Roles of cannabinoid CB1 and CB2 receptors in the modulation of psychostimulant responses

Abstract

Addiction to psychostimulant drugs, such as cocaine, D-amphetamine, and methamphetamine, is a public health issue that substantially contributes to the global burden of disease. Psychostimulant drugs promote an increase in dopamine levels within the mesocorticolimbic system, which is central to the rewarding properties of such drugs. Cannabinoid receptors (CB₁R and CB₂R) are expressed in the main areas of this system and implicated in the neuronal mechanisms underlying the rewarding effect of psychostimulant drugs. Here, we reviewed studies focusing on pharmacological intervention targeting cannabinoid CB₁R and CB₂R and their interaction in the modulation of psychostimulant responses.

Summation

The search identified studies that evaluated the rules of pharmacological and genetic modulation of CB1 and CB2 cannabinoid receptors in the regulation of psychostimulant responses. Most studies demonstrated that activation of CB2R and inhibition of CB1R inhibited behavioural and molecular effects induced by distinct psychostimulants.

Considerations

Although studies reported that blockade of CB1R inhibits psychostimulant effects, the incidence of serious psychiatric adverse events, limits the use of selective CB1R antagonists for treating psychostimulant addiction disorders. Drugs targeting CB2R signalling might represent a more promising approach.

Moreover, preclinical studies only have focused on male mice and rats, excluding female animals. As sexual dimorphism has been demonstrated in behavioural and molecular responses correlated to cannabinoid receptors, the role of CB1R and CB2R in regulation of psychostimulants in female animals should be explored in future studies.

Introduction

Psychostimulants are a broad class of drugs that englobe cocaine, amphetamine, and its derivatives [i.e., methamphetamine, N-methyl- 3,4-methylenedioxymethamphetamine (MDMA)]. Psychostimulant addiction is a public health issue that substantially contributes to the global burden of disease (UNODC, 2021). This chronic pathology is characterised by complex behavioural and neurobiological phenomena entailing the compulsive use of a substance (Wise and Koob, 2014, Volkow and Morales, 2015). The mechanisms for the addictive properties of drugs, including psychostimulants, involves the facilitation of reward centres in the brain, particularly the mesocorticolimbic dopaminergic pathways connecting the ventral tegmental area (VTA) with various limbic structures, such as nucleus accumbens (NAcc), prefrontal cortex (PFC), and hippocampus (Koob and Volkow, 2010). Acute and chronic exposure to psychostimulants cause both transient and persistent adaptations in regions of the mesocorticolimbic neurocircuitry, resulting in altered behavioural responses, ultimately, leading to drug addiction (Rothman and Baumann, 2003, Howell and Kimmel, 2008).

Several pieces of evidence show that the endocannabinoid system modulates the reward-related effects of dopamine and that this system might be involved in the neurobiological mechanism underlying psychostimulant addiction (Wiskerke et al., 2008, Manzanares et al., 2018). The endocannabinoid system comprises the endogenous ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG), the enzymes responsible for their synthesis and degradation and the cannabinoid receptors (Hillard, 2015). AEA and 2-AG are the main endocannabinoids, whose effects are mediated mainly by two metabotropic receptor termed CB₁R and CB₂R (Hillard, 2015, Lu and Mackie, 2016). Both cannabinoid receptors are expressed in mesolimbic pathways and can modulate excitability of dopaminergic neurons (Howlett et al., 1990, Onaivi et al., 2006, Covey et al., 2017). In accordance with their localisation, accumulating reports have pointed to the involvement of CB₁R and CB₂R in the main behavioural effects of psychostimulants (Wiskerke et al., 2008, Vlachou and Panagis, 2014). Moreover, cannabinoid receptors also display an important role in regulating molecular responses of these drugs (Wiskerke et al., 2008, Vlachou and Panagis, 2014, Parsons and Hurd, 2015). Interestingly, these studies suggest that cannabinoid receptors work with opposing functions to modulate certain behavioural- and molecular-related effects of these drugs, since an activation of CB₂R leads to similar results as compared to CB₁R blockade.

The focus of the present review is to discuss the distinct behavioural responses induced by psychostimulants and how a differential modulation of CB₁R and CB₂R can regulate them. The molecular mechanisms through which CB₁R and CB₂R change the neuroadaptations promoted by psychostimulants will also be explored. Finally, we will propose an overarching hypothesis integrating both receptors in the pharmacological modulation of psychostimulant effects.

