Enhancement of endocannabinoidneurotransmission through CB1 cannabinoidreceptors counteracts the reinforcingand psychostimulant effects of cocaine

Styliani Vlachou1, Fygaleia Stamatopoulou1, George G. Nomikos2 and George Panagis1

1 Laboratory of Behavioral Neuroscience, Department of Psychology, School of Social Sciences, University of Crete,

Rethymnon, Crete, Greece2 Cannasat Therapeutics, Toronto, Ontario, Canada

Abstract

Cannabinoids, in contrast to typical drugs of abuse, have been shown to exert complex effects on

behavioural reinforcement and psychomotor function. We have shown that cannabinoid agonists lack

reinforcing/rewarding properties in the intracranial self-stimulation (ICSS) paradigm and that the CB1

receptor (CB1R) agonist WIN55,212-2 attenuates the reward-facilitating actions of cocaine. We sought to

determine the effects of the endocannabinoid neurotransmission enhancer AM-404 (1, 3, 10, 30 mg/kg) on

the changes in ICSS threshold and locomotion elicited by cocaine and extend the study of the effects

of WIN55,212-2 (0.3, 1, 3 mg/kg) on cocaine-induced hyperlocomotion. AM-404 did not exhibit reward-

facilitating properties, and actually increased self-stimulation threshold at the highest dose. Cocaine

significantly reduced self-stimulation threshold, without altering maximal rates of responding. AM-404

(10 mg/kg) attenuated this action of cocaine, an effect which was reversed by pretreatment with the

selective CB1R antagonist SR141716A. WIN55,212-2 decreased locomotion at the two highest doses, an

effect that was blocked by SR141716A; AM-404 had no effect on locomotion. Cocaine caused a significant,

dose-dependent increase in locomotion, which was reduced by WIN55,212-2 and AM-404. SR141716A

blocked the effects of WIN55,212-2 and AM-404 on cocaine-induced hyperlocomotion. SR141716A alone

had no effect on ICSS threshold or locomotion. These results indicate that cannabinoids may interfere with

brain reward systems responsible for the expression of acute reinforcing/rewarding properties of cocaine,

and provide further evidence that the cannabinoid system could be explored as a potential drug discovery

target for the treatment of psychostimulant addiction and pathological states associated with psychomotor

overexcitability.

Received 19 December 2007 ; Reviewed 19 January 2008 ; Revised 11 February 2008 ; Accepted 17 February 2008 ;

First published online 1 April 2008

Key words : AM-404, cocaine, locomotion, reinforcement, WIN55,212-2.

Introduction

The endocannabinoid system consists of several

recently discovered lipid mediators that produce

their effects via interaction with specific cannabinoid

binding sites (Rodriguez de Fonseca et al., 2005).

Ample evidence indicates that endocannabinoids play

a significant modulatory role in the control of motor

behaviour, nociception, appetite, cognition, affect

and behavioural reinforcement/reward processes

(Chaperon and Thiebot, 1999). Most of the behavioural

studies have been conducted with direct cannabinoid

agonists, which are known for their undesirable psy-

chotropic effects.

Recently, interest has increased for substances that

enhance endocannabinoid neurotransmission through

inhibition of elimination, and therefore may lack

the adverse psychotropic effects induced by direct

stimulation of the cannabinoid CB1 receptor (CB1R;

Huestis et al., 2001). Although some of these en-

docannabinoid modulators have been examined in

Address for correspondence : Dr G. Panagis, University of Crete,

School of Social Sciences, Department of Psychology, Laboratory

of Behavioral Neuroscience, 74100 Rethymnon, Crete, Greece.

Tel. : (30) (28310) 77544 Fax : (30) (28310) 77548

E-mail : panagis@psy.soc.uoc.gr

International Journal of Neuropsychopharmacology (2008), 11, 905–923. Copyright f 2008 CINPdoi:10.1017/S1461145708008717

ARTICLE

CINP

various behavioural paradigms (see e.g. Compton and

Martin, 1997 ; de Lago et al., 2004 ; Kathuria et al.,

2003), only a few studies have examined their possible

reinforcing/rewarding properties (Bortolato et al.,

2006 ; Gobbi et al., 2005; Hansson et al., 2007 ; Vlachou

et al., 2006).

Cannabis and compounds that stimulate endo-

cannabinoid neurotransmission may arguably affect

the actions of typical drugs of abuse (del Arco et al.,

2002 ; Gallate et al., 1999 ; Solinas et al., 2005; Vigano

et al., 2004 ; Vlachou et al., 2003). Although interactions

with most drugs of abuse have been extensively

studied, little is known about the interaction between

cannabinoids and psychostimulants, possibly because

most psychostimulants, in contrast to other drugs

of abuse, affect the mesolimbic dopaminergic ter-

minals (increasing extracellular dopamine concen-

trations) directly (Lupica and Riegel, 2005). Human

and animal studies (De Vries et al., 2001 ; Foltin et al.,

1993 ; Lukas et al., 1994; Vinklerova et al., 2002) have

shown that cannabis enhances cocaine’s euphorigenic

and behavioural effects. Inversely, other studies reveal

a counteracting effect of cannabinoids on cocaine-

induced behaviour-stimulating actions (Chaperon

et al., 1998 ; Fattore et al., 1999; Martin et al., 2000;

Parker et al., 2004 ; Schenk and Partridge, 1999;

Vlachou et al., 2003). Inconsistent findings have

also been reported with cannabinoids and other psy-

chostimulants, e.g. amphetamine and MDMA (Braida

et al., 2005; Lamarque et al., 2001 ; Muschamp and

Siviy, 2002). Interestingly, intracerebroventricular co-

administration of the CB1R agonist CP55,940 and

MDMA reduces the mean lever presses for their

combined self-administration, compared to self-

administration for each drug alone (Braida and Sala,

2002). In this direction, we have recently shown that

the CB1R agonist WIN55,212-2, in a dose that does

not affect baseline self-stimulation threshold, reduces

cocaine’s reinforcing/rewarding actions (Vlachou

et al., 2003).

The psychomotor effects of cannabinoids have

attracted interest, since the endocannabinoid system

has been implicated in different psychomotor dis-

orders, such as Parkinson’s and Huntington’s disease

and Tourette’s syndrome (Pacher et al., 2006).

Cannabinoids show a biphasic effect on locomotion,

causing hyperactivity in low doses and hypoactivity

or even catalepsy in high doses (Drews et al., 2005;

Sanudo-Pena et al., 2000 ; Sulcova et al., 1998).

However, our knowledge of the effects of indirect

cannabinoid modulators on either spontaneous loco-

motion or hyperlocomotion induced by psychostimu-

lants remains rather limited.

Accordingly, we investigated the effects of the en-

docannabinoid neurotransmission enhancer AM-404

(Beltramo et al., 1997) on brain stimulation reinforce-

ment and on cocaine’s reward-facilitating action. To

assess the possible involvement of CB1R in these ef-

fects, we also examined whether they could be re-

versed by pretreatment with the selective CB1R

antagonist SR141716A (Rinaldi-Carmona et al., 1994),

since not all of the centrally mediated effects of

endocannabinoids occur through CB1R stimulation.

Additionally, we examined the effects of WIN55,212-2

and AM-404 on spontaneous locomotion and cocaine-

induced hyperlocomotion, and whether SR141716A

could counteract these effects.

