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Published in final edited form as: Alcohol Clin Exp Res. 2020 Oct 3;44(11):2158–2165. doi: 10.1111/acer.14456

COX-2 Inhibition Antagonizes Intra-Accumbens 2-Arachidonoylglycerol–Mediated Reduction in Ethanol Self-Administration in Rats

Francisco J Pavon 1, Ilham Polis 2, David G Stouffer 3, Marisa Roberto 4, Rémi Martin-Fardon 5, Fernando Rodriguez de Fonseca 6, Loren H Parsons 7, Antonia Serrano 8
PMCID: PMC7680444  NIHMSID: NIHMS1643678  PMID: 32944989

Abstract

Background:

Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB1 signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB1 receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB1 receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake.

Methods:

In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB1 inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide.

Results:

Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration.

Conclusions:

We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non–CB1 receptor mechanism involving the COX-2 pathway.

Keywords: 2-Arachidonoyl Glycerol, Nucleus Accumbens, Ethanol, Cyclooxygenase-2

ALCOHOL IS THE most widely used psychoactive drug in our society, and its harmful consumption is becoming a major public health concern. Alcohol exerts its behavioral and reinforcement effects through actions on multiple central neurotransmitter systems. Among them, the endogenous cannabinoid system (ECS) has been shown to play a prominent role in the modulation of some pharmacological and behavioral aspects of alcohol (Serrano and Parsons, 2011).

The main endocannabinoids, arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), are synthesized on demand and bind to cannabinoid receptors (CB1 and CB2) to exert their effects. The metabolism of endocannabinoids is mediated by multiple enzymatic pathways that are critical for the regulation of their endogenous levels. Inactivation of endocannabinoid signaling occurs by intracellular hydrolysis catalyzed by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the enzymes primarily responsible for the degradation of AEA and 2-AG, respectively (Cravatt et al., 1996; Dinh et al., 2002). In addition to these degrading enzymes, the cyclooxygenase-2 (COX-2) appears to play an important role in endocannabinoid metabolism to produce bioactive prostaglandin-like derivatives (Kozak et al., 2000; Yu et al., 1997).

A growing body of literature implicates the ECS in the modulation of ethanol (EtOH) consumption. For example, systemic administration of CB1 receptor agonists dose-dependently increases EtOH drinking and the reinforcing properties of EtOH in rodents (Colombo et al., 2002; Gallate et al., 1999). Conversely, the pharmacologic blockade of CB1 receptors by systemic administration of SR141716A decreases EtOH consumption in normal and alcohol-preferring rodent strains (Arnone et al., 1997; Colombo et al., 1998; Rodriguez de Fonseca et al., 1999; Wang et al., 2003). These findings are consistent with reports that mice lacking CB1 receptor consume less EtOH than do their wild-type counterparts (Hungund et al., 2003; Wang et al., 2003), and the CB1 antagonist–induced suppression of EtOH consumption is absent in these mice (Thanos et al., 2005). In addition to these observations, our previous studies suggest a regionally selective influence of brain CB1 receptors in the modulation of EtOH self-administration behavior (Alvarez-Jaimes et al., 2009a; Caillé et al., 2007). In fact, SR141716A infusions into the nucleus accumbens (NAc) shell or the ventral tegmental area reduce EtOH self-administration, while the administration of this antagonist into the medial prefrontal cortex has no effect on EtOH self-administration (Alvarez-Jaimes et al., 2009a). Consistent with this regional pattern is the observation that EtOH self-administration is accompanied by altered endocannabinoid levels in a regionally specific manner. Using in vivo microdialysis, we have reported that EtOH self-administration significantly increases 2-AG extracellular levels in the NAc shell of rats, but it has no effect on AEA concentrations (Alvarez-Jaimes et al., 2009b; Caillé et al., 2007).

