British Journal of British Journal of Pharmacology (2019) 176 1541–1551 1541

Pharmacology

Themed Section: 8th European Workshop on Cannabinoid Research

RESEARCH PAPER
Opposing roles of CB1 and CB2 cannabinoid
receptors in the stimulant and rewarding
effects of cocaine
Correspondence Pedro H Gobira and Fabricio A Moreira, Department of Pharmacology, Institute of Biological Sciences, Universidade
Federal de Minas Gerais, Av. Pres. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil. E-mail: gobirapedro@hotmail.com;
fabriciomoreira@icb.ufmg.br

Received 25 October 2017; Revised 19 July 2018; Accepted 24 July 2018

Pedro H Gobira1, Ana C Oliveira1, Julia S Gomes1, Vivian T da Silveira1, Laila Asth1, Juliana R Bastos1 ,
Edleusa M Batista2, Ana C Issy3, Bright N Okine4, Antonio C de Oliveira1, Fabiola M Ribeiro2, Elaine A Del Bel3,
Daniele C Aguiar1, David P Finn4 and Fabricio A Moreira1
1
Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 2Department of
Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 3Department of
Morphology, Stomatology and Basic Pathology, Faculty of Odontology, University of São Paulo, Ribeirão Preto, Brazil, and 4Department of
Pharmacology and Therapeutics, School of Medicine, National University of Ireland Galway, Galway, Ireland

BACKGROUND AND PURPOSE

The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) bind to CB1 and CB2 cannabinoid receptors in the brain
and modulate the mesolimbic dopaminergic pathway. This neurocircuitry is engaged by psychostimulant drugs, including
cocaine. Although CB1 receptor antagonism and CB2 receptor activation are known to inhibit certain effects of cocaine, they have
been investigated separately. Here, we tested the hypothesis that there is a reciprocal interaction between CB1 receptor blockade
and CB2 receptor activation in modulating behavioural responses to cocaine.
EXPERIMENTAL APPROACH
Male Swiss mice received i.p. injections of cannabinoid-related drugs followed by cocaine, and were then tested for cocaine-
induced hyperlocomotion, c-Fos expression in the nucleus accumbens and conditioned place preference. Levels of
endocannabinoids after cocaine injections were also analysed.
KEY RESULTS
The CB1 receptor antagonist, rimonabant, and the CB2 receptor agonist, JWH133, prevented cocaine-induced hyperlocomotion.
The same results were obtained by combining sub-effective doses of both compounds. The CB2 receptor antagonist, AM630,
reversed the inhibitory effects of rimonabant in cocaine-induced hyperlocomotion and c-Fos expression in the nucleus
accumbens. Selective inhibitors of anandamide and 2-AG hydrolysis (URB597 and JZL184, respectively) failed to modify this
response. However, JZL184 prevented cocaine-induced hyperlocomotion when given after a sub-effective dose of rimonabant.
Cocaine did not change brain endocannabinoid levels. Finally, CB2 receptor blockade reversed the inhibitory effect of rimonabant
in the acquisition of cocaine-induced conditioned place preference.

CONCLUSION AND IMPLICATIONS

The present data support the hypothesis that CB1 and CB2 receptors work in concert with opposing functions to modulate certain
addiction-related effects of cocaine.

LINKED ARTICLES
This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this
section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc

