Behavioural and neurochemical effects of combined MDMA and THC administration in mice
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- Robledo, P., Trigo, J.M., Panayi, F. et al. Psychopharmacology (2007) 195: 255. doi:10.1007/s00213-007-0879-8
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Cannabis is the most widely consumed drug associated with 3,4-methylenedioxymethamphetamine (MDMA) use.
This study examines whether low doses of MDMA and delta-9-tetrahydrocannabinol (THC) produce synergistic rewarding/reinforcing effects in mice using the conditioned place preference (CPP) and operant self-administration paradigms. Changes in dopamine (DA) outflow were monitored in the nucleus accumbens (NAC) after single or combined administration of these compounds.
MDMA induced a significant CPP at the dose of 10 mg/kg but not at the dose of 3 mg/kg. THC (0.3 mg/kg) by itself was also ineffective in this paradigm. The combined administration of the low dose of MDMA (3 mg/kg) and THC (0.3 mg/kg) produced CPP, whereas the combination of MDMA (10 mg/kg) and THC (0.3 mg/kg) significantly decreased CPP. Animals treated with THC self-administered a sub-threshold dose of MDMA (0.06 mg/kg per infusion), while animals receiving vehicle did not. However, THC did not modify the self-administration of an effective dose of MDMA (0.125 mg/kg per infusion). In microdialysis studies, a low dose of THC significantly increased DA outflow in the NAC, while a low dose of MDMA did not. When MDMA was administered before THC, DA levels decreased with respect to THC. However, when THC was administered before MDMA, DA levels were not significantly modified with respect to THC.
These results demonstrate that a low dose of THC modifies in different ways (increases and decreases) the sensitivity of animals to the behavioural effects of MDMA and that THC and MDMA converge at a common mechanism modulating DA outflow in the NAC of mice.
In the late 1990s, a survey performed among thousands of visitors to technoparties in several European cities showed that consumers of 3,4-methylenedioxymethamphetamine (MDMA) fitted the polydrug occasional user model characterised by the occasional use of diverse substances that are usually taken together. The sole use of MDMA was rare, and about 70% of MDMA users had additionally used cannabis within the defined time span where the pharmacological interaction between both drugs is expected (6 h before or after MDMA ingestion; Tossmann et al. 2001). Thus, cannabis is often associated with MDMA either to “bring-out” the high of MDMA or to alleviate the negative experience that arises as the MDMA-related euphoria wears off. A great deal of research is being conducted investigating the detrimental effects of this drug combination in humans, especially related to learning and memory impairments and to the possible development of neuropsychiatric disorders such as depression and anxiety. These studies reveal clear interactions between MDMA and cannabis in several processes, but both synergistic and antagonistic effects have been described (Gouzoulis-Mayfrank and Daumann 2006). On the other hand, little is known about how these two substances interact in terms of rewarding effects that contribute to addiction. One study in adolescents and young adults shows a greater onset of MDMA use in those subjects who had previously consumed cannabis, but the effects of concomitant use of MDMA and delta-9-tetrahydrocannabinol (THC) were not assessed (see Zimmermann et al. 2005). Both drugs given alone can produce rewarding effects in several species. Thus, MDMA-induced conditioned place preference (CPP) has been reported in rats (Bilsky et al. 1990) and mice (Salzmann et al. 2003; Robledo et al. 2004a), and MDMA self-administration has also been revealed in monkeys (Beardsley et al. 1986; Lamb and Griffiths 1987; Fantegrossi et al. 2002), rats (Ratzenboek et al. 2001; Schenk et al. 2003) and mice (Trigo et al. 2006; Trigo et al. 2007). For THC, CPP was demonstrated in mice and rats (Lepore et al. 1995; Valjent and Maldonado 2000) under certain experimental conditions. More recently, it has been reported that naive monkeys can learn to self-administer THC (Justinova et al. 2003). Interactions between the cannabinoid system and MDMA-rewarding effects have been suggested in studies showing that pre-treatment with the cannabinoid CB1 receptor antagonist, rimonabant blocked CPP induced by intracerebroventricular (ICV) injections of MDMA in rats (Braida et al. 2005). Using the ICV drug self-administration paradigm in rats, it was also shown that rimonabant increased, and the CB1 agonist CP 55,940 lowered the mean number of infusions of MDMA (Braida and Sala 2002).
