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The Journal of Neuroscience, February 1, 2002, 22(3):1146-1154
Motivational Effects of Cannabinoids Are Mediated by µ-Opioid
and  -Opioid Receptors
Sandy
Ghozland1,
Hans
W. D.
Matthes2,
Frederic
Simonin2,
Dominique
Filliol2,
Brigitte L.
Kieffer2, and
Rafael
Maldonado1
1 Laboratori de Neurofarmacologia, Facultat de
Ciéncies de la Salut i de la Vida, Universitat Pompeu Fabra,
08003 Barcelona, Spain, and 2 Centre National de la
Recherche Scientifique Unité Propre de Recherche 9050, Ecole
Superieure de Biotechnologie de Strasbourg, 67400 Illkirch, France
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ABSTRACT |
Repeated THC administration produces motivational and somatic
adaptive changes leading to dependence in rodents. To investigate the
molecular basis for cannabinoid dependence and its possible relationship with the endogenous opioid system, we explored
 9-tetrahydrocannabinol (THC) activity in mice lacking µ-,  - or
 -opioid receptor genes. Acute THC-induced hypothermia,
antinociception, and hypolocomotion remained unaffected in these mice,
whereas THC tolerance and withdrawal were minimally modified in mutant
animals. In contrast, profound phenotypic changes are observed in
several place conditioning protocols that reveal both THC rewarding and
aversive properties. Absence of µ receptors abolishes THC place
preference. Deletion of receptors ablates THC place aversion and
furthermore unmasks THC place preference. Thus, an opposing activity of
µ- and -opioid receptors in modulating reward pathways forms the
basis for the dual euphoric-dysphoric activity of THC.
Key words:
9-tetrahydrocannabinol; place preference; place
aversion; knock-out; tolerance; dependence; reward
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INTRODUCTION |
Cannabinoids and opioids are the
most widely consumed illicit drugs worldwide (Smart and Ogborne, 2000 ).
Both types of compounds mimic endogenous ligands and act through
distinct G-protein-coupled receptor families known as cannabinoid
(Felder and Glass, 1998) and opioid (Kieffer, 1995) receptors.
Pharmacological studies have shown functional interactions between the
two systems (Manzanares et al., 1999). Thus, cannabinoid and opioid
agonists share several pharmacological properties, including
antinociception and hypothermia (Narimatsu et al., 1987; Vivian et al.,
1998). Biochemical studies have revealed that repeated THC
administration increases opioid peptide gene expression (Corchero et
al., 1997a,b). Acute THC also increases extracellular levels of
endogenous enkephalins in the nucleus accumbens (Valverde et al.,
2001). The existence of cross-tolerance between opioid and cannabinoid
agonists has been supported by a variety of studies. Thus,
morphine-tolerant animals show decreased THC antinociceptive responses,
whereas THC-tolerant rodents show a decrease in morphine
antinociception (Hine, 1985; Thorat and Bhargava, 1994).
Cross-dependence between opioid and cannabinoid compounds has also been
reported. Indeed, the opioid antagonist naloxone precipitated a
withdrawal syndrome in THC-tolerant rats (Kaymakcalan et al., 1977),
whereas the cannabinoid antagonist SR171416A was able to precipitate
abstinence in morphine-dependent rats (Navarro et al., 1998).
Besides, the severity of opioid withdrawal was reduced by the
administration of THC (Hine et al., 1975; Valverde et al., 2001) or the
endogenous cannabinoid agonist anandamide (Vela et al., 1995). This
bidirectional cross-dependence has been recently confirmed by using
knock-out mice, because opioid dependence was reduced in mice lacking
the CB1 cannabinoid receptor (Ledent et al., 1999), whereas cannabinoid
dependence was reduced in mice lacking the preproenkephalin gene
(Valverde et al., 2000).
Another important aspect of marijuana activity is the complexity of
evoked emotional responses and in particular the possibility of dual
euphoria-dysphoria effects (Halikas et al., 1985). Electrical brain
stimulation (Gardner et al., 1988) and in vivo microdialysis (Chen et al., 1990; Tanda et al., 1997) have suggested that
cannabinoids produce their rewarding action by stimulating mesolimbic
dopaminergic transmission, a common substrate for the rewarding effects
of other substances of abuse (Koob, 1992), and that µ-opioid
receptors could be involved (Tanda et al., 1997). The endogenous
cannabinoid system participates in the rewarding effects of opioids,
because both morphine self-administration (Ledent et al., 1999) and
place preference (Martin et al., 2000) are decreased in mice lacking the CB1 receptor. However, the possible involvement of the endogenous opioid system in the different motivational responses induced by
cannabinoids remains to be clarified. GABAergic (Onaivi et al., 1990)
and corticotropin-releasing factor (Rodriguez de Fonseca et al., 1996)
systems have been suggested to be involved in the anxiogenic responses
induced by cannabinoids. These anxiogenic effects could have some
influence in the dysphoric properties of cannabinoids, but the
mechanisms that underlie the potential aversive effects of THC remain unexplored.