© 2018 The British Pharmacological Society DOI:10.1111/bph.14473

 P H Gobira et al.
Abbreviations
2-AG, 2-arachidonoylglycerol; ACEA, arachidonoyl 20 -chloroethylamide; AM630, 1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-
(4-methoxybenzoyl)-6-iodoindole; CPP, conditioned place preference; FAAH, fatty acid amide hydrolase; JWH133,
(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran; JZL184, 4-nitrophenyl-
4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate; MAGL, monoacyl-glycerol lipase; Rimonabant, 5-
(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide; URB597, [3-(3-
carbamoylphenyl)phenyl] N-cyclohexylcarbamate
Introduction
Cannabinoid CB1 and CB2 receptors are responsible for the
pharmacological effects of Δ9-tetrahydrocannabinol, the
main active compound from Cannabis sativa (Devane et al.,
1988; Munro et al., 1993; Pertwee 2008). The endogenous li-
gands of these receptors, termed endocannabinoids, are the
arachidonic acid derivatives, arachidonoyl ethanolamide
(anandamide) and 2-arachidonoylglycerol (2-AG) (Devane
et al., 1992; Sugiura et al., 1995). Each of these ligands has dif-
ferent degradation pathways. Fatty acid amid hydrolase
(FAAH) is responsible mainly for anandamide hydrolysis,
whereas the metabolism of 2-AG is facilitated by monoacyl-
glycerol lipase (MAGL) (Cravatt et al., 1996; Dinh et al.,
2002). Pharmacological and genetic manipulations of these
enzymes regulate the levels and consequently the action of
endocannabinoids (Petrosino and Di Marzo 2010; Batista
et al., 2014).
Among the brain circuits modulated by endo-
cannabinoids is the dopaminergic mesolimbic pathway,
which is engaged by several drugs of abuse, including
cocaine (Cheer et al., 2007). This drug inhibits dopamine
uptake and facilitates dopamine receptor-mediated signal-
ling, increasing c-Fos expression in the nucleus accumbens
(Valjent et al., 2000; Zhang et al., 2004). The presence of the
endocannabinoid system in the mesolimbic pathway is in
agreement with the evidence that endocannabinoids modu-
late behavioural responses to cocaine (Maldonado et al.,
2006). For instance, pharmacological blockade or genetic de-
letion of CB1 receptors inhibits the motor hyperactivity in-
duced by this psychostimulant (Poncelet et al., 1999;
Corbille et al., 2007; Li et al., 2009). Inhibition of CB1 receptor
signalling also reversed cocaine self-administration and re-
sponses in the conditioned place preference (CPP) paradigm
(Soria et al., 2005; Xi et al., 2008; Yu et al., 2011). Moreover,
blockade of CB1 receptors seems to modulate the neurochem-
ical effects of cocaine, as cocaine-induced increases in extra-
cellular dopamine levels in the nucleus accumbens were
inhibited following silencing of these receptors (Cheer et al.,
2007; Corbille et al., 2007; Li et al., 2009).
Regarding the CB2 receptor, early evidence suggested
that this receptor might be absent in brain and restricted
to peripheral tissues (Munro et al., 1993). However, recent
studies have challenged this view, detecting expression of
CB2 receptors in some encephalic structures (Gong et al.,
2006; Onaivi et al., 2008). Moreover, pharmacological and
genetic interventions at this receptor result in behavioural
changes, further indicating that this receptor is present
and functional in the brain (Ortega-Alvaro et al., 2011; Xi
et al., 2011). In agreement, recent findings show that the
CB2 receptor is involved in behavioural responses to
cocaine, although its functions tend to oppose those
1542 British Journal of Pharmacology (2019) 176 1541–1551
ascribed for CB1 receptors. For example, activation of CB2
receptors attenuated cocaine-induced hyperlocomotion,
self-administration and dopamine release in the nucleus
accumbens (Xi et al., 2011; Zhang et al., 2014; Zhang
et al., 2015; Zhang et al., 2017). Accordingly, transgenic
mice overexpressing CB2 receptors have reduced cocaine
self-administration and motor sensitization (Aracil-
Fernandez et al., 2012).
Although several studies provide evidence for a role of the
endocannabinoid system in the modulation of cocaine re-
sponses, a possible interaction between CB1 and CB2 recep-
tors in this context remains to be investigated. Both
receptors modulate the mesolimbic dopaminergic pathway
in the ventral tegmental area (Zhang et al., 2014; Wang
et al., 2015; Zhang et al., 2017). The CB1 receptor is expressed
in GABAergic terminals projecting onto dopaminergic neu-
rons, where its activation disinhibits the mesolimbic path-
way (Wang et al., 2015). The CB2 receptor, on the other
hand, has been identified in the dopaminergic cell bodies,
in which they may exert inhibitory functions (Zhang et al.,
2014; Zhang et al., 2017). Therefore, CB1 and CB2 receptors
can be simultaneously activated by endocannabinoids at a
given synapse in the ventral tegmental area, with opposing
effects on dopaminergic activity.
On the basis of such evidence, the working hypothesis
of this study was that endocannabinoids could either
facilitate or inhibit the behavioural effects of cocaine
through CB1 and CB2 receptors respectively. First, we inves-
tigated the role of each cannabinoid receptor and
endocannabinoid in cocaine-induced hyperlocomotion.
Second, we hypothesized that blockade of CB1 receptors
would ameliorate the effects of cocaine because
endocannabinoids would then act predominantly on CB2
receptors. Thus, we tested if CB2 receptor antagonists
would prevent the inhibitory effects of CB1 receptor antag-
onism on cocaine-induced hyperlocomotion and c-Fos
expression in the nucleus accumbens. We also analysed
the levels of brain endocannabinoids after cocaine
injection. Finally, we extended this analysis to the
rewarding effects of cocaine in the CPP test.
Methods
Compliance with design and statistical
analysis requirements
This study complies with the design and statistical analysis
requirements of the British Journal of Pharmacology (Curtis
et al., 2018). Specific details regarding ethics, experimental
designs and data analysis are provided in appropriate sections
below.