Materials and methods

Intracranial self-stimulation (ICSS) studies

Animals and surgery

Male Sprague–Dawley rats weighing 300–350 g at the

time of surgery were used. Before surgery they were

housed in groups of three under a 12-h light–dark

cycle (lights on 08:00 hours) with free access to food

and water. The animals were anaesthetized with in-

tramuscular (i.m.) injection of ketamine hydrochloride

(100 mg/kg) and xylazine (10 mg/kg). Atropine sul-

phate (0.6 mg/kg i.m.) was injected to reduce bron-

chial secretion. Movable monopolar stimulating

electrodes (Model SME-01, Kinetrods, Ottawa, ON,

Canada) were lowered into the medial forebrain

bundle (MFB) at the level of lateral hypothalamus

(coordinates AP x2.5 mm from bregma, L x1.7 mm

from the midline, VD x8.0 from a flat skull), accord-

ing to Paxinos and Watson (1998).

The electrodes consisted of a plastic guiding base

and a 0.25-mm diameter movable stainless-steel wire,

which were insulated with epoxylite except for the

conically shaped tip. The anode was an amphenol pin

connected to five miniature skull screws. Following

implantation and for the entire duration of the ex-

periments, the animals were housed individually.

Apparatus and procedures for self-stimulation

One week after surgery, the animals were tested for

self-stimulation in an operant chamber made of

transparent Plexiglas (25 cm wide, 25 cm deep and

30 cm high). A stainless-steel rodent lever protruded

2 cm from the left wall at a height of 4 cm from the

floor. Each lever press triggered a constant current

generator that delivered a 0.4-s train of rectangular

cathodal pulses of constant duration (0.1 ms) and

906 S. Vlachou et al.

intensity (250 mA) and variable frequency (15–100 Hz,

i.e. 6–40 number of pulses/0.4 s). The pulse frequency,

i.e. the number of pulses within a train, was pro-

gressively increased up to 40 per stimulation train

until the subject showed vigorous self-stimulation.

During the acquisition phase the animals were trained

to self-stimulate for at least three consecutive days

(1 h daily), using stimulation parameters that main-

tained near maximal lever-pressing rates. After self-

stimulation had been acquired and stabilized for a

given pulse frequency, animals were trained to self-

stimulate using four alternating series of ascending

and descending pulse frequencies. The pulse fre-

quency was varied by steps of y0.1 log units. Each

frequency was tested within trials of 60 s duration,

followed by an extinction period of 30 s. At the be-

ginning of each trial, the animals received three

trains of priming stimulation, at the frequency of the

stimulation, which was available for that trial. Thus,

drug-induced changes on the rewarding efficacy of

self-stimulation were inferred using the curve-shift

paradigm. This method, which appears to have the

reward selectivity that is required in psychopharma-

cological research, enabled us to distinguish between

reward and performance, while allowing quantifi-

cation of the drug effect.

Drugs

Cocaine hydrochloride (National Monopoly for

Narcotics, Ministry of Health, Greece) was dissolved

in 0.9% NaCl and injected intraperitoneally (i.p.) at a

volume of 1 ml/kg of body weight. AM-404 (Tocris

Bioscience, Ellisville, MO, USA) and SR141716A

(synthesized by Lilly Research Laboratories, Indian-

apolis, IN, USA) were dissolved into a vehicle solu-

tion that consisted of 5% dimethylsulfoxide, 5%

cremophor EL and 90% of 0.9% NaCl and injected i.p.

at a volume of 3 ml/kg of body weight. The doses

of the cannabinoid compounds tested were within

the range of doses regularly used in a plethora of

functional studies (see e.g. Compton andMartin, 1997 ;

de Lago et al., 2004 ; Kathuria et al., 2003).

Experimental procedure

Drug testing began for each animal when the function

relating lever-pressing rate to pulse frequency (the

rate-frequency function) was stable for at least three

consecutive days. The criterion for stability was met

when the frequency thresholds did not vary by more

than 0.1 log unit. Each drug or vehicle self-stimulation

test consisted of a baseline and a drug rate-frequency

function determination (for 45 min each). Following

the baseline period, each animal was injected with the

drug or its vehicle. The animals were tested 10 min

after the last injection.

In the present study we used a mixed design, i.e.

some animals received only one treatment, whereas

other animals received all doses for only one drug

treatment tested. In this case, the sequence of injec-

tions for the different drug doses was counterbalanced

with respect to order and a 3-d period was allowed

between injections. As we have observed in our pre-

vious studies, this period is considered sufficient

for the behaviour of the animals to return to stable,

pre-treatment levels, and not be affected by prior can-

nabinoid administration.

Experiment 1. Effects of systemically administered AM-

404 on brain stimulation reward. Twenty-seven rats were

used. Seven of them received all doses of AM-404 (1, 3,

10, 30 mg/kg i.p.) or its vehicle in a randomized order,

while 20 received only one drug treatment.

Experiment 2. Effects of the CB1R antagonist SR141716A

on AM-404-induced changes in brain stimulation reward.

Fifteen rats were used. Three of them received i.p.

SR141716A (0.02 mg/kg) or vehicle followed 5 min

later by AM-404 (30 mg/kg) or its vehicle i.p. in a

randomized order, while 12 received only one combi-

nation of SR141716A and AM-404 (or their respective

vehicles).

Experiment 3. Effects of AM-404 on the cocaine-induced

lowering of brain reward thresholds. Sixteen rats were

used. Four of them received AM-404 (0, 10 mg/kg i.p.)

followed 5 min later by cocaine (0, 5 mg/kg i.p.), while

12 received only one combination of AM-404 and

cocaine.

Experiment 4. Reversal of the action of AM-404 on the

cocaine-induced lowering of brain reward thresholds by

pretreatment with SR141716A. Fifteen rats were used.

Three of them received all combinations of SR141716A

(0, 0.02 mg/kg i.p.) and AM-404 (0, 10 mg/kg i.p.),

followed 5 min later by cocaine (0, 5 mg/kg i.p.), while

12 received only one combination of SR141716A, AM-

404 and cocaine.

Data analysis and statistics

Data gathered from pre- and post-injection portions of

each session were curve-fitted and threshold and

asymptote estimates were obtained using the

Gompertz sigmoid model (Coulombe and Miliaressis,

1987) :

f (X)=aexeb(xixx)

:

CB1R stimulation counteracts cocaine’s behavioural actions 907

In this equation, a represents the maximum rate

(asymptote), whereas xi (X at inflection) represents the

threshold frequency. The latter is the pulse number

producing 36.7% of the asymptotic rate, i.e. the rate

lying on the fastest accelerating region of the curve.

Parameter b represents an index of the slope whereas

e is the base of natural logarithms.

The post-treatment threshold and asymptote values

were expressed as percentage of pre-drug values.

The results were statistically evaluated using one-way

(effects of cannabinoid agonists alone) or two-way

(effects of combined administration of cannabinoid

agonists and antagonists) or three-way (effects of com-

bined administration of cocaine, cannabinoid agonists

and antagonists) analyses of variance (ANOVA) fol-

lowed, whenever appropriate, by the LSD test for

multiple contrasts. It should be noted that in our de-

sign, some animals received all treatments and others

received only one (see above). In order to combine

these two datasets, for all rats we expressed drug ef-

fects as a percentage of pre-injection baseline, which

was established independently for each drug or ve-

hicle treatment. Each drug test was thus treated as an

independent measure for statistical analysis.

Histology

At the end of the experiment, the animals were given

a lethal dose of sodium pentothal. The location of the

terminal stimulation site was then marked according

to the protocol described by Vlachou et al. (2006). The

brains were then removed and stored in a solution of

10% formalin until sectioned and stained for verifi-

cation of the electrode tips. Only the rats in which

tracks from the electrode were verified to be located in

the MFB were included in this study.

Spontaneous motor activity

Animals

Male Sprague–Dawley rats (n=576 total) weighing

300–350 g were used for all experiments. Animals

were housed three per cage under a 12-h light–dark

cycle (lights on 08:00 hours) with free access to food

and water.

