Involvement of the opioid system in the anxiolytic-like effects induced by D9-tetrahydrocannabinol
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Psychopharmacology (2002) 163:111–117
DOI 10.1007/s00213-002-1144-9
ORIGINAL INVESTIGATION
Fernando Berrendero · Rafael Maldonado
Involvement of the opioid system in the anxiolytic-like effects induced
by D9-tetrahydrocannabinol
Received: 24 February 2002 / Accepted: 8 May 2002 / Published online: 27 June 2002
4 Springer-Verlag 2002
Abstract Rationale: Recent studies have shown that Introduction
several pharmacological actions induced by cannabinoids,
including antinociception and reward, involve the partic- D9-Tetrahydrocannabinol (THC), the main psychoactive
ipation of the endogenous opioid system. Objectives: The constituent of Cannabis sativa, produces a wide spectrum
present study was designed to examine the possible of central and peripheral actions, including antinocicep-
involvement of the different opioid receptors in the tion, hypothermia, immunomodulation, inhibition of
anxiolytic-like responses induced by D9-tetrahydrocan- locomotor activity, catalepsy, memory disruption and
nabinol (THC). Methods: The administration of a low alleviation of both intraocular pressure and emesis
dose of THC (0.3 mg/kg) produced clear anxiolytic-like (Dewey 1986). The central nervous system (CNS)
responses in the light-dark box, as previously reported. responses to cannabinoids are mediated by the CB1
The effects of the pretreatment with the CB1 cannabinoid cannabinoid receptor (Devane et al. 1988; Matsuda et
receptor antagonist, SR 141716A (0.5 mg/kg), or the >- al. 1990), which is a member of the family of G-protein-
opioid receptor antagonist, b-funaltrexamine (5 mg/kg), coupled receptors. Within the brain, high levels of CB1
the d-opioid receptor antagonist, naltrindole (2.5 mg/kg) receptors are found in the basal ganglia, limbic structures
and the k-opioid receptor antagonist, nor-binaltorphimine and cerebellum (Herkenham et al. 1991; Mailleux and
(2.5 mg/kg) were evaluated on anxiolytic-like responses Vanderhaeghen 1992). Cannabinoid receptors, located in
induced by THC. Results: SR 141716A completely the limbic system, could participate in regulating a variety
blocked the anxiolytic-like response induced by THC, of emotional responses. Thus, several studies have shown
suggesting that this effect is mediated by CB1 cannabi- that cannabinoid agonists can induce anxiogenic and
noid receptors. The >-opioid receptor antagonist b- anxiolytic-like responses in rodents, depending on the
funaltrexamine and the d-opioid receptor antagonist dose and the environmental conditions (RodrBguez de
naltrindole, but not the k-opioid receptor antagonist nor- Fonseca et al. 1996; Valjent et al. 2002). Low doses of
binaltorphimine, abolished THC anxiolytic-like effects, cannabinoids usually induce an anxiolytic-like effect,
suggesting an involvement of >- and d-opioid receptors in whereas higher doses cause the opposite response. In
this behavioural response. Conclusions: These results addition, the endogenous cannabinoids through the acti-
demonstrate that the endogenous opioid system is vation of CB1 receptors seem to be involved in the
involved in the regulation of anxiety-like behaviour by regulation of emotional behaviour, as revealed by: (i) the
cannabinoids and provide new findings to clarify further anxiogenic-like response induced in rats by the acute
the interaction between these two neuronal systems. administration of the CB1 cannabinoid receptor antago-
nist, SR 141716A (Navarro et al. 1997) and (ii) the
Keywords D9-Tetrahydrocannabinol · CB1 cannabinoid increase in the basal level of anxiety during the light-dark
receptor · Opioid system · Anxiety · Light-dark box test observed in CB1 knockout mice (Martin et al. 2002).
However, the mechanisms by which cannabinoids would
control anxiety-related behaviour remain to be elucidated.
F. Berrendero · R. Maldonado ())
Opioid compounds induce their pharmacological ef-
Laboratori de Neurofarmacologia, fects by activating >-, d- and k-opioid receptors (Thomp-
Facultat de CiEncies de la Salut i de la Vida, son et al. 1993; Mansour et al. 1995) and share several
Universitat Pompeu Fabra, C/Doctor Aiguader 80, actions with cannabinoids such as hypothermia, sedation,
08003 Barcelona, Spain hypotension, inhibition of intestinal motility, motor
e-mail: rafael.maldonado@cexs.upf.es depression and antinociception (Bloom and Dewey
Tel.: +34-93-5422845 1978; Dewey 1986). Pharmacological interactions be-
Fax: +34-93-5422802112
tween these two systems have been suggested, mainly administered 30 min and 24 h before THC (0.3 mg/kg), respec-
concerning antinociception and addictive properties tively. All drugs were injected IP in a volume of 10 ml/kg.