To investigate these major aspects of cannabinoid-opioid interactions,
we have examined whether the genetic ablation of µ-opioid (Matthes et
al., 1996), -opioid (Filliol et al., 2000), or -opioid (Simonin
et al., 1998) receptors in mice has any influence on THC tolerance,
physical dependence, and motivational responses.
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MATERIALS AND METHODS |
Mice. The generation of mice lacking either
µ-opioid (MOR  /), -opioid (DOR /), or -opioid (KOR
/) receptors has been described previously (Matthes et al., 1996;
Simonin et al., 1998; Filliol et al., 2000). Mice weighing 22-24 gm at
the start of the study were housed, grouped, and acclimatized to the
laboratory conditions (12 hr light/dark cycle, 21 ± 1°C room
temperature, 65 ± 10% humidity) 1 week before the experiment
with ad libitum access to food and water. All animals were
1:1 hybrids from 129/SV and C57B1/6 mouse strains. Wild-type
littermates were used for the control groups in all experiments.
Mutants and their wild-type littermates showed comparable spontaneous
locomotor activity, except for DOR / mice, which displayed
significant hyperlocomotion (increase of 161.85 ± 19.58%
comparing with wild-type controls, F(1,23) = 6.277, p < 0.05) as previously reported (Filliol et al., 2000). Behavioral tests
and animal care were conducted in accordance with the standard ethical
guidelines (National Institutes of Health, 1995; Council of Europe,
1996) and approved by the local ethical committee. The observer was
blind to the genotype and treatment in all experiments.
Drugs. THC (Sigma, Poole, UK) was dissolved in a
solution of 5% ethanol, 5% cremophor El, and 90% distilled water,
and injected in a volume of 0.1 ml per 10 gm body weight. The selective
CB1 cannabinoid receptor antagonist SR141716A was dissolved in a
solution of 10% ethanol, 10% cremophor El, and 80% distilled water,
and injected by intraperitoneal route in a volume of 0.2 ml per 10 gm
body weight.
Tolerance and withdrawal. Animals were injected
intraperitoneally twice daily at 9:00 A.M. and 7:00 P.M. for 5 d
with THC (20 mg/kg) or vehicle. On day 6, mice only received the
morning injection. Four different responses were measured once a day
during the chronic THC treatment: body weight, rectal temperature,
antinociception, and locomotor activity. Body weights were recorded for
each animal, using an electronic balance (Mettler PM 4800; sensitive to
0.01 gm), once a day before morning injections. Locomotor
measurements for each mouse were taken 20 min after morning injections
by placing animals in individual actimeters (9 × 20 × 11 cm) (Imetronic, Bordeaux, France) equipped with two lines of six
infrared beams for 10 min, and recording both horizontal and vertical
activity, under a dim light (<20 lux). Antinociceptive measurements
for each mouse were taken 30 min after morning injection by using the
tail immersion assay as described previously (Janssen et al., 1963).
Antinociceptive responses were also evaluated in the hot plate test
(Columbus Instruments, Columbus, OH) on the first day. For the tail
immersion, the time to withdraw the tail from the bath was registered
(50 ± 0.5°C), with a cutoff latency of 15 sec to prevent tissue
damage. For the hot plate (52 ± 0.5°C), two different
nociceptive thresholds were measured: paw licking (cutoff latency of 30 sec) and jumping (cutoff latency of 240 sec). Rectal temperature was
measured in each mouse using an electronic thermocouple flexible rectal
probe (Panlab, Madrid, Spain). The probe was placed 3 cm into
the rectum of the mice for 20 sec before the temperature was recorded,
and measures were taken 40 min after morning injection.
