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Pharmacol Res. Author manuscript; available in PMC Sep 1, 2012.
Published in final edited form as:
PMCID: PMC3140828
NIHMSID: NIHMS295614

Enhancement of endocannabinoid signaling with JZL184, an inhibitor of the 2-arachidonoylglycerol hydrolyzing enzyme monoacylglycerol lipase, produces anxiolytic effects under conditions of high environmental aversiveness in rats

Abstract

Dysregulation in signaling of the endocannabinoid 2-arachidonoylglycerol (2-AG) is implicated in hyperresponsiveness to stress. We hypothesized that blockade of monoacylglycerol lipase (MGL), the primary enzyme responsible for 2-AG deactivation in vivo, would produce context-dependent anxiolytic effects in rats. Environmental aversiveness was manipulated by varying illumination of an elevated plus maze. Percentage open arm time and numbers of open and closed arm entries were measured in rats receiving a single intraperitoneal (i.p.) injection of either vehicle, the MGL inhibitor JZL184 (1–8 mg/kg), the benzodiazepine diazepam (1 mg/kg), the cannabinoid CB1 receptor antagonist rimonabant (1 mg/kg), or JZL184 (8 mg/kg) coadministered with rimonabant (1 mg/kg). JZL184 (8 mg/kg) produced anxiolytic-like effects (i.e. increased percentage open arm time and number of open arm entries) under high, but not low, levels of environmental aversiveness. Diazepam produced anxiolytic effects in either context. Rimonabant blocked the anxiolytic-like effects of JZL184, consistent with mediation by CB1. Anxiolytic effects of JZL184 were preserved following chronic (8 mg/kg per day × 6 days) administration. Chronic and acute JZL184 treatment similarly enhanced behavioral sensitivity to an exogenous cannabinoid (WIN55,212-2; 2.5 mg/kg i.p.) 24 or 72 h following the terminal injection, suggesting a pervasive effect of MGL inhibition on the endocannabinoid system. We attribute our results to alterations in emotion rather than locomotor activity as JZL184 did not alter the number of closed arm entries in the plus maze or produce motor ataxia in the bar test. Our results demonstrate that JZL184 has beneficial, context-dependent effects on anxiety in rats, presumably via inhibition of MGL-mediated hydrolysis of 2-AG. These data warrant further testing of MGL inhibitors to elucidate the functional role of 2-AG in controlling anxiety and stress responsiveness. Our data further implicate a role for 2-AG in the regulation of emotion and validate MGL as a therapeutic target.

Keywords: anxiety, 2-arachidonoylglycerol (2-AG), endocannabinoid, environmental aversiveness, JZL184, monoacylglycerol lipase (MGL)

1. Introduction

The endocannabinoid system is a neuromodulatory system that controls principal psychophysiological processes, including regulation of emotion and responses to stress. Dysregulation of endocannabinoid signaling is implicated in emotional disturbances, such as mood and anxiety disorders (for review see 1, 2). Blockade of cannabinoid CB1 receptors produces anxiogenic effects (38), although atypical anxiolytic effects have also been reported (9, 10). Agents that prevent deactivation of the endocannabinoid anandamide exhibit psychotherapeutic efficacy in animal models (for review see 11) while minimizing psychological, physiological, and motoric side effects (1215) associated with direct activation of cannabinoid CB1 receptors (see also 16).

The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are deactivated by transport into cells followed by enzymatic hydrolysis (for review see 17). Anandamide is preferentially degraded by fatty-acid amide hydrolase (FAAH) (1820), whereas 2-AG is preferentially degraded by monoacylglycerol lipase (MGL) (2123). Selective inhibition of these hydrolytic pathways may elucidate the contributions of each endocannabinoid ligand in emotion regulation. For example, FAAH inhibitors produce anxiolytic effects in rodents (6, 15, 2428) that are moderated by environmental aversiveness (29).

People bearing low levels of FAAH through a common single nucleotide polymorphism (C385A) in the FAAH gene exhibit diminished threat-related amygdala reactivity, a measure that negatively predicts trait anxiety (30). Yet, whether people suffering from anxiety disorders exhibit dysregulated 2-AG signaling is unknown. Individuals suffering from major depression, an affective disorder comorbid to anxiety, exhibit a selective elevation in 2-AG, but not AEA, content in serum after social stress (31). Moreover, motion (32) and orthostatic (33) stress increased plasma levels of 2-AG, but not AEA, in normal individuals after and during stressor exposure. By contrast, stress-related imagery has been reported to decrease AEA, but not 2-AG, in plasma of healthy individuals (34). These results indicate that AEA and 2-AG have distinct roles in regulating the response to stress and stress-related pathology.