Psychostimulants: mechanisms of action and behavioural responses

Psychostimulants are a broad class of psychotropic substances with the capacity to stimulate various functions of the central nervous system, including attention, vigilance, alertness, arousal, and locomotion (Favrod-Coune and Broers, 2010). Intense hedonic feelings characterised as a “rush” are also described (Boutrel and Koob, 2004, McCreary et al., 2015). Most of them act by directly facilitating mesocorticolimbic terminals by either inhibiting the dopamine transporter (DAT) or facilitating the release of dopamine (Harris and Baldessarini, 1973, Nestler, 2004). While cocaine binds predominantly to DAT and inhibits dopamine reuptake, amphetamine has two more complementary action mechanisms to elevate dopamine levels (Sulzer et al., 2005). This drug can act by reversing the vesicular monoamine transporter, leading to a large release of the cytoplasmic and vesicular stores of dopamine (Robertson et al., 2009). An additional mechanism of action for amphetamines is the facilitation of the output of dopamine from vesicles into the cytoplasm (Sulzer et al., 2005). Both mechanisms also lead to an enhanced dopaminergic signalling in the mesocorticolimbic circuitry.

The use of experimental animal models is an important strategy to obtain direct insights into the molecular and behavioural effects promoted by psychostimulants (McCreary et al., 2015). Administration of cocaine and amphetamine in laboratory animals induces a dose-dependent increase in locomotor activity. Moreover, repeated exposure to these drugs leads to the development of behavioural sensitisation, which is characterised by a progressively increasing behavioural response to repeated drug exposure (Sanchis-Segura and Spanagel, 2006, Steketee and Kalivas, 2011). This phenomenon is observed, for example, as an increase in locomotor activity, which becomes even more pronounced in animals previously exposed to single or repeated administration of the same psychostimulant (Shuster et al., 1977, Steketee and Kalivas, 2011).

The rewarding properties of cocaine and amphetamine are largely assessed using the conditioned place preference (CPP) paradigm (Bardo and Bevins, 2000, Wiskerke et al., 2008). CPP is generally performed in a box containing two distinct compartments with different contextual cues, which are equally explored by the rodents in a pre-test session. The conditioning phase is performed by administering the psychostimulants and keeping the animal confined to one compartment (the conditioned stimulus), whereas vehicle injection is paired with the other compartment, in alternate periods. After the conditioning phase, the test session is performed in the absence of the drug, and the animals can explore both sides of the box. An increase in the time exploring the drug-paired compartment, compared to time spent in the vehicle-paired side, is suggestive of the rewarding effect of the drugs (Bardo and Bevins, 2000, Sanchis-Segura and Spanagel, 2006).

Drug self- administration has been one of the most direct approaches to study the rewarding properties of cocaine and amphetamine in experimental animals (Gardner, 2000). In this behavioural model, rodents are trained to perform an operant response (e.g., a lever press or nose poke) for an infusion of drug, typically accompanied by a concurrently-delivered, discrete cue such as a light and a tone. Different kinds of schedule might be used to obtain the drug, being the most largely used the fixed ratio (FR) schedule and progressive ratio (PR) schedule (Gardner, 2000, Farrell et al., 2018). Briefly, under a FR schedule, the psychostimulant is delivered every time that a pre-selected number of responses are completed. Conversely, under a PR schedule, the required ratio increases following a predefined progression, which usually is an arithmetic one. Breakpoints in this schedule, which reflects the motivation for the drug, can be defined as the maximum response rate achieved to obtain a single infusion of psychostimulant before the animal fails to complete the next ratio requirement (Gardner, 2000, Panagis et al., 2014).

Both CPP and self-administration paradigms can be used to assess relapse, another important property of psychostimulants. Once self-administration and place preference behaviours are established, animals undergo extinction training during which they are re-exposed to the drug environment in the absence of psychostimulants. After these extinction processes, animals can be tested for reinstatement, which are often precipitated by exposure to a small priming dose of drug, experiencing acute stress, or encountering discrete or contextual cues previously paired with drug use (Shaham et al., 2003, Farrell et al., 2018).

Over the years, the use of preclinical models has helped to elucidate the cellular and molecular aspects regarding the neurobiology of psychostimulant drugs, as well as new potential strategies for the pharmacological modulation of psychostimulant actions including, compounds targeting CB₁R and CB₂R.

Overview of CB₁R and CB₂R

Cannabis is one of the first plants to be used as a medicine and a drug of abuse by the humankind (Zuardi, 2006). This plant is the source of a set of more than 100 compounds, among which is Δ9-tetrahydrocannabinol (Δ9-THC), the main responsible for the psychoactive effects of the plant (Mechoulam and Hanus, 2000, Hanus et al., 2016). After the identification of Δ9-THC, researchers focused their efforts in elucidating the pharmacological mechanism underlying its effects (Mechoulam and Hanus, 2000, Pertwee, 2006). Complementary studies with Δ9-THC synthetic derivatives, including radioactive ligands, provided convincing evidence regarding the existence of specific cannabinoid receptors (Devane et al., 1988, Pertwee, 2006). Currently, two major types of receptors have been characterised and cloned, CB₁R and CB₂R (Devane et al., 1988, Munro et al., 1993, Pertwee, 2010).

