Effects of a cannabinoid receptor (CB) 1 antagonist AM251 on behavioral sensitization to nicotine in a rat model of novelty-seeking behavior: correlation with hippocampal 5HT
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- Bhatti, A.S., Aydin, C., Oztan, O. et al. Psychopharmacology (2009) 203: 23. doi:10.1007/s00213-008-1366-6
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There are marked individual differences in the efficacy of mainstream nicotine cessation agents in preventing relapse. A rat model of novelty-seeking phenotype was reported to have predictive value for psychostimulant taking behavior where locomotor reactivity to novelty is used to rank high (HR, highest 1/3) versus low (LR, lowest 1/3) responsiveness to novelty in outbred rats. We tested the hypothesis that a cannabinoid receptor (CB) 1 antagonist that is in clinical trials for smoking cessation may reverse behaviorally sensitizing effects of nicotine in HRs and repeated nicotine-induced elevations in hippocampal 5HT.
Materials and methods
Adolescent LRHR rats underwent intermittent behavioral sensitization to nicotine regimen with or without a CB1 receptor antagonist AM251 or bupropion treatment following nicotine training during 1 week of nicotine-free period. Expression of behavioral sensitization to nicotine was assessed in response to a low-dose nicotine challenge. Using the same sensitization regimen and therapeutic treatments, hippocampal 5HT levels were measured via in vivo microdialysis in response to the nicotine challenge.
HR but not LR animals showed behavioral sensitization to a low-dose nicotine challenge following intermittent nicotine training and 1 week of injection-free period. AM251 (5 mg/kg, i.p.) but not bupropion administration during injection-free period successfully reversed locomotor sensitization to nicotine challenge in HRs. AM251 treatment also reversed nicotine-induced elevations in extracellular 5HT in the HR hippocampal hilus.
These data suggest that CB1 antagonists may prevent locomotor sensitization to nicotine and reverse nicotine-induced elevations in hippocampal 5HT in high novelty seekers.
KeywordsCannabinoid receptorHippocampusNovelty seekingNicotine sensitizationBupropionSerotonin
Both the acquisition of nicotine dependence and response to nicotine cessation agents show vast individual differences (Cryan et al. 2003; Lerman and Berrettini 2003). Nicotine-based therapies such as the nicotine patch are successful in weaning some individuals from nicotine, even though relapse rates as high as 80% are reported within the first year (Murray et al. 1997). A widely used, FDA-approved, non-nicotine-based therapeutic agent, bupropion, is effective in smoking cessation in a subgroup of individuals with propensity for negative affect (Markou and Kenny 2002; Dwoskin et al. 2006); even though over 70% of those individuals still relapse within 1 year (Cryan et al. 2003). Lack of a therapeutic agent that is efficacious for majority of users and that is able to sustain quitting underlines the importance of devising treatment methods specific for different types of nicotine users.
A naturally occurring individual difference that may have predictive value for propensity to psychostimulant taking is the novelty-seeking phenotype in the rat (Piazza et al. 1989; Hooks et al. 1991), which was initially proposed to model aspects of human sensation-seeking trait (Zuckerman 1984). Experimentally naïve rats, when exposed to the stress of a novel environment, vary in their exploratory activity. Some rats display high rates of locomotor reactivity (HR), whereas others display low rates (LR). The LRHR phenotype is useful for predicting propensity to self-administer nicotine (Suto et al. 2001). We have recently shown that adolescent HR rats show expression of behavioral sensitization to nicotine after a single nicotine exposure, and lidocaine inactivation of hippocampus enhances this behavior (Bhatti et al. 2007).
Present experiments compare behavioral and neurochemical outcomes of a novel cannabinoid receptor 1 (CB1) antagonist, AM251 (an analog of Rimonabant; phase III clinical trials for smoking cessation), and bupropion following an intermittent behavioral sensitization to nicotine regimen in the LRHR rats. For clinical relevance, the peripubertal-juvenile period was chosen for nicotine exposure because the majority of nicotine-addicted humans self-report having acquired the habit as adolescents (Mansvelder and McGehee 2002). Nicotine alters 5HT neurotransmission and 5HT1A messenger RNA expression in hippocampus (Kenny et al. 2000a; Kenny et al. 2001), all of which are implicated in depressive symptoms exacerbated by nicotine abstinence. We used hippocampal 5HT microdialysis to assess neurochemical adaptations following behaviorally sensitizing nicotine regimen either with or without therapeutic intervention. The central hypothesis is that the HR phenotype will show inhibited expression of behavioral sensitization to nicotine and associated reversal of nicotine-induced elevations in hippocampal 5HT following the CB1 receptor antagonist treatment.
