Psychopharmacology (2020) 237:385–394

https://doi.org/10.1007/s00213-019-05370-5

ORIGINAL INVESTIGATION

The roles of cannabinoid CB1 and CB2 receptors in cocaine-induced

behavioral sensitization and conditioned place preference in mice
Jadna B. Lopes 1 & Juliana R. Bastos 1 & Rayssa B. Costa 1 & Daniele C. Aguiar 1 & Fabrício A. Moreira 1

Received: 20 October 2018 / Accepted: 30 September 2019 / Published online: 30 October 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Rationale Cocaine is a psychostimulant drug that facilitates monoaminergic neurotransmission. The endocannabinoid system,
comprising the cannabinoid receptors (CB1R and CB2R), the endocannabinoids, and their metabolizing-enzymes, modulates the
mesolimbic dopaminergic pathway and represents a potential target for the treatment of addiction.
Objectives Here, we tested the hypothesis that the cannabinoid receptors are implicated in cocaine-induced motor sensitization,
conditioned place preference (CPP), and hippocampal activation.
Methods Male Swiss mice received injections of AM251 (CB1R antagonist; 0.3–10 mg/kg) or JWH133 (CB2R agonist; 1–10
mg/kg) before acquisition or expression of cocaine (20 mg/kg)-induced sensitization and CPP. After the CPP test, cFos-staining
was employed as a marker of neuronal activation in the hippocampus.
Results AM251 inhibited the acquisition (0.3, 1, and 3 mg/kg) and expression (1 and 3 mg/kg) of sensitization, as well as the
acquisition (10 mg/kg) of CPP. JWH133 inhibited the acquisition (0.3 and 1 mg/kg) and expression (1 and 3 mg/kg) of both
sensitization and CPP. JWH133 effects were reversed by AM630 (CB2R antagonist; 5 mg/kg). AM251 and JWH133 also
prevented neuronal activation (c-Fos expression) in the hippocampus of CPP-exposed animals.
Conclusions CB1R and CB2R have opposite roles in modulating cocaine-induced sensitization and CPP, possibly by preventing
neuronal activation in the hippocampus.

Keywords Psychostimulants . Reward . Drug abuse . Addiction . Memory . Endocannabinoids

