Progress in Neuro-Psychopharmacology & Biological Psychiatry 51 (2014) 65–71

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Basolateral amygdala CB1 cannabinoid receptors mediate nicotine-induced place preference Shiva Hashemizadeh a, Maryam Sardari a, Ameneh Rezayof a,b,⁎ a b

Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran School of Cognitive Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran

a r t i c l e

i n f o

Article history: Received 27 October 2013 Received in revised form 10 January 2014 Accepted 16 January 2014 Available online 24 January 2014 Keywords: Basolateral amygdala CB1 cannabinoid receptors Nicotine Rat(s) Reward

a b s t r a c t In the present study, the effects of bilateral microinjections of cannabinoid CB1 receptor agonist and antagonist into the basolateral amygdala (intra-BLA) on nicotine-induced place preference were examined in rats. A conditioned place preference (CPP) apparatus was used for the assessment of rewarding effects of the drugs in adult male Wistar rats. Subcutaneous (s.c.) administration of nicotine (0.2 mg/kg) induced a significant CPP, without any effect on the locomotor activity during the testing phase. Intra-BLA microinjection of a non-selective cannabinoid CB1/CB2 receptor agonist, WIN 55,212-2 (0.1–0.5 μg/rat) with an ineffective dose of nicotine (0.1 mg/kg, s.c.) induced a significant place preference. On the other hand, intra-BLA administration of AM251 (20–60 ng/rat), a selective cannabinoid CB1 receptor antagonist inhibited the acquisition of nicotine-induced place preference. It should be considered that the microinjection of the same doses of WIN 55,212-2 or AM251 into the BLA, by itself had no effect on the CPP score. The administration of a higher dose of AM251 (60 ng/rat) during the acquisition decreased the locomotor activity of animals on the testing phase. Interestingly, the microinjection of AM251 (20 and 40 ng/rat), but not WIN55,212-2 (0.1–0.5 μg/rat), into the BLA inhibited the expression of nicotine-induced place preference without any effect on the locomotor activity. Taken together, these findings support the possible role of endogenous cannabinoid system of the BLA in the acquisition and the expression of nicotine-induced place preference. Furthermore, it seems that there is a functional interaction between the BLA cannabinoid receptors and nicotine in producing the rewarding effects. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Nicotine, the principal psychoactive component of tobacco, is responsible for addictive properties of cigarettes (Goniewicz and Delijewski, 2013). Several studies have demonstrated that mesolimbic dopaminergic pathways, which originate from the ventral tegmental area (VTA) and project to forebrain structures such as the nucleus accumbens (NAc) and the prefrontal cortex, play a critical role in reinforcing effects of drug abuse, including nicotine (for review see Mark et al., 2011). Neurotoxic lesion in mesolimbic dopamine (DA)-containing neurons or pretreatment by dopamine antagonist attenuates intravenous self-administration as well as reward preference of nicotine in animal models (Corrigall et al., 1992; Pak et al., 2006). Moreover, nicotine

Abbreviations: AM251, N-(piperidin-1-yl)-5-(4-isodophenyl)-1-(2,4-dichlorophenyl)4-methyl-1H-pyrazole-3-carboxamide; ANOVA, Analysis of variance; BLA, Basolateral amygdala; CB, Cannabinoid; CNS, Central nervous system; CPP, Conditioned place preference; DA, Dopamine; DMSO, Dimethyl sulphoxide; NAc, Nucleus accumbens; nAchRs, Nicotinic acetylcholine receptors; s.c, Subcutaneous; VTA, Ventral tegmental area; WIN 55,212-2, WIN55,212-2 mesylate. ⁎ Corresponding author at: Department of Animal Biology, School of Biology, College of Science, University of Tehran, P. O. Box 4155-6455, Tehran, Iran. Tel.: +98 21 61112483; fax: +98 21 66405141. E-mail address: [email protected] (A. Rezayof). 0278-5846/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2014.01.010