(Pugh et al. 1996, 1997; Gardner and Vorel 1998;
Manzanares et al. 1999a; Navarro et al. 2001). Although Light-dark box paradigm
the biochemical mechanisms involved in these interac-
tions remain unclear several hypotheses have been The possible involvement of the endogenous opioid system on THC
anxiolytic-like responses was evaluated in the light-dark box
formulated. First, cannabinoids and opioids might interact (Filliol et al. 2000). Mice were individually exposed for 5 min to a
at the level of their signal-transduction mechanisms box consisting of a small compartment (15M20M25 cm) with black
(Manzanares et al. 1999a), since opioid and cannabinoid walls and black floor dimly lit (5 lux) connected by a 4 cm long
receptors are coupled to similar intracellular signalling tunnel to a large compartment (30M20M25 cm) with white walls and
systems, i.e. reduction in adenylyl cyclase activity and floor, under intense illumination (500 lux). Lines were drawn on the
floor of both compartments to allow measurement of locomotor
blockage of calcium currents, through activation of G activity by counting the number of squares (5M5 cm) crossed. Floor
proteins (Childers et al. 1992; Howlett 1995; Reisine et al. lines separated the lit compartment into three equal zones, from the
1996). Interestingly, co-expression of CB1 cannabinoid tunnel to the opposite wall, designated as proximal, median and
receptors and >-opioid receptors in the same striatal cells distal zone. The time spent in each compartment, locomotor
activity and number of visits into each zone of the lit compartment
has been recently reported (RodrBguez et al. 2001). The were recorded. Each animal was placed in the dark compartment
nature of cannabinoid-opioid interaction might also facing the tunnel at the beginning of each session, which started
involve the existence of a direct effect of cannabinoid 30 min after the acute injection of THC.
compounds on the synthesis and release of endogenous In a first experiment, the effects of SR 141716A administration
(0.5 mg/kg) on THC (0.3 mg/kg) responses were evaluated
opioid peptides (Corchero et al. 1997a, 1997b; Man- (vehicle, n=9; THC, n=9; SR 141716A, n=10; SR
zanares et al. 1998; Valverde et al. 2001). 141716A+THC, n=10). In a second experiment, the effects of b-
The aim of the present study was to explore the funaltrexamine administration (5 mg/kg) on THC (0.3 mg/kg)
possible involvement of the endogenous opioid system in responses were evaluated (vehicle, n=13; THC, n=14; b-funaltrex-
the anxiolytic-like responses induced by cannabinoids. amine, n=14; b-funaltrexamine+THC, n=14). In a third experiment,
the effects of naltrindole administration (2.5 mg/kg) on THC
For this purpose, the effects of the pretreatment with (0.3 mg/kg) responses were evaluated (vehicle, n=19; THC, n=17;
selective antagonists of the opioid receptors were evalu- naltrindole, n=19; THC+naltrindole, n=18). Finally, the effects of
ated on the anxiolytic-like responses induced by low nor-binaltorphimine administration (2.5 mg/kg) on THC (0.3 mg/
doses of THC in the light-dark box. In this model of kg) responses were evaluated (vehicle, n=11; THC, n=12; nor-
binaltorphimine, n=12; THC+nor-binaltorphimine, n=10). Different
anxiety, mice are exposed to a conflict represented by the groups of animals were used for each experiment.
novelty and aversive characteristics of the lit compart-
ment of the box.
Statistical analysis
One-way analysis of variance ANOVA (between subjects) followed
Materials and methods by a Newman-Keuls post-hoc comparison was used. Differences
were considered significant if the probability of error was113 Fig. 1A, B Effects of pretreatment with SR 141716A on the anxiolytic-like responses induced by THC in the light-dark box. The following results are shown: A time spent in the dark compartment; B time spent in the lit compartment. SR 141716A (0.5 mg/kg) and THC (0.3 mg/kg) were administered 35 and 30 min before the test, respectively. Data are expressed as mean€SEM (n=9–10 mice for each group). Black stars P
114
Fig. 4A–C Effects of pretreatment with nor-binaltorphimine on the
anxiolytic-like responses induced by THC in the light-dark box.