Experimental animals
All animal care and experimental procedures were in accor-
dance with the Brazilian Society of Neuroscience and
Behaviour Guidelines for the Care and Use of Laboratory An-
imals and were approved by the local ethics committee
(CEUA–UFMG) under the protocol 242/2013. Animal studies
are reported in compliance with the ARRIVE guidelines
(Kilkenny et al., 2010; McGrath et al., 2010). Male Swiss mice
(25–30 g) were group-housed (5 per cage) in a room
maintained at 25°C with a 12 h light/dark cycle (lights on at
8 a.m.). Food and water were available ad libitum. Each animal
was used only once..
Role of the endocannabinoid system in
cocaine-induced hyperlocomotion
All the experiments were conducted in the light phase,
between 8.00 a.m. and 16.00 p.m. in an isolated, sound-
attenuated room. The experiments measuring locomotion
were carried out in a square open field (40 cm × 40 cm
with a 50 cm high Plexiglas wall). The animals received
injections of one of the cannabinoid-related drugs and
20 min later were habituated in the open field for
10 min. Next, they received cocaine (20 mg·kg1) injection
and were immediately placed back in the open field. The
distance moved was analysed for 10 min with the help of
Any Maze software (Stoelting Co).
Roles of CB1 and CB2 receptors in
cocaine-induced c-Fos expression
The animals were subjected to the same injection and be-
havioural protocols as described in the previous section.
Two hours after exposure to the arena, the animals were
anaesthetized with an overdose of urethane and perfused
transcardially with saline (200 mL) followed by paraformal-
dehyde (4%) in 0.1 M phosphate buffer (150 mL, pH 7.4).
Brains were removed and post-fixed over 2 h in paraformal-
dehyde (4%) and stored for 36 h in 30% sucrose for
cryoprotection. Coronal sections (40 μm) of nucleus ac-
cumbens core and shell, as identified with the help of the
atlas of the mouse brain (Paxinos and Watson 1997), were
obtained in a cryostat. The slices were stored in triplicate
and processed for immunohistochemistry, as previously de-
scribed (Vilela et al., 2015). Briefly, tissue sections were
washed with phosphate buffer in saline and incubated
overnight at room temperature with rabbit IgG antibody
in phosphate buffer in saline (1:1000, from Santa Cruz,
Dallas, TX, USA). The sections were washed in phosphate
buffer in saline and incubated with a biotinylated anti-
rabbit IgG (1:1000, Santa Cruz, Dallas, TX, USA). Fos-like
immunoreactivity was revealed by the addition of the chro-
mogen diaminobenzidine (Sigma, St. Louis, MO, USA) and
visualized as a brown precipitate inside the neuronal nu-
clei. The images from the slices were captured with the
auxiliary of the microscope (Zeiss, Oberkochen, Germany)
and an observer blind to group assignment performed the
analysis of the number of Fos-like immune-reactivity man-
ually counted with the help of a computerized image anal-
ysis system (Image Pro-Plus 4.0, Media Cybernetics). The
nucleus accumbens sites were identified with the help of
the atlas Paxinos and Watson (1997) at the anteroposterior
Cannabinoid receptors and cocaine
(AP) localization from bregma AP = 1.18 mm, and one
section from each animal for each group was evaluated.
Cocaine effects on endocannabinoid levels
LC–tandem MS was used to determine levels of the endog-
enous cannabinoids anandamide and 2-AG in hippocam-
pus, striatum and prefrontal cortex as described
previously (Ford et al., 2011). Immediately after testing in
the arena, animals were killed by decapitation, and regions
of interest were immediately dissected and snap-frozen on
dry ice. Then, samples were homogenized in 400 μL of
100% acetonitrile containing deuterated internal standards
(0.014 nmol AEA-d8, 0.48 nmol 2-AG-d8). Homogenates
were centrifuged at 14 000 g for 15 min at 4°C, and the su-
pernatant was collected and evaporated to dryness in a
centrifugal evaporator. Samples were resuspended in
40 μL of 65% acetonitrile and separated on a Zorbax®
(Santa Clara, CA, USA) C18 column (150 × 0.5 mm inter-
nal diameter; Agilent Technologies Ltd, Cork, Ireland) by
reversed-phase gradient elution. Analyte detection was
carried out in electrospray-positive ionization and multiple
reaction monitoring mode on an Agilent 1100 HPLC sys-
tem coupled to a triple quadrupole 6460 mass spectrome-
ter (Agilent Technologies Ltd). Quantification of each
analyte was done by ratiometric analysis and expressed as
pmol or nmol·g1 (anandamide and 2-AG, respectively)
of tissue. The limit of quantification was 1.32 pmol·g1
for anandamide and 12.1 pmol·g1 for 2-AG.
Roles of CB1 and CB2 receptors in
cocaine-induced CPP
Conditioned place preference (CPP) was assessed in an acrylic
box consisting of two chambers of equal size (20 cm long, 15
wide and 10 cm high) with doors (5 × 5 cm) connecting them
to a central compartment (6 cm long, 15 cm wide and 10 cm
high). The walls of the lateral chambers had interspersed
black and white stripes, and the floors consisted of removable
metal surfaces. In one of the chambers (chamber A), the walls
were painted with vertical stripes, and the floor consisted of a
metal grid with parallel, equally-spaced rods. The other
(chamber B) had walls painted with horizontal stripes and a
metal floor with circular holes. The light intensity was similar
among the three compartments. The CPP protocol was based
in previous studies (Yu et al., 2011; Almeida-Santos et al.,
2014). In the pre-test phase (first day), each mouse was placed
in the central compartment of the box, with the doors open,
and could freely explore during 15 min. The time spent in
each compartment was registered and automatically analysed
with the AnyMaze software (Stoelting Co®). In the condi-
tioning phase (days 2–7), the animals were randomly
assigned to one of the experimental treatments. They re-
ceived cocaine injections on days 2, 4 and 6 and were imme-
diately confined to one of the chambers (drug-paired side) for
30 min. On alternate days (3, 5 and 7), mice were injected
with saline and confined to the other compartment of the
chamber for 30 min. We designed a counterbalance protocol,
meaning that each group contained animals receiving to co-
caine injections in chamber A, but saline in chamber, as well
as animals assigned to the opposite pairing. Cannabinoid an-
tagonists were co-administered on days 2, 4 and 6, 30 min
British Journal of Pharmacology (2019) 176 1541–1551 1543
 P H Gobira et al.