Introduction to various molecular, cellular, and behavioral changes. In ex-

Cocaine addiction is a chronic disorder for which pharmaco-
logical treatments are limited (Penberthy et al. 2010). This
psychostimulant drug blocks the reuptake of monoamines,
including dopamine, and increases the levels of this neuro-
transmitter in some brain regions (Heikkila et al. 1975).
Dopaminergic neurons project from the substantia nigra and
the ventral tegmental area (VTA) to forebrain regions
(Bjorklund and Dunnett 2007), comprising distinct neuronal
pathways linked to cognition, mood, and reward (Bressan and
Crippa 2005; Volkow et al. 2017). After continuous use, co-
caine and other drugs Bhijack^ brain reward systems, leading
* Fabrício A. Moreira
fabriciomoreira@icb.ufmg.br
1 tinct compartments with different contextual cues, which are
Department of Pharmacology, Institute of Biological Sciences,
Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos
6627, Belo Horizonte, MG 31270-901, Brazil
perimental animals, one major effect of repeated cocaine ad-
ministration is an increase in motor stimulant responses,
termed behavioral sensitization (Shuster et al. 1977; Steketee
and Kalivas 2011). Several protocols have been developed to
study this response, with previous studies showing that a sin-
gle dose of cocaine is sufficient to induce sensitization
(Marinho et al. 2015; Valjent et al. 2010). Behavioral sensiti-
zation is relevant for studying both the neurobiology and treat-
ment of addiction, since it is a robust protocol and responds
well to pharmacological interventions. However, its use is
limited due to some confounding factors, particularly when
the testing compounds reduce spontaneous locomotion
(Steketee and Kalivas 2011).
Another experimental approach to the study of cocaine
addiction is the conditioned place preference (CPP) test. The
CPP test is generally performed in a box containing two dis-
equally explored by the rodents in a pre-test session. The
conditioning session consists of injecting the drug of interest
386
and confining the animal in one compartment (the conditioned
stimulus), whereas vehicle injection is paired with the other
compartment, in alternate days. After the conditioning phase,
the test session is performed in absence of the drug (i.e., the
unconditioned stimulus). If there is an increase in the time
exploring the paired compartment, as compared to the
vehicle-paired side, the drug is considered as a rewarding
stimulus. (Bardo and Bevins 2000; Mucha et al. 1982;
Sanchis-Segura and Spanagel 2006). One major advantage
of CPP is its sensitivity to manipulations that affect the acqui-
sition and the expression of the conditioned response.
However, the interpretations of this test are limited by the fact
that the animal does not self-administer the drug
(Cunningham et al. 2006). Another limitation is that the neural
circuitry underlying CPP is only partially understood. It is
proposed to involve brain regions related to rewarding re-
sponses, particularly the mesolimbic dopaminergic system,
as well as structures underlying contextual learning, such as
the hippocampus. This structure has been proposed as a key
brain region responsible for the formation of memories related
to addictive drugs, possibly through efferent connections to
the brain rewarding system in the ventral striatum (Hitchcock
and Lattal 2018; Meyers et al. 2006; Sjulson et al. 2018;
Trouche et al. 2016).
The endocannabinoid system, comprising the cannabinoid
receptors, the endocannabinoids, and the enzymes responsible
for their synthesis and degradation, modulates dopaminergic
reward pathways (Covey et al. 2017; Pan et al. 2008; Riegel
and Lupica 2004). The physiological effects of the main
endocannabinoids, anandamide and 2-arachidonoylglycerol
(2-AG), are mediated by two Gi/o protein-coupled receptors,
CB1R (Devane et al. 1988; Matsuda et al. 1990) and CB2R
(Munro et al. 1993). The CB1R is densely expressed in vari-
ous brain regions, including the nucleus accumbens, the pre-
frontal cortex, the hippocampus, and the VTA, among others
involved in reward and addiction (Herkenham 1992). The
CB2R, first described as a peripheral receptor, seem to be also
expressed in the central neural system (CNS), with recent
evidence suggesting its localization in dopaminergic VTA
neurons (Zhang et al. 2014; Zhang et al. 2017) and in the
hippocampus (Brusco et al. 2008a; Stempel et al. 2016).
Both cannabinoid receptors may influence the behavioral
and molecular responses to psychostimulant drugs (Covey
et al. 2017; Moreira et al. 2015). For instance, CB1R antago-
nists attenuate behavioral sensitization (Corbille et al. 2007;
Marinho et al. 2015; Mereu et al. 2015), acquisition of CPP
(Hu et al. 2015; Yu et al. 2011), and relapse to self-
administration (De Vries et al. 2001) induced by cocaine.
CB1R antagonists also reduce neuronal activation after
cocaine-induced sensitization (Marinho et al. 2015).
However, there are conflicting results showing that cocaine-
induced self-administration (Cossu et al. 2001), sensitization,
and CPP (Martin et al. 2000) remain unaltered in CB1R
Psychopharmacology (2020) 237:385–394
knockout mice. CB2Rs seem to play an opposite role on the
rewarding effects of cocaine, since their activation leads to
similar results compared to CB1R antagonism on cocaine
self-administration (Zhang et al. 2015) and CPP
(Ignatowska-Jankowska et al. 2013). Moreover, the overex-
pression of CB2R also attenuates cocaine-induced responses
(Aracil-Fernandez et al. 2012). Finally, studies that directly
compared the effects of CB1R antagonists and CB2R agonists
observed that both classes of compounds inhibit the hyperac-
tivity and the CPP induced by cocaine in rats and mice (Delis
et al. 2017; Gobira et al. 2019). However, it is still unclear if
the same applies to cocaine sensitization. In addition, the brain
regions implicated in the cannabinoid modulation of cocaine-
induced CPP remains to be investigated.
Here, we tested the hypothesis that CB1R and CB2R facil-
itate and inhibit the behavioral effects of cocaine, respectively.
We compared the effects of three doses of the CB1R antago-
nist, AM251, and the CB2R agonist, JWH133, on motor sen-
sitization and CPP induced by cocaine in mice. Moreover, we
investigated the effects of these drugs on cocaine-induced
neural activation (c-Fos expression) in the hippocampus after
the CPP protocol. Our data support the hypothesis that CB1R
and CB2R have opposite roles in modulating some addiction-
related effects of cocaine.
Methods