The following results are shown: A time spent in the dark
Fig. 3A–C Effects of pretreatment with naltrindole on the anxio- compartment; B time spent in the lit compartment; C percentage of
lytic-like responses induced by THC in the light-dark box. The visits to the zones of the lit compartment. Nor-binaltorphimine
following results are shown: A time spent in the dark compartment; (2.5 mg/kg) and THC (0.3 mg/kg) were administered 60 and 30 min
B time spent in the lit compartment; C percentage of visits to the before the test, respectively. Data are expressed as mean€SEM
zones of the lit compartment. Naltrindole (2.5 mg/kg) and THC (n=10–12 mice for each group). Black stars P115
changes were observed in the number of squares crossed pituitary-adrenal axis activity (RodrBguez de Fonseca et
in the dark compartment indicating that locomotor al. 1997). In our experimental conditions, we also
activity was not altered as a result of drug treatment observed the same anxiogenic-like effect of SR141716A
[F(3,41)=1.518, P=0.2241] (data not shown). when administered at the dose of 1 mg/kg, as revealed by
a decrease in the time spent in the lit compartment
(46.94% of decrease compared to saline control group,
Discussion P-opioid receptor antagonist b-funaltrexamine and
support for the involvement of the endogenous opioid the d-opioid antagonist naltrindole, but not the k-opioid
system in regulating this cannabinoid mediated emotional antagonist nor-binaltorphimine, also abolished the anxi-
response. However, the k-opioid antagonist nor-binaltor- olytic-like responses displayed by THC. Numerous stud-
phimine failed to modify the decrease in the level of ies have reported a pharmacological interaction between
anxiety induced by THC administration. opioids and cannabinoids, mainly in antinociception,
Previous studies have shown that cannabinoid agonists tolerance, dependence and rewarding properties (for
produce a dose-dependent biphasic effect in anxiety-like review, see Manzanares et al. 1999a). A high density
responses depending on the environmental conditions and/or functional activity of CB1 cannabinoid receptors
(Onaivi et al. 1990; RodrBguez de Fonseca et al. 1996; has been reported in the limbic system and other brain
Valjent et al. 2002). Thus, low doses of HU-210 (4 >g/kg) areas related to emotional responses, such as hypothala-
displayed anxiolytic-like effects in a novel environment mus and hippocampus (Herkenham et al. 1991; Mailleux
in the defensive withdrawal test whereas under familiar and Vanderhaeghen 1992; Tsou et al. 1997). Some of
conditions HU-210 induced a dose-dependent anxiogenic these brain structures, like the hypothalamus, have a high
effect (RodrBguez de Fonseca et al. 1997). Similar level of cannabinoid-activated G proteins despite low
biphasic responses were obtained by Valjent et al. cannabinoid receptor density (Breivogel et al. 1997).
(2002) using THC in the light-dark box. This test has Furthermore, the co-localization of both cannabinoid and
shown to be the most reliable and sensitive to analyze the opioid receptors in several limbic structures including the
anxiolytic-like effects of THC in mice (Valjent et al. nucleus accumbens and the amygdala (Mansour et al.
2002). In this test, an anxiolytic-like response was 1995), raises the hypothesis of an interaction between
induced by the administration of 0.3 mg/kg of THC, opioid and cannabinoid systems to control anxiety-like
whereas no responses were found using lower or higher responses.
doses of this compound (0.03, 0.1, 1 and 2.5 mg/kg). In The role of the endogenous opioid system in regulating
contrast, an anxiogenic-like effect was reported in this emotional-like behaviour has been recently studied by
paradigm when THC was administered at the dose of evaluating the spontaneous responses of mice lacking >-,
5 mg/kg (Valjent et al. 2002). This biphasic response has d- or k-opioid receptors in different models of anxiety
been already observed in the case of other pharmacolog- (Filliol et al. 2000). In agreement with our results, >- and
ical actions induced by cannabinoids, such as motor d-, but not k-opioid receptor knockout mice, showed
activity (SaSudo-PeSa et al. 2000) and aggressiveness changes in basal levels of anxiety (Filliol et al. 2000).
(Sulcova et al. 1998). The neurobiological mechanisms Thus, d-opioid receptor knockout mice showed anxio-
involved in the anxiogenic effects of cannabinoids are genic-like responses in the elevated plus maze and the
well known, and a crucial role of central corticotropin- light-dark box suggesting that the spontaneous activity of
releasing factor system has been reported (Weidenfel et d-opioid receptors seems to contribute to decrease levels
al. 1994; RodrBguez de Fonseca et al. 1996; Manzanares of anxiety. In line with these data, we have observed that
et al. 1999b; ArEvalo et al. 2001). However, the possible d-opioid receptors mediate the anxiolytic-like effects of
mechanisms involved in the anxiolytic-like responses THC. However, mice lacking >-opioid receptors showed
displayed by these compounds have not been yet studied. lower levels of anxiety in the elevated plus maze (Filliol
The CB1 antagonist SR 141716A reversed the anxiolytic- et al. 2000), although no significant changes were found
like effects induced by a low dose of THC, providing in the light-dark box. Besides d-opioid receptors, we have
clear evidence for the specific involvement of CB1 also observed that the activity of >-opioid receptors is
cannabinoid receptors in this behavioural response. necessary for THC to produce its anxiolytic-like effects.
SR141716A has been reported to produce an anxiogenic Although the mechanism of the cannabinoid-opioid
response on its own without affecting the hypothalamic- interaction is not clear yet, one possibility to explain the116
present findings is that THC, through CB1 receptors, for delta and mu-opioid receptors exhibit opposing alterations
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Acknowledgements This work has been supported by grants from cannabinoid receptor-mediated regulation of the increase in
Plan Nacional Sobre Drogas, European Communities BIOMED2 adrenocorticotropin hormone and corticosterone plasma con-
(98-2227), Human Frontier Science Program Organization centrations induced by central administration of D9-tetrahydro-
(RG0077/2000-B), Generalitat de Catalunya (Research Distinction) cannabinol in rats. Brain Res 839:173–179
and Laboratorios Dr. Esteve. The technical assistance of Ms. Martin M, Ledent C, Parmentier M, Maldonado R, Valverde O
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