before each cocaine injection. Finally, on the test phase (day 8), Results
mice were tested for the expression of cocaine-induced CPP
under drug-free conditions identical to those described in Role of the endocannabinoid system in
the preconditioning test. The CPP score was defined as the
cocaine-induced hyperlocomotion
time spent in the drug-paired chamber minus the time spent
First, we tested the effects of CB1 receptor blockade on
in the saline-paired chamber.
cocaine-induced hyperlocomotion. A dose response curve
with rimonabant (1, 3 and 10 mg·kg1) revealed that this
compound was effective at the highest dose (Figure 1A). To
Data and statistical analysis explore the role of the CB2 receptor, we tested the effects of
Sample sizes appropriate for each type of experiment were es-
the selective CB2 agonist, JWH133 (20 mg·kg1) and found it
timated based on pilot studies and were calculated based on
to attenuate cocaine-induced hyperlocomotion (Figure 1B)].
the equation (Eng, 2003): CI95 = 1.96 s/√n, where CI stands
Next, we examined whether the combined administration of
for the confidence interval, 1.96 is the corresponding tabu-
sub-effective doses of these drugs would attenuate cocaine
lated value for CI95, s is the SD of the mean and n is the sample
hyperlocomotion. A combination of rimonabant (3 mg·kg1)
size. Sample sizes may differ slightly between groups in each
and JWH133 (10 mg·kg1) inhibited cocaine effects (Figure 1C).
experiment, as not all animals in a batch provided by the an-
We also investigated if CB1 receptor blockade by rimonabant
imal facility satisfied the criteria for the experiments (e.g.
would shift endocannabinoid actions to CB2 receptors.
high body weight or age).
Supporting this hypothesis, pretreatment with a CB2 receptor
The animals were randomized for experimental treat-
antagonist, AM-630 (10 mg·kg1), reversed the inhibitory effect
ments. The distances moved in the open field and the num-
of rimonabant (10 mg·kg1) on hyperlocomotion induced by
ber of c-Fos positive cells were subjected to ANOVA
cocaine (Figure 1D). The next experiments were designed to
followed by the Newman–Keuls test. To test for a linear corre-
check the selectivity of AM630 for the CB2 over the CB1 receptor
lation, the individual values of distance moved and c-Fos pos-
at the current dose (10 mg·kg1). If this is the case, rimonabant
itive cells for each animal were subjected to the Pearson
(10 mg·kg1), but not AM630, would prevent the effects of a
correlation analysis. Endocannabinoid levels were analysed
selective CB1 receptor agonist. Accordingly, only rimonabant
by Student’s t-test. Drug effects on CPP were analysed by com-
prevented ACEA-induced hypolocomotion (Figure 1E. Impor-
paring CPP scores in the test session through ANOVA
tantly, none of the cannabinoid-related compounds, at the doses
followed by the Newman–Keuls test. The level of significance
used, interfered with basal locomotion (Figure 1F)].
was set at P < 0.05. Post hoc tests were run only if F achieved
To evaluate the participation of endocannabinoids in
P < 0.05, and there is no significant variance in homogeneity.
modulation of cocaine-induced responses, we injected ani-
All data are presented as scattered dot plots, with the mean
mals with URB597 (0.1, 0.3 and 1.0 mg·kg1) or JZL184 (1, 3
and SEM shown.
and 10 mg·kg1), which inhibit anandamide and 2-AG hy-
drolysis respectively. Neither URB597 (Figure 2A)] nor
JZL184 (Figure 2B) modified the effects of cocaine. Inhibiting
Materials endocannabinoid hydrolysis might not prevent the effects of
Cocaine hydrochloride (20 mg·kg1, Merck, Kenylworth, NJ,
cocaine because they activated both cannabinoid receptors,
USA) was dissolved in physiological saline. The CB1 receptor
which cancelled each other’s effects. Thus, we hypothesized
antagonist/inverse agonist, rimonabant (1, 3, and 10-
that a subthreshold dose of rimonabant (3 mg·kg1) com-
mg·kg1; Cayman Chemical, Ann Arbor, USA), was dissolved
bined with an ineffective dose of endocannabinoid hydroly-
in cremophor–ethanol–saline (1:1:18, v/v). A similar solution
sis inhibitor would selectively facilitate CB2 receptor
was used to dissolve the CB1 receptor agonist arachidonoyl 20 -
signalling and thus inhibit cocaine’s effects. However, con-
chloroethylamide (ACEA, 5 mg·kg1; Tocris, Bristol, UK), the
trary to this prediction, combining rimonabant (3 mg·kg1)
FAAH inhibitor URB597, (0.1, 0.3 and 1.0 mg·kg1; Cayman
with URB597 (1 mg·kg1) failed to change responses to co-
Chemical, Ann Arbor, USA) and the MAGL inhibitor JZL184
caine (Figure 2C). However, when the same subthreshold
(1.0, 3.0 and 10 mg·kg1; Cayman Chemical, Ann Arbor,
dose of rimonabant was combined with the2-AG hydrolysis
USA). The CB2 receptor antagonist, AM630 (1, 3 and 10-
inhibitor, JZL184 (10 mg·kg1), the hyperlocomotor effect
mg·kg1; Tocris, Bristol, UK), and the CB2 receptor agonist,
of cocaine was prevented (Figure 2D)].
JWH133 (1, 3 and 10 mg·kg1; Cayman Chemical, Ann
Arbor, USA), were dissolved in physiological saline contain-
ing Tween 80 (5%) and DMSO (5%). The solutions were pre- Roles of CB1 and CB2 receptors in
pared immediately before use and injected i.p. in a volume cocaine-induced c-Fos expression
of 10 mL·kg1. We then tested our hypothesis on the cellular effects of co-
caine. In accordance with the behavioural results,
rimonabant (10 mg·kg1) prevented cocaine-induced in-
Nomenclature of targets and ligands creases in c-Fos expression in the nucleus accumbens, and
Key protein targets and ligands in this article are hyperlinked to pretreatment with AM-630 (10 mg·kg1) reversed this inhibi-
corresponding entries in http://www.guidetopharmacology. tory effect. This occurred in both the shell (Figure 3A) and the
org, the common portal for data from the IUPHAR/BPS Guide core (Figure 3B) parts of the nucleus accumbens. The total dis-
to PHARMACOLOGY (Harding et al. 2018), and are perma- tance moved correlated with the number of c-Fos immuno-
nently archived in the Concise Guide to PHARMACOLOGY stained nuclei in the shell (Figure 2C) and the core
2017/18 (Alexander et al. 2017a,b) [r = 0.5742, P < 0.05; Figure 2D]. Representative