The CB₁R is one of the most abundant Gi protein alpha subunit (Gi/o) protein-coupled receptors in the brain (Howlett et al., 2002). The activation of these presynaptic receptors leads to inhibition of neurotransmitter release by a mechanism that involves inhibition of voltage-gated calcium (Ca²⁺) channels and activation of inwardly rectifying potassium (K⁺) channels via the stimulated cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway (Kano, 2014, Howlett and Abood, 2017). CB₁R expression was described in distinct regions of mesocorticolimbic dopaminergic pathways, including hippocampus, PFC, and NAcc (Howlett et al., 2002). These regions are involved in motivational and reward processes, which are modulated by endogenous and exogenous CB₁R ligands (Koob and Volkow, 2010, Wenzel and Cheer, 2018).

Regarding the CB₂R, early evidence suggested that this receptor might be absent in brain and restricted to peripheral tissues. However, after the development of more selective and sensitive tools, it was possible to identify CB₂R in the central nervous system (Van Sickle et al., 2005, Onaivi et al., 2006). Indeed, CB₂R is distributed extensively in different brain areas, such as hippocampus, PFC, amygdala, olfactory nucleus, striatum, and thalamus (Chen et al., 2017). The CB₂R shares 44% homology with the CB₁R and also is coupled to Gi/o protein (Howlett et al., 2002). CB₂R also modulates the activity of Ca²⁺ and K⁺ channels, and recent electrophysiological and biochemistry findings confirmed the functionality of these receptors in mesocorticolimbic pathways (Zhang et al., 2014, Howlett and Abood, 2017, Jordan and Xi, 2019).

This convergence of cannabinoid receptors in the central nervous system, especially in mesolimbic circuitry, is consistent with the reward effects of synthetic and natural cannabinoids (Gessa et al., 1998, Zhang et al., 2014, Li et al., 2021). In addition, CB₁R and CB₂R are crucial mediators of synaptic plasticity in mesolimbic pathways, an important component in the control of motivated behaviour promoted by drugs that promote addiction (Xi et al., 2011, Garcia-Gutierrez et al., 2013, Zlebnik and Cheer, 2016). Therefore, CB₁R and CB₂R not only underlie the rewarding effects of cannabis, but can also interact with other drugs of abuse, including psychostimulants (Wiskerke et al., 2008, Zhang et al., 2014).

Role of CB₁R in psychostimulant responses

Activation of CB₁R is essential for the establishment of addiction to cannabinoid drugs (Wenzel and Cheer, 2018). Moreover, both genetic and pharmacological approaches strongly suggest a role for CB₁R signalling on responses to other drugs of abuse, including psychostimulants (Wiskerke et al., 2008).

CB₁R and psychostimulant motor effects

Acute administration of cocaine or amphetamine induces a robust increase in locomotor activity (hyperlocomotion) in animals exposed to an open field. Administration of rimonabant, a CB₁R antagonist/inverse agonist, dose-dependently inhibits the hyperlocomotion induced by d-amphetamine and cocaine in rodents previously exposed to the open field (Poncelet et al., 1999, Gobira et al., 2019). A similar effect has been observed after pharmacological blockade of CB₁R by AM251, a more selective CB₁R antagonist/inverse agonist (Corbille et al., 2007). Accordingly, locomotor responses to cocaine were also significantly reduced in CB₁R knockout mice (Li et al., 2009).

Converging evidence also supports that blockade of CB₁R regulates behavioural sensitisation induced by psychostimulants. The development of single-trial cocaine- and amphetamine-induced locomotor sensitisation was impaired in CB₁R knockout (KO) mice or after CB₁R pharmacological blockade in wild-type mice (Corbille et al., 2007, Mereu et al., 2015, Delis et al., 2017, Lopes et al., 2019). The sensitised locomotor response to a single cocaine challenge was also reduced in rats pretreated with rimonabant (Filip et al., 2006). These pharmacological studies evaluated the expression of psychostimulant locomotor sensitisation, since the blockade of CB₁R was performed before the cocaine challenge. By injecting the CB₁R antagonism on the first day of test, a recent work demonstrated that the acquisition of motor sensitisation also was impaired by blockade of these receptors (Lopes et al., 2019).

