Adolescent exposure to cannabinoids enhances the behavioural effects of cocaine, and high novelty-seeking trait predicts greater sensitivity to the conditioned place preference (CPP) induced by this drug. Our aim was to evaluate the influence of novelty-seeking on the effects of adolescent cannabinoid exposure. Adolescent male mice were classified as high or low novelty seekers (HNS and LNS) in the hole-board test. First, we evaluated the CPP induced by the cannabinoid agonist WIN 55212-2 (0.05 and 0.075 mg/kg, i.p.) in HNS and LNS mice. Then, HNS and LNS mice were pretreated i.p. with vehicle, WIN 55212-2 (0.1 mg/kg), or cannabinoid antagonist rimonabant (1 mg/kg) and were subsequently conditioned with WIN 55212-2 (0.05 mg/kg, i.p.) or cocaine (1 or 6 mg/kg, i.p.). Only HNS mice conditioned with the 0.075 mg/kg dose acquired CPP with WIN 55212-2. Adolescent exposure to this cannabinoid agonist increased the rewarding effects of 1 mg/kg of cocaine in both HNS and LNS mice, and in HNS mice it also increased the reinstating effect of a low dose of cocaine. Our results endorse a role for individual differences such as a higher propensity for sensation-seeking in the development of addiction.
Drug addiction is a multifactorial disorder caused by the interaction of individual and environmental factors. Among the underlying factors that contribute to an enhanced predisposition to drug addiction are the existence of a vulnerable personality or phenotype [
One of the most recognised factors facilitating the transition from voluntary, recreational drug use to dependence and addiction is exposure to drugs of abuse early on in life [
Consumption of cannabis, the most used illegal drug, usually begins during adolescence, and an increase in the problematic use of this drug among adolescents has been reported in recent years [
Regarding the individual factors that contribute to drug addiction, differences in response to novelty and impulsivity that exist before the first experience of the drug have been related to differences in sensitivity to drug reward and vulnerability to addiction [
Thus, the first aim of the present study was to evaluate the influence of the novelty-seeking phenotype on the sensitivity of adolescent mice to the rewarding effects of low doses of the CB1 agonist WIN 55212-2 in the CPP paradigm. We hypothesised that only HNS mice would acquire CPP after conditioning with a low dose of WIN 55212-2, as occurs with other drugs of abuse. The second aim was to study whether the stimulation or blockade of CB1 receptors during adolescence modifies the conditioned rewarding effects of WIN 55212-2 or cocaine and if such effects are modulated by the novelty-seeking phenotype. We expected preexposure to a cannabinoid agonist during adolescence to increase the CPP induced by low doses of WIN 55212-2 and cocaine and for this effect to be more pronounced in HNS mice.
A total of 250 male mice of the OF1 strain were acquired commercially from Charles River (Barcelona, Spain) at 21 days of age. They were housed in groups of four in plastic cages (25 × 25 × 14.5 cm) for 5 days before experiments were initiated, under the following conditions: constant temperature (21°C), a reversed light schedule (white lights on 19.30–07.30 h), and food and water available ad libitum, except during behavioural tests. Animals were handled on each of the 3 days immediately prior to the preconditioning (Pre-C) phase in order to reduce their stress levels in response to experimental manipulations. Procedures involving mice and their care were conducted in conformity with national, regional, and local laws and regulations, which are in compliance with the European Directive 2010/63/EU.
The hole-board test was carried out in a square box (28 × 28 × 20.5 cm) with transparent Plexiglas walls and 16 equidistant holes of 3 cm in diameter in the floor. Photocells below the surface of the holes detected the number of times mice performed a head dip. Frequency of head dips was recorded automatically by the apparatus (CIBERTEC, SA, Spain).
For place conditioning, we employed twelve identical Plexiglas boxes with two equal sized compartments (length 30.7 cm, width 31.5 cm, and height 34.5 cm) separated by a grey central area (length 13.8 cm, width 31.5 cm, and height 34.5 cm). The compartments of these boxes had different coloured walls (black versus white) and distinct floor textures (fine grid in the black compartment and wide grid in the white one). Four infrared light beams in each compartment of the box and six in the central area allowed the position of the animal and its crossings from one compartment to the other to be recorded. The equipment was controlled using three PCs and MONPRE 2Z software (CIBERTEC, SA, Spain).