Animals
Male Swiss mice (20–25 g) were housed in groups of five
animals per box in a room under constant temperature (24 ±
2 °C) and a 12 h light/dark cycle, with free access to water and
food. The experiments were conducted with a total of 598
animals during the light phase of the cycle, in accordance with
previous protocols (Cunningham et al. 2006; Shuster et al.
1977). All procedures used in the present study were approved
by the Ethics Committee on the Use of Animals (CEUA) from
the Federal University of Minas Gerais (UFMG), protocol
number 55/2016, in accordance with Brazilian and interna-
tional guidelines for the use of laboratory animals.
Drugs
Sensitization and CPP were induced with a dose of 20 mg/kg
of cocaine (Merck & Co., Inc.) diluted in saline (NaCl 0,9%).
To dilute the cannabinoid-related drugs, a solution was pre-
pared with 5% ethanol, 5% Cremophor®, and 90% saline.
They were administrated before the acquisition or expression
phases of sensitization and CPP. AM251 (Tocris), an
antagonist/inverse agonist of CB1R, and JWH133 (Tocris), a
CB2R agonist, were administered at the doses of 0.3–10
mg/kg. AM630 (Tocris), a CB2R antagonist, was administered
Psychopharmacology (2020) 237:385–394
at the doses of 5 or 10 mg/kg. All drugs were intraperitoneally
administered in a volume of 10 ml/kg. The dose ranges were
selected based on previous studies (Xi et al. 2011; Zhang et al.
2015; Zhang et al. 2014).
Behavioral sensitization
Cocaine-induced sensitization was evaluated in a cylindrical
arena (30 cm diameter and 50 cm high). In both days, the
animals were initially habituated to the arena for 10 min.
After habituation, they received a 20 mg/kg dose of cocaine
and were immediately placed back to the arena, where loco-
motion was measured for 10 min (test phase). Cannabinoid
drugs were administrated either on day 1 or day 2, to test for
their effects on the acquisition and expression of cocaine-
induced sensitization, respectively. AM251 and JWH133
were administered 30 min before cocaine (i.e., 20 min before
the habituation phase), whereas AM630 was administered
40 min before cocaine injection (i.e., 30 min before the habit-
uation phase). Mouse activity was recorded with a video cam-
era (Microsoft LifeCam®) and the total distance traveled was
analyzed with ANY-maze (4.5 version) software. The data are
presented as the total distance traveled (in meters) on each day.
Conditioned place preference
Cocaine-induced CPP was evaluated using an acrylic box
with two compartments (15 × 12 × 12 cm each) connected
by a central corridor (9.5 × 5 × 12 cm) with removable doors
between each compartment. The walls and floors had distinct
color and texture. On day 1, each animal was placed in the
central corridor with free access to the box compartments for
15 min. Animals spending more than 70% of the time in one
of the compartments were excluded from the experiment.
During the conditioning phases (days 2–7), they received co-
caine and vehicle injections, alternately, immediately before
exposure to a specific compartment of the box for 30 min.
Half of the animals had cocaine paired with one compartment
and the rest of the animals with the opposite compartment. On
the test day (day 8), the same procedure described for day 1
was repeated.
To test for drug effects on CPP acquisition, AM251 and
JWH133 were administrated 30 min before each cocaine in-
jection, whereas AM630 was administered 40 min before. To
test for drug effects on CPP expression, the cannabinoid drugs
were administrated only on the test day. The experiments were
recorded with a video camera (Microsoft LifeCam®) and the
time spent in each compartment was analyzed using ANY-
maze (4.5 version) software. The data are expressed as CPP
scores (in seconds), which is defined as the time spent in the
drug-paired compartment minus the time spent in the vehicle-
paired compartment (Cunningham et al. 2006).
387
Immunohistochemistry
Two hours after the CPP test, the animals were anesthe-
tized with ketamine (80 mg/kg) and xylazine (15 mg/kg)
and transcardially perfused with saline and paraformalde-
hyde 4% (PFA) in PB 0.1 M (pH 7.4). The brains were
fixed in PFA for 2 h, cryoprotected in sucrose 30% for 48
h, frozen in isopentane solution, and kept at − 80 °C.
Coronal slices (25 μm) were obtained with cryostat
(Leica CM1850) and immediately transferred to PBS
0.01 M (pH 7.4). The quantification of c-Fos expression
in the hippocampus of mice exposed to CPP was per-
formed using a procedure previously described (Batista
et al. 2016). Slices were incubated with hydrogen perox-
ide 3% in PBS 0.01 M for 30 min. Epitopes were blocked
with bovine serum 1% in PBS 0.01 M with Triton X-100
0.1%. Incubation with anti c-Fos primary antibody (sc-52,
rabbit polyclonal IgG, Santa Cruz Biotechnology), diluted
at 1:500 in the blocking solution, was performed for 44 h
at room temperature. After washing, the slices were incu-
bated with secondary biotinylated antibody (goat anti-
rabbit IgG 1:1000 in PBS with BSA 1% and Triton X-
100 0.4%). The avidin-biotin-peroxidase complex
(Vectastain ABC kit, Vector Lab) was used to amplify
the target antigen signal. To reveal c-Fos staining, 3,3′-
diaminobenzidine (DAB) was used. We counted positive
c-Fos cells, manually (ImageJ 1.51j8 version), in slices
between − 1.82 and − 2.06 mm from bregma (Paxinos
and Franklin 2004). The images were obtained with a
light microscope (Zeiss, Oberkochen, Germany) and Zen
software (× 10 objective). In order to analyze the whole
hippocampus in each slice, four images per slice were
taken. We selected for cFos analysis only those groups
in which we detected a behavioral effect of treatments
with cannabinoid-related drugs. The results are presented
as the number of positive cells per hippocampus slice.
Statistical analysis
All statistical analyses and graphic elaborations were per-
formed with GraphPad Prism 5 software. Results from sen-
sitization protocol were subjected to two-way analysis of
variance (ANOVA), considering drug treatments and days
as experimental factors, followed by Bonferroni post-hoc
test. Cocaine-induced CPP scores were analyzed by one-
way ANOVA followed by the Newman-Keuls test. The
control experiment for the effects of cannabinoid drugs
on CPP were subjected to one-sample t test, in which the
CPP score of each group was compared to zero (neither
preference nor aversion). The significance level was con-
sidered as p < 0.05. Data are presented as mean and stan-
dard error of mean (s.e.m.).
388 Psychopharmacology (2020) 237:385–394