Despite this evidence, other studies have demonstrated that neither genetic silencing nor pharmacological inhibition of CB₁R altered psychostimulant ability to induce motor sensitisation (Martin et al., 2000, Lesscher et al., 2005). In addition to distinctions in animal species and strains, these discrepancies might result from differences in the dose of psychostimulant and number of injections during acquisition phase (single or repeated drug injection). The context of CB₁R antagonist administration also appears to be important in the regulation of behavioural sensitisation. For instance, Gerdeman and co-workers observed that rimonabant did not diminish the established cocaine sensitisation if delivered in the home cage, but only if the rimonabant-injected mice were exposed to activity chambers previously paired with cocaine (Gerdeman et al., 2008).

In accordance with behavioural responses, CB₁R also appears to be relevant in psychostimulant-activated signalling pathways. cAMP-dependent phosphorylation of glutamate receptor 1, promoted by cocaine, was altered in the striatum of CB₁R -null mice (Corbille et al., 2007). Moreover, phosphorylation of extracellular signal-regulated kinase (ERK) promoted by cocaine and D-amphetamine were prevented in the dorsal striatum, as well as in the NAcc core and shell of CB₁R mutant mice (Corbille et al., 2007). Corroborating these findings, blockade of CB₁R prevented cocaine-induced increased in c-Fos expression in the shell and core portions of NAcc, and (Gobira et al., 2018). Altogether, these results provide evidences that CB₁R is essential for biochemical responses to psychostimulants that are intrinsically correlated with locomotor behavioural effects.

CB₁R and psychostimulant reward and reinforcement

The endocannabinoid signalling has also been implicated in the modulation of psychostimulant-induced reward, as evaluated in the CPP test. Administration of CB₁R antagonist before cocaine or methamphetamine injections, in the conditioning phase, impaired dose-dependently the acquisition of CPP (Yu et al., 2011, Delis et al., 2017, Lopes et al., 2019). CB₁R antagonist also decreased the expression of cocaine-induced CPP. Blockade of CB₁R also prevented neuronal activation in the hippocampus of animals exposed to cocaine-CPP (Lopes et al., 2019). Interestingly, no effect was observed when the CB₁R blockade was performed only on the test day (Chaperon et al., 1998, Lopes et al., 2019). These data suggest that CB₁R might be important in the consolidation of psychostimulant-paired memories, but is not involved in the retrieval of these memories (Lopes et al., 2019). Despite pharmacological findings indicating that CB₁Rs are involved in psychostimulant reward memory, cocaine-induced CPP was unaffected in CB1R KO mice (Martin et al., 2000, Houchi et al., 2005). These differences are not clear, but might be explained by compensatory changes in the CB1R KO mice, since CB1R is important during development for establishing proper neuronal connectivity in brain regions related to memory and reward (Berghuis et al., 2007).

Intravenous drug self-administration is one of the most used approaches for studying drug reinforcement. Experiments using this paradigm also provide evidence that CB₁Rs play a critical role in psychostimulant-induced reinforcing properties. A significant reduction in acquisition of cocaine self-administration was observed in CB₁R KO mice compared with wild type (Soria et al., 2005). The number of sessions required to CB₁R null mice to achieve this behaviour was increased (Soria et al., 2005). Pharmacological blockade of CB₁R with SR141716A in wild-type mice promoted similar effects (Soria et al., 2005). Evidence also pointed that the maximal effort to obtain a cocaine infusion, in PR reinforcement schedule, was significantly reduced after the genetic and pharmacological ablation of CB₁R (Soria et al., 2005, Xi et al., 2008). Treatment with CB₁R antagonists, in a dose-dependent manner, lowered the break point for cocaine self-administration under a PR reinforcement schedule in rats (Xi et al., 2008). Similarly, the blockade of CB₁R suppressed the intake of methamphetamine in rats trained to self-administer this drug (Vinklerova et al., 2002).

Intriguingly, these reports are countered by studies which demonstrated that pharmacological and genetic inactivation of CB₁R were ineffective to modulate cocaine and amphetamine self-administration under FR schedules (Cossu et al., 2001, Lesscher et al., 2005). Besides the differences in ratio schedule, the extension in the period of cocaine self-administration also appears to be important to the effect of CB₁R in modulation of drug-intake. For example, blockade of CB₁R reduces the breakpoint for cocaine self-administration in rats that had 6 h to access the drug. On the other hand, a lower efficacy of CB₁R antagonist was observed in rats that access cocaine only 1 h daily (Orio et al., 2009). In accordance with those findings, the levels of both phosphorylated and total CB₁R protein were increased only in the NAcc of rats given extended daily access to cocaine (Orio et al., 2009). In the extended access regimen, the intake of the drug gradually increases over days, on the other hand the consume of cocaine remains stable in animals under short access protocol (Ahmed and Koob, 1998, Wee et al., 2007). This escalated drug intake also is associated with increased breakpoints or responding for cocaine under a progressive ratio (PR) schedule of reinforcement, both processes that demonstrated pivotal role of CB₁R in modulating psychostimulant behaviour (Wee et al., 2007). Therefore, this evidence suggested that the capacity of CB1R to regulate the rewarding properties of psychostimulants might influence the motivation to obtain these drugs.