modulates reward pathways through nicotinic acetylcholine receptors (nAChRs) in the VTA (Mansvelder et al., 2002). Activation or desensitization/inactivation of these ionotropic pentameric receptors is important in nicotine addictive properties (for review see Changeux, 2010). For example, the blockade of nAChRs in the VTA decreased systemic nicotine-evoked DA release in the NAc (Gotti et al., 2010) and also intravenous self-administration of nicotine- induced rewarding effect in rats (Kenny and Markou, 2006). Recent evidence indicated that the motivation effect of nicotine in reward circuitry is modulated by the endocannabinoid system (Forget et al., 2005). It is well recognized that there are functional and structural interactions between nicotine and cannabinoid receptors (LópezMoreno et al., 2008). Available data suggest that co-abuse of nicotine and cannabinoid share pharmacological properties such as antinociception (Valjent et al., 2002), anxiety-like behavior (Biala et al., 2009), learning and memory (Alijanpour and Rezayof, 2013). Cannabinoid influences physiological functions in the central nervous system (CNS) via well characterized CB1 cannabinoid receptors (Litvin et al., 2013; McLaughlin et al., 2013). These receptors belong to the Gprotein coupled receptor family (Shim et al., 2013). Neuroanatomical studies have reported a high density of CB1 cannabinoid receptors in the neurons of brain regions such as the NAc, the VTA and the amygdala reflecting reward motivational behaviors (Mackie, 2005).

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The amygdala complex anatomically divides into more than a dozen nuclei and has a critical role in mood and emotional behavior (for review see Sah et al., 2003). The basolateral nucleus (BLA), one of the important nuclei of the amygdala, has connectivity with reward circuitry and is well known to be necessary in reward pathways that play a central role in reward seeking behavior, emotion and reward-associated memory (Heinrichs et al., 2013; Wassum et al., 2012). It seems that there is an overlapping distribution of CB1 cannabinoid receptors and nACh receptors in the BLA (McDonald and Mascagni, 2001; Zhu et al., 2005). Previous studies have suggested a possibility of functional interactions between these two systems that regulate emotional responses, cognition (Pessoa, 2010) and reward (Kodas et al., 2007) in this region of the brain. These findings suggested that the endocannabinoid system modulates cortico-limbic circuitry for motivational properties of nicotine. Previous reports indicated that drug abuse has a dual motivational effect that conditioned place preference (CPP) paradigm which is widely used to evaluate both reinforcing and aversive effects of drug abuse including nicotine and cannabis in laboratory animals (Brielmaier et al., 2008; Cheer et al., 2000). It has been shown that systemic administration of CB1 receptor antagonists such as rimonabant (Fang et al., 2011) and AM251 (Budzyńska et al., 2009) inhibited nicotine-induced place preference. Nicotine-induced CPP also inhibited by a selective CB2 receptor antagonist, SR144528 (Ignatowska-Jankowska et al., 2013), indicating that CB1/CB2 receptors play a critical role in nicotine reward and may be a target for relapsing nicotine addiction. Considering that previous studies suggested that the specific brain circuits may be involved in the functional interaction between nicotine and cannabis in rewarding processes (for review see Vlachou and Panagis, 2014) and also that the basolateral amygdala (BLA) is a key component of the reward circuit (Stuber et al., 2011; Wassum et al., 2012), the present study was designed to investigate the role of CB1 receptors of the BLA in mediating nicotine reward. Therefore, this study highlights whether the acquisition and expression of nicotine-induced CPP could be affected by intra-BLA microinjections of CB1-receptor agonist and/or antagonist.

guide cannulas in order to prevent clogging until each animal was given the BLA injections. All animals were allowed a seven day recovery period from surgery to clear the anesthetic effects. During the recovery period, rats were handled about 5 min each day prior to the behavioral testing. For drug injection, the stylets were gently removed from the guide cannulas and replaced by 27-gauge injection needles. Considering that the guide cannulas were implanted 1 mm above the BLA, the injection needles were 1 mm longer than those. Each injection unit was connected by polyethylene tubing to 2 μl Hamilton syringes. The left and right BLA were injected with a 0.3 μl solution on each side (0.6 μl/rat) over a 60 s period. The injection needles were left in place an additional 60 s to allow diffusion. The stylets were subsequently reinserted into the guide cannulas. 2.3. Drugs In the present study, the drugs were nicotine hydrogen tartrate (Sigma, Poole, Dorset, UK), WIN55,212-2 mesylate (Tocris Cookson, Bristol, UK) and AM251 (N-(piperidin-1-yl)-5-(4-isodophenyl)-1(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; Tocris, Bristol, UK). Nicotine was dissolved in sterile saline and then the pH of the solution was adjusted to 7.2 with NaOH (0.1 normal solution). Nicotine was injected subcutaneously (s.c.) at a volume of 1 ml/kg. WIN55,212-2 and AM251 were dissolved in dimethyl sulphoxide (DMSO; up to 10% v/v) and sterile 0.9% saline and a drop of Tween 80, which also was used as DMSO (10% DMSO; 0.6 μl/rat) or vehicle respectively. WIN55,212-2 and AM251 were injected into the BLA at a volume of 0.6 μl/rat. In the experiments where the animals received one or two injections, the control groups also received one or two saline or vehicle injections. The time intervals of drug administrations and the drugs' doses were based on our pilot experiments and previous studies (Rezayof et al., 2011; Walters et al., 2006). 2.4. Conditioned place preference apparatus