The neurochemical basis for a possible interaction between MDMA and THC has been investigated in rats only in relation to anxiety processes. In that study, THC partially reduced 5-HT depletion and anxiety produced by repeated low doses of MDMA but through a mechanism that appears to be independent of cannabinoid CB1 receptors (Morley et al. 2004). On the other hand, the mesolimbic dopamine (DA) system is a likely candidate for interactions to occur between MDMA and THC in terms of reward-related behaviours, as both compounds activate this system (Chen et al. 1990; Tanda et al. 1997; Yamamoto and Spanos 1988; Marona-Lewicka et al. 1996; White et al. 1996; Kankaanpää et al. 1998; Robledo et al. 2004b), which has been shown to mediate the rewarding/reinforcing properties of different drugs of abuse. However, no studies have been provided correlating changes in dopamine activity with the behavioural effects of combined treatment with MDMA and THC, the principal active ingredient of Cannabis sativa.
Therefore, in this study, we evaluated whether low doses of MDMA and THC produced synergistic rewarding effects in mice using the CPP and MDMA self-administration paradigms and whether these effects could be related to changes in extracellular concentrations of DA in the nucleus accumbens (NAC) after single or combined administration of MDMA and THC using the in vivo microdialysis technique.
Materials and methods
Animals and drugs
Experiments were performed in CD1 male mice, weighing 25 to 30 g at their arrival in the laboratory. Mice were initially housed five per cage in a temperature (21 ± 1°C) and humidity (55 ± 10%) controlled room. In the self-administration paradigm, mice were housed individually after surgery with a reversed light/dark cycle (lights off from 0800 to 2000 hours) with ad libitum food and water. Mice used in the CPP and in microdialysis experiments were housed in a different room with a normal light/dark cycle (lights on from 0800 to 2000 hours). MDMA (Lipomed AG Switzerland) was dissolved in 0.9% saline solution. In the CPP and microdialysis experiments, where THC (THC Pharm GmbH, The Health Concept, Frankfurt) was administered intraperitoneally, the vehicle solution was composed of 5% ethanol, 5% cremophor and 90% distilled water (vehicle 1). In the self-administration studies, the percentage of ethanol was reduced to 1% combined with 1% Tween 80 and 98% saline (vehicle 2) to avoid the possible intrinsic effects of intravenously injected 5% ethanol. Microdialysis studies took place during the light phase and self-administration experiments during the dark phase. Behavioural tests and animal care were conducted in accordance with the standard ethical guidelines (National Institutes of Health 1995; European Communities Directive 86/609 EEC) and approved by the local ethical committee (CEEA-IMAS-UPF). Wherever possible, experiments were performed under blind conditions.
Conditioned place preference paradigm
The place conditioning apparatus consisted of two main square conditioning compartments (15 × 15 × 15 cm) separated by a triangular central area (Robledo et al. 2004a). The light intensity within the conditioning chambers was 30 ± 5 lx. During the pre-conditioning phase, drug-naive mice were placed in the middle of the central area and had free access to both compartments (striped and dotted) of the apparatus for 20 min. The time spent in each compartment was recorded by computerised monitoring software (Videotrack; View Point, Lyon, France). During the conditioning phase, mice received MDMA (0, 3 and 10 mg/kg, i.p.) 15 min before and THC (0 and 0.3 mg/kg, i.p.) immediately before being confined into one of the two conditioning compartments for 45 min. The order of administration of these two drugs was chosen because previous studies have shown that the maximal CPP scores in mice were obtained when MDMA was administered from 15 to 30 min before conditioning (Robledo et al. 2004a) and THC just before conditioning (Valjent and Maldonado 2000). Four pairings were carried out with the drugs and four pairings with the vehicle solutions. Drugs and saline were administered on alternate days, and the animals were exposed to only one pairing per day. 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. free access to each compartment for 20 min. A CPP score was calculated for each mouse by subtracting the time spent in the drug-paired compartment during the pretest phase from the time spent in the drug-paired compartment during the test phase. The doses of MDMA (3 and 10 mg/kg) were chosen based on previous studies showing that MDMA at 10 mg/kg is effective in the CPP paradigm, while 3 mg/kg is not (Robledo et al. 2004a). The sub-threshold dose of THC (0.3 mg/kg) was chosen, as it does not produce rewarding effects in the CCP paradigm (Valjent et al. 2002).