Increases in dopamine extracellular levels in the NAcc have been related to the primary reinforcing effects of psychostimulants (Di Chiara, 1998). Similar to observed in the modulation of self-administration, the importance of silencing of CB₁R in regulation of levels of dopamine in the NAcc also appears to be complex. Striatal extracellular dopamine response to acute cocaine was reduced in CB₁R KO mice (Li et al., 2009). A similar result was obtained after the pharmacological blockade of these receptors in wild-type mice (Li et al., 2009). Although this is consistent with findings that rimonabant inhibits cocaine- and amphetamine-induced dopamine release in rats (Cheer et al., 2007, Covey et al., 2016), both basal and cocaine-induced increase in extracellular levels of dopamine in the NAcc were unaffected in CB₁R KO mice (Soria et al., 2005) or after treatment with CB₁R antagonist (Caille and Parsons, 2006). Differences in the genetic background of the KO animals and methods to evaluate dopamine levels might explain these discrepant results. For example, while no changes in cocaine-enhanced dopamine release were observed in KO mice from CD1 background (Soria et al., 2005), alterations in dopamine levels following cocaine injections were obtained in CB₁R -null mice with a C57BL/6J genetic background (Li et al., 2009).

A recent study, using modern molecular tools to selectively ablate CB₁R on specific subtypes of neurons, provided interesting novel insights that clarified the role of these receptors in the regulation of dopamine levels in the NAcc in animals submitted to self-administration paradigm. A lower training dose was required to acquire cocaine self-administration for the mutant mouse lines with CB₁R deletion targeted in forebrain GABAergic (GABA-CB1-KO) neurons, suggesting an increased sensitivity to the aversive effect of high unit drug doses (Martin-Garcia et al., 2016). Conversely, at low doses, GABA-CB₁R-KO mice self-administered more than the wild type, confirming an increased sensitivity to the positive reinforcing effects of cocaine (Martin-Garcia et al., 2016). A dopaminergic mechanism appears to be involved in this behavioural response, since naïve GABA-CB₁R-KO mice showed increased cocaine-induced dopamine release in the NAcc (Martin-Garcia et al., 2016). Authors also observed that silencing of cortical glutamatergic neurons did not change cocaine’s primary reinforcing effects as revealed by the similar dose-response curves for cocaine self-administration in this genotype compared to wild type (Martin-Garcia et al., 2016).

Overall, blockade of CB₁R might curb behavioural and dopaminergic responses correlated to psychostimulant reward. However, these effects are sensitive to variations in the experimental protocol, such as dose of psychostimulant, ratio schedule, and the extension in the period of drug self-administration. Moreover, recent evidence demonstrated that CB₁R located on glutamatergic and in GABAergic neurons contribute differentially to the effect of psychostimulants.

CB₁R and psychostimulant reinstatement

A major feature of psychostimulants use disorder is the risk of relapse in drug use even after long periods of withdrawal (Le Moal and Koob, 2007, Wise and Koob, 2014). Reinstatement episodes might be triggered by re-exposure to the drug itself or even to previously drug-associated contextual cues, as well as exposure to stressful stimuli (Shaham et al., 2003, Steketee and Kalivas, 2011). De Vries and co-workers provided evidence for a pivotal role of CB₁R signalling in psychostimulant reinstatement. They found that a single injection of the CB₁R agonist HU-210 reinstated drug-seeking following the extinction of cocaine self-administration, an effect reversed by co-administration of a CB₁R antagonist (De Vries et al., 2001). The authors also showed that rimonabant by itself prevented drug-induced reinstatement of cocaine seeking (De Vries et al., 2001). These findings were replicated by other studies using AM251 (Xi et al., 2006, Adamczyk et al., 2012) and ORG 27,569, a CB₁R negative allosteric modulator (Jing et al., 2014). Similarly, methamphetamine- and MDMA-induced reinstatement were prevented by both CB₁R antagonism and by allosteric modulation of these receptors (Jing et al., 2014, Nawata et al., 2016). Accordingly, CB₁R antagonism also impaired cocaine and methamphetamine-induced reinstatement in the CPP paradigm (Yu et al., 2011).