Materials and methods
Behavioral sensitization to nicotine
Animal housing and the LRHR phenotype screening
Animals were treated in accordance with the National Institute of Health guidelines on laboratory animal use and care. A total of 162 male Sprague–Dawley rats (Charles River, Wilmington, MA, USA) were used for the dose–response assessment. An additional 162 Sprague–Dawley males were used for each therapeutic intervention study (per different doses of AM251 and bupropion). Male rats arrived at weaning and were housed three per cage in 43 × 21.5 × 25-cm Plexiglas cages and kept on a 12-h light/dark cycle (lights on at 7:00 a.m.). Food and water were available ad libitum. On PN 24, animals were screened for locomotor reactivity to a novel environment for 60 min using commercially available locomotion chambers (San Diego Instruments, San Diego, CA, USA; Bhatti et al. 2007). Locomotor reactivity to novelty was tested in 43 × 43 × 24.5-cm (high) clear Plexiglas cages with stainless steel grid flooring. Horizontal locomotion and rearing were monitored by two banks of photocells (a total of X = 16 by Y = 16 photocells) 2.5 cm above the grid floor and equally spaced along the sides of the box. At the end of a 60-min screening session, total locomotion (i.e., X, Y, and Z locomotion) was pooled and the rats were ranked as HRs (i.e., rats that exhibited locomotor scores in the highest third of the sample) or LRs (i.e., rats that exhibited locomotor scores in the lowest third of the sample). The population distribution of locomotor scores fitted a normal distribution. For each behavioral study (dose–response assessment and each dose of therapeutics), 54 HRs and LRs are screened.
Bupropion (Sigma-Aldrich, MO, USA), an FDA-approved, non-nicotinic therapeutic agent for nicotine cessation, was included in therapeutic studies for comparison to the CB1 receptor antagonist. AM251 (Tocris, MO, USA) is a commercially available CB1 receptor antagonist. Nicotine bitartrate was obtained from a commercial supplier (Sigma). The doses for nicotine, bupropion, and AM251 were chosen based on effective doses used in the literature in numerous studies (Miller et al. 2001; Suto et al. 2001; Shoaib et al. 2003; Le Foll and Goldberg 2004). Three different doses of nicotine (0.35, 0.70, and 1.0 mg/kg) were used for nicotine training, and a low-dose nicotine (0.10 mg/kg) is used for challenge. Nicotine was dissolved in 0.9% NaCl and the pH is adjusted to 7.4. Since the CB1 receptor ligands available for research are insoluble in water, organic solvents were required as vehicle. The AM251 was administered via intraperitoneal (i.p.) injection at doses of 1 and 5 mg/kg dissolved in a vehicle solution consisting of Tween 80, DMSO, 0.9% NaCl (1:2:7; Xi et al. 2008). Bupropion was administered via subcutaneous (s.c.) injection at doses of 40 and 60 mg/kg dissolved in 0.9% NaCl (Wing and Shoaib 2007).
Nicotine training and challenge
Hippocampal 5HT following nicotine sensitization
Animals and stereotaxic surgery
For the initial study investigating effects of nicotine on hippocampal 5HT, a total of 150 Sprague–Dawley male rats were phenotype-screened and tested in response to various doses of nicotine challenge (0.10, 0.35, 0.70 mg/kg, s.c.) following saline or nicotine pre-training (0.70 mg/kg, s.c.). The behavioral sensitization procedure employed was identical to the one outlined in Table 1. A total of 47 LRs and 44 HRs were included in the final 5HT assessment after cannulae placements were confirmed. Additional set of 150 rats were phenotype-screened for each therapeutic study which used the same nicotine training and challenge doses as above. During injection-free period, saline pre-trained animals for each phenotype were divided into animals that received saline (1 ml/kg, s.c.), DMSO (Tween 80, DMSO, 0.9% NaCl; 1:2:7, i.p.), BP (40 or 60 mg/kg dissolved in saline, s.c.), or AM251 (1 or 5 mg/kg dissolved in DMSO vehicle, i.p.; see Table 1 for injection days). Nicotine pre-trained animals for each phenotype were assigned to receive either saline, BP, or AM251. During challenge and sample collection for 5HT, half of the saline pre-trained animals that received saline during the injection-free period and all of the nicotine pre-trained animals for both phenotypes received the low-dose nicotine injection. Remaining saline pre-trained rats received a saline injection during challenge, with the exception of the DMSO vehicle group that received DMSO during challenge. For all doses of therapeutics combined, 85 LRs and 90 HRs were included in the final 5HT assessment.