Self-administration training was carried out in 16 operant chambers (Model ENV-307A-CT, Medical Associates, Georgia, VT, USA) equipped with two holes, one was selected as the active hole for delivering the drug and the other as the inactive hole. Acquisition of drug self-administration was performed using a fixed ratio 1 (FR1) schedule of reinforcement such that one nose-poke in the active hole resulted in one MDMA infusion, while nose-poking in the inactive hole had no consequences. The side of the active nose poke was counterbalanced across animals. The chambers were housed in sound- and light-attenuated boxes equipped with fans to provide ventilation and ambient noise. A stimulus light, located above the active hole, was paired contingently with the delivery of the drug. Infusions were delivered in a volume of 23.5 μl over 2 s. MDMA was infused via a syringe that was mounted on a microinfusion pump (PHM-100A, Med-Associates, Georgia, USA) and connected via Tygon tubing (0.96 mm o.d., Portex Fine Bore Polythene Tubing, Portex, Kent, England) to a single channel liquid swivel (375/25, Instech Laboratories, Plymouth USA) and to the mouse intravenous catheter. The swivel was mounted on a counterbalanced arm above the operant chamber.
Mice were anesthetised with a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg) injected in 0.2 ml/10 g of body weight, i.p. and then implanted with indwelling i.v. silastic catheters in the right jugular vein, as previously described (Soria et al. 2005). Briefly, a 5 cm length of silastic tubing (0.3 mm inner diameter, 0.6 mm outer diameter) (Silastic®, Dow Corning, UK) was fitted to a 22-gauge steel cannula (Semat, England) bent at a right angle and then embedded in a cement disk (Dentalon Plus, Heraeus Kulzer, Germany) with an underlying nylon mesh. The external jugular vein was isolated, and the catheter was inserted 1.3 cm into the vein and anchored with sutures. The remaining tubing ran subcutaneously to the cannula, which exited at the midscapular region. All incisions were sutured and coated with antibiotic ointment (Bactroban, GlaxoSmithKline, Spain). After surgery, animals were individually housed and allowed 4 days for recovery before the behavioural testing. The patency of the catheters was evaluated periodically (once a week) and whenever drug self-administration behaviour appeared to deviate dramatically from the one previously observed, by infusing 0.1 ml of thiopental (5 mg/ml) through the catheter. If prominent signs of anaesthesia were not apparent within 3 s of the infusion, the animal was removed from the experiment.
Four days after surgery, mice were trained to nose-poke for MDMA (0.06 or 0.125 mg/kg per infusion) infusions during 10 consecutive days. These doses were chosen from previous studies where it was demonstrated that outbred mice self-administer the dose of 0.125 mg/kg per infusion but not 0.06 mg/kg infusion (Trigo et al. 2006). Fifteen minutes before every daily session, animals received either THC (0.3 mg/kg in 0.15 ml) or vehicle (0.15 ml) through the intravenous catheter. The order of administration of THC and MDMA in this experiment is contrary to the one used in the CPP paradigm due to the methodological constraints of this particular operant procedure to evaluate the acquisition of MDMA self-administration behaviour. Every session begun with a priming injection of the drug and was carried out for 120 min 6 days per week. After each session, mice were returned to their home cages. The maximum number of infusions was limited to 100 infusions per session. Each infusion was followed by a 30 s timeout period during which an active nose-poke had no consequence. The criteria for stable acquisition of self-administration behaviour was to achieve all of the following conditions: (1) less than 20% deviation from the mean of the total number of infusions earned in three consecutive sessions (80% stability), (2) at least 65% responding on the active hole and (3) a minimum of eight infusions earned per session.