This set of experimental evidence suggests that this increase in 2-AG transmission induced by EtOH self-administration may be promoting the motivation for EtOH consumption. Thus, to further explore this hypothesis, we investigated whether pharmacologically driven enhancement of 2-AG levels in the NAc shell is involved in the EtOH consumption using the 2-AG clearance inhibitors URB602 (this compound inhibits MAGL, the main enzyme involved in the degradation of 2-AG) and nimesulide (an inhibitor of COX-2, an alternative pathway for 2-AG inactivation), and 2-AG itself. To further pharmacologically characterize the endocannabinoid signaling in the action of 2-AG, we also evaluated the effects of the intra-NAc shell administration of AEA and the CB1 receptor inverse agonist SR141716A on operant self-administration. In addition to the fact that the NAc shell is a brain region critically involved in mediating the rewarding properties of EtOH, this region was chosen based on our previous studies describing that EtOH self-administration significantly increases 2-AG extracellular levels in the NAc shell of rats (Alvarez-Jaimes et al., 2009b; Caillé et al., 2007). Our findings show that infusions of 2-AG into the NAc shell induce a dose-dependent reduction in EtOH self-administration through a non–CB1 receptor mechanism.

MATERIALS AND METHODS

Animals

Male Wistar rats (Charles River, Wilmington, MA, USA) weighing 220 to 250 g at their arrival were housed in groups of 3 in a humidity and temperature-controlled (22°C) vivarium with a reversed 12-hour light/dark cycle (lights off at 8:00 am) and food and water ad libitum. Rats were given at least 1 week to acclimate to the new environment before any experimental procedure was performed. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute. Animal studies are reported in compliance with the ARRIVE guidelines (McGrath et al., 2015). All efforts were made to minimize unnecessary pain and/or distress.

Drugs and Reagents

EtOH (2% and 10% w/v) was prepared with 95% ethyl alcohol and water. Saccharine (Sigma, St. Louis, MO) was dissolved in water.

SR141716A was generously provided by the National Institute of Mental Health Chemical Synthesis and Drug Supply Program (Washington, DC). The endogenous cannabinoid receptor agonists N-arachidonoyl ethanolamine (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), the metabolically stable form of anandamide methanandamide (Me-AEA), the selective MAGL inhibitor URB602, and the selective inhibitor of COX-2 nimesulide were obtained from Cayman Chemical (Ann Arbor, MI). All the compounds were mixed in a vehicle of EtOH: emulphor: saline (1:1:18 v/v/v). They were infused intra-NAc shell, but nimesulide that was administered intraperitoneally (i.p) (Table 1).

Table 1.

Doses and Pretreatment Time of the Different Drugs

Drug Doses Pretreatment time (min)
SR141716A 1 and 3 μg/side 30
AEA 0.1, 1, and 10 μg/side 0
2-AG 30,100,300, and 3000 ng/side 0
Me-AEA 30 μg/side 0
URB602 3 and 10 μg/side 15
Nimesulide 10 mg/kg (i.p.) 20
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EtOH Self-Administration Training

Self-administration training and testing were performed in standard operant chambers housed in sound-attenuated and ventilated cubicles (Coulbourn Instruments, Allentown, PA). Rats were trained to self-administer an EtOH solution using a sweet solution fading procedure slightly modified from Samson (1986) in 30-minutes daily operant sessions conducted during the dark phase of the light cycle. The chambers were equipped with 2 retractable levers (the active and the inactive) located on either side of drinking dipper. The position of the active lever was altered between left and right on consecutive days to avoid the development of location preferences. Operant EtOH self-administration was established under a fixed ratio 1 (FR-1) schedule of reinforcement, where active lever responding resulted in the delivery of 0.1 ml aliquots of liquid to a sipper cup for oral consumption, and the final EtOH concentration for operant training was 10% (w/v). The inactive lever responses were also recorded during the entire session as a measure of nonspecific behavioral activation but had no programmed consequences. The baseline criterion for stable operant responding was not more than 20% variance in the total number of reinforcers obtained per session for 3 consecutive sessions. Another group of rats consuming 0.0031% saccharin was used for comparison to determine whether the actions of the different drugs were specific for EtOH.

In addition to the number of reinforcers delivered for 30 minutes (10% EtOH), EtOH intake (g/kg) was also determined from body weight data and included in the figures. The amount of EtOH consumed was calculated by an estimation based on the number of lever presses. In addition, after each session, the liquid receptacle was checked to confirm the consumption of the total volume.

Intracerebral Surgery

Once animals acquired a stable operant self-administration behavior, they were anesthetized with isoflurane (1.5 to 2.0% vapor) and placed in a stereotaxic apparatus. Each rat was implanted with bilateral 22-gauge, 12-mm stainless guide cannulae that were positioned to end within 2 mm above the NAc shell (coordinates from bregma: AP, +1.7 mm; ML, ±0.9 mm; and DV, −5.0 mm (from Dura)) (Paxinos and Watson, 1998) and were secured to the skull with dental cement. Both guide cannulae were protected through stylets, and a minimum of 7 postoperative recovery days was allowed before experimentation. Postsurgical self-administration training sessions continued until stable self-administration behavior was achieved prior to the start of treatment drug testing.