Assessment of locomotor activity

Spontaneous motor activity was measured using an

activity recording system (Model 7445, Ugo Basile).

Each system consists of an animal cage and an elec-

tronic unit incorporating a counter and a printer. The

rectangular animal cage (56r56r30 cm) has trans-

parent sides and lid to allow observation. The cage

floor has horizontal and vertical infrared sensors. The

counter sums up the photocell disruptions, and a

printer displays the results at preset intervals. In our

studies, a summation of photocell disruptions of

locomotor activity and rearing, for each 5-min interval

period, during the 1 h observation period was

registered. The behavioural testing was performed

between 08:00 and 16:00 hours. All animals were

gently handled for 15 min each for 1 wk before the

beginning of the experimental procedure. Two days

before the drug testing each rat was gently handled for

15 min, injected i.p. with saline and habituated to the

experimental room for 3 h in the home cage. The rats

were also accustomed to the experimental room, for

1 h prior to the experiment. Each animal received only

one drug treatment.

Drugs

Cocaine hydrochloride (National Monopoly for Nar-

cotics, Ministry of Health, Greece) was dissolved in

0.9% NaCl and injected i.p. at a volume of 1 ml/kg

of body weight. WIN55,212-2 (Tocris Bioscience),

AM-404 (Tocris Bioscience) and SR141716A (syn-

thesized by Lilly Research Laboratories) were dis-

solved into a vehicle solution that consisted of 5%

dimethylsulfoxide, 5% cremophor EL and 90% of

0.9% NaCl and injected i.p. at a volume of 3 ml/kg

of body weight.

Study 1

Experiment 1. Effect of WIN55,212-2 on locomotor

activity. Animals (n=44) were injected with

WIN55,212-2 (0.3, 1, 3 mg/kg) or its vehicle and after

10 min placed in the centre of the chamber for 1 h.

Experiment 2. Reversal of WIN55,212-2-induced hypolo-

comotion with the CB1R antagonist SR141716A. Animals

(n=40) were injected with SR141716A (0.02 mg/kg)

or its vehicle, and after 5 min with WIN 55,212-2

(3 mg/kg) or its vehicle, and 10 min later they were

placed in the centre of the chamber for 1 h.

Experiment 3. Effect of WIN55,212-2 on cocaine-induced

hyperlocomotion (5 mg/kg). Animals (n=32) were in-

jected with WIN55,212-2 (1 mg/kg) or its vehicle and

after 5 min with cocaine (5 mg/kg) or saline ; im-

mediately thereafter the animals were placed in the

locomotor activity chambers.

Experiment 4. Reversal of the action of WIN55,212-2

on cocaine-induced hyperlocomotion (5 mg/kg) with

SR141716A. Animals (n=72) were injected with

908 S. Vlachou et al.

SR141716A (0.02 mg/kg) or its vehicle and WIN55,

212-2 (1 mg/kg) or its vehicle, followed after 5 min by

cocaine (5 mg/kg) or saline.

Experiment 5. Effect of WIN55,212-2 on cocaine-induced

hyperlocomotion (10 mg/kg). Two separate experiments

(expts 5 and 6) with a higher dose of cocaine (10 mg/

kg) than the dose used for the ICSS experiments

(5 mg/kg) were added in the study to ascertain the

robustness and the reproducibility of the effects of

WIN55,212-2 on counteracting cocaine-induced hy-

perlocomotion, as cocaine clearly increases locomotion

in a dose-dependent manner. Animals (n=36) were

injected with WIN55,212-2 (1 mg/kg) or its vehicle

and after 5 min with cocaine (10 mg/kg) or saline.

Experiment 6. Reversal of the action of WIN55,212-2

on cocaine-induced hyperlocomotion (10 mg/kg) with

SR141716A. Animals (n=64) were injected with

SR141716A (0.02 mg/kg) or its vehicle and

WIN55,212-2 (1 mg/kg) or its vehicle, followed after

5 min by cocaine (10 mg/kg) or saline.

Study 2

Experiment 1. Effect of AM-404 on locomotor activity.

Animals (n=36) were injected with AM-404 (3, 10,

30 mg/kg) or its vehicle and after 10 min placed in the

centre of the chamber for 1 h.

Experiment 2. Effect of AM-404 (10 mg/kg) on cocaine-

induced hyperlocomotion (5 mg/kg). Animals (n=32)

were injected with AM-404 (10 mg/kg) or its vehicle

and after 5 min with cocaine (5 mg/kg) or saline.

Experiment 3. Reversal of the action of AM-404 (10 mg/kg)

on cocaine-induced hyperlocomotion (5 mg/kg) with

SR141716A. Animals (n=80) were injected with

SR141716A (0.02 mg/kg) or its vehicle and AM-404

(10 mg/kg) or its vehicle, followed after 5 min by co-

caine (5 mg/kg) or saline.

Experiment 4. Effect of AM-404 (10 mg/kg) on cocaine-

induced hyperlocomotion (10 mg/kg). Similarly to the

experiments with WIN55,212-2 (see above), three ad-

ditional experiments (expts 4–6) were run with a

higher dose of AM-404 (30 mg/kg) and a higher dose

of cocaine (10 mg/kg) than the respective doses used

in the ICSS paradigm to confirm that AM-404 had

indeed a robust and reproducible action on cocaine-

induced hyperlocomotion. Animals (n=32) were in-

jected with AM-404 (10 mg/kg) or its vehicle and after

5 min, with cocaine (10 mg/kg) or saline.

Experiment 5. Effect of AM-404 (30 mg/kg) on cocaine-

induced hyperlocomotion (10 mg/kg). Animals (n=36)

were injected with AM-404 (30 mg/kg) or its vehicle

and after 5 min with cocaine (10 mg/kg) or saline.

Experiment 6. Reversal of the action of AM-404 (30 mg/

kg) on cocaine-induced hyperlocomotion (10 mg/kg) by

SR141716A. Animals (n=72) were injected with

SR141716A (0.02 mg/kg) or its vehicle and AM-404

(30 mg/kg) or its vehicle, followed after 5 min by

cocaine (10 mg/kg) or saline.

Statistical analysis

The total locomotor activity and rearing counts (de-

pendent variables) over the 1 h observation period

were statistically evaluated by using one-way (effects

of compounds alone), two-way (effects of combined

administration of two compounds) or three-way (ef-

fects of combined administration of three compounds)

ANOVA, followed by post-hoc analysis using the LSD

multiple comparison test in order to reveal specific

differences between groups.

All animal experiments were carried out in accord-

ancewith theNational Institute of Health Guide for the

Care and Use of Laboratory Animals (NIH publication

no. 86-23, revised 1996). Efforts were made in order to

minimize the number of animals used and reduce

their suffering.

Results

ICSS studies

Experiment 1. Effects of systemically administered

AM-404 on brain stimulation reward. The changes

in self-stimulation threshold and asymptotic rate of

responding after systemic injection of the endo-

cannabinoid neurotransmission enhancer AM-404

are presented in Figure 1(a, c), respectively. AM-404

(1, 3, 10, 30 mg/kg i.p.) significantly increased self-

stimulation thresholds at the highest dose tested

[F(4, 50)=4.239, p=0.005], while it had no effect in

the asymptotic rate of responding [F(4, 50)=1.100,

p=0.367]. Post-hoc analysis with the LSD test showed

that these effects on the self-stimulation thresholds

were significant at the highest dose tested (30 mg/

kg i.p.), compared with the other groups (p=0.011,

p=0.024 and p=0.012, respectively) and with the

vehicle-treated group (p<0.001).