Stereotaxic surgeries were conducted following phenotype screening, and animals recovered for 3 days. Specifically, on the day of the surgery, rats were anesthetized with a combination of xylazine (6 mg/kg, i.p.) and ketamine (80 mg/kg, i.p.) and mounted in a stereotaxic apparatus (David Kopf Instruments, Tujinga, CA, USA). A 22-gauge stainless steel guide cannula 10 mm in length was implanted bilaterally 2 mm below the surface of the skull and anchored to the skull with three stainless steel screws and dental cement and protected with stylets after implantation. The target coordinates for the tip of the probe were in the hilus region of the dorsal hippocampus (AP −3.0 mm to the bregma, ML ±1.8 mm and DV 4.1 mm; Paxinos and Watson 1982). The evening before the 5HT microdialysis assessment, concentric microdialysis probes (exchange surface 1.0 mm in length; 200 μm i.d., 13,000 MW cutoff; Spectrum Medical Industries, Los Angeles, CA, USA) were inserted through the guide cannulae. Rats were then placed in the test chamber and attached to a perfusion apparatus (Raturn™; Bioanalytical Systems, West Lafayette, IN, USA). The dialysis probes were perfused overnight with a buffered Ringer solution (140 mM NaCl, 3.0 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 0.27 mM NaH2PO4, 1.2 mM Na2HPO4, pH 7.4) at a rate of 1.0 μl/min.
5HT sample collection and detection
Sample collection started 24 h after the last injection-free day, and in the case of therapeutics studies, corresponding to 2 days after the last therapeutic injection. After four consecutive baseline sample collections, six experimental samples were collected in response to the saline vehicle (1 ml/kg of 0.9% NaCl, s.c.), DMSO vehicle (Tween 80, DMSO, 0.9% NaCl; 1:2:7; 1 ml/ kg, i.p.), or nicotine (0.10 mg/kg, 0.35 mg/kg or 0.70 mg/kg, s.c.) at 15, 30, 45, 60, 75, and 90 min at a volume of 15 μl per sample per hemisphere. Samples were pooled from two hemispheres for each animal. Separation of 5HT was achieved by high-performance liquid chromatography (HPLC) with electrochemical detection (HTEC-500; EiCOM, Japan). The mobile phase composition was 0.1 M phosphate buffer at pH 6.0, 500 mg/L 1-decanesulfonic acid, 50 mg/L EDTA, and 1.0% methanol and pumped at a rate of 0.40 ml/min. The applied potential was set at 400 mV vs. an Ag/AgCl reference electrode. The amount of 5HT was calculated by measurement of peak areas with comparison to known amounts in external standards using PowerChrom (AD Instruments, Boston, MA, USA).
Animals were killed via rapid decapitation following 5HT sample collection. Brains were harvested, frozen in isopentane (−30°C), and sectioned coronally at 20 μm through the dorsal hippocampus. Slide-mounted sections were then stained with cresyl violet. Animals that had one cannula or both cannulae tips outside of the hilus region were eliminated from analyses (34 out of 300).
In dose–response studies, nicotine training data for each training dose (0.35, 0.70, 1.0 mg/kg) were analyzed by a repeated-measures analysis of variance (ANOVA): phenotype (LR, HR) × drug (SAL, NIC) × injection days (INJ 1, INJ 2, INJ 3, INJ 4). In the same studies, nicotine challenge data were analyzed by two-way ANOVAs: phenotype (LR, HR) × pre-training (SAL, NIC). Nicotine challenge data for each therapeutic agent were analyzed by a three-way ANOVA: phenotype (LR, HR) × pre-training (SAL, NIC) × therapeutics (SAL, BP −40 mg, BP −60 mg) or (SAL, AM251 –1 mg, AM251 –5 mg). For each therapeutic agent, SAL controls from different doses were pooled in the final analysis. 5HT responses to various doses of nicotine challenge were analyzed by a three-way ANOVA: phenotype (LR, HR) × pre-training (SAL, NIC) × challenge (SAL, 0.1, 0.35, 0.70 mg/kg nicotine). The data from therapeutics in 5HT response to nicotine challenge were analyzed by a four-way ANOVA per each therapeutic agent employed: phenotype (LR, HR) × pre-training (SAL, NIC) × therapeutics (SAL, BP −40 mg, BP −60 mg) or (DMSO, AM251 −1 mg, AM251 −5 mg) × challenge (SAL, NIC) or (DMSO, NIC), respectively. For each therapeutic agent, control animals from different doses were pooled in the final analysis. Significant main effects and interactions were followed by Fisher’s least significant difference tests, and significance was set at p = 0.05.