CB₁R has also been found to play a critical role in mediating reinstatement of previously extinguished drug-seeking behaviour upon re-exposure to the drug-associated cues. The increase in operant self-administration response induced by re-exposure to cues previously paired with methamphetamine, MDMA, and cocaine infusion was blocked by CB₁R antagonist (Anggadiredja et al., 2004, Ward et al., 2009, Adamczyk et al., 2012, Nawata et al., 2016). Reinstatement to psychostimulant-seeking induced by different types of stressors was also inhibited by blockade of CB₁R. For instance, forced swim or restraint stress-induced reinstatement of extinguished cocaine-CPP was suppressed by systemic CB₁R antagonism (Vaughn et al., 2012, Tung et al., 2016, Guzman et al., 2021). Moreover, restraint stress-induced cocaine seeking was not observed in CB₁R-deficient mice (Tung et al., 2016). Reinstatement to cocaine-seeking promoted by injection of pharmacological stressor corticotrophin-releasing factor also was prevented by blockade of CB₁R (Kupferschmidt et al., 2012). The exposure to various types of stress events potentiated other relapse-promoting stimuli (e.g., cues, drug re-exposure), augmenting their proneness to elicit drug seeking (Mantsch et al., 2016, McReynolds et al., 2018). CB₁R played an important role in both stress- and drug-induced reinstatement, blockade of these receptors prevented the ability of stress to potentiate low-dose cocaine-induced reinstatement (McReynolds et al., 2016). In addition, a similar modulatory role of CB₁R has consistently been found with respect to cocaine- and amphetamine reinstatement induced by exposure to cues previously associated with these drugs.

Although it is recognised that CB₁Rs are important to the behavioural effects of psychostimulants-seeking, few studies have focused on understanding the neural substrates involved in these processes. The NAcc is an important neuroanatomical locus of the reinstatement-preventing effects of CB₁R antagonists. Local injections of this CB₁R antagonist into the NAcc inhibited cocaine-induced reinstatement of drug-seeking behaviour (Xi et al., 2006). The antagonism of CB₁R in the portion core of the NAcc, but not in the shell, dose-dependently prevented restraint stress-induced reinstatement of cocaine-CPP, while activation of CB₁R potentiated this behaviour (Guzman et al., 2021). Alteration of glutamate release within NAcc appears to be involved in these effects of CB₁R, since pharmacological modulation of CB₁R in the NAcc regulates extracellular levels of this neurotransmitter under cocaine-reinstatement conditions (Xi et al., 2006, Guzman et al., 2021). CB₁R expressed in VTA and in prelimbic (PL) cortex also appear to be involved on stress-induced cocaine reinstatement. CB₁R antagonist microinjected bilaterally into the VTA inhibited the capacity of the restraint stress to reinstate extinguished cocaine CPP (Tung et al., 2016). The activation of CB₁R inhibits GABA release leading to VTA dopaminergic disinhibition and reinstatement of cocaine CPP (Tung et al., 2016). Regarding PL, both stress- and corticosterone-potentiated cocaine reinstatement were prevented by intra-PL administration of the CB₁R antagonist in this region (McReynolds et al., 2018). Similarly, to observed in the VTA, a CB₁R-dependent attenuation of GABAergic neurotransmission in the PL seems to be involved in this process (McReynolds et al., 2018).

In summary, although the effect of CB₁R blockade in modulation of psychostimulant reward is still controversy, more robust behavioural and molecular evidence indicate that CB₁R is a required element in the ability of drug, stress and cue to reinstate psychostimulants seekingbehaviour.

Role of CB₂R in psychostimulant responses

Early evidence suggested that expression of CB₂R could be absent in encephalic structures and restricted to peripheral tissues (Munro et al., 1993). More recently, their expression and function were detected in the brain through molecular, genetic, behavioural, and pharmacological approaches (Gong et al., 2006, Jordan and Xi, 2019). Among other regions, CB₂Rs have been identified in the cell bodies of dopaminergic neurons in mesocorticolimbic pathway, indicating that these receptors might modulate the effects of psychostimulant drugs (Gong et al., 2006, Chen et al., 2017). Indeed, the presence of the CB₂R in mesocorticolimbic neurocircuitry is in conformity with findings that modulation of these receptors regulates behavioural and molecular responses to cocaine and amphetamine (Xi et al., 2011, Canseco-Alba et al., 2019, Jordan and Xi, 2019). Interestingly, the roles for CB₂R in the effects of psychostimulants seem to be opposite to those ascribed to CB₁R.

Systemic administration of the CB₂R agonist, JWH133, dose-dependently inhibited cocaine-enhanced locomotion in wild-type mice, but not in CB₂R KO animals (Xi et al., 2011, Gobira et al., 2019, Lopes et al., 2019). Local administration of CB₂R agonist into NAcc also resulted in attenuation of cocaine hyperlocomotion, confirming that this effect is mediated by activation of brain CB₂R (Xi et al., 2011). Consistently with these pharmacological data, findings obtained with genetically modified mice also support the importance of CB₂R to regulate psychostimulant responses. Transgenic mice overexpressing CB₂R were less responsive to cocaine-induced motor hyperactivity than wild-type mice (Aracil-Fernandez et al., 2012). Corroborating these findings, specific deletion of CB₂R in dopamine neurons increased the responsivity to acute administration of amphetamine, methamphetamine, and cocaine (Canseco-Alba et al., 2019).