Intra-NAc Shell Drug Testing

The effects of local administration of the different drugs on EtOH or saccharin self-administration were evaluated in separate groups of animals (the dose order presentation was randomized between animals following a Latin square design). After the establishment of stable self-administration behavior, the animals received an initial microinjector insertion (no liquid infusion) immediately before self-administration to acclimate them to the procedure and to produce the initial tissue damage from injector insertion. Subsequently, vehicle and compound infusions were made via bilateral 30-gauge stainless steel microinjectors that extended 2 mm beyond the tip of the guide cannulae. The injection volumes were 0.5 μl per side, infused over 30 seconds. Following drug delivery, the injectors were left in place for 30 seconds to allow drug diffusion, and then replaced with the stylets.

Histological Verification of Infusion Cannulae Placement

At the end of the experiment, rats were euthanized by isoflurane overdose. The brains were removed and sectioned to verify the correct position of the probe with the help of a rat atlas (Paxinos and Watson, 1998). Only those subjects with accurate bilateral placements were included in the final data analyses.

Statistical Analyses

All the data in the graphs are expressed as the mean ± SEM. The effects of different drug administration into the NAc shell on EtOH self-administration were evaluated using a within-subjects design with repeated-measures analysis of variance (ANOVA). The statistical analysis was performed using GraphPad Prism version 5.04 (GraphPad Software, San Diego, CA). A p-value of <0.05 was considered statistically significant. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018).

RESULTS

Intra-NAc Shell CB1 Receptor Blockade and 2-AG Infusions Induce a Dose-Dependent Reduction in EtOH Self-Administration

First, we examined the effects on operant self-administration induced by intra-NAc shell infusions of SR141716A and 2-AG. The average of EtOH reinforcers obtained by each group of animals during the final 3 self-administration sessions before the pretreatment tests was considered as baseline values: 23.89 ± 2.32 (0.58 ± 0.08 g/kg EtOH intake) and 23.78 ± 3.11 (0.57 ± 0.10 g/kg EtOH intake) reinforcers per session were used as baseline for the intra-NAc SR141716A test and the intra-NAc 2-AG test, respectively.

As shown in Fig. 1A, we found no differences in the number of reinforcers between the vehicle group (V) and baseline data. However, a repeated-measures ANOVA of bilateral infusions of the CB1 inverse agonist SR141716A (1 and 3 μg/side) into the NAc shell revealed a significant main effect on the self-administration of 10% EtOH, F(2, 16) = 11.626, p < 0.001. Thus, there was a significant decrease in the number of reinforcers in rats treated with both 1 and 3 μg/side of SR141716A compared with the vehicle group (p < 0.05) [EtOH intake: 0.52 ± 0.08 g/kg (V), 0.34 ± 0.04 g/kg (dose 1), and 0.30 ± 0.08 g/kg (dose 3)].

Fig. 1.

Fig. 1.

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Effects of intra-NAc shell infusions of SR141716A and 2-AG on 10% ethanol (EtOH) and saccharin self-administration. Effects of vehicle and the CB1 antagonist/inverse agonist SR141716A (1 and 3 μg/side) on 10% EtOH self-administration [EtOH intake: 0.58 ± 0.08 g/kg (baseline), 0.52 ± 0.08 g/kg (V), 0.34 ± 0.04 g/kg (dose 1 μg/side), and 0.30 ± 0.08 g/kg (dose 3 μg/side)] (A). Effects of vehicle and 2-AG (30, 100, 300, and 3,000 ng/side) on 10% EtOH self-administration [EtOH intake: 0.57 ± 0.10 g/kg (baseline), 0.54 ± 0.13 g/kg (V), 0.44 ± 0.15 g/kg (dose 30 ng/side), 0.39 ± 0.15 g/kg (dose 100 ng/side), 0.25 ± 0.11 g/kg (dose 300 ng/side), and 0.22 ± 0.08 g/kg (dose 3,000 ng/side)] (B). Effects of vehicle and SR141716A (1 and 3 μg/side) on 0.0031% saccharin self-administration (C). Effects of vehicle and 2-Ag (30, 100, 300, and 3,000 ng/side) on 0.0031% saccharin self-administration (D). Bars represent the mean ± SEM (n = 9 animals per group) of reinforcers obtained by active lever pressing (top panel) and responses on the inactive lever (bottom panel). Data were analyzed by repeated-measures ANOVA. *p < 0.05 and ***p < 0.001 denote significant differences compared with their respective vehicle group (V).