In vivo microdialysis
Mice were anesthetised with a ketamine/xylazine mixture (0.2 ml/10 g body weight i.p) and placed in a stereotaxic apparatus with a flat skull (Paxinos and Franklin 1997). For evaluation of DA extracellular levels, guide cannula (CMA/7, CMA Microdialysis, Stockholm, Sweden) were implanted vertically 1 mm above the NAC (AP, +1.5 mm; ML, ±0.8 mm; DV, −3.8 mm from bregma), as previously described (Robledo et al. 2004b). Two days after the surgery, the analytical probe (CMA/7/1 mm, CMA Microdialysis, Stockholm, Sweden) was inserted into the guide cannula. One day after probe implantation the animals were habituated to the experimental environment overnight. The following morning, a ringer solution was pumped through the dialysis probe (NaCl, 148 mM; KCl, 2.7 mM; CaCl2,1.2 mM and MgCl2, 0.8 mM, pH 6.0) at a constant rate of 1 μl/min, and baseline samples were taken during 2 h. Subsequently, mice were allocated to different experimental groups. Group 1 received MDMA (3 mg/kg, i.p.) first followed 15 min later by THC (0.3 mg/kg, i.p.); group 2 received THC (0.3 mg/kg, i.p) first followed 15 min later by MDMA (3 mg/kg, i.p.); group 3 received saline followed by THC (0.3 mg/kg, i.p.); group 4 received saline followed by vehicle and group 5 received MDMA (3 mg/kg, i.p.) followed by vehicle. Collection of samples was continued for 4 h following the last injection.
Dialysates (15 μl) were injected without any purification into a high performance liquid chromatography (HPLC) system that consisted of a pump linked to an automatic injector (Agilent 1100, Palo Alto, USA), a reverse-phase column (Zorbax SB C18, 3.5 μm, 150 × 4.6 mm, Agilent Technologies, Palo Alto, USA) and a coulometric detector (Coulochem II, ESA, Chelmsford, USA) with a 5011A analytical cell. DA was quantified as previously described (Robledo et al. 2004b). Briefly, the first electrode was fixed at −100 mV and the second electrode at +300 mV. The gain of the detector was set at 20 nA. The composition of the mobile phase was 50 mM NaH2PO4, 0.1 mM Na2EDTA, 0.65 mM octyl sodium sulfate and 15% (vol/vol) methanol, pH 3.5. The flow rate was set at 1 ml/min, and the sensitivity of the assay for DA was 1 pg/15 μl.
For the CPP data, a two-way analysis of variance (ANOVA) was used to compare the CPP scores with two between subject factors (dose of MDMA × THC treatment) followed by the least significant difference (LSD) post-hoc test for comparisons between doses of MDMA, and a Student’s t test for comparisons between vehicle-treated animals and THC-treated animals receiving the different doses of MDMA. The self-administration results were analysed with a three-way repeated measures ANOVA for the number of infusions of MDMA (day of acquisition × dose of MDMA × THC treatment). Additionally, discrimination between holes was analysed with two-way repeated measures ANOVAs comparing active vs inactive responses during the 10 days of training, followed by a Student’s t test for each dose and for each day of acquisition. Changes in DA outflow after drug administration were analysed using separate three-way repeated measures ANOVA (time as within subject factor; MDMA and THC treatments as between subject factors) for each order of administration. The area under the curve (AUC) data was analysed using one-way ANOVA followed by the LSD and Student’s t test post-hocs for individual comparisons.
Conditioned place preference paradigm
To examine whether THC modified the acquisition of MDMA self-administration, two-way repeated measures ANOVAs were performed comparing the discrimination between the active and inactive holes during the 10 days of training for each dose of MDMA. Animals treated with vehicle and trained to self-administer MDMA at the dose of 0.06 mg/kg per infusion did not learn to discriminate between the active and inactive holes (Fig. 3a), in line with our previous results showing that this dose is not sufficiently reinforcing in CD1 outbred mice (Trigo et al. 2006). A main effect of day of training [F(9,108) = 5.105, p < 0.001], a non-significant effect of hole and a significant interaction between these two factors were observed [F(9,108) = 2.822, p < 0.01]. No significant differences were observed in responding between holes at any of the days of acquisition. In contrast, animals trained with the dose of 0.06 mg/kg per infusion receiving THC pre-treatment discriminated between the active and inactive holes from day 8 of training (Fig. 3b), indicating that THC increases the threshold of MDMA reward. A significant main effect of day of training [F(9,108)=2.984, p < 0.01], of hole [F(1,12) = 10.608, p < 0.01] and a significant interaction between these two factors were observed [F(9,108) = 2.059, p < 0.05]. Significant differences between active and inactive responses were observed on days 8 (t < 0.05), 9 (t < 0.01) and 10 (t < 0.01). Mice pre-treated with vehicle learned to self-administer MDMA at the dose of 0.125 mg/kg per infusion (Fig. 3c). A significant main effect of day of training [F(9,126) = 5.025, p < 0.001], a non-significant effect of hole and a significant interaction between factors [F(9.126) = 5.855, p < 0.001] were observed. Significant differences were observed between active and inactive responses on days 9 and 10 (t < 0.05). In mice trained to self-administer the dose of 0.125 mg/kg per infusion previously receiving THC (0.3 mg/kg per infusion), a significant main effect of day of training [F(9,180) = 8.600, p < 0.001], a non-significant effect of hole and a significant interaction between these factors [F(9,180) = 2.933, p < 0.01] were observed. Significant differences between active and inactive responses were observed on day 10 (t < 0.05; Fig. 3d).