Therapeutic intervention during the injection-free period
5HT response to acute and repeated nicotine in LRs and HRs
5HT response to therapeutic intervention
Our results showed that even though at 0.70-mg/kg nicotine training dose both phenotypes showed progressive increase in nicotine-induced locomotion across injection days, only the HR phenotype displayed expression of behavioral sensitization to a low-dose nicotine challenge (0.10 mg/kg) following nicotine training with 0.35- and 0.70-mg/kg training doses (Fig. 1). Systemic treatment with the CB1 antagonist AM251 at 5-mg/kg dose during the injection-free period completely reversed the expression of behavioral sensitization to the low-dose nicotine challenge. Even though bupropion treatment at 40 mg/kg resulted in attenuation of nicotine-induced locomotion at subsequent naïve nicotine exposure in both phenotypes, bupropion at both 40- and 60-mg/kg doses were ineffective in reversing behavioral sensitization induced by repeated nicotine in the HRs (Fig. 2). In accordance with the behavioral findings, a sensitization was detected in extracellular 5HT levels from hippocampal hilus in HR animals in response to a low-dose (0.10 mg/kg) nicotine challenge following pre-training with repeated nicotine (0.70 mg/kg) compared to animals pre-trained with saline (Fig. 4). Challenge nicotine-induced elevations in hippocampal 5HT levels in HRs were significantly reversed with systemic AM251 at 5-mg/kg dose, but not with bupropion treatment (Fig. 5). It should be noted that there was a trend in HRs towards higher 5HT levels in response to acute injection of 0.1 mg/kg nicotine in saline pre-trained condition compared to LRs (Fig. 4); however, this was not significant. In the same vein, higher locomotor response to saline injections were observed in HR animals compared to LRs in some training days and doses (“Results”, Dose–response assessments), reconfirming the phenotype separation. In particular to the expression of sensitization following training, these results indicate that in the HR phenotype, a low dose of nicotine challenge (0.10 mg/kg) can induce sensitized levels of hippocampal 5HT and associated expression of behavioral sensitization to nicotine, both of which AM251 may effectively reverse.
HR animals acquire nicotine self-administration more readily and are willing to work more to obtain nicotine compared to LRs as tested using the progressive ratio schedule of reinforcement (Suto et al. 2001). Locomotor sensitization to psychostimulants was thought to be relevant to addiction in humans because of the assumption that the neural substrate that mediates these effects is the same as, or at least overlaps with, the neural substrate responsible for the reinforcing effects of drugs (Wise and Bozarth 1987). There are a large number of converging molecular, cellular, and neuroanatomical substrates between addiction and memory (Nestler 2002). The hippocampus is one such area that interacts with the mesolimbic reward circuitry (Brog et al. 1993; Wright and Groenewegen 1995; Pierce et al. 1998). We have shown that a single nicotine injection was sufficient to develop locomotor sensitization to a low-dose nicotine challenge following the injection-free period in HR animals, which was enhanced by lidocaine inactivation of the hippocampal hilus (Bhatti et al. 2007). A positive correlation was observed between challenge nicotine-induced locomotion and the mossy fiber terminal field volume. In the current experiment, we have shown that hippocampal 5HT neurochemistry is implicated in the expression of behavioral sensitization to nicotine in the LRHR phenotype.