Similarly to the intra-NAc SR141716A test, there were no differences in the number of reinforcers between the vehicle group and baseline data and the statistical analysis of bilateral infusions of 2-AG (30 to 3,000 ng/side) into the NAc shell also revealed a significant main effect on EtOH self-administration after, F(4, 32) = 3.146, p < 0.05, (Fig. 1B). The post hoc comparisons showed a dose-dependent reduction in the number of reinforcers, and high doses (300 and 3,000 ng/side) caused a significant decrease compared with the vehicle group (p < 0.001) [EtOH intake: 0.54 ± 0.13 g/kg (V), 0.25 ± 0.11 g/kg (dose 300), and 0.22 ± 0.08 g/kg (dose 3,000)]. Complementarily, the administration of lower doses of 2-AG (0.5 to 3 ng/side) did not significantly alter EtOH self-administration or EtOH intake (Fig. S1).

In order to determine whether the effect of 2-AG on EtOH self-administration was dependent on the concentration of EtOH consumed, a third group of rats were trained to self-administer 2% EtOH. However, intra-NAc shell infusions of 2-AG doses that significantly reduced the self-administration of 10% EtOH (300 and 3,000 ng/side) did not alter the self-administration of 2% EtOH or EtOH intake (Fig. S2).

Finally, no significant differences were found in inactive lever pressing among the different groups in both SR141716A and 2-AG tests. Furthermore, both compounds appear to selectively modify EtOH self-administration as there were no effects on saccharin self-administration (Fig. 1C,D).

EtOH Self-Administration is Unaltered by Intra-NAc Shell Administration of AEA or Methanandamide, but is Reduced by the Administration of the MAGL Inhibitor URB602

Besides 2-AG, we examined the effects on EtOH self-administration of the other main endogenous CB1 receptor agonist AEA and its synthetic analog Me-AEA. For this test, the baseline value was observed at 23.00 ± 2.66 EtOH reinforcers per session (0.57 ± 0.09 g/kg EtOH intake). As shown in Fig. 2A, there were no differences in the number of reinforcers between the vehicle group (V) and baseline data. In contrast to the effects produced by 2-AG, the intra-NAc administration of AEA (0.1, 1, and 10 μg/side) or Me-AEA (30 μg/side) did not alter EtOH self-administration as compared with the vehicle group.

Fig. 2.

Fig. 2.

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Effects of intra-NAc shell infusions of AEA, methanandamide, and URB602 on 10% ethanol (EtOH) self-administration. Effects of vehicle, AEA (0.1, 1, and 10 μg/side) and its metabolically stable form Me-AEA (30 μg/side) on 10% EtOH self-administration [EtOH intake: 0.57 ± 0.09 g/kg (baseline), 0.54 ± 0.09 g/kg (V), 0.49 ± 0.13 g/kg (dose 0.1 μg/side), 0.52 ± 0.06 g/kg (dose 1 μg/side), 0.49 ± 0.07 (dose 10 μg/side), and 0.49 ± 0.14 g/kg (dose 30 μg/side)] (A). Effects of vehicle and the MAGL inhibitor URB602 (3 and 10 μg/side) on 10% EtOH self-administration [EtOH intake: 0.69 ± 0.13 g/kg (baseline), 0.57 ± 0.16 g/kg (V), 0.42 ± 0.16 g/kg (dose 3 μg/side), and 0.30 ± 0.12 g/kg (dose 10 μg/side)] (B). Bars represent the mean ± SEM (n = 8 to 9 animals per group) of reinforcers obtained by active lever pressing (top panel) and responses on the inactive lever (bottom panel). Data were analyzed by repeated-measures ANOVA. **p < 0.01 denotes significant differences compared with the vehicle group (V).