Results
Effects of CB1R antagonism and CB2R agonism
on cocaine-induced sensitization
The administration of the CB1R antagonist AM251 (0.3–3
mg/kg), on day 1, prevented the acquisition of cocaine sensi-
tization (treatment: F4,70 = 9.444, p < 0.0001; day: F1,70 =
24.03, p < 0.0001; interaction: F4,70 = 1.350, n.s.; Fig. 1a).
Moreover, when administered on the second day, AM251 (1
and 3 mg/kg) inhibited the expression of cocaine-induced be-
havioral sensitization (treatment: F4,66 = 12.19, p < 0.0001;
day: F1,66 = 45.93, p < 0.0001; interaction: F4,66 = 3.024, p =
0.0237; Fig. 1b). Similar to AM251, the CB2R agonist,
JWH133, prevented the acquisition of cocaine-induced sensi-
tization, except at the highest dose (drug: F4,54 = 6.609, p =
0.0002; time: F1,54 = 9.965, p = 0.0026; interaction: F4,54 =
2.124, n.s.; Fig. 2a). JWH133 (1 and 3 mg/kg) also inhibited
the expression of cocaine-induced sensitization, when admin-
istered on the second day (drug: F4,49 = 4.385, p = 0.0041;
time: F1,49 = 5.583, p = 0.0221; interaction: F4,49 = 1.290, n.s.;
Fig. 2b).
The CB2R antagonist, AM630 (5 mg/kg), reverted the ef-
fect of JWH133 on both acquisition (drug: F4,50 = 12.21, p <
0.0001; time: F1,50 = 50.05, p < 0.0001; interaction: F4,50 =
4.034 , p = 0.0065; Fig. 3a) and expression phases (drug: F4,50
= 7.136, p = 0.0001; time: F 1,50 = 21.11, p < 0.0001;
interaction: F4,50 = 2.185, n.s.; Fig. 3b). As a control for drug
effects on basal motor activity, we analyzed the effects of
AM630 (5 mg/kg), JWH133 (0.3, 1, and 3 mg/kg), and
AM251 (0.1, 0.3, 1, 3, and 10 mg/kg) in animals that did
not receive cocaine administration. Importantly, none of these
drugs modified the total distance moved (F9,49 = 0.7124, n.s.),
discarding stimulant or depressing motor effects as potential
confounding factors (Table 1).
Effects of CB1R antagonism and CB2R agonism
on cocaine-induced CPP
The highest dose of AM251 (10 mg/kg), administrated before
cocaine injections, inhibited the acquisition of CPP (F4,47 =
7.741, p < 0.0001; Fig. 4a). This compound, however, failed
to prevent the expression of CPP (F4,44 = 4.956, p = 0.0022;
Fig. 4b). The CB2R agonist, JWH133, also modified cocaine
effect in the CPP paradigm. JWH133 (10 mg/kg) prevented
the development of cocaine-induced CPP when administrated
during the acquisition (F4,46 = 4.168, p = 0.0058; Fig. 5a) and
in the expression phase (F4, 44 = 4.843, p = 0.0025; Fig. 5b).
Next, we investigated if AM630 (10 mg/kg) prevents
JWH133 (10 mg/kg) effects. This compound reverted
JWH133 effects on both acquisition (F4,42 = 7.650, p =
0.0001; Fig. 6a) and expression (F4,49 = 6.222, p = 0.0004;
Fig. 6b) of cocaine-induced CPP. To test if the cannabinoid
drugs induce conditioned place preference or aversion,

