Effect of the CB1 cannabinoid agonist WIN 55212-2 on the acquisition and reinstatement of MDMA-induced conditioned place preference in mice

Numerous reports indicate that MDMA users consume other psychoactive drugs, among which cannabis is one of the most common. Findings regarding concomitant abuse of MDMA and cannabis are similar in different countries, ranging from between 73% and 100% [for review see [1, 2]]. Several studies of the prolonged combined use of MDMA and cannabis have highlighted an association with a variety of psychological problems, including elevated impulsiveness, anxiety and psychotic behavior [3].

MDMA is an indirect monoaminergic agonist, [4, 5]. Cannabinoids exert their effect through interactions with specific endogenous CB1 and CB2 cannabinoid receptors [6, 7] that are present in mammalian tissues. Many of the physiological responses provoked by MDMA are modulated by the endocannabinoid system [8, 9]. This system interacts with a variety of neurotransmitters, including DA and 5-HT [9, 10], and represents a common neurobiological substrate for the addictive properties of different drugs of abuse [11, 12].

Both MDMA and cannabinoid agonists such as WIN 55212-2 (WIN) produce rewarding effects in mice [13, 14] and rats when administered alone [15]. The few previous studies carried out to clarify the nature of the effects of exposure to cannabinoids on MDMA abuse liability, all of which were performed in rats, suggest that cannabinoid agonists potentiate the rewarding effects of MDMA [16] and that cannabinoid antagonists exhibit an opposite action [17]. However, a recent study by Robledo and co-workers [18] using mice has demonstrated that THC modifies the sensitivity of these animals to the behavioral effects of MDMA in different ways (increase/decrease) depending on the dose employed. The complex relation between cannabinoids and MDMA requires further study, especially regarding the potential mutual strengthening of their rewarding effects. The aim of the present study was to use the conditioned place preference (CPP) procedure to evaluate the influence of the cannabinoid agonist WIN 55212-2 on the rewarding effects of MDMA and the reinstatement of an extinguished preference in adolescent mice. This work may help to better understand the effects of polydrug abuse, particularly as we have chosen to study two of the most frequently used substances in adolescents. Firstly, we studied the rewarding effects of both drugs on the CPP and the ability of MDMA to reinstate the extinguished preference. The doses employed were chosen on the basis of previous work in our laboratory [13, 14]. In order to assess whether or not the stimulation of the cannabinoid system increases the rewarding properties of MDMA, the effects of co-administration of WIN with rewarding and non-rewarding doses of MDMA were evaluated in the CPP model. The role of the CB1 cannabinoid receptor in the potentiating effects of WIN was tested using the CB1 antagonist SR 141716 (SR). In these experiments the ability of a priming dose (50% of that used for conditioning) of MDMA to reinstate the extinguished preference was evaluated. In a second set of experiments the ability of a priming dose of WIN to reinstate an MDMA-induced CPP (cross-reinstatement) was tested. Additionally, levels of brain monoamines and their metabolites were determined in the striatum, hippocampus and cortex of some of the experimental groups.

The results of the present study confirm the complex interaction between the cannabinoid system and MDMA. The cannabinoid agonist WIN 55212-2 can increase or decrease the acquisition of MDMA-induced CPP depending on the dose employed. The CB1 receptor is not involved in the potentiating effect of WIN. We have also observed cross-reinstatement, since MDMA was capable of reinstating the WIN-induced CPP. Additionally, in contrary to the presumed neuroprotective role of cannabinoids, the present results show that WIN 55212-2 induces a decrease in the concentrations of dopamine in the striatum and serotonin in the cortex when administered with a non-neurotoxic dose of MDMA.

We have confirmed that MDMA is capable of inducing CPP even at low doses (2.5 mg/kg), and that reinstatement of the CPP can be achieved with priming doses 50% lower than that used for conditioning. In addition, the CB1 agonist WIN also induced CPP with the highest dose employed (0.5 mg/kg). Cross-reinstatement was observed, since the WIN-induced preference was reinstated after a priming dose of MDMA (5 mg/kg). The results obtained in these experiments confirm and extend those of a previous report by our group [14]. On the other hand, the induction of place preference with cannabinoid agonists and antagonists has been reported in several studies [20‐23]. Specifically, WIN 55212-2 induces CPP when administered peripherically [13] or injected into the hippocampus [15]. We have no knowledge of previous reports of cross-reinstatement between a cannabinoid agonist-induced CPP and MDMA. The strength of this reinstatement in the WIN-induced CPP group was similar to that in the group conditioned with MDMA, or even higher. Moreover, more time was required for the reinstated preference to be extinguished in mice conditioned with the cannabinoid agonist. The most important implication of the cross reinstatement observed is that exposure to MDMA can produce an intense craving and relapse to drug-taking behavior in cannabinoid-dependent humans.