2. Materials and methods 2.1. Subjects Male Wistar rats (Pasteur Institute; Tehran, Iran) weighing 240–280 g, at the time of surgery, were used. Each cage contained four animals and they could access water and food freely except during the time of experiments. The animals were kept under a 12-h light–dark cycle (lights on at 07:00 h) and controlled temperature (22 ± 2 °C). For adaptation to the laboratory conditions all animals were allowed to adapt for at least 1 week before surgery and before starting the experiments. Besides, each animal was handled for 5 min every day. All experiments were done during the light phase of the cycle. Each group of experiments contained six animals and each animal was analyzed once. All procedures for the treatment of animals were approved by the Research and Ethics Committee of the School of Biology, University of Tehran and were done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Moreover, all efforts were made to minimize the number of animals used and limiting their suffering.

The place preference apparatus and procedure were conducted as described previously, using a minor modification of a procedure according to Carr and White (1983), and with minor modification. Briefly, two large conditioning compartments A and B (40 × 30 × 30 cm) were connected by a communicating tunnel (compartment C: 40 × 15 × 30 cm) that differ in color and flour texture. The compartment A was white with black horizontal stripes 2 cm wide on the walls and also had a textured floor. The other compartment (B) was black with vertical white stripes 2 cm wide and also had a smooth floor. Compartment C was painted red and this smaller tunnel allows animals access to both compartments. It had removable wooden partition that separated it from the other compartments and could be opened allowing animals entrance into each of the two compartments (A and B). 2.5. Place conditioning In this experiment, we used the unbiased procedure of CPP paradigm. The experiments took place on 5 consecutive days involving three distinct phases: pre-conditioning (introduction session), conditioning (acquisition sessions) and post-conditioning (testing session).

2.2. Surgery and drug microinjection Under deep anesthesia (50 mg/kg of ketamine and 5 mg/kg of xylazine), the animals were placed in a stereotaxic frame. The animals were bilaterally implanted with 22-gauge guide steel cannulas into the basolateral nucleus of the amygdaloid complex (BLA) according to the atlas of Paxinos and Watson (2007). Stereotaxic coordinates for the BLA were AP: − 2.8; ML: ±4.6; DV: − 8.6. The guide cannulas were anchored by jeweler's screws, and the incision was closed with dental cement. Stainless steel stylets (27 gauge) were placed in the

2.5.1. Pre-conditioning On day 1, each animal was placed into the compartment C and the guillotine door was removed. The animal was allowed to move freely between the compartments for 15 min. The time spent by the animals in each compartment was computed to assess any baseline preferences for A or B compartment prior to nicotine administration. In the particular experimental setup used in this study, the animals did not show an unconditioned preference for either of the compartments. Therefore, the animals were randomly assigned to one of two groups for place

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conditioning and a total of six animals were used for each subsequent experiments. 2.5.2. Conditioning After preconditioning phase, the conditioning phase took place in three days, from day 2 to day 4. The experiments consisted of six, 30min sessions (three vehicle and three drug pairing) with a 4-h interval. On each of the days, separate groups of animals received one conditioning session with nicotine and one with vehicle. During these sessions, the animals were confined to one compartment by closing the removable wall. Animals of each group were injected with nicotine and were immediately confined to one compartment of the apparatus for 30 min. Following administration of vehicle, the animals were confined to the other compartment for 30 min. Treatment compartment and order of presentation of nicotine and vehicle were counterbalanced for each group. 2.5.3. Post-conditioning On the last day (of testing phase), each animal was allowed to move freely into all compartments for 15 min without any drug injection on the test day. The time spent in the drug paired compartment was recorded for each animal and the change of preference was calculated as the difference (in seconds) between the time spent in the drugpaired compartment on the testing day, and the time spent in this compartment in the pre-conditioning session. 2.6. Locomotor activity During the testing phase in a nicotine-free state, locomotor activity was measured for each animal, based on a method used previously (Rezayof et al., 2011). The floor of each compartment (A & B) was separated into four equal squares. The entrance of the animal into each of the square was considered as an index of locomotor activity in 15 min. 2.7. Experimental design 2.7.1. Dose–response curve for nicotine-induced place preference This experiment was designed to examine the effect of nicotine on conditioned place preference. Different doses of nicotine (0.1, 0.2 and 0.3 g/kg, s.c.) were selected for evaluating place preference. Another group of animals received vehicle (1 ml/kg, s.c.) in two compartments (A and B) as control group. Locomotor activity was also measured in the testing phase.