On the sixth day, 4 hr after the last THC or vehicle injection, mice
were placed in a circular clear plastic observation area (30 cm
diameter, 80 cm height) for a 15 min period of observation. Body weight
and rectal temperature were consecutively measured, and animals
received administration of SR141716A (10 mg/kg, i.p.). Mice were then
replaced in the observation area and observed for 45 min. Measurement
of somatic signs before and after SR 141716A challenge were divided in
5 min time intervals, as previously described (Hutcheson et al., 1998).
The number of bouts of sniffing, writhing, wet dog shakes, and front
paw tremor were counted. Penile licking or erection, ataxia, hunched
posture, tremor, ptosis, and piloerection were scored 1 for appearance
and 0 for nonappearance within each 5 min time period. Scores for the
level of activity were made by giving in each 5 min period a value of
0 = low activity (less than five complete crossings of the
observation area), 1 = normal activity (between five and twenty
complete crossings of the observation area), or 2 = increased
activity (more than twenty complete crossings of the observation area).
A quantitative value was calculated in each animal for the different
checked signs by adding the scores obtained in each 5 min time period. A global withdrawal score, ranging from 0 to 100, was calculated for
each animal by giving to each individual sign a relative weight, as
previously described (Koob et al., 1992): 0.9 point for the appearance of each checked sign in each 5 min time period; 0.4 point for each bout of counted sign.
Statistics. Acute effects and global withdrawal scores
were compared by using two-way ANOVA (genotype and treatment) between subjects followed by one-way ANOVA for individual differences. Values
of tolerance studies were compared by using three-way ANOVA (genotype
and treatment as between groups factors and day as within group
factor), followed by corresponding two-way and one-way ANOVAs and
post hoc comparisons when applied.
Place conditioning. An unbiased place conditioning procedure
was used to evaluate both rewarding and aversive properties of THC
(Valjent and Maldonado, 2000) in animals lacking each opioid receptor.
The apparatus consisted of two main square conditioning compartments
(15 × 15 × 15 cm) separated by a triangular central division (Maldonado et al., 1997). The light intensity within the
conditioning chambers was 50 ± 5 lux. The movement and location of the mice were recorded by computerized monitoring software (Videotrack; View Point, Lyon, France) with images relayed from a
camera placed above the apparatus. During the preconditioning phase,
drug-naive mice were placed in the middle of the central division and
had ad libitum access to both compartments (striped and
dotted compartment) of the conditioning apparatus for 20 min, with the
time spent in each compartment being recorded. The conditioning phase
consisted of five pairings with THC and five pairings with vehicle for
a 45 min conditioning time in all experiments. Mice were injected with
vehicle or THC (1 and 5 mg/kg, i.p.) and then immediately confined to
the conditioning compartment. Treatments were counterbalanced as
closely as possible between compartments. Control animals received
vehicle every day. The test phase was conducted exactly as the
preconditioning phase, i.e., ad libitum access to each
compartment for 20 min. Mice conditioned with the dose of 1 mg/kg of
THC received a single THC (1 mg/kg, i.p.) injection in the home cage 24 hr before starting the conditioning procedures, to avoid the dysphoric
effects of the first drug exposure (Valjent and Maldonado, 2000). As
previously described (Stinus et al., 1990; Valverde et al., 1996;
Maldonado et al., 1997), the time in central area was proportionally
shared and added to the time value of each conditioned compartment, as
follows: the time spent in each compartment is multiplied by the total
time (1200) and divided by the total time minus the time spent in the
central area:
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The total amount of time spent in the central area was similar
in all groups of the different experiments (see Statistics). A place
conditioning score was calculated for each animal as the difference
between time spent in the drug-paired compartment during the test and
preconditioning phases.
Statistics. Raw time score values were used for statistical
analysis in all the experiments. These values were compared by using
two-way ANOVA between subjects (genotype and treatment) followed by
one-way ANOVA for individual differences. Time spent in the drug-paired
compartment during preconditioning in the different groups was compared
by a one-way ANOVA (experimental group as between subjects factor) to
ensure use of an unbiased procedure (experiment with MOR / mice:
F(5,75) = 0.414, p = 0.837; experiment with DOR / mice:
F(5,64) = 0.753, p = 0.587; experiment with KOR / mice:
F(5,60) = 0.420, p = 0.833; experiment with KOR / mice without priming:
F(3,37) = 0.05, p = 0.985). To verify any possible influence of the mutation in
mouse spontaneous behavior in the place conditioning paradigm, time
spent in the central compartment during preconditioning in the
different groups was also compared by two-way ANOVA between subjects
(genotype and treatment). No significant effect of genotype (experiment
with MOR / mice: F(1,81) = 0.017, p = 0.897; experiment with DOR / mice:
F(1,70) = 1.136, p = 0.290; experiment with KOR / mice:
F(1,68) = 1.377, p = 0.260), treatment (experiment with MOR / mice:
F(2,81) = 1.757, p = 0.180; experiment with DOR / mice:
F(2,70) = 0.372, p = 0.372; experiment with KOR / mice:
F(2,68) = 3.905, p = 0.053) nor interaction between these two factors (experiment with MOR
/ mice: F(2,81) = 0.504, p = 0.606; experiment with DOR / mice:
F(2,70) = 0.657, p = 0.522; experiment with KOR / mice:
F(2,68) = 2.060, p = 0.136) was observed in any experiment. Individual comparisons of time
spent in the drug-paired compartment during preconditioning and test phases were made with paired two-tailed Student's t test.