On the other hand, the MDMA-induced CPP was not reinstated by priming injections of WIN (second experiment). Thus, cross reinstatement was not observed in this case. One possible justification for this lack of reciprocity is that these drugs have a particular neurotransmitter activation profile. MDMA is a DA releaser that affects other systems, such as the serotoninergic system, but cannabinoids seem to play a modulating role in DA transmission. Another possible explanation is that, in animals conditioned with MDMA, the first exposure to the cannabinoid agonist induces an aversive effect that does not recall the preference induced by MDMA. Conditioned place aversion induced by WIN has previously been reported [24]. We have recently induced reinstatement of MDMA-induced CPP by priming doses of WIN (0.1 mg/kg) in mice previously exposed to this cannabinoid agonist [25], which gives support to the latter hypothesis.

On the other hand, the CB1 agonist WIN exerted a dual effect on the MDMA-induced CPP. The highest WIN dose (0.5 mg/kg), which in itself induced CPP, diminished preference when administered with an effective dose of MDMA (2.5 and 5 mg/kg). On the other hand, a low dose of this CB1 agonist (0.1 mg/kg), inactive when administered alone, strengthened the rewarding effects of an inactive dose of MDMA (1,25 mg/kg) and induced CPP. Moreover, the time needed for the preference to be extinguished was longer in the groups treated with the lowest dose of WIN 55212-2. Our results are in accordance with those of Robledo et al [18], who reported that a sub-threshold dose of THC produced CPP in mice when combined with a non-rewarding dose of MDMA (3 mg/kg), but decreased the CPP induced by an effective dose of MDMA (10 mg/kg).

Self-administration studies have also confirmed that the endogenous cannabinoid system influences the mechanism that regulates MDMA's rewarding effect. Braida and Sala [16] demonstrated that the cannabinoid agonist CP 55,940 altered i.c.v. MDMA self-administration and significantly reduced MDMA intake, suggesting a synergistic action of cannabinoid agonists on the rewarding properties of MDMA. On the other hand, in a report by Robledo et al [18], the heightened operant response induced by the cannabinoid antagonist SR in MDMA self-administration indicated a decrease in sensitivity to the motivation for obtaining the drug. In addition, THC increased the threshold for MDMA when self-administrated.

Cannabinoids participate in the regulation of dopamine synthesis, release and turnover [26]. The overlapping expression of cannabinoid and dopamine receptors in some brain areas, such as the NAC [27], may represent a neuroanatomical substrate for such an interaction. The complex relation between cannabinoids and MDMA is also observed in microdialysis studies of DA release into the NAC. A low dose of THC was shown to significantly increase DA outflow in the NAC, while a low dose of MDMA did not. When MDMA was administered before THC, DA levels were lower with respect to those of THC. However, when THC was administered before MDMA, DA levels were not significantly modified with respect to those of THC [18]. If we apply these results to our experiment in which both drugs (MDMA and WIN) were administered together, we can hypothesize that doses of MDMA or WIN that in themselves can increase DA release would exert an antagonistic action on DA; i.e, DA would not be released.

The cannabinoid-selective CB1 antagonist SR 141716 did not affect the CPP or reinstatement of the preference induced by 5 mg/kg of MDMA when administered alone or with WIN. In the groups conditioned with 1.25 mg/kg of MDMA, which was not effective by itself, CPP was observed when administered with the CB1 antagonist. Moreover, when this sub-threshold dose of MDMA was administered with 0.1 mg/kg of WIN, the CPP induced was not affected by blockade of the CB1 receptor. These results can be explained if the sense that the CB1 cannabinoid agonist WIN exerts its effects in a way that does not involve the CB1 receptors. Although WIN is a CB1 receptor agonist, some of its effects seem to be mediated by mechanisms that do not involve cannabinoid receptors, and which could be calcium-dependent mechanisms [28]. On the hand, SR141716 is capable of producing conditioned place preference in rats [29, 21], although studies performed in our laboratory failed to detect an motivational action of this CB1 antagonist [13]. In a similar way to WIN, SR 141716 is reported to exhibit an independent CB1 activity; for instance, it exerts an anxiolytic effect on CB1 knockout mice [30] The aversive effects of cannabinoid agonists observed in many studies, and the rewarding effect of cannabinoid antagonists are suggestive of a cannabinergic tone in the rat brain. Cannabinoid receptors, while present in areas of the brain that support electrical self-stimulation, such as the nucleus accumbens or the lateral septum [31], are also localized in the central and basolateral amygdaloid nuclei and in the periaqueductal gray, two areas that support electrically-induced fear responses and are thought to be the sites of action of certain anxiogenic drugs. The observation by Herkenham and Brady [32] that D9-THC induces c-Fos expression in stress-responsive nuclei of the rat brain further supports an anxiogenic role of cannabinoids. Recently, Wu and co-workers [33] have demonstrated that SR141716A employed at the dose used in the present study (3 mg/kg, i.p.) produces an increase of DA release in the dorsal and ventral striatum, although inhibition of dopamine release induced by nicotine, ethanol, and cocaine has been described for other authors [34]. The present data suggest that endogenous cannabinoids both inhibit reward and induce aversion by actions exerted within these circuits [29].