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intra-BLA microinjections of DMSO (10% DMSO; 0.6 μl/rat) or different doses of AM251 (20, 40 and 60 ng/rat). After 5 min, they received vehicle (1 ml/kg, s.c.) or nicotine (0.2 mg/kg, s.c.), during the conditioning phase. All animals were tested 24 h after the last conditioning session, with no preceding injection. Locomotor activity of the animals was measured during the testing phase. 2.7.4. Effects of WIN 55,212-2 or AM251 on the expression of conditioned place preference induced by nicotine In this experiment, during the conditioning phase, the animals received nicotine (0.2 mg/kg, s.c.) once per day for 3 days without any microinjection of drug into the BLA. On the testing day, they received intra-BLA microinjections of DMSO (10% DMSO; 0.6 μl/rat), WIN 55,212-2 (0.1, 0.3 and 0.5 μg/rat) or AM251 (10, 20 and 40 ng/rat) 5 min before testing. The conditioning scores and the locomotor activity of the animals were measured during the testing phase. 2.8. Histology After completion of the behavioral sessions, each rat was deeply anesthetized with carbon dioxide and 1 μl of a 1% methylene-blue solution was bilaterally injected into the BLA (0.5 μl/side), then decapitated and its brain removed and placed in a 10% formalin solution. After 10 days, the brains were sliced and the sites of injections were verified according to the atlas of Paxinos and Watson (2007). Data from the animals with injection sites located outside the target regions (less than %10) were not used in the analysis. Therefore, a total of 162 animals with correct cannula placements were included in the data analysis. 2.9. Statistics In all experiments, the conditioning scores are expressed as differences in the time spent on the drug-associated side between the preconditioning and the testing phases. Locomotor activities are expressed as crossing of lines in both of the main compartments during the testing phase. Data are expressed as mean ± S.E.M. (n = 6). Analysis of data was performed using one-way or two-way ANOVA. Following a significant F-value, post-hoc analyses (Tukey's test) were performed for assessing specific group comparisons. The level of statistical significance was set at P b 0.05. 3. Results 3.1. Dose–response curve for nicotine-induced place preference

2.7.2. Effects of intra-BLA microinjection of WIN 55,212-2 with or without nicotine on the acquisition of CPP Effects of microinjection of different doses of WIN 55,212-2, an agonist of the CB1 receptor, into the BLA (intra-BLA) with or without nicotine on the acquisition of the conditioned place preference were determined as follows. The animals received nicotine (0.1 mg/kg, s.c.) or vehicle (1 ml/kg, s.c.) once daily in a 3-day schedule of conditioning. WIN 55,212-2(0.1, 0.3 and 0.5 μg/rat; intra-BLA) or DMSO (10% DMSO; 0.6 μl/rat) was injected into the BLA once per day for 3 days, 5 min before the administration of nicotine (three sessions); the conditioning scores then were measured in a drug-free state (testing day). Intra-BLA microinjections of the same (abovementioned) doses of all drugs without nicotine, during conditioning, were also used to assess their effects on CPP. The conditioning scores were then measured in a drugfree state on the test day. Locomotor activity was also evaluated during testing. 2.7.3. Effects of intra-BLA microinjection of AM251 with or without nicotine on the acquisition of CPP In order to test the effects of microinjection of AM251, a selective antagonist of the CB1 receptor, into the BLA on the acquisition of nicotine-induced conditioned place preference, all animals received