Experiment 2. Effects of SR141716A on AM-404-induced

changes in brain stimulation reward. Figure 1(b, d) pre-

sents the changes in self-stimulation threshold and

asymptotic rate of responding after systemic injection

CB1R stimulation counteracts cocaine’s behavioural actions 909

of SR141716A or its vehicle and AM-404 or its vehicle.

Two-way ANOVA showed that AM-404 (30 mg/kg)

produced an increase in self-stimulation threshold

[F(1, 20)=16.467, p<0.001]. The administration of

SR141716A (0.02 mg/kg) blocked this effect of AM-404

[F(1, 20)=7.996, p=0.010]. Two-way ANOVA also

showed that neither AM-404 (30 mg/kg), nor

SR141716A (0.02 mg/kg), nor their combined admin-

istration affected the asymptotic rate of responding.

Experiment 3. Effects of AM-404 on cocaine-induced

lowering of brain reward thresholds. The changes in self-

stimulation threshold and asymptotic rate of respond-

ing after systemic injection of the endocannabinoid

neurotransmission enhancer AM-404 or its vehicle and

cocaine or saline are presented in Figure 2(a, c), re-

spectively. Two-way ANOVA showed that cocaine

(5 mg/kg) produced a significant decrease in self-

stimulation threshold [F(1, 24)=11.976, p=0.002],

while AM-404 (10 mg/kg i.p.) had no effect on the

self-stimulation threshold per se [F(1, 24)=3.867,

p=0.061]. Pretreatment with AM-404 significantly

blocked the facilitation of brain stimulation reward

caused by cocaine [F(1, 24)=10.252, p=0.004]. Two-

way ANOVA also showed that neither AM-404

(10 mg/kg), nor cocaine (5 mg/kg), nor their com-

bined administration affected the asymptotic rate of

responding.

Experiment 4. Reversal of the action of AM-404 on cocaine-

induced lowering of brain reward thresholds by pretreat-

ment with the CB1R antagonist SR141716A. Figure 2(b, d)

presents the changes in self-stimulation threshold and

asymptotic rate of responding after systemic injection

of SR141716A or its vehicle, AM-404 or its vehicle and

cocaine or saline. Three-way ANOVA showed that

SR141716A (0.02 mg/kg) [F(1, 32)=3.806, p=0.060]

and AM-404 (10 mg/kg) [F(1, 32)=1.034, p=0.317]

had no effect on the self-stimulation by itself,

while cocaine (5 mg/kg) produced a decrease in

0

40

80

120

160

200

240

(a) (c)

(d)(b)

Threshold

#***

Pre

-dru

g v

alu

e (%

)P

re-d

rug

val

ue

(%)

Pre

-dru

g v

alu

e (%

)P

re-d

rug

val

ue

(%)

0

40

80

120

160

200Asymptote

AM-404AM-404

0

40

80

120

160

200

240

SR141716A + AM-404 SR141716A + AM-404

Threshold

###***

0

40

80

120

160

200Asymptote

Vehicle AM1 AM3 AM10 AM30

Veh + Veh Veh + AM30 SR0.02 + Veh SR0.02 + AM30

Figure 1. Changes in self-stimulation threshold (expressed as percentage of pre-drug values) (a, b) and asymptotic rate

of responding (c, d), following acute i.p. AM-404 (0, 1, 3, 10, 30 mg/kg) or SR141716A (0.02 mg/kg) and AM-404 (30 mg/kg)

administration. Vertical bars represent the SEMs. The asterisks (*) signify an intracranial self-stimulation (ICSS) threshold

significantly different from the control group: *** p<0.001, # p<0.05, ### p<0.001 compared to the other groups.

910 S. Vlachou et al.

self-stimulation threshold [F(1, 32)=32.291, p<0.0001].

Co-administration of SR141716A and AM-404 had no

effect on the self-stimulation threshold [F(1, 32)=2.372, p=0.133]. Co-administration of AM-404 and

cocaine significantly blocked the facilitation of brain

stimulation reward produced by cocaine [F(1, 32)=5.670, p=0.023]. It was found that the CB1R antagonist

SR141716A (0.02 mg/kg) completely reversed the

inhibitory action of AM-404 (10 mg/kg) on the co-

caine-induced lowering of brain-reward thresholds

[F(1, 32)=5.322, p=0.028]. Three-way ANOVA also

showed that neither SR141716A (0.02 mg/kg), nor

AM-404 (10 mg/kg), nor cocaine (5 mg/kg), nor their

combined administration affected the asymptotic rate

of responding (Figure 3).

Spontaneous motor activity

Study 1

Experiment 1. Effect of WIN55,212-2 on locomotor ac-

tivity. The effect of WIN55,212-2 on locomotor activity

and rearing is shown in Figure 4(a, c), respectively.

WIN55,212-2 decreased spontaneous locomotor ac-

tivity [F(3, 40)=3.955, p=0.015]. Further analysis with

the LSD test showed that this effect was statistically

significant at the highest dose tested (3 mg/kg),

compared to the vehicle group (p=0.002). WIN55,212-

2 also reduced rearing at all doses tested [F(3, 40)=4.231, p=0.011]. Further analysis with the LSD test

showed that this effect was statistically significant at

all doses tested (0.3, 1, 3 mg/kg) compared to the

vehicle group [p=0.029, p=0.025 and p=0.001,

respectively].

Experiment 2. Reversal of WIN55,212-2-induced hypolo-

comotion with the CB1R antagonist SR141716A. The effect

of SR141716A on WIN55,212-2-induced hypolocomo-

tion and rearing is shown in Figure 4(b, d), respect-

ively. Two-way ANOVA showed that WIN55,212-2

decreased spontaneous locomotor activity [F(1, 36)=3.831, p=0.05] and rearing [F(1, 36)=8.496, p=0.006]

at the highest dose tested (3 mg/kg). The CB1R antag-

onist SR141716A (0.02 mg/kg) reversed this effect

both on locomotion [F(1, 36)=12.250, p=0.001] and

rearing [F(1, 36)=3.664, p=0.044], while it had no ef-

fect by itself.

Experiment 3. Effect of WIN55,212-2 on cocaine-induced

hyperlocomotion (5 mg/kg). The effect of WIN55,212-2

on cocaine-induced hyperlocomotion and rearing is

0

40

80

120

160

200

240

(a)

(b)

(c)

(d)

###***

AM-404 + Cocaine AM-404 + Cocaine

ThresholdP

re-d

rug

val

ue

(%)

Pre

-dru

g v

alu

e (%

)

Pre

-dru

g v

alu

e (%

)P

re-d

rug

val

ue

(%)

0

40

80

120

160

200Asymptote

0

40

80

120

160

200

240

SR141716A + AM-404 + Cocaine SR141716A + AM-404 + Cocaine

Threshold

###***

###***

###***

0

40

80

120

160

200Asymptote

Vehicle + Saline

Key: panels (a), (c)

Vehicle + CocaineAM10 + SalineAM10 + Cocaine

Veh + Veh + SalVeh + Veh + CocVeh + AM10 + SalVeh + AM10 + CocSR0.02 + Veh + SalSR0.02 + Veh + CocSR0.02 + AM10 + SalSR0.02 + AM10 + Coc

Key: panels (b), (d)

Figure 2. Changes in self-stimulation threshold (expressed as percentage of pre-drug values) (a, b) and asymptotic rate

of responding (c, d), following acute i.p. AM-404 (30 mg/kg) and cocaine (5 mg/kg) or SR141716A (0.02 mg/kg), AM-404

(10 mg/kg) and cocaine (5 mg/kg) administration. Vertical bars represent the S.E.M. The asterisks (*) signify an intracranial

self-stimulation (ICSS) threshold significantly different from the control group: *** p<0.001, ### p<0.001 compared to the other

groups.