The role of CB1 cannabinoid receptors in the rewarding effects of drugs of abuse is not altogether clear [24, 35‐37]. Although SR reduces the acquisition of CPP induced by cocaine, morphine or food in a dose-depend manner, it does not affect the expression of the cocaine-induced CPP [24]. More recently, studies performed in CB1 knockout mice [38] have shown that they develop a normal CPP despite the fact that many of the effects of MDMA are diminished in these animals, among them self-administration. The CB1 cannabinoid receptor is essential in the acute rewarding effects of several drugs of abuse that produce their effect on the ventral tegmental area, such as nicotine, ethanol and morphine. However, psychostimulant drugs produce their effect directly on the nucleus accumbens DA terminals [11]. For that reason, the CB1 cannabinoid receptor does not seem to contribute to their acute rewarding effects when evaluated in the CPP. Accordingly, previous studies have reported that cocaine-induced CPP but not self-administration is preserved in CB1 knockout mice [39]. In the study of Braida et al [17], SR blocked the MDMA-induced CPP, although there are several and important differences between this and the present study, including the species used and the form of MDMA administration (intracerebroventricular). Moreover, SR increased the rewarding effects of 1.25 mg/kg MDMA. We can hypothesize that, when SR is administered jointly with WIN, the antagonist blocks the CB1 receptor and in that way inhibits WIN action, while at the same time decreasing the cannabinoid tone and indirectly increasing the rewarding action of MDMA.

As we have previously observed [14], MDMA did not affect the concentration of DA or serotonin, or their metabolites in the striatum or cortex at the doses employed during the CPP procedure. However, an increase in serotonin concentration (5 mg/kg) and decreases in the levels of 5-HIAA were observed in the hippocampus. The CB1 agonist WIN did not affect the brain monoamines, with the exception of an increase in the level of 5-HIAA in the hippocampus with the lowest dose (0.1 mg/kg). Nevertheless, in the striatum of the animals treated with the highest doses of MDMA and WIN, a decrease in the DA concentration (over 40%) was observed. A reduction in cortical serotonin occurred in the same group (over 28%) and in the groups treated with the highest dose of MDMA plus WIN 0.1 (in which there was also an increase in the levels of 5-HIAA in the hippocampus) or the lowest dose of MDMA plus the highest dose of WIN (over 20% of a reduction of serotonin in both groups).

The anti-inflamatory and anti-oxidative properties of cannabinoids point to their possible beneficial effects with respect to limiting neurological damage, particularly by decreasing neuroinflamation [40, 41]. However, animal studies have revealed that chronic administration of the cannabinoid THC causes hippocampal damage [42], and several reports have highlighted that exposure to low concentrations of cannabinoids over a prolonged period is likely to have a neurotoxic effect [43, 44]. Acute administration at high concentrations of cannabinoids at the time of trauma, on the other hand, may be neuroprotective. The decrease in striatal DA and cortical serotonin in animals treated with 5 mg/kg of MDMA plus the highest dose of WIN (0.5 mg/kg) could explain the surprising lack of CPP observed in this group. With a daily conditioning schedule we have seen that 5 mg/kg of MDMA induces a similar decrease in cortical serotonin and striatal DA and does not achieve preference in the Post-C test [14]. However, these results should be interpreted with caution, as the measurements were obtained only 48 h after the last drug administration.

The results of the present study confirm that the cannabinoid system plays a role in the rewarding effects of MDMA. A high proportion of MDMA users also consume cannabis, which makes the study of the interaction between the two drugs highly relevant. Moreover, the observed cross-reinstatement between these drugs highlights the importance of sporadic drug use in relapse to dependence. To conclude, our results do not support a neuroprotective action of cannabinoids; the present data show that WIN 55212-2 induces a decrease in the concentrations of dopamine and serotonin in the striatum and cortex 48 hours after administration in conjunction with a non-neurotoxic dose of MDMA.