1544 British Journal of Pharmacology (2019) 176 1541–1551

 Cannabinoid receptors and cocaine

Figure 1
1
Roles of CB1 and CB2 receptors in cocaine-induced hyperlocomotion. (A) The CB1 receptor antagonist, rimonabant (Rim; 10 mg·kg ), prevented
1 1
the hyperlocomotion induced by cocaine 20 mg·kg (n = 8, 10, 8, 8, 12). (B) The CB2 receptor agonist, JWH133 (JWH; 20 mg·kg ), prevented
1
cocaine-induced hyperlocomotion (n = 7, 7, 7, 8, 8). (C) A combination of sub-effective doses of rimonabant (3 mg·kg ) and JWH133
1 1
(10 mg·kg ) prevented cocaine hyperlocomotion (n = 7, 8, 7, 7, 8). (D) The CB2 receptor antagonist, AM630 (AM; 10 mg·kg ), reversed the
1 1
inhibitory effect of rimonabant (10 mg·kg ) on cocaine-induced hyperlocomotion (n = 8, 10, 8, 8, 12). (E) Rimonabant (10 mg·kg ), but
1 1 1
not AM630 (10 mg·kg ), prevented ACEA (5 mg·kg )-induced hypolocomotion (n = 7,6,6,7). (F) Rimonabant (10 mg·kg ), AM630 (10-
1 1
mg·kg ) and JWH133 (20 mg·kg ) did not interfere with basal locomotor activity as compared to the vehicle (Veh; cremophor–ethanol–saline,
1:1:18); n = 6. Data shown are individual values with means ± SEM; n as indicated. *P < 0.05, significantly different from vehicle-vehicle group;
#
P < 0.05, significantly different from vehicle-cocaine or vehicle-ACEA groups; ANOVA followed by Newman–Keuls test.

photomicrographs of c-Fos expression are shown in Figure 4.
Cannabinoid-related compounds did not alter basal levels of
c-Fos counting. The values in the shell portion were as follows
(mean ± SEM; n = 6 per group). Vehicle: 32 ± 4.7; rimonabant
(10 mg·kg1): 39.5 ± 3.8; AM630 (10 mg·kg1): 30.5 ± 5.6. The
values for c-Fos counting in the core portion were as follows
(mean ± SEM; n = 6 per group). Vehicle: 18.4 ± 3.1;
rimonabant (10 mg·kg1): 22.2 ± 4.2; AM630 (10 mg·kg1):
17.1 ± 3.3.
Cocaine effects on endocannabinoid levels
We also investigated whether endocannabinoid levels in
the mesolimbic system were increased after cocaine admin-
istration. Cocaine (20 mg·kg1) administration did not
change anandamide levels in the striatum (Figure 5A),

British Journal of Pharmacology (2019) 176 1541–1551 1545

 P H Gobira et al.