CB₂R also is involved in regulation of behavioural sensitisation induced by psychostimulants. A decrease in motor sensitisation to cocaine was observed in mice overexpressing the CB₂R and after treatment with an agonist of these receptors during the acquisition phase of sensitisation (Aracil-Fernandez et al., 2012, Delis et al., 2017, Lopes et al., 2019). Similarly, when a CB₂R agonist was injected on the test day, the expression of cocaine sensitization in both mice and rats also was inhibited (Delis et al., 2017). Interestingly, compared to the wild type, mice with a selective deletion of CB₂R in dopamine neurons did not develop behavioural sensitisation when exposed to repeated treatment with cocaine, amphetamine, and methamphetamine (Canseco-Alba et al., 2019).

CB₂R seems to be also involved in the regulation of psychostimulant-rewarding responses. Transgenic mice overexpressing CB₂R show an impairment in the acquisition of cocaine self-administration (Aracil-Fernandez et al., 2012). Similarly, both acquisition and expression of cocaine-induced CPP were inhibited by previous pharmacological treatment with CB₂R agonist (Delis et al., 2017, Lopes et al., 2019). These effects, were inhibited by blockade of CB₂R, supporting the involvement of these receptors in regulation of cocaine rewarding (Lopes et al., 2019).

Intriguingly, some studies have observed opposite results. For example, systemic blockade of CB₂R inhibited intravenous cocaine self-administration and shifted cocaine dose-response curves downward in rats and wild type, but not in CB₂R KO, mice (Jordan et al., 2020). Similarly pharmacological silencing of CB₂R reduced the reinstatement of cocaine-seeking behaviour (Adamczyk et al., 2012). Although the reasons for these discrepancies remain unclear, species difference in CB₂R expression could play a role. For instance, activation of CB₂R inhibited cocaine self-administration under a FR in mice, but not in rats (Zhang et al., 2015). However, under a PR schedule of reinforcement, activation of CB₂R increased breakpoint for cocaine self-administration in rats (Zhang et al., 2015). Beyond differences between species, the multifaceted pattern of CB₂R suggested by these studies may be due to the doses and the pattern of psychostimulants administration as well as by the differences in behavioural protocol used in the experiments.

Evidence from molecular assays suggests that brain CB₂R modulates the effects of psychostimulants by a dopaminergic mechanism. Indeed, while activation of these receptors reduces the cocaine-induced enhancement of dopamine levels in the NAcc, the blockade of CB₂R elevated basal extracellular dopamine levels in this brain region (Xi et al., 2011, Zhang et al., 2014). Moreover, electrophysiological studies showed that treatment with CB₂R agonists leads to a decrease in VTA’s dopamine neuronal firing (Zhang et al., 2014). Finally, a reduction in dopamine active transporter gene expression and enhanced in tyrosine hydroxylase activity were observed in the midbrain after selective deletion of CB₂R in dopamine neurons (Canseco-Alba et al., 2019).

In summary, the data reviewed here provided evidence that CB₂R modulates behavioural and molecular responses to psychostimulants. Considering the important limitation for the therapeutic development of CB₁R antagonists, which cause unwanted serious psychiatric adverse events, modulation of CB₂R might be an interesting target to treat psychostimulant addiction (Moreira and Crippa, 2009).

Integrating CB₁R and CB₂R functions in the modulation of psychostimulant effects

As discussed throughout this review, either CB₁R blockade or CB₂R activation inhibits the molecular and behavioural responses to psychostimulant drugs. Their diametrically opposite roles might be explained by differences in the expression patterns in mesolimbic pathways modulating drug reward and reinforcement. CB₁Rs are expressed in GABAergic neurons and glutamate presynaptic terminals in the VTA, while CB₂Rs are located direct in dopaminergic VTA neurons (Kortleven et al., 2011, Zhang et al., 2014, Wang et al., 2015). This differential expression leads to a distinct regulation in the function of dopamine neurons. CB₁R might fine-tune GABA and glutamate inputs onto mesolimbic dopaminergic neurons, predominantly increasing dopaminergic activity, whereas CB₂R might directly inhibit VTA neurons and reduce dopamine release (Zhang et al., 2014, Wang et al., 2015).