CB1R stimulation counteracts cocaine’s behavioural actions 911

presented in Figure 5(a, c), respectively. WIN55,212-2

(1 mg/kg) did not produce any significant change

on motor activity. Cocaine (5 mg/kg) produced

a statistically significant increase in spontaneous

motor activity [F(1, 28)=5.030, p=0.033] and rearing

[F(1, 28)=4.067, p=0.053], compared to the vehicle

group. WIN55,212-2 (1 mg/kg) completely blocked

this effect on locomotion [F(1, 28)=3.820, p<0.05] and

rearing [F(1, 28)=14.005, p=0.001], while it had no

effect per se.

Experiment 4. Reversal of the action of WIN55,212-2

on cocaine-induced hyperlocomotion (5 mg/kg) with

SR141716A. Figure 5(b, d) presents the reversal of the

Vehicle

0.8 1.0 1.2 1.4 1.6 1.8

0

40

80

120

160

(n = 124) (n = 124)

(n = 119)

(n = 119)

(n = 113)

(n = 104)

Log n pulses/train

Log n pulses/train Log n pulses/train

Log n pulses/trainLog n pulses/train

Log n pulses/train

Leve

r p

ress

ing

/min

Leve

r p

ress

ing

/min

Leve

r p

ress

ing

/min

Leve

r p

ress

ing

/min

Leve

r p

ress

ing

/min

Leve

r p

ress

ing

/min

AM30

0.8 1.0 1.2 1.4 1.6 1.8

0

40

80

120

160

Vehicle + Cocaine

0.8 1.0 1.2 1.4 1.6 1.8

0

40

80

120

160 AM10 + Cocaine

0.8 1.0 1.2 1.4 1.6 1.8

0

40

80

120

160

SR0.02 + Vehicle + Cocaine

0.8 1.0 1.2 1.4 1.6 1.8

0

40

80

120

160 SR0.02 + AM10 + Cocaine

0.8 1.0 1.2 1.4 1.6 1.8

0

40

80

120

160

Figure 3. Rate-frequency functions (rate of lever pressing as a function of stimulation frequency) taken from representative

animals for each drug treatment. Each plot represents data from a single animal under pre-drug and drug conditions.

Rate frequency functions were obtained by logarithmically decreasing the frequency of the stimulation pulses

from a value that sustained maximal lever pressing to one that failed to sustain lever pressing. –#–, Pre-injection ;

–$–, post-injection.

912 S. Vlachou et al.

action of WIN55,212-2 on the cocaine-induced hyper-

locomotion and rearing, respectively, by SR141716A.

Cocaine (5 mg/kg) produced a statistically significant

increase in spontaneous motor activity [F(1, 64)=110.959, p<0.0001] and rearing [F(1, 64)=31.443,

p<0.0001]. SR141716A and WIN55,212-2 had no effect

per se on either motor activity or rearing. SR141716A

(0.02 mg/kg) completely reversed the effect of

WIN55,212-2 on the cocaine-induced hyperlocomotion

[F(1, 64)=7.596, p=0.008] and increased rearing

[F(1, 64)=6.182, p=0.016].

Experiment 5. Effect of WIN55,212-2 on cocaine-induced

hyperlocomotion (10 mg/kg). The effect of WIN 55,212-2

on cocaine-induced hyperlocomotion and rearing is

presented in Figure 6(a, c), respectively. Two-way

ANOVA showed that cocaine (10 mg/kg) produced a

statistically significant increase in spontaneous motor

activity [F(1, 28)=27.049, p<0.0001], compared to the

vehicle group, while it did not affect rearing [F(1, 28)=3.604, p=0.068]. WIN55,212-2 (1 mg/kg) completely

blocked this effect on locomotion [F(1, 28)=7.515,

p=0.011] and rearing [F(1, 28)=4.855, p=0.036], while

it had no effect per se [F(1, 28)=3.510, p=0.071].

Experiment 6. Reversal of the action of WIN55,212-2

on cocaine-induced hyperlocomotion (10 mg/kg) with

SR141716A. Figure 6(b, d) presents the reversal of the

action of WIN55,212-2 on the cocaine-induced hyper-

locomotion and rearing, respectively, by SR141716A.

Cocaine (10 mg/kg) significantly increased spon-

taneous motor activity [F(1, 56)=186.407, p<0.0001]

and rearing [F(1, 56)=46.455, p<0.0001]. SR141716A

and WIN55,212-2 had no effect per se on either

locomotor activity [F(1, 56)=3.141, p=0.082 and

F(1, 56)=2.761, p=0.102, respectively] or rearing

WIN 55,212-20

1000

2000

3000

4000

5000

6000

(a) (c)

(d)(b)

**

Mo

bili

ty c

ou

nts

WIN 55,212-20

400

800

1200

1600

2000

Vehicle WIN0.3 WIN1 WIN3

****

*

Rea

rin

g c

ou

nts

SR141716A + WIN 55,212-20

1000

2000

3000

4000

5000

6000

Veh + Veh Veh + WIN3 SR0.02 + Vehicle SR0.02 + WIN3

++###***

Mo

bili

ty c

ou

nts

SR141716A + WIN 55,212-20

400

800

1200

1600

2000

+++###***

Rea

rin

g c

ou

nts

Figure 4. Effects of WIN55,212-2 (0, 0.3, 1 and 3 mg/kg) on locomotor activity and effect of SR141716A (0.02 mg/kg) on the

WIN55,212-2 (3 mg/kg)-reduced locomotion. Histograms represent the photocell disruptions caused by the animals’ locomotion

(a, b) and rearing (c, d) (mean¡S.E.M.). The asterisks (*) show a statistically significant effect compared to the vehicle group:

* p<0.05, ** p<0.01, *** p<0.001 ; ### p<0.001 compared to the Veh+WIN3 group; ++ p<0.01, +++ p<0.001 compared to the

SR0.02+Veh group.

CB1R stimulation counteracts cocaine’s behavioural actions 913

[F(1, 56)=1.122, p=0.294 and F(1, 56)=3.067, p=0.085,

respectively]. SR141716A (0.02 mg/kg) completely

reversed the effect of WIN55,212-2 on the cocaine-

induced hyperlocomotion [F(1, 56)=7.170, p=0.010]

and increased rearing [F(1, 56)=4.855, p=0.036].

Study 2

Experiment 1. Effect of AM-404 on locomotor activity.

AM-404 did not affect significantly locomotion

[F(3, 32)=0.701, p=0.559] or rearing [F(3, 32)=0.995,

p=0.408], although there was a tendency for a re-

duction in both indices at all doses tested, as shown in

Figure 7(a, b), respectively.

Experiment 2. Effect of AM-404 (10 mg/kg) on cocaine-

induced hyperlocomotion (5 mg/kg). Cocaine (5 mg/kg)

produced a statistically significant increase in spon-

taneous motor activity [F(1, 28)=14.535, p=0.001]

and rearing [F(1, 28)=14.141, p=0.001]. AM-404

(10 mg/kg) significantly blocked this effect on

locomotion [F(1, 28)=5.319, p=0.029] and rearing

[F(1, 28)=6.974, p=0.013], as presented in Figure 8(a,

c), respectively.

Experiment 3. Reversal of the action of AM-404 (10 mg/kg)

on cocaine-induced hyperlocomotion (5 mg/kg) with

SR141716A. Cocaine (5 mg/kg) produced a significant

increase in motor activity [F(1, 72)=119.549, p<0.0001] and rearing [F(1, 72)=48.882, p<0.0001].