Figure 2
Effect of inhibitors of endocannabinoid hydrolysis, alone or in combination with a subthreshold dose of the CB1 receptor antagonist, rimonabant,
1
on cocaine-induced hyperlocomotion. (A) URB597 (URB; 0.1, 0.3 and 1.0 mg·kg ), the anandamide hydrolysis inhibitor, did not change cocaine
1
effect (n = 7, 6, 8, 6, 8). (B) A similar pattern was observed after treatment with the 2-AG hydrolysis inhibitor, JZL184 (JZL; 1.0, 3.0 and 10 mg·kg )
1 1
(n = 6, 7, 6, 7, 7). (C) Combined treatment with a subthreshold dose of rimonabant (Rim; 3 mg·kg ) and URB597(1 mg·kg ) did not change
1
cocaine-induced hyperlocomotion (n = 7, 7, 6, 6, 7). (D) Combined treatment with a subthreshold dose of rimonabant (3 mg·kg ), and
1
JZL184 (10 mg·kg ), prevented cocaine-induced hyperlocomotion (n = 8,8,10,8,9). Data shown are individual values with means ± SEM; n as
#
indicated. *P < 0.05, significantly different from vehicle-vehicle; P < 0.05, significantly different from vehicle-cocaine group; ANOVA followed
by Newman–Keuls test.

prefrontal cortex (Figure 5B) and hippocampus (Figure 5C). Discussion

2-AG levels also remained unchanged in these regions: stri-
atum (Figure 5D), prefrontal cortex (Figure 5E) and hippo-
campus (Figure 5F).
Roles of CB1 and CB2 receptors in
cocaine-induced CPP
We also tested if our hypothesis of opposing roles for CB1 and
CB2 receptors would extend to the rewarding effect of co-
caine. Thus, we confirmed that CB2 receptor antagonism
would reverse the inhibitory effect of CB1 receptor antago-
nism on cocaine-induced CPP (Figure 6A). ANOVA of CPP
scores in the test session revealed a significant overall drug
effect. Post hoc Newman–Keuls analysis showed that
rimonabant pretreatment abolished the cocaine effect, as it
prevented the increase in CPP score compared to the
vehicle-cocaine group. Consistent with our hypothesis, pre
treatment with a CB2 receptor antagonist reversed the inhib-
itory effect of rimonabant. This result mimics our observation
in cocaine-induced hyperlocomotion. Rimonabant or
AM630 did not change induced place preference or aversion
on their own (Figure 6B).
The present study provided evidence for a reciprocal interac-
tion between CB1 receptor blockade and CB2 receptor activa-
tion in inhibiting behavioural responses to cocaine. In
addition to studying each receptor separately, we have also
found that the ameliorating effects of CB1 receptor antago-
nists was reversed by CB2 receptor antagonists. Thus, CB1 re-
ceptor antagonists inhibit cocaine effects, possibly because
endocannabinoid actions are diverted to the CB2 receptor.
The endocannabinoid involved in this mechanism might be
2-AG, because a MAGL inhibitor reduced cocaine-induced
hyperlocomotion when combined with a low-dose of a CB1
receptor antagonist.
Corroborating previous data, we observed that CB1 recep-
tor blockade with rimonabant prevented various responses to
cocaine. CB1 receptor antagonists are well-known to inhibit be-
havioural and neurochemical responses to psychostimulant
drugs (Cheer et al., 2007; Li et al., 2009; Hernandez et al.,
2014; Mereu et al., 2015; Wang et al., 2015). However, their
clinical applications are limited by their potential CNS side ef-
fects, such as anxiety and depression (Moreira and Crippa,
2009). More recently, the CB2 receptor agonists has been

1546 British Journal of Pharmacology (2019) 176 1541–1551

 Cannabinoid receptors and cocaine

Figure 3
1
Opposing roles for CB1 and CB2 receptors on c-Fos expression induced by cocaine. (A) Rimonabant (Rim; 10 mg·kg ) prevented the increase in
1
c-Fos expression induced by cocaine (20 mg·kg ) on the shell region of the nucleus accumbens, an effect reversed by previous treatment with
1
AM630 (AM; 10 mg·kg ) (n = 6/group). (B) The number of c-Fos cells in the shell region of the nucleus accumbens correlated with the total
distance moved in the open field. The best fit and confidence intervals are represented by continuous and dashed lines respectively.
1
r = 0.6019, P < 0.05; n = 24. (C) Rimonabant (10 mg·kg ) prevented cocaine-induced increased in c-Fos expression on the core portion of
nucleus accumbens, an effect reversed by previous treatment with AM630 (n = 6/group). (D) The number of c-Fos cells in the core regions of
the nucleus accumbens correlated with the total distance moved in the open field The best fit and confidence intervals are represented by
#
continuous and dashed lines respectively. r = 0.5742, P < 0.05 (n = 24). *P < 0.05, significantly different from vehicle-vehicle; P < 0.05,
significantly different from vehicle-cocaine group; ANOVA followed by Newman–Keuls test.