Environmental stressors (i.e., maternal deprivation, cat odor) alter expression of enzymes involved in 2-AG biosynthesis and degradation in brain regions controlling emotion, such as the amygdala, hippocampus, and periaqueductal grey area of rats (35, 36). Exposure to a footshock stressor also mobilizes 2-AG in the periaqueductal gray coincident with the expression of stress-induced analgesia (37). This phenomenon is also enhanced by local inhibition of MGL, which elevates 2-AG at the same site (37). Thus, 2-AG may act in the brain to regulate physiological responses induced by aversive environmental situations. For example, social isolation is an environmental manipulation associated with symptoms of psychiatric disorders (38), whereas handling is an environmental intervention that improves many of these symptoms (39). Chronic handling attenuates both anxiety and heightened emotion induced by social isolation and concomitantly elevates 2-AG accumulation in the prefrontal cortex (40). In line with these observations, 2-AG, but not AEA, levels in the ventral striatum negatively correlate with the hyperlocomotor response elicited by the stress of exposure to a novel open field (41). The relationship between novelty-induced locomotion and 2-AG is also abolished by subjecting rats to olfactory bulbectomy, which deafferents the ventral striatum, and produces both a hyperactive response to novelty stress and a dysregulated affective state (41). These data collectively suggest a role for 2-AG in regulating responsiveness to environmental stressors.

The recent development of MGL inhibitors (37, 42, 43) has enabled the role of 2-AG in emotion regulation to be directly investigated. A single preclinical study in mice suggests that inhibition of 2-AG degradation produces anxiolytic-like effects (44). Under baseline conditions (i.e., no stressor exposure), pharmacological inhibition of MGL with JZL184, administered acutely, produced a CB1-dependent reduction in marble burying, a compulsive behavior exhibited in the obsessive-compulsive anxiety disorder (44). It remains unknown whether the efficacy of MGL inhibition extends across different features of emotional behavior or is dependent upon the environmental context. Moreover, whether the anxiolytic profile of a MGL inhibitor, administered acutely, persists after chronic administration has yet to be investigated. Such a demonstration is critical for validating of the therapeutic potential of MGL inhibitors for the treatment of anxiety in humans.

We tested the hypothesis that inhibition of 2-AG deactivation with the MGL inhibitor JZL184 would produce anxiolytic-like responding in the elevated plus maze in rats. We postulated that the anxiolytic efficacy of the MGL inhibitor would be dependent upon the aversiveness of the environmental context. Finally, we evaluated whether behavioral efficacy of JZL184 would be preserved following chronic administration.

2. Materials and methods

2.1. Subjects

One hundred sixty two adult male Sprague Dawley rats (270–350 g) were used. Rats from Harlan (Indianapolis, IN, USA) were used in all studies employing repeated injections of drug or vehicle. Other studies used rats from Charles River (Wilmington, MA, USA) and Harlan (Indianapolis, IN, USA). The supplier was changed from Harlan to Charles River when institutional furloughs prevented receipt of animals from Harlan. Different experimental groups were always tested together and statistical analyses were performed to verify that it was appropriate to pool animals for each experimental condition from the two suppliers. Upon arrival, rats were singly housed and kept under a 12:12 light:dark cycle (lights on at 7 AM) and temperature of 23 ± 1 °C. Rats were given ad libitum access to food and water and allowed at least 6 days to acclimate to the facility before testing. Cages were cleaned and refilled with sawdust bedding weekly and, therefore, this maintenance was the only handling rats received before testing. All experiments were carried out in accordance with the National Institute of Health guide for the care and use of laboratory animals and procedures were approved by the institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, the number of animals used, and to utilize alternatives to in vivo techniques, if available.

2.2. Drugs

JZL184 used in single injection studies was purchased from Caymen Chemical (Ann Arbor, MI, USA). Rimonabant and JZL184 used in chronic injection studies were obtained from the National Institute on Drug Abuse Chemical Synthesis and Drug Supply Program (Bethesda, MD, USA). WIN55,212-2 and diazepam were purchased from Sigma Aldrich (St. Louis, MO, USA). All drugs were dissolved in a vehicle containing 20% DMSO and 80% emulphor: ethanol: saline in a 1:1:8 ratio. Drugs, administered either alone or in combination, were always dissolved in a volume of 1 ml/kg bodyweight to ensure that all studies employed a uniform injection volume and handling manipulation for i.p. drug administration.