In vivo microdialysis
In this study, we show a complex interaction between MDMA and THC in the CPP paradigm. Thus a sub-threshold dose of THC (0.3 mg/kg) produced CPP when combined with a non-rewarding dose of MDMA (3 mg/kg) but decreased CPP induced by an effective dose of MDMA (10 mg/kg). The present results indicate that a low dose of THC can modify the behavioural effects of MDMA and suggest an involvement of the cannabinoid system in the rewarding effects of MDMA in mice. Consistent with this idea, the CB1 cannabinoid antagonist, rimonabant-antagonised CPP induced by ICV administration of MDMA at the dose of 10 ng/kg in rats (Braida et al. 2005). To study whether THC might have similar effects on the reinforcing properties of MDMA, we used the self-administration paradigm in mice pre-treated with a low dose of THC (0.3 mg/kg) or vehicle. Under these conditions, mice acquired MDMA self-administration at a dose of MDMA (0.06 mg/kg per infusion) previously reported not to produce reinforcing effects in mice (Trigo et al. 2006), while the acquisition of an effective dose of MDMA (0.125 mg/kg per infusion) was not modified. These results indicate that a sub-effective dose of THC is able to increase the perception threshold of MDMA. These data are in agreement with the results obtained in the CPP experiment with the low dose of MDMA but not with those obtained with a higher dose. This apparent discrepancy may be due to the different order of THC administration or to the different range of doses employed in each paradigm. Nonetheless, the self-administration data also support an involvement of the cannabinoid system in the reinforcing effects of MDMA in mice, agreeing with data in rats showing that lower doses of MDMA were necessary to perceive its reinforcing effects after the administration of a cannabinoid agonist, and conversely, higher doses were necessary to obtain the same reinforcing effects after the administration of a cannabinoid antagonist (Braida and Sala 2002).
The effects of THC observed in these experiments may also be related to unspecific actions of THC affecting MDMA reward and reinforcement. Thus, THC can facilitate drug-seeking behaviour by reducing the anxiety-like effects associated with MDMA administration. Indeed, the dose of THC used (0.3 mg/kg) can produce anxiolytic-like effects in mice (Berrendero and Maldonado 2002). However, the low doses of MDMA used in the CPP study (3 mg/kg) and in the self-administration paradigm (0.06 mg/kg) have not been shown to be anxiogenic in mice. Other unspecific effects of THC include decreased locomotor activity and cognitive impairments, both of which may influence the rewarding/reinforcing properties of MDMA. It is unlikely that locomotor effects could have contributed to our results, as THC at the dose of 0.3 mg/kg per infusion has no effect on locomotion (unpublished observations). In addition, although lower levels of response in the inactive nose-poke were observed the first day of THC administration, in subsequent sessions, mice did acquire the self-administration of a non-reinforcing dose of MDMA, ruling out any persistent locomotor effects. THC might also unspecifically modulate the general rate of operant responding. However, this possibility can be refuted, as responding for an effective dose of MDMA was not modified by THC with respect to vehicle administration. Similarly, if this low dose of THC caused learning/memory impairments, it would have produced a decrease in active vs inactive hole discrimination in mice responding for the sub-threshold dose of MDMA, when in fact the opposite was observed.