considered as a potential alternative, mainly after studies sug-
gesting CB2 receptor expression in the brain and its function
in inhibiting responses to drugs of abuse (Xi et al., 2011;
Aracil-Fernandez et al., 2012; Zhang et al., 2014; Zhang et al.,
2015; Zhang et al., 2017). Accordingly, the present study shows
that activation of CB2 receptors with JWH133 inhibited co-
caine effects. In addition, a similar result was obtained by com-
bining ineffective doses of a CB1 receptor antagonist and a CB2
receptor agonist. We also found that the CB2 receptor antago-
nist, AM630 (10 mg·kg1), reversed the inhibitory effect of
the CB1 receptor antagonist, rimonabant, in cocaine-induced
hyperlocomotion and c-Fos expression in the nucleus accum-
bens. At this dose, AM630 is unlikely to bind significantly to
CB1 receptors, as it did not interfere with the effect of ACEA, a
selective CB1 receptor agonist. Finally, the effects on the dis-
tance moved correlated with the effects on c-Fos expression
in both the shell and core regions of the nucleus accumbens.
These data reveal a functional interaction between subtypes
of cannabinoid receptors in modulating cocaine responses,
suggesting that CB1 receptor antagonists ameliorate cocaine ef-
fects by diverting endocannabinoid actions to the CB2 recep-
tor. A possible site of action is the ventral tegmental area,
where CB1 and CB2 receptors are proposed to be located in
GABAergic terminals and dopaminergic cell bodies,
respectively (Zhang et al., 2014; Wang et al., 2015; Zhang
et al., 2017).
To investigate which endocannabinoid might be in-
volved in this process, we tested selective inhibitors of anan-
damide and 2-AG hydrolysis. However, selective inhibition
of FAAH or MAGL failed to modify cocaine-induced
hyperlocomotion. In fact, these results are in agreement
with previous data (Luque-Rojas et al., 2013) and might be
explained by a simultaneous activation of both CB1 and
CB2 signalling, which modulates cocaine responses in oppo-
site ways. Thus, to selectively increase activation of CB2 re-
ceptors by endocannabinoids, we administered an
ineffective dose of rimonabant before the endocannabinoid
hydrolysis inhibitors. Remarkably, treatment with an MAGL

British Journal of Pharmacology (2019) 176 1541–1551 1547

 P H Gobira et al.

Figure 4
Representative photomicrographs of coronal sections (40 μm) of the nucleus accumbens showing c-Fos immunoreactivity. For each treatment,
(vehicle,Veh; rimonabant, Rim; cocaine, Coca) the total area of this structure is presented at a 10× magnification, whereas the shell and core sub-
regions are presented at a 63× magnification. c-Fos positive cells are identified as dark dots. Scale bars: 100 μm.

Figure 5
1
Effect of cocaine on endocannabinoid levels. Treatment with cocaine (20 mg·kg ) did not change the levels of anandamide in the (A) striatum,
(B) hippocampus or (C) prefrontal cortex. Data shown are individual values with means ± SEM; n = 5. A similar lack of effect was observed for 2-AG
levels in these structures (D, E, and F). Data shown are individual values with means ± SEM; n = 5. Student’s t-test.

inhibitor, but not with a FAAH inhibitor, attenuated
cocaine-induced hyperlocomotion when CB1 receptors were
blocked by low-dose rimonabant. The possible explanation
for this difference might depend on the distinct affinities
and efficacies of anandamide and 2-AG at cannabinoid re-
ceptors (Di Marzo and De Petrocellis, 2012). Anandamide
exhibits higher affinity for CB1 , as compared to CB2 recep-
tors, whereas the opposite seems to be the case for 2-AG
(Di Marzo and De Petrocellis, 2012). Similarly, as observed
on a variety of in vitro functional assays, 2-AG acts as a full
agonist, whereas anandamide acts as a partial agonist at
CB2 receptors (Sugiura et al., 2000). Therefore, the role of
2-AG may depend on which cannabinoid receptor is pre-
dominantly activated. Previous studies found that 2-AG me-
diates cocaine effects through CB1 receptors in the ventral
tegmental area (Wang et al., 2015). The mechanism involves
the inhibition of GABAergic terminals, with subsequent dis-
inhibition of dopaminergic pathways that project to the nu-
cleus accumbens (Zhang et al., 2014; Wang et al., 2015). The
present study expands these mechanisms and proposes a

