB1 receptor antagonist, AM251, on the acquisitionof conditioned place preference in the absence or presence ofmorphine

Fig. 4A indicates the effects of bilateral intra-NAc adminis-tration of AM251 with or without morphine (5 mg/kg; sc) onthe acquisition of CPP. Two-way ANOVA followed by Bonfer-roni’s test indicates significant differences between response todifferent doses of AM251 as a cannabinoid receptor antagonistand morphine treatments compared to those of saline-treatedgroup [factor cannabinoid drugs: F(3.56) = 9.64, P < 0.0001; factormorphine treatments: F(1.56) = 33.57, P < 0.0001; interaction:F(3.56) = 0.7295, P = 0.5387]. Data indicated significant interaction

between AM251 and morphine on the acquisition of CPP. Itrevealed that solely administrating AM251 into the NAc could sig-nificantly induce place aversion in the highest dose (Fig. 4A; rightpanel), and intra-NAc injection of AM251 also reduces the effects of

S. Karimi et al. / Behavioural Brain Research 247 (2013) 125– 131 129

Fig. 2. The effects of bilateral intra-accumbal injection of WIN 55,212-2 as a cannabi-noid receptor agonist on the acquisition of conditioned place preference in theabsence or presence of morphine. Animals received intra-accumbal injection ofeither vehicle (0.5 �l DMSO) or three doses of WIN 55,212-2 (1, 2 and 4 mmol/0.5 �lDMSO), just prior to systemic saline (1 ml/kg; sc) or the ineffective (2 mg/kg;sc)/effective (5 mg/kg; sc) dose of morphine in a 3-day schedule of conditioningin saline- or morphine-treated animals, respectively. Values are the mean ± SEM of6–8 rats per group. (A) Conditioning scores and (B) locomotor activity for all of theseg*†

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Fig. 3. Effects of intra-accumbal injections of WIN 55,212-2 as a cannabinoid recep-tor agonist on the expression of conditioned place preference in the absence orpresence of morphine in rats. All animals received systemic saline (1 ml/kg; sc) or theineffective (2 mg/kg; sc)/effective (5 mg/kg; sc) dose of morphine in a 3-day scheduleof conditioning in saline- or morphine-treated animals, respectively. On the test day(post-conditioning phase), vehicle (0.5 �l DMSO) or different doses of WIN 55,212-2(1, 2 and 4 mmol/0.5 �l DMSO) were bilaterally injected into the nucleus accumbens.

roups were tested 24 h after the last conditioning session.P < 0.05, **P < 0.01, ***P < 0.001 different from the vehicle respective group.

P < 0.05, †† P < 0.01 different from the respective group in saline-treated animals.

orphine-induced place preference (Fig. 4A; left panel). Further-ore, as shown in the left panel, a comparison of conditioning

core between saline- and morphine-treated animals revealedhat a increase in the dose of AM251 could significantly reducehe rewarding effects of morphine. On the other hand, all differentoses of AM251 in saline- and morphine-treated groups didot change the locomotor activity during the 10 min test periodFig. 4B).

.4. Effects of CB1 receptor antagonist on the expression oforphine-induced place preference

Fig. 5 shows the effects of bilateral intra-NAc injection of AM251n the expression of morphine-induced CPP. Two-way ANOVA fol-owed by Bonferroni’s test [factor cannabinoid drugs: F(3.46) = 0.31,

= 0.818; factor morphine treatments: F(1.45) = 39.28, P < 0.0001;nteraction: F(3.46) = 0.3666, P = 0.7674] indicated that intra-ccumbal administration of different doses of AM251 had no effectsn morphine-induced place preference (right panel). Additionally,

Values are the mean ± SEM of 5–8 rats per group.*P < 0.05, **P < 0.01, ***P < 0.001 different from the respective group in saline-treatedanimals.

data showed that administration of AM251 into the NAc alone hadno effects on conditioned place preference (Fig. 5; left panel). Asshown in electronic supplementary Fig. 4 in this set of experi-ments, data analysis indicated that all different doses of AM251 insaline- and morphine-treated groups did not change the locomotoractivity.

4. Discussion

The present study investigated the role of cannabinoid CB1receptors within the NAc in development of morphine-induced CPPin rats. In addition, the functional interaction between opioid andintra-accumbal cannabinoid receptors in the morphine-inducedCPP was considered. CPP is a sensitive behavioral morphine showa reduction in some, but not all, of the model to evaluate therewarding (or aversive) effects of drugs. In the case of morphinea large number of studies have indicated that a CPP can be readilyproduced to this drug [6]. In this present study, major findingswere: (I) Intra-accumbal administration of WIN 55,212-2 as acannabinoid agonist alone, during the acquisition period, couldinduce place preference in rats. (II) CB1 receptors in the NAc couldpotentiate the effects of morphine on reward-related behaviors.(III) Solely administrating AM251 as a CB1 receptor antagonistinto NAc, during the acquisition period, induces place aversion.(IV) Blockade of intra-accumbal CB1 receptor could reduce theacquisition but not expression of morphine-induced CPP in rats. Onthe other hand, none of the drugs microinjected into the NAc hadsignificant effects on the locomotor activity. It is noticeable that,previous studies had indicated that subcutaneous administrationof morphine could produce a significant CPP in a dose-dependentmanner. Furthermore, the critical role of the NAc in producingmorphine reward and causing an interaction between endo-cannabinoid and opioid systems in the brain has been confirmedby previous studies. These investigations have revealed thatcannabinoid administration modulates both dopaminergic and

glutamatergic neurotransmission involved in the reward process[40]. The release of dopamine into the NAc from VTA is the causefor reward. Cannabinoid agonist increases the firing of dopamineneurons and increases dopamine in brain regions associated