In addition, we examined the effect of the inhibition of the MAGL, the primary 2-AG clearance mechanism, on EtOH self-administration using the MAGL inhibitor URB602 (Fig. 2B). The baseline value was 27.63 ± 3.50 EtOH reinforcers per session (0.69 ± 0.13 g/kg EtOH intake), and there were no significant differences with the group of rats treated with intra-NAc vehicle (V). A repeated-measures ANOVA revealed that intra-NAc URB602 treatment (3 and 10 μg/side) induced a significant main effect on 10% EtOH self-administration, F(2, 14) = 7.565, p < 0.01. There was a significant decrease in the number of reinforcers in rats treated with 10 μg/side of URB602 compared with the vehicle group (p < 0.01) [EtOH intake: 0.57 ± 0.16 g/kg (V) and 0.30 ± 0.12 g/kg (dose 10)].

The number of inactive lever responses was unaltered by the pharmacological intra-NAc treatment of AEA/Me-AEA or URB602.

The Effect of Intra-NAc shell 2-AG on EtOH Self-Administration is Blocked by Systemic Pretreatment With the COX-2 Inhibitor Nimesulide

Because endocannabinoids can also be metabolized by COX-2, we evaluated the effects of intra-NAc 2-AG treatment (300 ng/side) in combination with a selective COX-2 inhibitor pretreatment using nimesulide (10 mg/kg) on EtOH self-administration. In this experiment, the number of EtOH reinforcers per session under baseline conditions was 25.67 ± 4.86 (0.64 ± 0.17 g/kg EtOH intake), and once again, there were no differences as compared with the vehicle group (V). As shown in Fig. 3, the statistical analysis of the 10% EtOH self-administration revealed significant differences among groups, F(3, 24) = 3.45, p < 0.05, and the post hoc comparisons showed a significant decrease in the number of reinforcers in rats pretreated with vehicle and treated with intra-NAc 2-AG compared with the vehicle group (p < 0.05) [EtOH intake: 0.54 ± 0.17 g/kg (V/V) and 0.22 ± 0.12 g/kg (V/2AG)]. While EtOH self-administration was unaltered by systemic nimesulide pretreatment alone (N/V), the inhibitory effect of intra-NAc shell 2-AG on the number of reinforcers (V/2AG), F(1, 8) = 26.755, p < 0.001, was abolished by pretreatment with nimesulide (N/2AG), F(1, 8) = 20.126, p < 0.01.

Fig. 3.

Fig. 3.

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Effects of systemic pretreatment with nimesulide and intra-NAc shell infusions of 2-aG on 10% ethanol (EtOH) self-administration. Effects of pretreatment with vehicle or the COX-2 inhibitor nimesulide (10 mg/kg, i.p.), and intra-NAc shell treatment with vehicle or 2-AG (300 ng/side) on 10% EtOH self-administration [EtOH intake: 0.64 ± 0.17 g/kg (baseline), 0.54 ± 0.17 g/kg (V/V), 0.52 ± 0.09 g/kg (N/V), 0.22 ± 0.12 g/kg (V/2-AG), and 0.41 ± 0.13 g/kg (N/2-AG)]. Bars represent the mean ± SEM (n = 9 animals per group) of reinforcers obtained by active lever pressing (top panel) and responses on the inactive lever (bottom panel). Data were analyzed by repeated-measures ANOVA. *p < 0.05 denotes significant differences compared with the vehicle/vehicle group (V/V).

As expected, inactive lever responses were low and unaltered by treatment/pretreatment.

DISCUSSION

The major finding of this study is the demonstration that site-specific infusions of 2-AG into the NAc shell selectively reduced EtOH self-administration (but not saccharin), and this suppressive effect was dependent on the concentration of EtOH self-administered. EtOH self-administration was also reduced by inhibition of MAGL, the primary 2-AG clearance mechanism, as demonstrated by its inhibition by intra-NAc shell administration of the selective MAGL inhibitor URB602. However, the effect of intra-NAc shell 2-AG on EtOH self-administration was significantly reversed by pretreatment with the COX-2 inhibitor nimesulide, suggesting that the inhibitory effect of 2-AG may result from its metabolic conversion to prostaglandin glycerol esters via COX-2. Furthermore, the enhancement of cannabinoid CB1 receptor signaling through the AEA pathway was unable of reducing alcohol self-administration, as demonstrated using local infusion of AEA.