Fig. 1 Effects of the CB1R

antagonist, AM251 (0.3, 1, and 3
mg/kg), on cocaine (20 mg/kg)
sensitization in mice. a AM251,
injected 30 min before cocaine on
day 1, prevented the acquisition
of sensitization. *p < 0.05
compared to vehicle +
vehicle∣cocaine; #p < 0.05
compared to vehicle +
cocaine∣cocaine (n = 8/group). b
AM251, injected 30 min before
cocaine on day 2, prevented the
expression of sensitization. *p <
0.05 compared to vehicle∣vehicle
+ cocaine; #p < 0.05 compared to
cocaine∣vehicle + cocaine. (n =
7, 8, 7, 8, 8). Each bar represents
the mean and s.e.m. The data
were analyzed by two-way
ANOVA followed by Bonferroni
post-hoc test
Psychopharmacology (2020) 237:385–394 389

Fig. 2 Effects of the CB2R

agonist, JWH133 (0.3, 1, and 3
mg/kg), on cocaine (20 mg/kg)
sensitization in mice. a JWH133,
injected 30 min before cocaine on
day 1, prevented the acquisition
of sensitization. *p < 0.05
compared to vehicle +
vehicle∣cocaine; #p < 0.05
compared to vehicle +
cocaine∣cocaine (n = 6, 6, 7, 7,
6). b JWH133, injected 30 min
before cocaine on day 2,
prevented the expression of
sensitization. *p < 0.05 compared
to vehicle∣vehicle + cocaine; #p <
0.05 compared to
cocaine∣vehicle + cocaine. (n =
7, 8, 7, 8, 8). Each bar represents
the mean and s.e.m. The data
were analyzed by two-way
ANOVA followed by Bonferroni
post-hoc test

AM251, JWH133, and AM630 injections were paired with from zero, discarding any potential source of confounding
the context with no subsequent cocaine treatment. The CPP factors (AM251: t8 = 0.4825, n.s.; AM630: t8 = 1.341, n.s.;
score obtained after treatment with these drugs did not differ JWH133: t8 = 0.9380, n.s.; one sample t test; Table 2).