130 S. Karimi et al. / Behavioural Brain Research 247 (2013) 125– 131

Fig. 4. The effects of bilateral intra-accumbal injection of AM251 as a CB1 receptorantagonist on the acquisition of morphine-induced conditioned place preference.Animals received intra-accumbal injection of either vehicle (0.5 �l DMSO) or threedoses of AM251 (15, 45 and 90 mmol/0.5 �l DMSO), just prior to systemic saline(1 ml/kg; sc) or the effective dose of morphine (5 mg/kg; sc) in a 3-day scheduleof the conditioning phase. Values are the mean ± SEM of eight rats per group. (A)Conditioning scores and (B) locomotor activity for all of these groups were tested24 h after the last conditioning session.*†

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Fig. 5. Effects of bilateral intra-accumbal injections of AM251 as a CB1 receptorantagonist on the expression of morphine-induced conditioned place preferencein rats. All animals received systemic saline (1 ml/kg; sc) or the effective dose ofmorphine (5 mg/kg; sc) in a 3-day schedule of the conditioning phase. On the testday (post-conditioning phase), vehicle (0.5 �l DMSO) or different doses of AM251(15, 45 and 90 mmol/0.5 �l DMSO) were injected into the nucleus accumbens. Values

Acknowledgements

P < 0.05, **P < 0.01 different from the vehicle respective group. P < 0.05, †† P < 0.01 different from the respective group in saline-treated animals.

ith reward and drug addiction [16]. This elevation in DA levelsincreased transmission of dopamine in midbrain via cannabinoid)s a consequence of the reduction of the GABA-mediated inhibitoryost-synaptic current on VTA projections to the NAc in rats [41].

It is remarkable that the rewarding properties of cannabinoidsnd opioids might be functionally linked. Particular previoustudies confirmed that the endogenous cannabinoid system hasn important role in the modulation of opioid reward and itsddictive effects; also we know that CB1 as a cannabinoid receptors essential for modulation of morphine’s rewarding effects [42].t is of interest to note that CB1 and mu receptors are not onlyo-localized in many brain areas but also they may act on theame neurons in the NAc [43,44], in fact, this is a key site for

he mediation of the rewarding actions of substance abuse [45].onsidering that cannabinoids and opioids share an enhancement

n mesolimbic dopamine activity, one possible hypothesize can

are the mean ± SEM of 6–8 rats per group.*P < 0.05, **P < 0.01 different from the respective group in saline-treated animals.

be that the potentiation of morphine-induced place preference byintra-accumbal microinjection of WIN55,212-2 [22,46] might berelated to the increase of dopamine release through the VTA andinto the NAc via the disinhibition of GABA transmission in the VTA.On the other hand, the glutamatergic system which has an impor-tant role in mediating morphine reward may also be involved inthe functional interaction between the opioidergic system and thecannabinoid CB1 receptors in the nucleus accumbens.

Consequently in order to show the involvement of cannabinoidCB1 receptors within the NAc in the development and expressionof morphine-induced place preference, different doses of AM251an antagonist of CB1 receptor, alone or in combination with theeffective dose of morphine have been injected into the NAc duringthe conditioning or post-conditioning phase. The results showeda significant place aversion for treatment with the high dose ofAM251 by itself. Furthermore, the concurrent administration ofthis antagonist with effective dose of morphine could decreasemorphine-induced place preference. In agreement with this result,our previous study showed that injection of AM251 into thenucleus accumbens decreased morphine-induced place preferencein morphine sensitized rats [18]. In addition, microdialysis studiesrevealed that the systemic administration of morphine could notincrease dopamine in the NAc in cannabinoid CB1 receptor knock-out mice, in comparison with wild-type mice [47]. Gathering allthis together it sounds like our results could confirm previous data.It seems that cannabinoidergic and opioidergic systems are collab-orating on GABAergic system and they have interaction on this site.Hence, it is possible that presynaptic CB1 receptors in the NAc affectthe rewarding effects of drugs of abuse like morphine via alterationin the releasing of GABA in this area. However, more investigationswith cellular and molecular approaches at the level of the nucleusaccumbens by micodialysis, immunohistochemistry and moleculartechniques are required.

This study was supported by a grant from the Rafsanjan Univer-sity of Medical Sciences, Rafsanjan, Iran.

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S. Karimi et al. / Behavioural B

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.bbr.2013.03.022.

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