1548 British Journal of Pharmacology (2019) 176 1541–1551


Figure 6
Effect of CB1 and CB2 receptor antagonism on cocaine-induced CPP.
1
(A) Rimonabant (Rim; 10 mg·kg ), prevented cocaine-induced
place preference, an effect reversed by previous treatment
1
with AM-630 (10 mg·kg ). Data shown are individual values
with means ± SEM; n = 8,8,7. *P < 0.05, significantly different from
#
vehicle-vehicle-cocaine group; P < 0.05, significantly different from
vehicle-Rim-cocaine group; ANOVA followed by Newman–Keuls test.
(B) CB1 and CB2 receptor blockade did not induce either preference
or aversion. Data shown are individual values with means ± SEM;
n = 8, 7, 6; ANOVA followed by Newman–Keuls test.
new role of 2-AG, showing that this endocannabioid may
actually inhibit the effects of cocaine, if the CB1 receptors
are blocked.
This effect occurred even though cocaine did not change
the levels of anandamide and 2-AG in hippocampus, striatum
or prefrontal cortex. This finding is in agreement with earlier
reports of no changes in endocannabinoid levels in response
to cocaine (Gonzalez et al., 2002; Caille et al., 2007), although
other authors have shown that cocaine injection did increase
2-AG levels (Patel et al., 2003). Different experimental condi-
tions may explain these discrepancies. Other important fac-
tors may be the time point and the technique to measure
endocannabinoids. Finally, it may depend on the brain re-
gions, as we have analysed not only the nucleus accumbens
but also the whole striatum. Despite of these limitations,
our data suggest that 2-AG, but not anandamide, seems to
be the endocannabinoid responsible for mediating increases
in activation of CB2 receptors after blockade of CB1 receptors.
The preferential role of CB2 receptors in mediating 2-AG ef-
fects as compared to anandamide has also been observed in
other behavioural responses. For instance, the anxiolytic-like
effect induced by 2-AG hydrolysis inhibitors depend on acti-
vation of CB2 receptors, whereas the same effect induced by
anandamide hydrolysis inhibitors is mediated mainly by
CB1 receptors (Busquets-Garcia et al., 2011).
Cannabinoid receptors and cocaine
To extend these results to the rewarding response to co-
caine, we investigated the effects of cannabinoid antagonists
in CPP. This test is relevant as it allows the investigation of
the contextual memory mechanisms that trigger drug seek-
ing (Cunningham et al., 2006). Previous studies showed that
antagonism of CB1 receptors (Yu et al., 2011) and CB2 recep-
tor agonists (Delis et al., 2017) inhibited cocaine-induced
CPP. Here, we hypothesized that the inhibitory effect of a
CB1 receptor antagonist would be reversed by a antagonist
at CB2 receptors in the CPP test. In line with this possibility,
we reversed the inhibitory effect of rimonabant on cocaine-
induced CPP by pretreating mice with the CB2 receptor
antagonist AM630. This result is indicative of an interaction
between cannabinoid receptors in modulating the rewarding
memories induced by cocaine.
In conclusion, this study supports the hypothesis that
endocannabinoids facilitate the stimulant and rewarding
properties of cocaine through CB1 receptors but inhibit them
if CB2 receptor activation is favoured. Therefore, the amelio-
rating effects of CB1 receptor antagonists possibly occur by
redirecting 2-AG to CB2 receptor-mediated activities. More-
over, combining low doses of a CB1 receptor antagonist with
2-AG hydrolysis inhibitors represents a potential approach
to curb the responses to cocaine. These results advance our
understanding of endocannabinoid modulation of
psychostimulant activity and may inform the development
of new pharmacological treatments for drug addiction.
Acknowledgements
The authors thank Ms. Adriane Aparecida and Mr. Jorge
Ferreira for the technical support. This research was funded
by Fundação de Amparo à Pesquisa do Estado de Minas
Gerais, FAPEMIG (APQ-01728-13), Conselho Nacional de
Desenvolvimento Científico e Tecnológico, CNPq (477541/
2012-7) and Science Foundation Ireland (10/IN.1/B2976).
Author contributions
P.H.G. conducted most of the experiments as part of his
PhD thesis. A.C.O., J.S.G., V.T.d.S., L.A., J.R.B., E.M.B.,
F.R.S., A.C.I. and B.N.O. conducted experiments. A.C.d.O.,
F.M.R., E.A.D.B., D.C.A., D.P.F. and F.A.M. supervised the re-
search. All authors participated in interpreting the results
and revising the manuscript and approved the final version.
Conflict of interest
The authors declare no conflicts of interest.
Declaration of transparency and
scientific rigour
This Declaration acknowledges that this paper adheres to the
principles for transparent reporting and scientific rigour of pre-
clinical research recommended by funding agencies, publishers
and other organisations engaged with supporting research.
British Journal of Pharmacology (2019) 176 1541–1551 1549
 P H Gobira et al.
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