SR141716A (0.02 mg/kg) did not have any effect

on motor activity. There was a statistically signif-

icant interaction between AM-404 and cocaine in

motor activity [F(1, 72)=4.846, p=0.031] and rearing

[F(1, 72)=4.065, p=0.048]. This was due to the fact

that, AM-404 blocked the stimulatory effects of

cocaine. SR141716A (0.02 mg/kg) completely reversed

the effect of AM-404 on cocaine-induced hyper-

locomotion [F(1, 72)=5.439, p=0.022] and increased

rearing [F(1, 72)=5.188, p=0.026], as shown in

Figure 8(b, d), respectively.

Experiment 4. Effect of AM-404 (10 mg/kg) on cocaine-

induced hyperlocomotion (10 mg/kg). Figure 9(a, d)

shows the effect of AM-404 on cocaine-induced

hyperlocomotion and rearing, respectively. Cocaine

(10 mg/kg) produced a statistically significant

increase in locomotor activity [F(1, 28)=22.912, p<0.0001] and rearing [F(1, 28)=12.646, p=0.001].

WIN 55,212-2 + Cocaine0

1000

2000

3000

4000

5000

6000

(a) (c)

(d)(b)

+##**

Mo

bili

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ou

nts

WIN 55,212-2 + Cocaine0

400

800

1200

1600

2000

+++##***

Rea

rin

g c

ou

nts

SR141716A + WIN 55,212-2 + Cocaine0

1000

2000

3000

4000

5000

6000

###***

###***

*

###***

Mo

bili

ty c

ou

nts

SR141716A - WIN 55,212-2 + Cocaine0

400

800

1200

1600

2000

##**

##**

###***

Rea

rin

g c

ou

nts

Veh + Veh + Sal

Veh + Veh + Coc5

SR0.02 + Veh + Sal

SR0.02 + Veh + Coc5

Veh + WIN1 + Sal

Veh + WIN1 + Coc5

SR0.02 + WIN1 + Sal

SR0.02 + WIN1 + Coc5

Vehicle + Saline

Vehicle + Cocaine5

WIN1 + Saline

WIN1 + Cocaine5

Key: panels (a), (c)

Key: panels (b), (d)

Figure 5. Effects of WIN55,212-2 (1 mg/kg) and cocaine (5 mg) on locomotor activity and effect of SR141716A (0.02 mg/kg)

on the WIN55,212-2-induced reduction of cocaine’s hyperlocomotion. Histograms represent the photocell disruptions caused

by the animals’ locomotion (a, b) and rearing (c, d) (mean¡S.E.M.). The asterisks (*) show a statistically significant effect

compared to the vehicle group: ** p<0.01, *** p<0.001, ## p<0.01, ### p<0.001 compared to all other groups ; ++ p<0.01,+++ p<0.001 compared to the WIN1+Coc5 group.

914 S. Vlachou et al.

AM-404 (10 mg/kg), which had no effect per se, failed

to affect either the hyperlocomotion [F(1, 28)=0.106,

p=0.747] or the enhanced rearing [F(1, 28)=1.427,

p=0.242] induced by cocaine, while there was a tend-

ency for reduction in the increased rearing induced by

cocaine.

Experiment 5. Effect of AM-404 (30 mg/kg) on cocaine-

induced hyperlocomotion (10 mg/kg). Figure 9(b, e) shows

the effect of AM-404 (30 mg/kg) on cocaine-induced

hyperlocomotion (10 mg/kg) and rearing, respect-

ively. Two-way ANOVA showed that cocaine (10 mg/

kg) produced a statistically significant increase in

locomotor activity [F(1, 32)=175.820, p<0.0001] and

rearing [F(1, 32)=49.002, p<0.0001]. AM-404 (30 mg/

kg) significantly blocked the hyperlocomotion

[F(1, 32)=9.333, p=0.005] and rearing [F(1, 32)=5.231,

p=0.029] induced by cocaine.

Experiment 6. Reversal of the action of AM-404 (30 mg/kg)

on cocaine-induced hyperlocomotion (10 mg/kg) with

SR141716A. Cocaine (10 mg/kg) produced a signifi-

cant increase in motor activity [F(1, 64)=158.361,

p<0.0001] and rearing [F(1, 64)=86.983, p<0.0001].

There was a statistically significant effect of cocaine

after the administration of SR141716A and AM-404

on motor activity [F(1, 64)=4.599, p=0.036 and

F(1, 64)=6.600, p=0.013, respectively] and rearing

[F(1,64)=4.579, p=0.034 and F(1, 64)=5.381, p=0.024,

respectively], although it is clearly shown that AM-

404 reduced the hyperlocomotor effect of cocaine.

SR141716A (0.02 mg/kg) completely reversed the ef-

fect of AM-404 on the cocaine-induced hyperlocomo-

tion [F(1, 64)=5.153, p=0.027], while there seemed to

be no interaction on rearing [F(1, 64)=2.742, p=0.103],

as shown in Figure 9(c, f), respectively.

Discussion

The present study showed that the endocannabinoid

neurotransmission enhancer AM-404 at relatively

low doses (1, 3 and 10 mg/kg) did not affect in-

tracranial self-stimulation behaviour, whereas at

the highest dose (30 mg/kg) increased brain reward

thresholds. These changes were not accompanied

by significant changes in asymptotic rates of respond-

ing. The anhedonic-like effect of AM-404, observed

at the highest dose, was completely abolished by

pretreatment with the CB1R antagonist SR141716A.

However, since it is not clear whether AM-404 at the

WIN 55,212-2 + Cocaine0

1000

2000

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(a)

+###

#

Mo

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WIN 55,212-2 + Cocaine0

400

800

1200

1600

2000 ##**

(c)

SR141716A + WIN 55,212-2 + Cocaine0

1000

2000

3000

4000

5000

6000 ###***

(b)

###***

###***

###***

SR141716A + WIN 55,212-2 + Cocaine0

400

800

1200

1600

2000

++###***

(d)

++###*** +

###***

Rea

rin

g c

ou

nts

Rea

rin

g c

ou

nts

Veh + Veh + SalVeh + Veh + Coc10SR0.02 + Veh + SalSR0.02 + Veh + Coc10Veh + WIN1 + SalVeh + WIN1 + Coc10SR0.02 + WIN1 + SalSR0.02 + WIN1 + Coc10

Vehicle + SalineVehicle + Cocaine10WIN1 + SalineWIN1 + Cocaine10

Key : panels (b), (d)

Key : panels (a), (c)

Figure 6. Effects of WIN55,212-2 (1 mg/kg) and cocaine (10 mg) on locomotor activity and effect of SR141716A (0.02 mg/kg)

on the WIN55,212-2-induced reduction of cocaine’s hyperlocomotion. Histograms represent the photocell disruptions caused

by the animals’ locomotion (a, b) and rearing (c, d) (mean¡S.E.M.). The asterisks (*) show a statistically significant effect compared

to the vehicle group: ** p<0.01, *** p<0.001, # p<0.05, ## p<0.01, ### p<0.001 compared to all other groups ; + p<0.05,++ p<0.01 compared to the WIN1+Coc10 (a) or Veh+WIN1+Coc10 group (d).

CB1R stimulation counteracts cocaine’s behavioural actions 915

dose of 30 mg/kg can still selectively prevent ana-

ndamide elimination, the observed effects could be

due to a non-selective increase of extracellular con-

centrations of endocannabinoids other than ananda-

mide or due to unselective actions of this drug, such as

direct stimulation of the CB1R, the latter been more

probable since AM-404 action was reversed by

SR141716A (Dickason-Chesterfield et al., 2006). It is

worth noting that in our study SR141716A did not af-

fect ICSS threshold by itself, suggesting lack of a tonic

regulation of reward/reinforcement processes via

CB1R. The present results are in agreement with a re-

cent study from our laboratory (Vlachou et al., 2006),

in which the endocannabinoid neurotransmission

modulators PMSF, URB-597 and OMDM-2, depending

on the dose administered, either do not affect or de-

crease brain reward function. The finding that AM-404

lacks appetitive properties also agrees with other stu-

dies, where anandamide and URB-597 do not produce

conditioned place preference in male Wistar rats

(Gobbi et al., 2005 ; Mallet and Beninger, 1998).