Fig. 3 Effects of the CB2R

antagonist, AM630 (5 mg/kg), on
the inhibitory effect of JWH133
(1 mg/kg) on cocaine
sensitization in mice. a AM630
prevented the inhibitory effect of
JWH133 on the acquisition of
sensitization. *p < 0.05 compared
to vehicle + vehicle +
vehicle∣cocaine; #p < 0.05
compared to vehicle + vehicle +
cocaine∣cocaine; &p < 0.05
compared to vehicle + JWH133 +
cocaine∣cocaine (n = 6/group). b
AM630 prevented the inhibitory
effect of JWH133 on the
expression of sensitization; *p <
0.05 compared to vehicle∣vehicle
+ vehicle + cocaine; #p < 0.05
compared to cocaine∣vehicle +
vehicle + cocaine; &p < 0.05
compared to cocaine∣ vehicle +
JWH133 + cocaine (n = 6/group).
Each bar represents the mean and
s.e.m. The data were analyzed by
two-way ANOVA followed by
Bonferroni post-hoc test
390 Psychopharmacology (2020) 237:385–394

Table 1 Effects of AM630, JWH133, and AM251 on basal locomotion

in mice exposed to the arena during 10 min after habituation. Distances
traveled were not different from vehicle, indicating that none of the
compounds interfered with motor activity. Results are expressed as
mean ± SEM (n = 6 mice/group)

Treatment Dose Distance traveled (m)

Vehicle – 8.25 ± 1.14

AM630 5 mg/kg 8.49 ± 1.47
JWH133 0.3 mg/kg 9.85 ± 1.17
1 mg/kg 8.40 ± 0.84
3 mg/kg 10.29 ± 0.68
AM251 0.1 mg/kg 9.24 ± 1.96
0.3 mg/kg 8.90 ± 2.18
1 mg/kg 8.24 ± 0.37
3 mg/kg 6.44 ± 0.99
10 mg/kg 7.07 ± 1.48