2.3 General experimental methods

2.3.1. Experiment 1: Evaluation of anxiolytic effects produced by acute (i.p.) injection of JZL184 in the elevated plus maze under varying conditions of environmental aversiveness

Rats were randomly assigned to receive a single intraperitoneal (i.p.) injection of either vehicle, the MGL inhibitor JZL184 (1, 4, or 8, or 16 mg/kg), the benzodiazepine anxiolytic diazepam (1, 2 or 3 mg/kg), or the cannabinoid CB1 receptor antagonist rimonabant (SR141716; 1mg/kg) in the presence or absence of JZL184 (8 mg/kg). All drugs were administered 30 min before behavioral testing. After injections, rats were placed in a room adjacent to the experimental room until tested. All drugs were prepared fresh on the day of testing and dispersed in vehicle before use by vortexting. Doses of diazepam (4547) and JZL184 (42, 48) were selected based on previous studies. Pilot studies in our laboratory in rats indicated that 8 mg/kg was the maximum behaviorally effective dose of JZL184 on elevated plus maze behavior and tests of nociception (data not shown). Limitations in drug solubility prevented testing of doses exceeding 16 mg/kg i.p. Previous research has shown that MGL blockade using JZL184 produces robust increases in 2-AG, but not AEA, in mouse brain (43). Our laboratory previously validated that, in the rat, JZL184 (local) selectively suppresses MGL but not FAAH activity ex vivo (49) and produces non-overlapping, modality-specific, and pharmacologically distinct antinociceptive effects from that of the FAAH inhibitor URB597 (50).

Behavioral testing was conducted during the light phase of the light:dark cycle between 11:30 AM to 6:30 PM. The elevated plus maze (EPM) was a solid black “+” shaped structure with a black matte painted floor. The apparatus was elevated 50 cm above the floor and included two open (45 × 9 cm) and two closed (45 × 9 × 38 cm) arms extending from a central platform (9 × 9 cm). Rats were placed in the central platform of the plus-shaped maze, facing an open arm opposite to the experimenter. Test sessions of 5-min duration were digitally-recorded, as previously described (51). An experimenter blind to treatment conditions quietly remained in the room during testing, hidden behind a room divider, and monitored the session. Measured behaviors were the number of open and closed arm entries and the percentage of time spent in open arms (24). Between tests, the apparatus was wiped cleaned with a chlorhexidine diacetate solution and was allowed to dry.

To induce different levels of environmental aversiveness, fluorescent lighting in the testing room was adjusted to emit low and high levels of illumination in the maze. For the low environmental aversiveness condition, illumination in the open and closed arms of the maze was 15 and 0 lux, respectively. For the high environmental aversiveness condition, illumination in the open and closed arms was 890 and 480 lux, respectively. Testing was conducted in a sound-attenuated (79 dB) environment.

2.3.2. Experiment 2: Effects of chronic and acute administration of the MGL inhibitor JZL184 on anxiety-like behavior in the elevated plus maze

Rats received repeated once daily injections of either JZL184 (8 mg/kg i.p.) or vehicle for 6 days. To control for handling effects, a third group of rats (i.e. referred to here as the acute JZL184 condition) received five once daily injections of vehicle followed by a terminal injection of JZL184 (8 mg/kg i.p.) on day 6 only. All rats were tested on the elevated plus maze under the high-light condition 30 minutes after the last injection. This study employed the same elevated plus maze testing procedure described above.

2.3.3. Experiment 3: Effects of MGL inhibition with JZL184 on locomotor activity

Directly after elevated plus maze testing, all rats were tested for catalepsy using the bar test (52, 53). Forepaws were placed on a stainless steel bar (suspended 9 cm above a flat platform) when hindpaws remained in contact with a table, as described previously (54). Time spent immobile on the bar was measured in duplicate for each rat.

2.3.4. Experiment 4: Evaluation of behavioral sensitivity to WIN55,212-2, an exogenous cannabinoid, following chronic and acute administration of the MGL inhibitor JZL184

Rats receiving repeated daily injections of vehicle (6 days), JZL184 (8 mg/kg i.p. × 6 days) or vehicle (5 days) followed by a terminal injection of JZL184 (8 mg/kg i.p. on day 6 only) were used to assess behavioral sensitivity to cannabinoids in the tail-flick test. Antinociceptive effects of WIN55,212-2 (2.5 mg/kg i.p.) were assessed in the same animals used in the elevated plus maze either 24 or 72 h following the terminal injection of JZL184/vehicle. This study employed methods similar to that described previously (67). Tail-flick latencies were measured three times at 2 min intervals before (baseline) and after injection of WIN55,212-2 (28–32 min post drug).