Fig. 1 shows the effect of a 3-day schedule of conditioning with nicotine in a CPP apparatus. The animals, which received vehicle (1 ml/kg, s.c.) twice per day, during six sessions, exhibited no preference for either compartment. Systemic administration of 0.2 mg/kg of nicotine, during conditioning, induced CPP [one-way ANOVA; F(3,20) = 19.68, P b 0.001]. However, the higher dose of the drug (0.3 mg/kg, s.c.) did not cause significant changes in place preference (P N 0.05). One-way ANOVA also indicated that the administration of the different doses of nicotine (0.1, 0.2 and 0.3 mg/kg; Fig. 1B), during conditioning phase, alone had no effect on the locomotor activity during the testing phase [F (3, 20) = 0.3, P N 0.05]. 3.2. Effects of intra-BLA microinjection of WIN 55,212-2 with or without nicotine on the acquisition of CPP The effect of bilateral microinjection of different doses of WIN 55,212-2 into the BLA with or without an ineffective dose of nicotine on the acquisition of CPP and locomotor activity has been shown in Fig. 2. Two-way ANOVA indicated a significant difference between the effects of WIN 55,212-2 (0, 0.1, 0.3 and 0.5 μg/rat; intra-BLA) alone and WIN 55,212-2 plus nicotine (0.1 mg/kg, s.c.) on the acquisition of

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Nicotine (mg/kg) Fig. 1. Dose–response curve for nicotine in a CPP paradigm. Different doses of nicotine (0.1, 0.2 and 0.3 mg/kg) and vehicle (1 ml/kg) were administered subcutaneously (s.c.) in a 3-day schedule of conditioning. On the test day, the animals were observed for a 15-min period. The change of preference was assessed as the difference between the time spent on the day of testing and the time spent on the day of the pre-conditioning session (Graph A). Locomotor activity was also measured in the testing phase (Graph B). Data are expressed as mean ± S.E.M. of 6 animals per group. ***P b 0.001 different from the vehicle control group.

CPP [for treatment, F(1,40) = 4.6, P b 0.05; dose, F(3,40) = 19.0, P b 0.001; and treatment × dose interaction, F(3,48) = 10.05, P b 0.001], but not on the locomotor activity [for treatment, F(1,40) = 10.6, P b 0.01; dose, F(3,40) = 0.61, P N 0.05; and treatment × dose interaction, F(3,40) = 0.01, P N 0.05]. One-way ANOVA also revealed that in the animals which were conditioned by intra-BLA microinjection of WIN 55,212-2, no significant change was observed in the CPP [F(3,20) = 0.56, P N 0.05] and the locomotor activity [F(3,20) = 0.25, P N 0.05]. However, one-way ANOVA revealed that intra-BLA microinjection of the agonist with an ineffective dose of nicotine (0.1 mg/kg, s.c.) induced a significant place preference [F(3,20) = 17.6, P b 0.001] without any effect on the locomotor activity [F(3,20) = 0.4, P N 0.05].

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Fig. 2. Effects of bilateral microinjections of WIN55,212-2 into the basolateral amygdala (BLA) alone or with nicotine on the acquisition of conditioned place preference. The animals received intra- BLA microinjections of different doses of WIN55,212-2 (0.1, 0.3 and 0.5 μg/rat) or DMSO (10% DMSO, 0.6 μl/rat) with or without nicotine (0.1 mg/kg; s.c.) once daily in a 3-day schedule. The change of preference was assessed as the difference between the time spent on the day of testing and the time spent on the day of the pre-conditioning session (Graph A). Locomotor activity was also measured in the testing phase (Graph B). Data are expressed as mean ± S.E.M. of 6 animals per group. ***P b 0.01 different from DMSO/nicotine control group.

F(1,40) = 17.9, P b 0.001; dose, F(3,40) = 31.2, P b 0.001; and treatment × dose interaction, F(3,40) = 25.4, P b 0.001] and locomotor activity [for treatment, F(1,40) = 0.03, P N 0.05; dose, F(3,40) = 5.9, P b 0.01; and treatment × dose interaction, F(3,40) = 0.75, P N 0.05]. One-way ANOVA also revealed that in the animals which were conditioned by intra-BLA microinjection of AM251, no significant change was observed in the CPP [F(3,20) = 2.5, P N 0.05] and locomotor activity [F(3,20) = 2.0, P N 0.05]. Further analysis also revealed that intra-BLA administration of AM251 inhibited the acquisition of nicotine-induced place preference [F(3, 20) = 51.6, P b 0.001] and also decreased the locomotor activity on the testing phase [F(3,20) = 5.05, P b 0.01] (Fig. 3).