Neuronal activation in the hippocampus

after cocaine-induced CPP

After the CPP test, animals from vehicle + vehicle, vehicle +

cocaine, AM251 + cocaine, and JWH133 + cocaine groups
were randomly selected for c-Fos quantification in the hippo-
campus. In line with the behavioral results, the administration
of the CB1R antagonist during the acquisition phase (F2,11 =
31.84, p < 0.0001; Fig. 7) and the CB2R agonist during the
acquisition and the expression phases (A: F2,10 = 8.22, p =
0.0077, B: F2,9 = 23.01, p = 0.0003; Fig. 8) prevented the
increase in c-Fos expression in the hippocampus.
Discussion
Drugs that target CB1R and CB2R have been investigated in
animal models relevant to cocaine addiction. Herein, we tested
three doses of the CB1R antagonist, AM251, and the CB2R
agonist, JWH133, to provide a detailed analysis of their effects
on cocaine-induced sensitization and CPP in mice. We
showed that these compounds inhibit motor sensitization at
doses that preserve basal locomotion. Moreover,
complementing previous studies (Delis et al. 2017; Gobira
et al. 2019), we found that AM251 and JWH133 also
prevented cocaine-induced CPP. Finally, using cFos as a
marker of neuronal activation, we observed that these com-
pounds inhibit hippocampal activity in animals exposed to the
context previously paired with cocaine.
Behavioral sensitization protocols vary considerably in re-
gard to the time between acquisition and expression phases. In
the present experiments, we found that a two-injection proto-
col results in cocaine sensitization, in accordance with previ-
ous studies (Marinho et al. 2015; Valjent et al. 2010). Using
Fig. 4 Effects of the CB1R antagonist, AM251 (1, 3, and 10 mg/kg), on
CPP induced by cocaine (20 mg/kg) in mice. a AM251 inhibited the
acquisition of CPP (n = 11, 11, 10, 10, 10). b AM251 failed to inhibit
the expression of CPP (n = 12, 12, 8, 8, 9). *p < 0.05 compared to vehicle
+ vehicle group, #p < 0.05 compared to vehicle + cocaine group. Each bar
represents the mean and s.e.m. The data were analyzed by one-way
ANOVA followed by Newman-Keuls post-hoc test
this protocol, we demonstrated that AM251, a CB1R antago-
nist, inhibited both the acquisition (when administrated on day
1) and the expression (when administered on day 2) of
cocaine-induced sensitization. These results corroborate stud-
ies using rimonabant, another CB1R antagonist (Marinho et al.
2015; Mereu et al. 2015), although other authors did not ob-
serve any effect with AM251 (Corbille et al. 2007;
Kupferschmidt et al. 2012), rimonabant (Filip et al. 2006) or
in CB1R knockout mice (Martin et al. 2000). These discrep-
ancies might be explained by differences in animal species
and strains.
Regarding the role of CB2R, previous studies have focused
on the acute effects of cocaine on locomotion, but not on
behavioral sensitization (Delis et al. 2017; Gobira et al.
2019; Xi et al. 2011). Here, we show that the selective
CB2R agonist, JWH133, prevents the acquisition and expres-
sion of cocaine sensitization at doses that did not change the
acute response to this psychostimulant drug, corroborating
previous results in mice overexpressing the CB2R (Aracil-
Fernandez et al. 2012). The effects of JWH133 on the acqui-
sition of cocaine sensitization occurred only at the lowest and
intermediate doses. Although the reason for this is unclear, it is
Psychopharmacology (2020) 237:385–394
Fig. 5 Effects of the CB2R agonist, JWH133 (1, 3, 10 mg/kg), on CPP
induced by cocaine (20 mg/kg) in mice. a JWH133 inhibited the
acquisition of CPP (n = 10, 11, 11, 10, 9). b JWH133 inhibited the
expression of CPP (n = 9, 11, 9, 10, 10). *p < 0.05 compared to vehicle
+ vehicle group; #p < 0.05 compared to vehicle + cocaine group. Each bar
represents the mean and s.e.m. The data were analyzed by one-way
ANOVA followed by Newman-Keuls post-hoc test
unlike to depend on effects on motor activity, since JWH133
did not reduce spontaneous locomotion at any of the doses
tested. Regarding the antagonism of CB2R, AM630 did not
alter cocaine effects, in accordance with previous results
(Blanco-Calvo et al. 2014).
In addition to locomotor sensitization, we investigated the
role of cannabinoid receptors in cocaine-induced CPP, a
contextual-memory paradigm. Behavioral sensitization and
CPP have different implications for the various aspects of drug
addiction, each recruiting specific neural substrates.
Sensitization seems to depend on converging circuitries relat-
ed to motivational and motor responses (Steketee and Kalivas
2011), whereas CPP engages brain regions related to contex-
tual memory formation (Bardo and Bevins 2000). We ob-
served that the antagonism of CB1R (with AM251) prevented
the acquisition of cocaine-induced CPP, corroborating previ-
ous studies with rimonabant (Delis et al. 2017; Hu et al. 2015;
Yu et al. 2011). When administrated on the expression phase,
AM251 failed to revert cocaine effects. Thus, CB1R seems to
participate on the acquisition of the contextual memory related
to cocaine, although it is not necessary for the evocation of this
391
Fig. 6 Effects of the CB2R antagonist, AM630 (10 mg/kg), on the
inhibitory effect of JWH133 (10 mg/kg) on CPP induced by cocaine
(20 mg/kg) in mice. a AM630 reversed the inhibitory effect of JWH133
on the acquisition of CPP (n = 10, 10, 10, 9, 9). b AM630 reversed the
inhibitory effect of JWH133 on the expression of CPP (10, 11, 11, 11, 11).
*p < 0.05 compared to vehicle + vehicle + vehicle group; #p < 0.05
compared to vehicle + vehicle + cocaine group. Each bar represents the
mean and s.e.m. The data were analyzed by one-way ANOVA followed
by Newman-Keuls post-hoc test
memory. As for the CB2R, the selective agonist JWH133
inhibited both the acquisition and expression of cocaine-
induced CPP. These results indicate that the CB2R modulates
the rewarding memories formed after cocaine injections, cor-
roborating a recent study using rats as experimental subjects
(Delis et al. 2017).
Table 2 Effects of AM251 (10 mg/kg), AM630 (10 mg/kg), and
JWH133 (10 mg/kg) on CPP. CPP scores were not different from 0,
indicating that these compounds induced neither place preference nor
aversion. Results are expressed as mean ± SEM (n = 9 mice/group)
Treatment Dose CPP score (s)
AM251 10 mg/kg 16.87 ± 34.95
AM630 10 mg/kg 39.11 ± 29.17
JWH133 10 mg/kg 3.23 ± 40.28
392 Psychopharmacology (2020) 237:385–394