Nicotine can alter 5HT1A expression in the hippocampus (Kenny et al. 2000b, 2001), which is implicated in depressive symptoms exacerbated by nicotine withdrawal. Specifically, the serotonin-synthesizing cells located in the median raphe nucleus project to the dorsal hippocampus (Mokler et al. 1998). There is in vivo microdialysis evidence that both local infusion and systemic injection of nicotine alter extracellular 5HT levels in the hippocampus (Olausson et al. 2002; Seth et al. 2002). Neurochemical manipulations that increase or decrease the 5HT tone such as 5HT depletion or selective serotonin uptake inhibitors interfere with locomotor sensitization to nicotine (Seth et al. 2002), suggesting that a medium 5HT tone is necessary for sensitization. There is recent evidence suggesting that hippocampal 5HT is important for the novelty-seeking phenotype. For example, the 5HT7 receptor expression is inversely correlated with tendency for novelty-seeking in the dorsal hippocampus using the LRHR phenotype (Ballaz et al. 2007). Our data suggest that sensitized levels of hippocampal 5HT in response to nicotine challenge may be a neurochemical adaptation induced by repeated nicotine in the HRs. Others, however, have shown that consecutive nicotine administration at 0.4-mg/kg dose for 20 days can reduce the overflow of hippocampal 5HT, suggesting a depressogenic effect of nicotine (Balfour and Ridley 2000). Dosage of nicotine is a crucial parameter, as low doses of nicotine such as the present challenge dose (0.1. mg/kg) can induce anxiolytic effects, whereas high doses of nicotine can induce anxiogenic effects (File et al. 1998). Our intermittent nicotine administration (four injections at 3-day intervals) with 1 week of injection-free period followed by a low-dose challenge produced an increase in hippocampal 5HT, an effect opposite to the consecutive administration of 0.4 mg/kg of nicotine for 20 days followed by a test injection at the same dose (Balfour and Ridley 2000), suggesting that procedural differences in nicotine administration may be an important determinant of hippocampal 5HT response.
CB1 antagonists (e.g., Rimonabant) are presently in clinical trials for nicotine cessation. The CB1 receptors are expressed in the cerebral cortex and the hippocampus (Pertwee 1997), the classic mesolimbic system (Robbe et al. 2002) and the raphe nucleus (Gobbi et al. 2005; Haring et al. 2007). Endocannabinoids function as retrograde messengers acting on widely distributed CB1 receptors to suppress neurotransmitter release at both glutamatergic and GABAergic synapses (Wilson and Nicoll 2002; Maldonado et al. 2006). CB1 receptor antagonists reduce nicotine self-administration (Cohen et al. 2002), and the CB1 knockout mice demonstrate blunted rewarding effects of nicotine in the conditioned place preference task (Castane et al. 2002). Hippocampal 5HT levels can be modulated via activation of the CB1 receptors located on serotonergic neurons or on afferent fibers contacting serotonergic neurons in the raphe nucleus (Haring et al. 2007). CB1 protein has selective expression in hippocampal nerve fiber systems and axon terminals (Egertova and Elphick 2000), the same compartments that show expression of the nicotinic acetylcholine receptors (nAChR; Fabian-Fine et al. 2001), and most likely receive overlapping innervation from the median raphe (Mokler et al. 1998). Our data show that systemic therapeutic treatment with a CB1 antagonist during nicotine-free period could reverse challenge nicotine-induced increase in 5HT levels observed in the HR hippocampus. It is plausible that endocannabinoid binding to CB1 receptors present in the GABAergic nerve terminals that contact serotonergic cell bodies in the median raphe may serve to disinhibit serotonergic input to hippocampus, net result being an increase in 5HT release. Consequently, CB1 receptor antagonists may reverse this response. However, since a systemic injection was used, we cannot exclude other neural targets for the CB1 antagonist. Possible regulation of hippocampal 5HT by nicotine’s direct action on presynaptic nAChR is not overruled.
Bupropion is an atypical antidepressant that inhibits reuptake of dopamine and norepinephrine (Dwoskin et al. 2006). It is also used to alleviate withdrawal symptoms such as negative affect associated with nicotine abstinence, especially in tobacco smokers who also have a history of major depression (Covey 1999). There is controversial evidence as to the effectiveness of bupropion in regulating nicotine craving, some reports showing a reduction in craving (Hurt et al. 1997; Brody et al. 2004) while others showing no effect (Shiffman et al. 2000). In support of the latter, our data show that bupropion intervention during the injection-free period had no effect in regulating challenge nicotine-induced locomotion in HR animals pre-trained with repeated nicotine.
These findings suggest that the high novelty seekers may benefit most from CB1 receptor antagonists in reversing nicotine-induced adaptations in hippocampal 5HT levels, which may be implicated in effectiveness of CB1 receptor antagonists in counteracting incentive motivation.
This work is entirely supported by the Florida Department of Health grant 05NIR-5194 awarded to Dr. Isgor.