Numerous studies have described the involvement of CB1 receptors in the modulation of voluntary EtOH consumption and that disruption of CB1 signaling in rodents is associated with a decrease in voluntary EtOH intake (for review, see Henderson-Redmond et al., 2016). In addition to this role of CB1 receptors on voluntary EtOH drinking, it is known that CB1 receptors are also involved in the modulation of EtOH self-administration. In this regard, previous studies have reported that systemic administration or direct infusions into the NAc of the CB1 inverse agonist SR141716A decreases EtOH self-administration in Wistar rats and alcohol-preferring rats (Alvarez-Jaimes et al., 2009a; Caillé et al., 2007; Economidou et al., 2006; Malinen and Hyytia, 2008). In agreement with these reports, we have shown here that intra-NAc shell SR141716A infusions attenuated EtOH self-administration using a FR schedule in Wistar rats. SR141716A decreased selectively EtOH self-administration, since we did not find any effect on saccharin self-administration using the same dose range that was used for EtOH. By contrast, other reports have described that SR141716A treatment attenuates sucrose and saccharin self-administration (Cippitelli et al., 2005; Economidou et al., 2006; Malinen and Hyytia, 2008). However, it is possible that the different route of administration (i.p vs. intra-NAc shell) or the use of alcohol-preferring vs. outbred rats might contribute to these discrepancies among studies.

In addition to the evidence that EtOH self-administration is sensitive to disruption of CB1 signaling in the NAc, we have also described that EtOH consumption increases extracellular levels of 2-AG in this brain region (Alvarez-Jaimes et al., 2009b; Caillé et al., 2007), but the participation of 2-AG on the modulation of EtOH self-administration remains elusive. Recently, it has been described that the systemic treatment with an inhibitor of the biosynthesis of 2-AG is associated with a decrease in the responding for EtOH in mice, whereas a pharmacologically induced increase in 2-AG has no effect (Gianessi et al., 2019). To a better understanding of the role for 2-AG in the modulation of the motivation for EtOH, we first studied the effects of intra-NAc shell infusions of different doses of 2-AG on EtOH self-administration. We found that intra-NAc shell administration of high doses of 2-AG induced a reduction in EtOH self-administration. In addition of the 2-AG dose, this effect was also dependent on the concentration of EtOH self-administered, since the same doses that reduced the self-administration of 10% EtOH had no effect on 2% EtOH. Our results suggest the specificity of the effects of 2-AG on EtOH reinforcement in the NAc, since intra-NAc shell 2-AG produced no effect on saccharin self-administration. Similar to the results obtained using direct infusions of 2-AG into the NAc shell, we found that self-administration of 10% EtOH was dose-dependently reduced by intra-NAc shell infusions of the MAGL inhibitor URB602, suggesting that EtOH self-administration is sensitive to potentiation of EtOH-induced increases in 2-AG. However, although endogenous 2-AG acts as a CB1 receptor agonist, we found similar effects on EtOH self-administration with intra-NAc shell SR141716A. A possible explanation is that exogenous 2-AG could induce internalization of CB1 or changes in CB1/CB2 receptor balance, which produces effects similar to those of CB1 antagonists. In this regard, a previous study in a model of medium-spiny striatal neuron culture reported that exogenous 2-AG induces a fast arrestin-mediated CB1 receptor internalization of short duration without changes in the protein levels (Laprairie et al., 2014). Although the 2-AG–mediated internalization of CB1 receptor could explain similar alterations in the pattern of EtOH self-administration regarding SR1411716A, this 2-AG–induced cellular mechanism is not able of producing a permanent decrease in the CB1 receptors since it activates a rapid transcription and synthesis of CB1 receptors. In any case, additional studies are needed to confirm whether this internalization occurs for exogenous 2-AG in our EtOH self-administration model, which is a limitation for the present set of experimental data.

To further assess the participation of CB1 receptor on EtOH self-administration, we also evaluated the effects of AEA, the other best characterized endogenous CB1 agonist, and its synthetic analog Me-AEA, a potent and selective CB1 agonist. We found that neither AEA nor Me-AEA induced any effect on EtOH self-administration. This lack of effect by AEA together with the finding that EtOH self-administration has no effect on AEA concentrations in the NAc (Alvarez-Jaimes et al., 2009a; Caillé et al., 2007) suggests a role for 2-AG, but not for AEA, in the modulation of the motivation for EtOH in the NAc.