Fig. 7 Effects of the CB1R antagonist, AM251 (10 mg/kg), on c-Fos
expression in mice hippocampi after CPP induced by cocaine (20 mg/kg).
AM251 prevented the increase in c-Fos expression when injected in the
acquisition phase of CPP. Brains were randomly chosen from each group
of animals after the behavioral experiment (n = 4 mice/group). Positive
cells in both sides of the hippocampi were manually counted with ImageJ
software. *p < 0.05 compared to vehicle + vehicle group, #p < 0.05
compared to vehicle + cocaine group. Each bar represents the mean and
s.e.m. The data were analyzed by one-way ANOVA followed by
Newman-Keuls post-hoc test. Representative images obtained from mice
dentate gyrus are also presented

Fig. 8 Effects of the CB2R

agonist, JWH133 (10 mg/kg), on
c-Fos expression in mice hippo-
campi after CPP induced by co-
caine (20 mg/kg). JWH133
prevented the increase in c-Fos
expression when injected in the
acquisition (a) and expression (b)
phases of CPP. Brains were ran-
domly chosen from each group of
animals after the behavioral ex-
periment (n = 4 mice/group).
Positive cells in both sides of the
hippocampi were manually
counted with ImageJ software. *p
< 0.05 compared to vehicle + ve-
hicle group, #p < 0.05 compared
to vehicle + cocaine group. Each
bar represents the mean and s.e.m.
The data were analyzed by one-
way ANOVA followed by
Newman-Keuls post-hoc test.
Representative images obtained
from mice dentate gyrus are also
presented
Psychopharmacology (2020) 237:385–394
The brain regions in which CB1R and CB2R modulate
cocaine responses have been poorly investigated. CB1R is
expressed pre-synaptically in GABAergic terminals
projecting onto dopaminergic cell bodies in the VTA, where
it may modulate the increased dopaminergic activity after co-
caine administration (Wang et al. 2015). It is also densely
expressed in various subregions of the hippocampus
(Herkenham et al. 1991; Herkenham et al. 1990). As for the
CB2R, although it was initially believed to be restricted to the
periphery, later studies suggested its expression in the brain
(Onaivi et al. 2006), including in dopaminergic neurons of the
VTA (Zhang et al. 2014; Zhang et al. 2017) and in the hippo-
campus (Brusco et al. 2008a; Brusco et al. 2008b; Stempel
et al. 2016). Since CPP expression depends on neural circuits
responsible for spatial memory recovery (Hitchcock and
Lattal 2018; Meyers et al. 2006; Sjulson et al. 2018; Trouche
et al. 2016), we tested the effects of cannabinoid drugs on
hippocampal neural activation after cocaine-induced CPP.
AM251 and JWH133 prevented the increase in cFos expres-
sion in the hippocampus, mimicking their behavioral effects.
Although these results do not necessarily imply the hippocam-
pus as the direct site of action of these drugs, they suggest that
cannabinoid receptors interfere with the retrieval of drug-
related memories by inhibiting hippocampal activation during
exposure to contextual cues.
In conclusion, the present data extend previous studies that
directly compared CB1R antagonists and CB2R agonists in
animal models relevant to cocaine addiction (Delis et al.
2017; Gobira et al. 2019). We found that these compounds
inhibit the development of cocaine-induced sensitization and
CPP in mice. The inhibitory effects on CPP may depend on
the regulation of neuronal activity in the hippocampus. These
results imply a complex role for cannabinoid receptors in
modulating cocaine effects, with opposite functions of CB1R
and CB2R. Exploring these mechanisms may advance our
understanding of cannabinoid modulation of drug addiction.
Funding information This research was funded by Fundação de Amparo
à Pesquisa do Estado de Minas Gerais (FAPEMIG; APQ-02064-15),
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq; 406122/2016-4) and Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP; 2017/24304-0).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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