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RESULTS |
THC tolerance and withdrawal in MOR /, DOR /, and KOR
/ mice
To induce tolerance and dependence, we chronically injected a high
dose of THC (20 mg/kg). The first THC injection produced similar
antinociception in wild-type, MOR /, DOR /, and KOR / mice,
in both the hot plate (paw licking: mean value, 79.3 ± 3.26% of
analgesia, F(5,68) = 0.788, p = 0.562; jump: mean value, 95.9 ± 1.22% of
analgesia, F(5,68) = 0.913, p = 0.478) and the tail immersion (mean value,
47.8 ± 5.14% of analgesia;
F(5,68) = 1.386; p = 0.24). This first THC injection also produced hypolocomotion (mean
decrease, 66.2 ± 2.04%; F(5,67) = 0.867; p = 0.508) and hypothermia (mean decrease,
3.63 ± 0.34°C; F(5,67) = 2.091; p = 0.077) that did not differ between genotypes.
During repeated THC administration, a progressive decrease in the
antinociceptive, hypolocomotor, and hypothermic activity of the drug
was observed. This tolerance developed similarly in wild-type, MOR
/, and DOR / mice, whereas minor changes were observed in KOR
/ mice (Table 1, Fig.
1). Tolerance for locomotor responses was
developed on the second day of THC treatment in all groups. However,
habituation in vehicle-treated mice during the first 3 d of
testing was observed. This habituation can also be important for
reducing initial differences between THC and vehicle-treated groups.
Noticeable was the significant lower locomotion in THC-treated KOR
/ mice compared with THC-treated wild-type controls on day 5 (F(1,21) = 13.426; p < 0.005). Tolerance to the hypothermic effects developed
similarly in all mouse genotypes. Tolerance to
antinociceptive effects of THC was also similar in all genotypes (Table
1, Fig. 1).
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Table 1.
Three-way ANOVA of hypothermia, hypolocomotion, and
antinociception induced during chronic THC treatment in mice lacking
µ-, -, or -opioid receptor
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Figure 1.
Adaptive responses to chronic THC in KOR /
mice. Development of tolerance to the antinociceptive, hypothermic, and
hypolocomotor effects of chronic THC (20 mg/kg, i.p., twice daily) is
minimally affected in KOR / mice. Number of mice per group from 10 to 14. Values are expressed as means ± SEM.
 p < 0.05; p < 0.01;
p < 0.001; comparison between treatments
(one-way ANOVA); p < 0.01, comparison
between genotypes (one-way ANOVA).