Animals were injected i.p. with cocaine hydrochloride (Laboratorio Alcaliber SA, Madrid, Spain), WIN 55212-2 (Tocris, Biogen Científica, S.L., Madrid, Spain), or rimonabant (SR 141716A, Sanofi Recherche, Montpellier, France) in a volume of 0.01 mL/g. Control groups were injected with the physiological saline used to dissolve cocaine (NaCl 0.9%) or with Tween-80 (Sigma-Aldrich, Madrid, Spain), which was used to dissolve WIN 55212-2 and rimonabant (0.01%, 0.01 mL of Tween dissolved in 100 mL of saline). The doses of cocaine we administered were selected on the basis of previous studies showing that 1 mg/kg is a subthreshold dose for inducing CPP in naïve mice, while 6 mg/kg is effective in inducing CPP acquisition but not in producing reinstatement after extinction of CPP [
At the beginning of the test, mice were placed in the same corner of the box and were allowed to freely explore the apparatus for 10 min. The illumination in the experimental room consisted of four neon tubes fixed to the ceiling (light intensity of 110 lux at 50 cm above floor level). In all experiments, animals were first tested in the hole board on PND 26 (prior to any treatment) and defined as high novelty seekers (HNS) or low novelty seekers (LNS) according to whether the number of head dips they performed was higher or lower than the median of the group. We have previously used this median-split analysis to study the effects of novelty-seeking on the behavioural effects of different drugs of abuse [
Timeline of experiments.
Place conditioning, consisting of three phases, took place during the dark cycle. During the first phase, or preconditioning (Pre-C), mice were allowed access to both compartments of the apparatus for 15 min (900 s) per day for 3 days. On day 3, the time spent by the animal in each compartment during 900 s period was recorded. Animals showing strong unconditioned aversion for any compartment (less than 33% of the session time) were excluded from the rest of the procedure so that the CPP procedure was unbiased in terms of initial spontaneous preference [
In the second phase (conditioning), animals were conditioned with WIN 55212-2 or cocaine, as described in previous studies [
During the third phase (postconditioning, Post-C), which took place on day 8 (in the case of cocaine) or day 12 (in the case of WIN 55212-2), the guillotine door separating the two compartments was removed and the time spent by the untreated mice in each compartment was recorded during a 900 s observation period. The difference in seconds between the times spent in the drug-paired compartment in the Post-C versus Pre-C test is a measure of the degree of conditioning induced by the drug. If this difference is positive, then the drug is considered to have induced a preference for the drug-paired compartment.
Groups showing CPP in Post-C underwent an extinction session every three days (on Mondays, Wednesday, and Friday) during which they were placed in the apparatus (without the guillotine doors separating the compartments) for 15 min until the time spent by each group in the drug-paired compartment was similar to that of Pre-C and differed from that of Post-C (Student’s
In Study 1, two experiments were performed in order to study the influence of the novelty-seeking phenotype on the effects of cannabinoid exposure on the acquisition of the CPP induced by WIN 55212-2. In the first experiment, 60 male mice performed the hole-board test in order to be classified as HNS or LNS and were randomly assigned a drug treatment (0.075 or 0.05 mg/kg of WIN 55212-2). Thus, four groups of mice were formed according to novelty-seeking profile and the dose of WIN 55212-2 received during CPP conditioning (HNS+WIN 0.075, HNS+WIN 0.05, LNS+WIN 0.075, and LNS+WIN 0.05). The CPP procedure began on PND 32 and conditioning took place from PND 35 to PND 42. After the Post-C test, groups showing CPP underwent extinction and reinstatement tests. In the second experiment, 80 male mice performed the hole-board test in order to be classified as HNS or LNS and were randomly assigned a drug treatment (vehicle, 0.1 mg/kg of WIN 55212-2 or 1 mg/kg of rimonabant). Mice received one daily injection of their respective treatment for 5 days (PND 26–30) and, after an interval of 3 days without any treatment, underwent the CPP procedure following conditioning with the same dose of WIN 55212-2 (0.05 mg/kg). Thus, six groups of mice were formed according to novelty-seeking profile and the pretreatment received before conditioning with WIN 55212-2 (HNS-Veh-WIN, HNS-WIN-WIN, HNS+SR-WIN, LNS-Veh-WIN, LNS-WIN-WIN, and LNS-SR-WIN). The place conditioning procedure began on PND 34, and conditioning took place from PND 37 to PND 44.