Although 2-AG is mainly hydrolyzed by MAGL, there are other alternative degradative pathways involved in the inactivation of this endocannabinoid, such as the cyclooxygenases (Kozak et al., 2004), leading to the production of bioactive metabolites that may induce opposite effects than those induced by CB1 receptor agonists. Thus, 2-AG is oxygenated by COX-2 to produce prostaglandin glycerol esters as the primary products, which can modulate inhibitory neurotransmission via non–CB1 receptor mechanisms in an opposite manner to the effects induced by 2-AG through CB1 activation (Katona and Freund, 2012; Sang et al., 2006; Sang et al., 2007). Recently, it has been reported that 2-AG oxidative metabolites are produced after combination of an increase in both COX-2 expression and 2-AG levels in the mouse brain (Morgan et al., 2018). In this regard, it is known that EtOH increases COX-2 expression (Blanco et al., 2004). This might explain that the effects of 2-AG are dependent on the concentration of EtOH used in the self-administration sessions (present results). Notably, recent evidence suggests that COX-2 inhibitors might serve for the treatment of stress and/or alcohol withdrawal–associated behavioral symptoms (Dhir et al., 2005; Gamble-George et al., 2016) where 2-AG plays an important role (Serrano et al., 2018). Future studies using 2-AG–derived COX-2-generated metabolites are needed to further support the role of COX pathway and may provide a new target for the treatment of alcohol use disorders in humans.

In summary, our results suggest that the oral self-administration of 10% EtOH was inhibited by enhanced 2-AG levels in the NAc shell, but not the self-administration of lower EtOH concentration (2%). This inhibitory effect of 2-AG was prevented by pretreatment with the COX-2 inhibitor nimesulide, which suggests a potential role of the oxidative metabolites of 2-AG on EtOH self-administration in NAc shell.

Supplementary Material

Fig S1

Fig. S1. Effects of intra-NAc shell infusions of low doses of 2-AG on 10% ethanol self-administration.

Fig S2

Fig. S2. Effects of intra-NAc shell infusions of 2-AG on 2% ethanol self-administration.

Sup figure Legends

ACKNOWLEDGMENTS

This is manuscript number 29930 from The Scripps Research Institute. The present study has been supported by the following grants: National Institute on Alcohol Abuse and Alcoholism (AA006420, AA017447, AA020404, AA022249, and AA026999), Instituto de Salud Carlos III (ISCIII) and European Regional Development Funds-European Union (ERDF-EU; Subprograma RETICS Red de Trastornos Adictivos, RD16/0017/0001), Ministerio de Economía y Competitividad and ISCIII (PI16/01953, PI16/01698, and PI17/02026), Ministerio de Sanidad, Servicios Sociales e Igualdad and Plan Nacional sobre Drogas (PND2017/043, PND2018/033, and PND2018/044), and Consejería de Economía, Innovatión y Ciencia, Junta de Andalucía and ERDF-EU (CTS-433). FJP and AS hold a “Miguel Servet II” research contract funded by ISCIII and ERDF-EU (CPII19/00022 and CPII19/00031, respectively).

Footnotes

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.

Contributor Information

Francisco J. Pavon, Department of Neuroscience, The Scripps Research Institute, La Jolla, California; Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Regional Universitario de Málaga, Unidad de Gestión Clínica de Salud Mental, Malaga, Spain

Ilham Polis, Department of Neuroscience, The Scripps Research Institute, La Jolla, California.

David G. Stouffer, Department of Neuroscience, The Scripps Research Institute, La Jolla, California

Marisa Roberto, Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California..

Rémi Martin-Fardon, Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California..

Fernando Rodriguez de Fonseca, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Regional Universitario de Málaga, Unidad de Gestión Clínica de Salud Mental, Malaga, Spain.

Loren H. Parsons, Department of Neuroscience, The Scripps Research Institute, La Jolla, California

Antonia Serrano, Department of Neuroscience, The Scripps Research Institute, La Jolla, California; Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Regional Universitario de Málaga, Unidad de Gestión Clínica de Salud Mental, Malaga, Spain.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig S1

Fig. S1. Effects of intra-NAc shell infusions of low doses of 2-AG on 10% ethanol self-administration.

NIHMS1643678-supplement-Fig_S1.pdf (32.7KB, pdf)
Fig S2

Fig. S2. Effects of intra-NAc shell infusions of 2-AG on 2% ethanol self-administration.

NIHMS1643678-supplement-Fig_S2.pdf (33.4KB, pdf)
Sup figure Legends
NIHMS1643678-supplement-Sup_figure_Legends.docx (13.4KB, docx)

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