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We then administered the cannabinoid antagonist SR141716A (10 mg/kg) in
chronically THC-treated mice. A significant expression of
several signs of withdrawal, including wet dog shakes, paw tremor,
ptosis, piloerection, mastication, and sniffing, was detected in
wild-type, MOR /, DOR /, and KOR / mice. Expression of these signs was comparable in mutant animals and their respective wild-type controls, except for paw tremor that was significantly reduced in THC-treated DOR / mice (vehicle-treated DOR +/+ = 2.46 ± 1.39; vehicle-treated DOR / = 3.25 ± 1.13;
THC-treated DOR +/+ = 81.6 ± 9.78; THC-treated DOR / = 64.4 ± 13.4; two-way ANOVA, genotype:
F(1,38) = 76.97, p < 0.001; treatment: F(1,38) = 5.173, p < 0.05, genotype × treatment:
F(1,38) = 5.556, p < 0.05; one-way ANOVA for genotype comparison:
F(1,14) = 4.894, p < 0.05). Other signs of THC withdrawal such as body tremor, writhing,
hunched posture, and penile licking were less obvious, and no
difference was detectable in any of these signs when comparing mutant
and wild-type groups. We determined global withdrawal scores for each
MOR /, DOR /, and KOR / genotype and their wild-type controls. Total withdrawal score comparisons revealed no significant alteration of THC withdrawal in any group of mutant mice (Fig. 2). Indeed, two-way ANOVA revealed a
significant effect of THC treatment (experiment with MOR / mice:
F(1,41) = 123.2, p < 0.001; experiment with DOR / mice:
F(1,38) = 241.3, p < 0.001; experiment with KOR / mice:
F(1,36) = 44.31, p < 0.001), but not effect of genotype (experiment with MOR / mice:
F(1,41) = 0.122, p = 0.873; experiment with DOR / mice:
F(1,38) = 0.135, p = 0.715; experiment with KOR / mice:
F(1,36) = 0.092, p = 0.736) nor interaction between treatment and genotype (experiment with
MOR / mice: F(1,41) = 0.058, p = 0.810; experiment with DOR / mice:
F(1,38) = 0.525, p = 0.525; experiment with KOR / mice:
F(1,36) = 0.019, p = 0.891) in the three experiments.
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Figure 2.
Somatic expression of withdrawal from THC after SR
141716A administration (10 mg/kg, i.p.) is similar in MOR /, DOR
/, and KOR / mice and in their respective wild-type controls.
Mice received a chronic administration of vehicle or THC (20 mg/kg,
i.p., twice daily) for 6 d. Values are expressed as mean ± SEM of global withdrawal scores calculated by giving to each individual
sign a proportional weight. Number of mice per group from 10 to 14. p < 0.001, comparison between treatments
(one-way ANOVA).
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THC-induced place conditioning
We explored motivational responses to THC in MOR /, DOR /,
and KOR / mice, using several place conditioning protocols. We
first investigated the rewarding properties of THC, in a place conditioning protocol that minimizes the aversive activity of THC. Mice
received a single injection of a low dose of THC (1 mg/kg) in their
home cage and were subsequently conditioned to the same dose of the
drug. Time spent by all the mice in the two conditioning compartments
of the apparatus was similar during the preconditioning phase in the
different experiments (mean in striped compartment: 615.8 sec; mean in
dotted compartment: 582.6 sec). No initial place preference or aversion
was observed in any of the experiments. Under those conditions, all the
groups of wild-type mice showed a strong place preference, as
previously reported (Valjent and Maldonado, 2000). This was revealed by
significant increases in the time spent in the drug-paired compartment
from the preconditioning to the test phase in MOR +/+, DOR +/+, and KOR
+/+ mice (see Table 4), as well as by significant scores when comparing
THC-treated mice with the respective vehicle controls (Tables
2, 3, Fig.
3). A similar conditioned place
preference to THC (1 mg/kg) was observed in DOR / and KOR /
mutants receiving the previous THC single injection (Tables
2-4, Fig. 3). In contrast, this response
was completely absent in MOR / mice (Tables 2-4, Fig. 3),
demonstrating that µ-opioid receptors are essential for the rewarding
effects of THC.
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Figure 3.
THC place preference and aversion. A,
Place preference to THC is abolished in MOR / mice, whereas place
aversion is diminished (number of mice per group from 13 to 14);
B, DOR / mice show similar place conditioning to THC
than their wild-type controls (number of mice per group from 10 to 13);
and C, Place aversion to THC is abolished in KOR /
mice (number of mice per group from 11 to 12) mice. THC1 (1 mg/kg,
i.p.); THC5 (5 mg/kg, i.p.). Mice conditioned to the dose of 1 mg/kg
THC received a previous THC injection (1 mg/kg, i.p.) in their home
cage, 24 hr before the start of the conditioning phase. In wild-type
mice, THC1 conditions reveal THC place preference, whereas THC5
conditions show THC place aversion. Values are expressed as mean ± SEM. Scores were calculated as the difference between test and
preconditioning time spent in the drug-paired compartment.
p < 0.05, p < 0.01, comparison between treatments; p < 0.01, comparison between genotypes (one-way ANOVAs).
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Table 4.