A major challenge consists in tying together studies focusing on each cannabinoid receptor to postulate an integrative hypothesis on endocannabinoid modulation of psychostimulant effects. Recently, we found that CB₁R antagonists and CB₂R agonists prevent the hyperlocomotion and the CPP induced by cocaine in mice, as expected. More importantly, the ameliorating effects of CB₁R antagonism could be reversed by previous administration of CB₂R antagonist. Therefore, CB₁R antagonists could inhibit cocaine effects possibly because endocannabinoid actions are diverted predominantly to CB₂R. Moreover, although inhibition of the endocannabinoid hydrolysing enzymes FAAH (fatty acid amide hydrolase) and MAGL (monoacylglycerol lipase) failed to interfere with cocaine effects, inhibition of MAGL, which preferentially hydrolysis 2-AG, did prevent cocaine hyperlocomotion when combined with a low, ineffective dose of a CB₁R antagonist (Gobira et al., 2019). Accordingly, cocaine inhibition of norepinephrine uptake stimulates 2-AG release in the VTA, with subsequent inhibition of GABAergic terminals and facilitation of dopaminergic activity (Wang et al., 2015). Moreover, modulation of 2-AG levels in VTA and in prelimbic cortex also regulate cocaine-related responses (Tung et al., 2016, McReynolds et al., 2018). In summary, a distinct functional localisation of cannabinoid receptors in the mesocorticolimbic system might explain how CB₁R blockade and CB₂R activation exert opposite effects upon cocaine responses. In addition, the ameliorating effects of CB₁R antagonists might occur by redirecting 2-AG effects to predominantly facilitate CB₂R signalling (Fig. 1).

Fig. 1. CB1 and CB2 receptors differentially regulate the effects of psychostimulant drugs. Both CB₁R blockade and CB₂R activation inhibits the molecular and behavioural responses to psychostimulant drugs (Panel A). Blockade of CB₁R redirects 2-AG effects to predominantly facilitate CB2R signalling (Panel B).

The integrative response promoted by CB1R blockade and CB2R activation in modulation of other drugs of abuse have not been performed yet. However, the individual role of cannabinoid receptors in decreasing the effects induced by distinct classes of addictive drugs has already been demonstrated (Manzanares et al., 2018). For instance, pharmacological and genetic silencing of CB₁R reduced the hyperlocomotion and rewarding effects induced by alcohol, opiates, and nicotine (Navarro et al., 2001, Houchi et al., 2005, Simonnet et al., 2013, Marinho et al., 2015, Guegan et al., 2016). CB₁R also modulates the dopamine release in the NAcc elicited by these prototypical drugs (Cheer et al., 2007, Parsons and Hurd, 2015). Moreover, similarly to observed with psychostimulants, the activation of CB₂R reduced ethanol consumption and the ethanol-induced CPP (Al Mansouri et al., 2014). Treatment with CB₂R-agonist also decreases the responses promoted by opiates (Zhang et al., 2018, Iyer et al., 2020). Together these studies suggested that modulation of both cannabinoid receptors seems to be a common mechanism underlying the molecular and behavioural properties of different classes of drugs.

Concluding remarks and future perspectives

Preclinical studies implicate cannabinoid receptors in the modulation of behavioural and molecular responses induced by psychostimulant drugs. The consistent results showing that genetic and pharmacological blockade of CB₁R inhibits cocaine effects and could encourage the use of selective antagonists for treating psychostimulant addiction disorders. However, the incidence of serious psychiatric adverse events, such as anxiety and depression, limits the use of these compounds. In this context, drugs targeting CB₂R might represent a more promising approach. Thus, an increasing number of studies has focused on the effects of CB₂R agonists in modulation of psychostimulants responses. Combining inhibition of 2-AG hydrolysis with low doses of CB₁R antagonists and therefore favouring endocannabinoid facilitation of CB₂R signalling could also represent a new approach.

Despite this substantial evidence demonstrating the important roles of CB₁R and CB₂R in regulation of psychostimulant responses, so far the studies have focused on male mice and rats, excluding in female animals. Sexual dimorphism has been demonstrated in behavioural and molecular responses correlated to CB₁R and CB₂R. For example, expression and functionality of CB₁R were observed in the VTA and PFC of females compared to male animals, possibly providing a neural substrate for the existing sex differences to the rewarding effects of cannabinoids (Llorente-Berzal et al., 2013, Castelli et al., 2014). In fact, females self-administered more WIN55,212-2, a non-selective cannabinoid agonist, than male rats(Fattore et al., 2010). Regarding responses correlated to CB₂R, Onaivi and co-workers demonstrated that treatment with CB₂R agonist alters mouse spontaneous locomotor activities in a sex-dependent fashion (Onaivi et al., 2006). In the face with this evidence, the role of CB₁R and CB₂R in regulation of psychostimulants in female animals should be explored in future studies.

Table 1. Cannabinoid receptors influence on psychostimulant-related behaviours

*DAT-Cnr2 conditional knockout, ** Deleted CB₁R in cortical glutamatergic neurons.