However, it should be mentioned that anandamide

and methanandamide have been shown to be self-

administered by squirrel monkeys (Justinova et al.,

2005) and AM-404 induces conditioned place pre-

ference in rats living in an enriched environment

(Solinas et al., 2005). These seemingly contrasting re-

sults could be attributed to differences in the pharma-

cological properties and the dose range of the tested

compounds, the species and the strain of the animals

used or the methods followed; in addition, the route

of administration might be a determinant factor in

assessing appetitive actions of cannabinoids, as has

previously been argued (Vlachou et al., 2003, 2005,

2006, 2007).

Acute administration of AM-404 counteracted the

reward-facilitating effect of systemic cocaine at a dose

that by itself was ineffective in altering brain stimu-

lation reward thresholds. Cocaine (5 mg/kg) pro-

duced a significant reduction in self-stimulation

threshold, without altering maximal rates of respond-

ing, in agreement with previous studies (Maldonado-

Irrizary et al., 1994 ; Matthews et al., 1996; Panagis

et al., 2000; Panagis and Spyraki, 1996; Ranaldi et al.,

1997 ; Vlachou et al., 2003). AM-404 (10 mg/kg) atten-

uated the reward-facilitating action of cocaine, an

effect which was reversed by pretreatment with

SR141716A (0.02 mg/kg). The present results are in

agreement with a number of studies, indicating in-

teractions of cannabinoids with psychostimulants

(Braida et al., 2001 ; Braida and Sala, 2002 ; Cadoni et al.,

2001 ; De Vries et al., 2001 ; Fattore et al., 1999;

Gallate et al., 1999 ; Ghozland et al., 2002 ; Gonzalez

et al., 2002a,b ; Filip et al., 2006 ; Gonzalez-Cuevas et al.,

2007 ; Higuera-Matas et al., 2007 ; Lamarque et al.,

2001 ; Navarro et al., 2001 ; Pontieri et al., 2001a,b;

Tanda, 2007 ; Vlachou et al., 2003). We have recently

shown that the CB1R agonist WIN55,212-2 at a dose

that does not affect brain stimulation reward per se

counteracts the reward-facilitating effects of cocaine

(Vlachou et al., 2003). Fattore and colleagues have

shown that WIN55,212-2 reduces the intravenous

self-administration of cocaine (Fattore et al., 1999), a

finding also consistent with the most recent study

by Panlilio and colleagues, in which D9-tetra-

hydrocannabinol (THC) was found to reduce the self-

administration of cocaine under a progressive ratio

schedule of reinforcement (Panlilio et al., 2007), and

with another recent study by Cippitelli and colleagues,

in which AM-404 was found to reduce ethanol

self-administration (Cippitelli et al., 2007). On the other

hand, it has been found that the potent CB1R agonist

HU-210 induces relapse to cocaine-seeking after a pro-

longed withdrawal period (De Vries et al., 2001), and

CP55,940 does not seem to affect the arousal caused to

00 3

AM-404 (mg)

AM-404 (mg)

10 30

0 3 10 30

1000

2000

3000

4000

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6000

Mo

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0

400

800

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1600

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(b)

(a)

Rea

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Figure 7. Effects of AM-404 [0 (vehicle), 3, 10 and 30 mg] on

locomotor activity and rearing. Histograms represent the

photocell disruptions caused by the animals’ locomotion (a)

and rearing (b) (mean¡S.E.M.).

916 S. Vlachou et al.

Cebus monkeys by d-amphetamine (Madsen et al.,

2006). Moreover, it has recently been shown that CB1R

knockout mice respond to a lesser extent tococaine

self-administration (Soria et al., 2005 ; but see Cossu

et al., 2001). Conversely, cocaine self-administration

does not alter dialysate levels of anandamide or

2-arachidonoylglycerol) and SR141716A does not

affect cocaine self-administration (Caille et al., 2007).

The fact that there appear to be contrasting effects

of cannabinoids on the actions of cocaine supports

the hypothesis that there is a differential interaction

between these two classes of drugs depending

on context as well as on the pharmacological and

behavioural modalities under study (Chaperon et al.,

1998 ; De Vries et al., 2001 ; Lesscher et al., 2005 ;

Xi et al., 2007). Although the neurobiological mech-

anisms underlying this interaction remain unclear,

we speculate that, since there is co-existence of CB1

and dopamine receptors in brain-reward circuits

(Hermann et al., 2002), the CB1R stimulation could

act as a counteracting feedback mechanism for the

increased extracellular dopamine concentrations in

the nucleus accumbens produced by cocaine and other

psychostimulants (see e.g. Centonze et al., 2004 ;

Giuffrida et al., 1999) ; whether this results in a

modified dopamine response remains to be un-

equivocally shown.

WIN55,212-2 decreased spontaneous locomotor ac-

tivity at the highest dose tested (3 mg/kg), while AM-

404 (3, 10, 30 mg/kg) had no significant effect onmotor

function per se at any of the doses tested, although

there was a tendency for reduction of locomotion at

the highest dose administered. SR141716A (0.02 mg/

kg) blocked the effects of WIN55,212-2 on spontaneous

motor activity. It is well known that psychostimulant

drugs, increase spontaneous motor activity (Beninger,

1983 ; Fung and Lau, 1988 ; Iwamoto, 1984 ; Norton,

1973). Studies that have been conducted in the last

years with cannabinoids have shown that they have a

biphasic effect on neurotransmitter release (Tzavara

et al., 2003), as well as on locomotion, causing both

hyperlocomotion in low doses (Drews et al., 2005 ;

McGregor et al., 1996 ; Muschamp and Siviy, 2002; see

also, Introduction) and hypolocomotion or even cata-

lepsy at high doses (e.g. Meschler et al., 2001 ;

Rodriguez de Fonseca et al., 1998 ; Sanudo-Pena et al.,

2000). Since the CB1R are predominantly located in the

basal ganglia and associated structures with the high-

est density among all other receptors in the brain, their

motor effects have been hypothesized to be caused by

AM-404 + Cocaine0

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0

1000

2000

3000

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###***

(a)M

ob

ility

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un

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ob

ility

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AM-404 + Cocaine0

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###***

(c)

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SR141716A + AM-404 + Cocaine

###***

+##

(b)

###*** ###

***

SR141716A + AM-404 + Cocaine0

400

800

1200

1600

2000

###***

(d)

###***

###***

Vehicle + SalineVehicle + Cocaine5AM10 + SalineAM10 + Cocaine5

Veh + Veh + SalVeh + Veh + Coc5SR0.02 + Veh + SalSR0.02 + Veh + Coc5Veh + AM10 + SalVeh + AM10 + Coc5SR0.02 + AM10 + SalSR0.02 + AM10 + Coc5

Key : panels (b), (d)

Key : panels (a), (c)

Figure 8. Effects of AM-404 (10 mg/kg) and cocaine (5 mg) on locomotor activity and effect of SR141716A (0.02 mg/kg) on the

AM-404 induced reduction of cocaine’s hyperlocomotion. Histograms represent the photocell disruptions caused by the

animals’ locomotion (a, b) and rearing (c, d) (mean¡S.E.M.). The asterisks (*) show a statistically significant effect compared to the

vehicle group: *** p<0.001, ## p<0.01 compared to the SR0.02+AM10+Sal group (c) ; ### p<0.001 compared to all other groups ;+ p<0.05 compared to the Veh+AM10+Sal group.