Study 2 was performed in order to study the influence of the novelty-seeking phenotype on the effects of cannabinoid exposure on the acquisition of the CPP induced by cocaine. Eighty male mice performed the hole-board test and were defined as HNS or LNS and randomly assigned a drug treatment (vehicle, 0.1 mg/kg of WIN 55212-2 or 1 mg/kg of rimonabant). The animals received a daily injection of their respective treatment for 5 days (PND 26–30) and, after an interval of 3 days without any treatment, underwent the CPP procedure having been conditioned with the same dose of cocaine (1 mg/kg). Thus, six groups of mice were formed according to novelty-seeking profile and the pretreatment received before conditioning with cocaine (HNS-Veh-COC, HNS-WIN-COC, HNS+SR-COC, LNS-Veh-COC, LNS-WIN-COC, and LNS-SR-COC). Following the Post-C test, groups showing CPP underwent extinction and reinstatement tests. With the objective of corroborating the results obtained in the groups receiving pretreatment with WIN 55212-2 and conditioned with 1 mg/kg of cocaine, two additional groups were included in the procedure. Thirty mice performed the hole-board test in order to be classified as HNS or LNS and were then treated with 0.1 mg/kg of WIN 55212-2 for 5 days. After an interval of 3 days without any treatment, the mice underwent the CPP procedure having been conditioned with 6 mg/kg of cocaine (HNS-WIN-COC6 and LNS-WIN-COC6). In this study all the groups began the CPP procedure on PND 34, and conditioning took place from PND 37 to PND 40.
Differences between the number of dips performed by HNS and LNS groups were analysed with Student’s
The novelty scores for each mouse, identified by group, are represented in Figure
Novelty scores for each mouse, identified by group, in Study 1 ((a) experiment 1 and (b) experiment 2) and Study 2 ((c) mice conditioned with 1 mg/kg of cocaine and (d) mice conditioned with 6 mg/kg of cocaine).
Study 1 explains the influence of the novelty-seeking phenotype on the effects of cannabinoid exposure on acquisition of the CPP induced by WIN 55212-2.
Experiment
The ANOVA of the data obtained with the mice conditioned with 0.05 and 0.075 mg/kg of WIN 55212-2 revealed that the interaction “Days × Treatment × Novelty-Seeking” [
Effects of WIN 55212-2 (0.05 and 0.075 mg/kg) on the CPP paradigm in HNS and LNS adolescent mice (HNS 0.05,
Experiment
The ANOVA did not show any significant effect, thus indicating that pretreatment with a cannabinoid agonist or antagonist did not increase the sensitivity of mice to the conditioned rewarding effects of WIN 55212-2 (see Figure
Effects of WIN 55212-2 (0.05 mg/kg) on the CPP paradigm in HNS and LNS adolescent mice pretreated 6 days before initiation of conditioning with vehicle (HNS,
Study 2 explains the influence of the novelty-seeking phenotype on the effects of agonist and antagonist cannabinoid on acquisition of the CPP induced by cocaine.
The ANOVA of the data obtained with the mice conditioned with 1 mg/kg of cocaine revealed a significant effect of the interaction “Days × Pretreatment” [
CPP induced by cocaine (1 mg/kg) in HNS and LNS adolescent mice pretreated 6 days before initiation of conditioning with vehicle (HNS,
The ANOVA of the data obtained with the mice pretreated with WIN 55212-2 and conditioned with 6 mg/kg of cocaine revealed a significant effect of the variable “Days” [
CPP induced by cocaine (6 mg/kg) in HNS and LNS adolescent mice pretreated 6 days before initiation of conditioning with 0.1 mg/kg of WIN 55212-2 (HNS,
Effects of the novelty-seeking phenotype on the extinction of cocaine CPP. Mean number of days needed to achieve complete extinction of CPP in HNS and LNS mice. After conditioning with 6 mg/kg of MDMA, all groups showed CPP in the Post-C test and underwent daily extinction sessions. HNS mice required more extinction sessions to achieve complete extinction of CPP than LNS mice.
Linear and logistic regression analysis did not show any significant correlation between the novelty-seeking trait and development of the CPP induced by 1 or 6 mg/kg of cocaine, in accordance with a previous study carried out in our laboratory [
Animal models are a vital tool for increasing our understanding of the behavioural traits (e.g., novelty-seeking) and environmental events (e.g., early drug exposure) associated with the individual vulnerability of subjects to repeated drug consumption and how these factors interact to facilitate the development of drug addiction. The results of the present study demonstrate for the first time that adolescent HNS mice are more vulnerable to the rewarding effects of cannabinoids. Even more importantly, given the high risk of adverse effects associated with cocaine, there was some indication that adolescent mice with this phenotype are more vulnerable to the reinstating effects of a low dose of cocaine if they have been previously exposed to a cannabinoid agonist.