3.3. Effects of intra-BLA microinjection of AM251 with or without nicotine on the acquisition of CPP

3.4. Effects of WIN 55,212-2 or AM251 on the expression of conditioned place preference induced by nicotine

Fig. 3 shows the effects of bilateral microinjection of AM251 into the BLA in the absence or presence of nicotine (0.2 mg/kg, s.c.) on the acquisition of CPP. Two-way ANOVA indicated a significant difference between the effects of AM251 (20, 40 and 60 ng/rat; intra-BLA) alone and AM251 plus nicotine on the acquisition of CPP [for treatment,

Fig. 4 shows the effect of bilateral intra-BLA microinjection of WIN55,212-2 (0.1, 0.3 and 0.5 μg/rat) or AM251 (10, 20 and 40 ng/rat) on the expression of nicotine-induced CPP. One-way ANOVA indicated that intra-BLA injection of WIN55,212-2 had no effect on the expression of nicotine-induced place preference [F(3,20) = 0.94, P N 0.05], while

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Fig. 3. Effects of the microinjection of AM251 into the BLA in the absence or presence of nicotine on the acquisition of CPP. The animals received intra-BLA microinjections of different doses of AM251 (20, 40 and 60 ng/rat) or DMSO (10% DMSO, 0.6 μl/rat) with or without nicotine (0.2 mg/kg, s.c.) once daily in a 3-day schedule. The change of preference was assessed as the difference between the time spent on the day of testing and the time spent on the day of the pre-conditioning session (Graph A). Locomotor activity was also measured in the testing phase (Graph B). Data are expressed as mean ± S.E.M. of 6 animals per group. ***P b 0.001 different from DMSO/vehicle control group, ++P b 0.01, +++P b 0.001 different from the DMSO/nicotine control group.

the injection of AM251 into the BLA inhibited the expression of nicotine response [F(3,20) = 28.0, P b 0.001]. It is important to note that the treatments could not alter the locomotor activity [F(6,35) = 2.3, P N 0.05]. 4. Discussion In the present study, systemic administration of 0.2 mg/kg of nicotine, during three conditioning sessions, using an unbiased conditioned place preference (CPP) paradigm induced CPP. It should be considered that there was no significant place preference with the higher dose of the drug (0.3 mg/kg, s.c.). Our results also showed that the administration of the different doses of nicotine, during conditioning phase, alone had no effect on the locomotor activity during the testing phase. Nicotine influences both motivational and aversive effects mainly by activation of highly expressed neuronal nAChRs that are located on both dopamine (DA) and non-DA neurons of the ventral tegmental area (VTA; Hendrickson et al., 2013; Tan et al., 2009; Yin and French, 2000). In fact a functional balance between inhibitory and excitatory inputs to the VTA DA neurons results in the rewarding effect of nicotine (Mansvelder et al., 2002). Subsequent activation of cholinergic

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Fig. 4. Effects of bilateral microinjections of WIN55, 212–2 into the BLA on the expression of nicotine-induced place preference. The animals received nicotine (0.2 mg/kg, s.c.) once per day for 3 days without any microinjection of drug into the BLA. On the testing day, they received intra-BLA microinjection of DMSO (10% DMSO, 0.6 μl/rat), WIN55,212-2 (0.1, 0.3 and 0.5 μg/rat) or AM251 (10, 20 and 40 ng/rat) 5 min before testing. The change of preference was assessed as the difference between the time spent on the day of testing and the time spent on the day of the pre-conditioning session (Graph A). Locomotor activity was also measured in the testing phase (Graph B). Data are expressed as mean ± S.E.M. of 6 animals per group. ***P b 0.001 different from DMSO/nicotine control group.

receptors in the VTA regulates release of dopamine in the nucleus accumbens (NAc) to further cause a reinforcing effect (Pidoplichko et al., 2004). Our result is similar to a previously reported study which indicated that the moderate dose of nicotine (0.2 mg/kg) produced a significant place preference (Yararbas et al., 2010). Moreover, it has been shown that nicotine at doses of 0.4, 0.8 and 1.2 mg/kg induced conditioned place aversion, but it was significant only for the 0.8 mg/kg dose (Jorenby et al., 1990). In addition, Fudala et al. (1985) observed nicotine CPP over a wide range of doses (0.1–1.2 mg/kg) in Sprague–Dawley rats in a biased procedure. It seems that unlike other drugs of abuse such as morphine (Zarrindast et al., 2005), methamphetamine (Cunningham and Noble, 1992) and ethanol (Cunningham et al., 1997) nicotine could not induce place preference in a dose dependent manner. A review of the literature confirms variations between the results of studies on nicotine-induced CPP (Jackson et al., 2009; Pastor et al., 2013). Important factors that influence these discrepancies include strain of the rodent, the dose of nicotine used, pre-exposure to nicotine, different times spent in conditioning sessions, number of sessions, use of