Paired Student's t test comparisons of the
time spent in the drug-paired compartment during the preconditioning
and test phases, in mice lacking µ-, -, or -opioid receptor
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We then used another place conditioning protocol where animals received
no injection of THC before conditioning and were subjected to a high
dose of THC (5 mg/kg). Under those conditions only THC dysphoria is
detectable. In agreement with previous studies (Valjent and Maldonado,
2000), wild-type mice showed marked conditioned place aversion, as
revealed by significant decrease in time spent in the drug-paired
compartment in MOR +/+, DOR +/+, and KOR +/+ (Table 4), and in the
score values (Tables 2, 3, Fig. 3). In DOR / mice,
conditioned place aversion to THC (5 mg/kg) was comparable
with wild-type (Tables 2-4, Fig. 3). In MOR / mutants, the
aversive effects of THC were diminished but not abolished (Tables 2-4,
Fig. 3). In KOR / animals, THC place aversion was completely absent
(Tables 2-4, Fig. 3). The latter finding demonstrates that receptors are critically implicated in the dysphoric aspect of THC activity.
Because this is a first evidence for an involvement of receptors in
THC aversive properties, we further explored this finding using a third
place conditioning protocol. Mice were conditioned to 1 mg/kg of THC
without receiving any injection of the drug before the conditioning
period. We have previously shown that this protocol does not reveal any
motivational effect of THC in wild-type mice, most probably because THC
reward and dysphoria equally oppose each other (Valjent and Maldonado,
2000). Accordingly, wild-type mice showed no place conditioning, as
shown by comparable time spent in the drug-paired compartment between
preconditioning and test (t(1,9) = 0.680; p = 0.514) and comparable score values between
animals conditioned to THC or vehicle
(F(1,19) = 0.348; p = 0.562) (Fig. 4). In contrast, KOR /
mice conditioned to 1 mg/kg of THC showed under these
experimental conditions a significant place preference as seen by an
increase in the time spent in the drug-paired compartment
(t(1,9) = 2.770; p < 0.05) and a significantly different conditioning score from vehicle
controls (F(1,18) = 5.587;
p < 0.05). Also, although vehicle groups from both
genotypes showed similar scores
(F(1,18) = 0.006; p = 0.938), KOR / animals conditioned to THC displayed significantly
different scores from wild-type controls
(F(1,19) = 4.877; p < 0.05). Therefore, the lack of receptors in mutant mice reveals THC
place preference, suggesting that, under those conditions, receptor activity hinders the rewarding properties of THC in wild-type
mice.
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Figure 4.
KOR / mice show place preference to THC (1 mg/kg, i.p.), without priming exposure, whereas their wild-type
controls do not. Values are expressed as mean ± SEM. Number of
mice per group from 9 to 10. Scores calculated as the difference
between test and preconditioning time spent in the drug-paired
compartment. p < 0.05, comparison between
treatments; p < 0.05, comparison between
genotypes (one-way ANOVAs).
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DISCUSSION |
Bidirectional interactions between the opioid and cannabinoid
systems have been reported (Manzanares et al., 1999). Here we show that
disruption of µ-, -, or -opioid receptor gene does not modify
acute THC responses or the expression of THC withdrawal, and that the
development of THC tolerance is only slightly altered in KOR /
mice. Both µ- and -opioid ligands have been previously reported to
modulate cannabinoid antinociception (Manzanares et al., 1999). Thus,
THC antinociception was blocked in mice by the -selective opioid
antagonist norbinaltorphimine and by high doses of the nonselective
opioid antagonist naloxone (Welch, 1993; Smith et al., 1998). The
synergistic effects of morphine and THC on antinociception were also
blocked by both norbinaltorphimine and  -funaltrexamine, a µ selective opioid antagonist (Reche et al., 1996). The absence of
changes in THC antinociception in this study is unlikely attributable
to a ceiling effect, because a maximal response was observed in one
response (jump in the hot plate) over three nociceptive thresholds
evaluated. Rather, we may suggest that the ablation of one opioid
receptor only is not sufficient to reveal significant changes, and
indeed, high doses of opioid antagonists are usually required to block
THC antinociception (Manzanares et al., 1999). THC antinociception in
the tail immersion test was reduced in knock-out mice lacking the
preproenkephalin gene (Valverde et al., 2000), but derivatives from
proenkephalin are not selective agonists of any opioid receptor.
Besides, the deletion of µ- or -opioid receptors has no measurable
consequence on the development of THC tolerance, whereas tolerance was
reduced in KOR / mice on the last day of treatment. Although these
modifications were subtle, the data suggest that receptors could
contribute to the development of adaptive responses to chronic THC, in
agreement with the demonstration of cross-tolerance between THC and
-opioid agonists (Smith et al., 1994). Besides, THC-induced antinociceptive tolerance can be modified by antisense oligodeoxynucleotides to the receptor (Rowen et al., 1998), and mice tolerant to THC also displayed tolerance to the effects of selective agonists (U-50,488 and CI-977), but not of µ or selective agonists (Smith et al., 1994).