The first contribution of this study is the demonstration that the HNS phenotype increases the sensitivity of mice to the conditioned rewarding effects of the cannabinoid agonist WIN 55212-2. We have observed that HNS mice acquire CPP after conditioning with 0.075 mg/kg, a dose that is ineffective in LNS. Although no previous studies have evaluated the influence of the novelty-seeking trait on the rewarding effects of cannabinoids, our results are in accordance with those observed with other drugs of abuse, such as cocaine or MDMA, which have demonstrated that HNS mice are more sensitive to the conditioned rewarding effects of these drugs [
The second important result of the present study is that even though exposure to the cannabinoid agonist WIN 55212-2 during adolescence did not enhance the acquisition of CPP induced by WIN 55212-2 itself at the doses tested, it did enhance the acquisition of CPP induced by a low dose of cocaine. Mice pretreated with WIN 55212-2 exhibited CPP after conditioning with a low dose of cocaine that was ineffective in inducing CPP in animals pretreated with vehicle. Moreover, mice pretreated with WIN 55212-2 showed priming-induced reinstatement of the CPP induced by 6 mg/kg of cocaine, an effect that has not been observed in naïve mice [
ECS plays an important role in adolescent brain development, and the strong stimulation of this system by cannabinoids might induce long-lasting neurobiological consequences, such as alterations in emotional and cognitive performance, increased risk of developing schizophrenia, and enhanced vulnerability to the use of drugs of abuse [
On the other hand, neither exposure to the cannabinoid agonist WIN 55212-2 during adolescence nor pretreatment with rimonabant modified the subsequent effect of WIN 55212-2 in the CPP paradigm, in contrast with that observed with cocaine in this study or in previous studies with morphine or MDMA [
The main result of the present work is that the novelty-seeking phenotype determines the influence of adolescent cannabinoid exposure on the subsequent rewarding effects of cocaine in the CPP paradigm. Not all mice are equally vulnerable to the sensitization of the brain reward system induced by stimulation of the cannabinoid system during adolescence. HNS mice are particularly affected by pretreatment with WIN 55212-2, showing a priming-induced reinstatement of the CPP induced by 1 mg/kg of cocaine and an enhanced duration of the CPP induced by 6 mg/kg of this drug (effects that are not observed in LNS mice exposed to WIN 55212-2). Our results support the idea that exposure to cannabis during adolescence, though it can increase the rewarding effects of subthreshold doses of cocaine six days after pretreatment, is not enough to promote long-lasting brain changes that increase the likelihood of the development of cocaine addiction (which can be evaluated by the maintenance of CPP or its reinstatement after extinction). Genetic and behavioural predispositions—for example, a novelty-seeking phenotype—may underlie increased adolescent drug experimentation, enhanced reward when the subject is exposed to the drug, and the development of neuroadaptations that lead to later adult addiction.
Animal models allow us to answer questions that cannot be explored in human subjects due to ethical constraints and can be useful for analysing possible neurobiological substrates underlying interactions between environmental and biological factors that contribute to individual vulnerability to drug abuse and addiction. The phenotypic causation gateway hypothesis proposes a sequential progression of drug use in which early initiation of cannabis use is a risk factor for the future consumption of other drugs of abuse, such as cocaine [
There is a subpopulation of adolescents which engages in extremely risky cannabis and drug use early in life and which seems to run a greater risk of abuse and addiction later in life. The results of the present study suggest that there is not a direct causal mechanism between adolescent drug exposure and the subsequent development of addiction. Instead, there are individual brain and behavioural differences that are present prior to the onset of drug use, such as a higher propensity for sensation-seeking, which influence both the tendency to experiment with drugs of abuse early in life and the later development of addiction. These subjects appear to be more vulnerable to the appearance of permanent neurobiological changes following drug exposure that may lead to the transition from voluntary to compulsive drug use. Thus, specific preventive programs aimed at these more vulnerable subjects could reduce drug consumption and later addiction.
The authors are entirely responsible for the scientific content of the paper.
The authors declare no conflict of interests.
This work was supported by the following grants: Ministerio de Economía y Competitividad (MINECO), Dirección General de Investigación, PSI2011-24762 and PSI2014-51847-R; Instituto de Salud Carlos III, Red de Trastornos Adictivos (RTA) RD12/0028/0005, and Unión Europea, Fondos FEDER “una manera de hacer Europa”; Ministerio de Sanidad, Servicios Sociales e Igualdad; Delegación del Gobierno para el Plan Nacional Sobre Drogas, Proyectos de Investigación sobre Drogodependencias, 2014I007; Generalitat Valenciana, Conselleria de Educación, PROMETEOII/2014/063, Spain. The authors wish to thank Brian Normanly for editing the English language of the paper.