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a biased or unbiased procedure and route of nicotine administration (for review see Le Foll and Goldberg, 2005). It is an established fact that a functional inter-connectivity network exists between the BLA and two main regions of reward pathways including the VTA and the NAc (Ambroggi et al., 2008; Phillips et al., 2003). The NAc is situated in a proper place where received direct excitatory projections from the BLA (Floresco et al., 2001; Sugase-Miyamoto and Richmond, 2005) to response reward seeking behavior (Di Ciano and Everitt, 2004) as well as dopaminergic projection from the VTA modulate dopamine release from the NAc. Moreover behavioral studies assigned the BLA as an important site in emotional behaviors (Laviolette and Grace, 2006) and reward-related learning (Sun and Laviolette, 2012). Since the BLA has a critical role in reward and reward-related behaviors, the present study was designed to assess the modulatory role of the BLA CB1 cannabinoid receptors in nicotine-induced CPP. Intra-BLA microinjection of a non-selective cannabinoid receptor agonist, WIN 55,212-2 (0.1-0.5 μg/rat) during conditioning phase by itself could not induce CPP and had no effect on the locomotor activity. In reviewing the literature, controversial results have been reported regarding the effects of cannabinoid agonists on CPP paradigm. Some authors suggested that systemic administration of WIN55,212-2 (Chaperon et al., 1998) or Δ9-tetrahydrocannabinol (Sañudo-Peña et al., 1997) can induce conditioned place aversion (CPA), whereas others have found that intra-BLA (Rezayof et al., 2012), intra-CeA (Rezayof et al., 2011) or intra-dorsal hippocampal microinjection (Zarrindast et al., 2007) of the agonist of CB1 receptors induced significant CPP. In addition, it was reported that intraperitoneal injection of WIN 55,212-2 at doses of 0.1 mg/kg (i.p.) or higher doses could not induced a significant CPP (Manzanedo et al., 2004; Polissidis et al., 2009). Therefore, it seems that the rewarding effects of cannabinoids depend on the routes of administration and the dose range of the tested compounds. Our results also indicated that intra-BLA microinjection of the same doses of agonist with an ineffective dose of nicotine (0.1 mg/kg, s.c.) induced a significant place preference without any effect on the locomotor activity. Interactions between cannabinoids and nicotine have also been previously reported. For example, Valjent et al. (2002) reported that systemic co-administration of low doses of nicotine and THC induced CPP in mice without significant changes in locomotor activity. Moreover, nicotine did not produce a significant CPP in CB1 knockout mice (Castañé et al., 2002), confirming the role of CB1 receptors in the rewarding effects of nicotine. Chronically exposed to nicotine has also been reported to increase arachidonoylethanolamide (AEA), the endogenous ligand for cannabinoid receptors, in the limbic forebrain (González et al., 2002). It is important to note that the BLA endocannabinoid signaling may indirectly have a modulatory role in emotional behavior and reward process through mesolimbic dopaminergic pathways (Perra et al., 2008). Considering that CB1 receptors have predominantly expressed in the BLA (McDonald and Mascagni, 2001) and intra-BLA administration of WIN 55,212-2 increased rewarding properties of a sub-threshold dose of nicotine (0.1 mg/kg), one may suggest that cannabinoid CB1 receptors in the BLA have an important role in nicotine reward. In support of a functional interaction between the BLA endocannabinoid system and nicotine reward, we designed the second part of the study to investigate whether CB1 receptor blockade can also affect nicotine-induced place preference. With this purpose different cannabinoid CB1 receptors (AM251) doses alone or in combination with rewarding dose of nicotine have been injected into the BLA during the conditioning phase. The results showed no significant change in the CPP apparatus of the animals which were conditioned by intra-BLA microinjection of AM251. Furthermore, bilateral microinjection of CB1 cannabinoid antagonist receptor, AM251, into the BLA inhibited the acquisition of nicotine-induced place preference. On the other hand, intra-BLA injection of AM251 by itself had no effect on locomotor activity, while the administration of higher dose of AM251 (60 ng/rat) plus nicotine (0.2 mg/kg) decreased locomotor activity on the testing