CB1R stimulation counteracts cocaine’s behavioural actions 917

an interplay of actions on the dopaminergic, gluta-

matergic and GABAergic systems in these brain areas

(Sanudo-Pena et al., 1999, 2000). Therefore, the re-

duction in locomotor activity observed in our studies

could be attributed to stimulation of CB1R in the

striatum, which increases GABAergic neurotransmis-

sion in the globus pallidus or substantia nigra (Maneuf

et al., 1997). It could also be due to trans-synaptic

stimulation of GABAergic neurons, which project to

the substantia nigra and cause reduced stimulation of

the nigrostriatal dopaminergic neurons (Gueudet et al.,

1995).

Our results are in agreement with previous studies

showing that anandamide, THC or CB1R agonists re-

duce motor function (Darmani, 2001 ; Ferrari et al.,

1999 ; Romero et al., 2002 ; Singh et al., 2005 ; Valjent

et al., 2002). However, other studies have shown dif-

ferent results (Drews et al., 2005; Muschamp and

Siviy, 2002 ; Przegalinski et al., 2005). Przegalinski and

colleagues have shown that administration of 3 mg

and 6 mg WIN55,212-2 does not affect locomotion,

while in our study there was a clear hypolocomotion

and reduced rearing after administration of 3 mg

WIN55,212-2. Our results also differ from Muschamp

and Siviy (2002), where there was an increase in

locomotion with 1 mg/kg WIN55,212-2, although this

was shown in Lewis rats and only in the first 15 min

of the experimental session. It is probable that the

different results could be attributed to different ex-

perimental conditions.

Cocaine (5, 10 mg/kg) caused a significant, dose-

dependent increase in locomotor activity, which was

AM-404 + Cocaine0

1000

2000

3000

4000

5000

6000##**

(a)

##**

AM-404 + Cocaine0

400

800

1200

1600

2000

+###**

(d)

AM-404 + Cocaine0

1000

2000

3000

4000

5000

6000 ###***

(b)

+++###***

AM-404 + Cocaine0

400

800

1200

1600

2000###***

(e)

+++##**

Rea

rin

g c

ou

nts

Rea

rin

g c

ou

nts

SR141716A + AM-404 + Cocaine0

100020003000400050006000 ###

***

(c)

###***

###***

+++##**

Mo

bili

ty c

ou

nts

Mo

bili

ty c

ou

nts

Mo

bili

ty c

ou

nts

Rea

rin

g c

ou

nts

SR141716A + AM-404 + Cocaine0

400

800

1200

1600

2000

###***

(f)

###***###

*** +++#*

Vehicle + SalineVehicle + Cocaine10AM10 + SalineAM10 + Cocaine10

Vehicle + SalineVehicle + Cocaine10AM30 + SalineAM30 + Cocaine10

Veh + Veh + SalVeh + Veh + Coc10SR0.02 + Veh + SalSR0.02 + Veh + Coc10Veh + AM30 + SalVeh + AM30 + Coc10SR0.02 + AM30 + SalSR0.02 + AM30 + Coc10

Key: panels (c), (f)

Key: panels (b), (e)

Key: panels (a), (d)

Figure 9. Effects of AM-404 (10 and 30 mg/kg) and cocaine (10 mg) on locomotor activity and effect of SR141716A (0.02 mg/kg)

on the AM-404 (30 mg)-induced reduction of cocaine’s hyperlocomotion. Histograms represent the photocell disruptions caused

by the animals’ locomotion (a–c) and rearing (d–f) (mean¡S.E.M.). The asterisks (*) show a statistically significant effect

compared to the vehicle group: * p<0.05, ** p<0.01, *** p<0.001, # p<0.05, ## p<0.01, ### p<0.001 compared to AM10 or

AM30+Sal (a, b, d, e), or compared to all other groups (c, f) ; + p<0.05, compared to the AM10+Coc10 group; +++ p<0.001

compared to the Veh+Coc10 (e) or all the cocaine groups (c, f).

918 S. Vlachou et al.

reduced by WIN55,212-2 (1 mg/kg) and AM-404 (10,

30 mg/kg, respectively). AM-404 (10 mg/kg) did not

affect the hyperolocomotor effects of cocaine (10 mg/

kg), although it did reduce cocaine’s stimulatory ac-

tion on rearing. A higher dose of AM-404 (30 mg/kg)

was found to reduce cocaine (10 mg/kg)-induced hy-

peractivity. SR141716A (0.02 mg/kg) blocked the ef-

fects of WIN55,212-2 and AM-404 on spontaneous

motor activity and on the hyperlocomotion induced by

cocaine, while it had no effect on motor function per

se. The above finding shows that WIN55,212-2 and

probably AM-404 exhibited their actions through

CB1R stimulation. Ferrari and colleagues have also

shown that HU-210 reduces the increased locomotion

and rearing caused by cocaine (Ferrari et al., 1999), as

it was also recently shown for morphine and alcohol

(Hagues et al., 2007). Similarly, Muschamp and Siviy

(2002) have shown that acute administration of

WIN55,212-2 (1 mg/kg), in Lewis rats inhibits the

hyperlocomotion and increased rearing induced by

cocaine. Our results also agree with other studies

involving THC and its counteracting action on am-

phetamine-induced hyperlocomotion (Gorriti et al.,

1999). Importantly, in a very recent study Rodvelt and

colleagues (2007) have shown that WIN55,212-2 at-

tenuates nicotine-induced hyperactivity. It is probable

that WIN55,212-2 and AM-404 alter the hyperactivity

produced by cocaine through an indirect mechanism,

such as alteration in endocannabinoid tone of motor

circuits. A possible mechanism could involve a nega-

tive feedback mechanism induced by cannabinoids in

the striatum, as mentioned above (see Centonze et al.,

2004, 2007 ; Giuffrida et al., 1999). A second alternative

mechanism of action of at least WIN55,212-2 could be

the inhibition of dopamine transporter, which has

been shown in the case of WIN55,212-2 resulting in

competition with cocaine that also inhibits the dop-

amine transporter (Steffens and Feuerstein, 2004).

In conclusion, acute systemic administration of the

direct CB1R agonist WIN55,212-2 and the endo-

cannabinoid neurotransmission enhancer AM-404,

even at high doses, did not have reinforcing effects,

but rather reversed the reward-facilitating effects of

cocaine in the ICSS paradigm. Furthermore, the same

compounds either did not affect, or (at high doses)

decreased locomotor activity. Interestingly, both

WIN55,212-2 and AM-404 in doses that did not affect

spontaneous motor activity reduced the hyperloco-

motion induced by cocaine. These effects are mediated

through CB1R stimulation, since they were reversed by

pretreatment with the CB1R antagonist SR141716A.

Therefore, cannabinoids clearly play a modulatory

role in cocaine’s psychostimulant actions. Based upon

the present findings, we speculate that drugs increas-

ing the cannabinergic tone could be further tested as a

potential treatment for various aspects of psycho-

stimulant addiction and psychomotor excitability. In

this regard, it is worth-noting that Tzavara et al. (2006)

have recently shown that endocannabinoids acting

through vanilloid1 receptors reduce hyperdopamin-

ergia-related hyperactivity in a relevant genetic animal

model. Thus, endocannabinoid neurotransmission

modulators appear to offer the potential of a dual

receptor action through both CB1R and non-CB1R

counteracting overt psychomotor stimulation without

affecting basal function and with seemingly low

overall psychotropic liability.

Acknowledgements

This study was supported by a grant from the

Research Committee (KA 2303) and the Department

of Psychology of the University of Crete. Styliani

Vlachou was supported by a scholarship from Pro-

pondis Foundation.

Statement of Interest

None.

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