To investigate whether the behavioural interaction observed between THC and MDMA was paralleled by changes at the level of the DA mesolimbic system, we carried out in vivo microdialysis experiments evaluating changes in extracellular concentrations of DA in the NAC after single and combined administration of MDMA and THC. The single administration of MDMA at a dose which does not induce CPP (3 mg/kg) did not significantly increase DA levels in the NAC, while the sub-threshold dose of THC (0.3 mg/kg) did. The finding that the THC-induced DA increase in the NAC was not related to the induction of CPP was unexpected. Indeed, most drugs of abuse at doses that induce CPP and support self-administration behaviour have been shown to increase DA levels in the NAC, including cocaine (Carboni et al. 2001), THC (Chen et al. 1990) and MDMA (Yamamoto and Spanos 1988; Marona-Lewicka et al. 1996; White et al. 1996; Kankaanpää et al. 1998; Robledo et al. 2004b). Our results suggest that at least in the case of mice, DA release in the NAC produced by low doses of THC may not necessarily be associated to reward mechanisms. In the case of MDMA, an enhancement in DA extracellular concentrations in the NAC of rats (Kankaanpää et al. 1998) has only been observed at doses (3 and 9 mg/kg) similar to those which induce CPP (5 and 10 mg/kg; Bilsky et al. 1990; Marona-Lewicka et al. 1996). Correspondingly in mice, DA release in the NAC is increased by an effective dose of MDMA (10 mg/kg; Robledo et al. 2004a, b) but not by an ineffective dose (3 mg/kg). However, a recent study using the self-administration technique showed that although serotonin transporter (SERT) knockout mice did not acquire MDMA self-administration, MDMA (10 mg/kg) increased extracellular levels of DA in the NAC of wild-type and SERT mice equally (Trigo et al., 2007), suggesting that MDMA-induced reward may not be exclusively related to increased DA activity in the NAC. In fact, the results of the latter study as well as other data suggest that the serotonergic system may also participate in mediating the rewarding/reinforcing properties of MDMA (Bilsky and Reid 1991; Fantegrossi et al. 2002).
When MDMA and THC were combined, the order of administration was comparable to the procedure followed in both the CPP and self-administration paradigms. In the group of mice receiving MDMA (3 mg/kg) first, followed 15 min later by THC (0.3 mg/kg), cumulative DA levels in the NAC for the entire sampling period were reduced with respect to THC alone. However, if we consider only the time points corresponding to the period of drug-paired conditioning in the CPP (45 min), no significant differences were observed between MDMA plus THC and THC alone. Therefore, the pre-treatment with MDMA has minor consequences on the facilitating effect of THC on DA transmission observed immediately after its administration but attenuates the global effect of THC on this monoaminergic system. On the other hand, in the group of mice receiving THC before MDMA, as in the self-administration paradigm, DA levels were increased to the same degree as in mice receiving THC alone when considering the entire sampling period and the period equivalent to the conditioning session in the CPP. Together, these results indicate that MDMA can modulate the THC-associated increase in DA levels in the NAC depending on whether it is already present before THC administration and suggest that these two substances may interact on common mechanisms related to the mesolimbic DA system. A likely substrate for this interaction may be the serotonergic system. In this sense, previous studies show that DA release induced by THC in the striatum of rats is modulated by serotonergic changes induced by the serotonin re-uptake inhibitor, fluoxetine, an effect which depends on the time of its administration relative to that of THC (Malone and Taylor 1999).
In conclusion, we show that a low dose of THC can modulate the rewarding properties of MDMA, and that the interaction between these two drugs of abuse is more complex than just a simple synergism. The neurochemical data substantiate that THC and MDMA converge at a common mechanism modulating DA outflow in the NAC, although further studies are required to determine the nature of this mechanism.
The authors would like to thank Ms. Dulce Real Muñoz for her expert help in the microdialysis experiments, and Mr. Jordi Ortuño for his assistance with the HPLC methodology. This work was supported by FIS grant number 03/0305, Plan Nacional Sobre Drogas 2005, NIH-NIDA (USA), Extra-mural research project (#5 R01 DA016768), I.S. CARLOS III Redes de grupos ISCIII (# RTA G03/005), Ministerio de Ciencia y Tecnología (# BFU2004–00920/BFI and # GEN2003–20651), Generalitat de Catalunya 2005SGR00131 and GENADDICT LSHM-CT-2004–05166.