phase. Previously, it has been reported that systemic administration of rimonabant, a selective cannabinoid CB1 receptor antagonist, prevented nicotine-induced place preference (Forget et al., 2005; Le Foll and Goldberg, 2004). Moreover, intra-BLA administration of rimbonant dose-dependently reduced nicotine-seeking behavior (Kodas et al., 2007) and systemic administration of this drug blocked nicotineinduced dopamine in the NAc (Cheer et al., 2007). Systemic administration of SR-141716A (selective cannabinoid antagonist) also decreased nicotine-induced dopamine release in the NAc (Cohen et al., 2002). The rewarding properties of nicotine have been shown to abolish in CB1 knockout mice (Castañé et al., 2002). Interestingly, our results indicated that the microinjection of AM251, but not WIN55,212-2, into the BLA before testing, inhibited the expression of nicotineinduced place preference. The drugs had no effect on the locomotor activity. Thus, the BLA CB1 receptors are involved in mediating the acquisition and the expression of nicotine-induced place preference. 5. Conclusion From the present data, we can conclude that cannabinoid CB1 receptors of the BLA are involved in mediating nicotine reward. Moreover, considering the effects of intra-BLA microinjection of WIN55,212-2 (enhancement of CPP), and the effects of intra-BLA microinjection of AM251 (prevention of CPP) when co-administered with nicotine, it can be suggested that nicotine-induced place preference may be related to activation of the endocannabinoid system. It is also reasonable to conclude that there is a functional correlation between the endocannabinoid system of the BLA and nicotine in the acquisition and expression of CPP which may be a reason for the tendency toward co-administration of cannabis and nicotine. Acknowledgments The authors would like to thank Dr. Mahmoud Efatmaneshnik for his assistance in the preparation of the manuscript. References Alijanpour S, Rezayof A. Involvement of dorsal hippocampal and medial septal nicotinic receptors in cross state-dependent memory between WIN55, 212–2 and nicotine or ethanol in mice. Neuroscience 2013;245:61–73. Ambroggi F, Ishikawa A, Fields HL, Nicola SM. Basolateral amygdala neurons facilitate reward-seeking behavior by exciting nucleus accumbens neurons. Neuron 2008;59: 648–61. Biala G, Kruk M, Budzynska B. Effects of the cannabinoid receptor ligands on anxiety-related effects of D-amphetamine and nicotine in the mouse elevated plus maze test. J Physiol Pharmacol 2009;60:113–22. Brielmaier JM, McDonald CG, Smith RF. Nicotine place preference in a biased conditioned place preference design. Pharmacol Biochem Behav 2008;89:94–100. Budzyńska B, Kruk M, Biała G. Effects of the cannabinoid CB1 receptor antagonist AM 251 on the reinstatement of nicotine-conditioned place preference by drug priming in rats. Pharmacol Rep 2009;61:304–10. Carr GD, White NM. Conditioned place preference from intra-accumbens but not intra-caudate amphetamine injections. Life Sci 1983;33:2551–7. Castañé A, Valjent E, Ledent C, Parmentier M, Maldonado R, Valverde O. Lack of CB1 cannabinoid receptors modifies nicotine behavioural responses, but not nicotine abstinence. Neuropharmacology 2002;43:857–67. Changeux JP. Nicotine addiction and nicotinic receptors: lessons from genetically modified mice. Nat Rev Neurosci 2010;11:389–401. Chaperon F, Soubrié P, Puech AJ, Thiébot MH. Involvement of central cannabinoid (CB1) receptors in the establishment of place conditioning in rats. Psychopharmacology (Berl) 1998;135:324–32. Cheer JF, Kendall DA, Marsden CA. Cannabinoid receptors and reward in the rat: a conditioned place preference study. Psychopharmacology (Berl) 2000;151:25–30. Cheer JF, Wassum KM, Sombers LA, Heien ML, Ariansen JL, Aragona BJ, et al. Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation. J Neurosci 2007;27:791–5. Cohen C, Perrault G, Voltz C, Steinberg R, Soubrié P. SR141716, a central cannabinoid (CB(1)) receptor antagonist, blocks the motivational and dopamine-releasing effects of nicotine in rats. Behav Pharmacol 2002;13:451–63. Corrigall WA, Franklin KB, Coen KM, Clarke PB. The mesolimbic dopaminergic system is implicated in the reinforcing effects of nicotine. Psychopharmacology (Berl) 1992;107:285–9. Cunningham CL, Noble D. Methamphetamine-induced conditioned place preference or aversion depending on dose and presence of drug. Ann N Y Acad Sci 1992;654:431–3.

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Basolateral amygdala CB1 cannabinoid receptors mediate nicotine-induced place preference.

In the present study, the effects of bilateral microinjections of cannabinoid CB1 receptor agonist and antagonist into the basolateral amygdala (intra...
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