The lack of alteration of THC withdrawal was unexpected, because clear
interactions between opioid and cannabinoid dependence have been
reported. Thus, naloxone, the prototypic nonselective opioid
antagonist, precipitates an opioid-like withdrawal syndrome in
cannabinoid-dependent rodents (Kaymakcalan et al., 1977; Navarro et
al., 1998) and conversely, the CB1 cannabinoid receptor antagonist SR
141716A induces withdrawal in morphine-dependent rats (Navarro et al.,
1998). This suggests that simultaneous activation of the two endogenous
systems could participate to both opioid and cannabinoid dependence.
Consistent with this notion, gene-targeting experiments have shown
attenuated naloxone-precipitated opioid withdrawal in
morphine-dependent CB1 knock-out mice (Ledent et al., 1999) and
attenuated SR141716A-precipitated cannabinoid withdrawal in THC-dependent mice lacking preproenkephalin gene (Valverde et al.,
2000). Pretreatment with THC (Hine et al., 1975; Valverde et al., 2001)
or anandamide (Vela et al., 1995), have been also shown to decrease
morphine withdrawal. Our finding that the disruption of a single opioid
receptor gene has no major consequences on the somatic expression of
THC withdrawal may indicate that concomitant changes in the activity of
several opioid receptors are implicated in the expression
of THC withdrawal. Possibly the analysis of combinatorial double or
triple opioid receptor-deleted mice may reveal a concerted implication
of several components of the opioid system in the development of THC
physical dependence.
The suppression of THC rewarding effects in MOR / mice provides a
clear genetic evidence to clarify the crucial role of µ receptors in
THC motivational responses. Gene targeting experiments have recently
demonstrated the central role of µ-opioid receptors in mediating
rewarding properties of several drugs of abuse, including morphine
(Matthes et al., 1996), ethanol (Roberts et al., 2000), and now THC
(this study). µ receptors, therefore, seem to represent a convergent
substrate for positive reinforcement. A possible mechanism to explain
the involvement of µ receptors in cannabinoid rewarding effects could
be caused by their effects on the mesolimbic dopaminergic system. Thus,
THC-induced increase in mesolimbic dopaminergic activity (Navarro et
al., 1993) was reversed by naloxone (Chen et al., 1990), or
naloxonazine, a µ1 antagonist (Tanda et al.,
1997). However, another study showed no effect of naloxone on THC
excitatory activity on dopamine neurons from the ventral tegmental area
(French, 1997). Therefore, other mechanisms could also mediate the
implication of µ-opioid receptors in cannabinoid motivational
properties. Interestingly, morphine-induced rewarding effects were
suppressed in mice deficient in CB1 cannabinoid receptors (Ledent et
al., 1999), suggesting a bidirectional influence of µ-opioid and CB1
cannabinoid receptors on reward processes.
-opioid receptors do not seem to be involved in the motivational
responses to THC. The differential location of CB1 cannabinoid and opioid receptors may be important for this result. Although both
receptors are localized in the nucleus accumbens (Mansour et al., 1995;
Robbe et al., 2001), they might not be colocalized in other brain
regions implicated in the rewarding effects of cannabinoids. The
present results demonstrate a crucial involvement of -opioid
receptors in the aversive properties of THC. Thus, THC-induced
conditioned place aversion was abolished in KOR / mice. In
addition, the lack of -opioid receptors reveals THC place preference
in mutant mice, suggesting that, under those conditions, receptor
activity hinders the rewarding properties of THC in wild-type mice.
These observations indicate that an endogenous receptor tone
underlies THC aversion, a hypothesis that has not been explored by
pharmacological studies. Several peripheral and somatic effects of
THC can participate in its aversive properties, as it has
been previously reported with agonists (Bechara et al., 1987).
Indeed, THC produces hypothermia and hypolocomotion that can induce a
discomfort state in the mouse, leading to the aversive response. The
exact involvement of these somatic responses remains to be clarified.
A possible limitation in the interpretation of these results is the
required use of ethanol to dissolve THC and SR 141716A. Indeed, ethanol
is also a psychoactive compound sharing some common pharmacological
properties with cannabinoids and opioids. The pharmacological responses
induced by these vehicle solutions of cannabinoid agents have been
reported to be exclusively caused by the activation of CB1 cannabinoid
receptors because both acute and chronic THC effects were abolished in
knock-out animals deficient in these CB1 receptors (Ledent et al.,
1999). However, THC behavioral effects can be modulated by the presence
of ethanol, and this modulation may be different in µ-, -, or
-opioid receptor knock-out mice. Therefore, a possible contribution
of ethanol in the behavioral responses observed in these knock-out mice
cannot be excluded. Ethanol was also present in the SR 141716A solution
used to precipitate THC withdrawal. The behavioral signs of cannabinoid
withdrawal observed when using this procedure are caused by the
blockade of CB-1 cannabinoid receptors because they were abolished in
CB-1 knockout mice (Ledent et al., 1999), and these behavioral signs were not elicited by the ethanol-containing vehicle alone (Cook et al.,
1998). However, the presence of ethanol could attenuate the behavioral
expression of cannabinoid withdrawal, considering the common
pharmacological effects of both compounds. This observation must be
considered for the interpretation of the cannabinoid withdrawal results. Besides, the possibility of developmental compensations after
the deletion of the different opioid receptors and/or influences of the
hybrid genetic background (Kelly et al., 1998) cannot be discarded.
However, no homologous compensatory changes have been reported in these
knock-out mice. Indeed, the abolition of µ-opioid (Matthes et al.,
1996), -opioid (Filliol et al., 2000), or -opioid (Simonin et
al., 1998) receptors has no influence on the binding properties and
distribution of the other opioid receptors, and did not modify the
expression of the different opioid peptide precursors.
The present novel finding highlights the involvement of homeostatic
opioid mechanisms in cannabinoid motivational responses. We propose
that opposing µ-opioid and -opioid receptor activities mediate the
dual euphoric-dysphoric effects of THC. This hypothesis is consistent
with the proposed opposite role of the two opioid receptors in
modulating mesolimbic dopaminergic activity (Di Chiara, 1995) and
suggests that THC influences both players within the reward circuitry.
A possible mechanism could be that cannabinoid receptor activation
modifies endogenous opioid peptide levels in mesolimbic areas that
would, in turn, modulate dopaminergic activity. This is supported by
findings of increased opioid peptides levels in the hypothalamus
(Corchero et al., 1997a,b), or preproenkephalin mRNA levels in the
striatum and nucleus accumbens (Manzanares et al., 1998) after
cannabinoid treatment. Cannabinoids and opioids might also interact at
the level of their signaling activity. Both receptor types are coupled
to similar intracellular effectors via Gi/Go-proteins, modulating cAMP
levels, K+ and
Ca2+ channel activities, and MAP kinase
phosphorylation (Bouaboula et al., 1995; Fukuda et al., 1996;
Manzanares et al., 1999), and cross-talk could occur downstream of
receptors coexpressed in specific mesocorticolimbic neurons (Mansour et
al., 1995; Breivogel and Childers, 1998).
In conclusion, we found that single disruption of µ-opioid,
-opioid, or -opioid receptors has minimal influence on somatic adaptations to prolonged cannabinoid exposure. In contrast, we provide
strong evidence for the involvement of µ-opioid and
-opioid receptors in motivational responses to THC. These data shed
a new light on mechanisms underlying the possible addictive potential of marijuana, highlighting a major implication of opioid
receptor-mediated mechanisms essentially in THC
euphoria-dysphoria.
| |
FOOTNOTES |
Received July 23, 2001; revised Nov. 7, 2001; accepted Nov. 15, 2001.
This work was supported by the European Commission (Biomed-2 Grant
98-2227, to R.M.), Dr. Esteve S. A. Laboratories (R.M.), Generalitat de Catalunya (Research Distinction, to R.M.), the Spanish
Ministry of Health (Fondo de Investigación Sanitaria Grant
99/0624, to R.M.), the Mission Interministerielle de Lutte contre la
Drogue et la Toxicomanie (B.K.), and the Centre National de la
Recherche Scientifique (B.K.). We thank J. F. Poirier and N. Scallon for animal care.
Correspondence should be addressed to Rafael Maldonado,
Laboratori de Neurofarmacologia, Facultat de Ciéncies de la Salut i de la Vida, Universitat Pompeu Fabra, c/o Dr Aiguader 80, 08003 Barcelona, Spain. E-mail: rafael.maldonado{at}cexs.upf.es.
| |
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ABSTRACT
INTRODUCTION