2.4. Statistics

Homogeneity of variance and group normality were validated using the Levene and Kolmogorov-Smirnov statistics, respectively. Data were analyzed by multivariate and one-way analysis of variance (ANOVA), as appropriate. Subsequent multiple comparison post hoc tests were performed, as required, to evaluate statistical significance of comparisons to control (Dunnett) and all other treatments (Fisher’s Protected LSD). Planned comparison t-tests were used to subsequently compare effects of chronic and acute JZL184 in the elevated plus maze and determine whether effects of acute JZL184 treatment were blocked by rimonabant. Groups that received rimonabant either alone or coadministered with JZL184 did not differ and were pooled for subsequent comparisons to avoid a type II statistical error. Directionality of the tests was based upon predicted hypotheses. Comparisons that did not meet the equal variance assumption were corrected by fractional adjustment of degrees of freedom. Interclass correlation coefficients using Cronbach alpha were calculated across groups and behaviors to verify strong intra-rater reliability in behavioral coding, which ranged from α = 0.87 – 1.0. Classic eta squared (η2) effect size calculations were performed to gauge the amount of variance our manipulations accounted for in the dependent measures evaluated. Using Cohen’s standards, eta squared values above 0.0099, 0.058, and 0.1379 can be considered small, medium, and large effects, respectively (55), although limitations of these stated criteria (e.g., overestimation of population association, dependence upon sample size) must be acknowledged (56, 57). Eta squared was calculated for ANOVAs (SS Factor / SS Corrected Total or SS Factor a / SS Corrected Total - SS Factor a - SS axb), and for t-tests (t2 / [t2 + [n1 + n2 – 2]). All other analyses were performed using SPSS statistical software (version 16.0; SPSS Incorporated, Chicago, IL, USA).

Antinociception in tail flick test was calculated as the percentage of maximum possible effect (% MPE), using the following formula: (Post - drug tail - flick latency - baseline) / (cut - off value - baseline) *100.

3. Results

3.1. Experiment 1

3.1.1. Under low environmental aversiveness, diazepam, but not JZL184, produces anxiolytic-like responding in the elevated plus maze

A priori analyses revealed that diazepam-treated rats spent a greater percentage of time on the open arms compared to vehicle-treated rats under low light conditions (t15 = -1.781, p = 0.048; η2 = 0.175; Fig. 1a). In addition, drug treatment increased the number of open arm entries (F3,30 = 5.458, p = 0.004; η2 = 0.353; Fig. 1b) without altering the number of closed arm entries (p > 0.05; Fig. 1c). Diazepam-treated rats (1 mg/kg) exhibited a greater number of open arm entries compared to vehicle- (p = 0.009; η2 = 0.235; Fig. 1b), JZL184- (4 mg/kg) (p = 0.004; η2 = 0.235; Fig. 1b), and JZL184 (8 mg/kg) -treated rats (p = 0.005; η2 = 0.224; Fig. 1b). JZL184-treated rats were no different from vehicle-treated rats in the number of open arm entries (p > 0.05; Fig. 1b).

Figure 1
Under conditions of low environmental aversiveness, diazepam, but not JZL184, produced anxiolytic-like responding in the elevated plus maze

3.1.2. Under high environmental aversiveness both JZL184 and diazepam produce anxiolytic-like responding in the elevated plus maze

Under high light, drug treatment increased the percentage of time spent on the open arms of the maze (F4,51 = 2.914, p = 0.030; η2 = 0.186; Fig. 2a). Both JZL184 (8 mg/kg) (p = 0.039; η2 = 0.159; Fig. 2a) and diazepam (1mg/kg) (p = 0.013; η2 = 0.182; Fig. 2a) increased the percentage of open arm time compared to vehicle-treated rats. In addition, the drug treatments increased the number of open arm entries (F4,51 = 2.495, p = 0.054; η2 = 0.164; Fig. 2b) without altering the number of closed arm entries (p > 0.05; Fig. 2c). Both JZL184 (8 mg/kg) (p = 0.059; η2 = 0.159; Fig. 2b) and diazepam (1 mg/kg) (p = 0.051; η2 = 0.182; Fig. 2b) increased the number of open arm entries compared to vehicle-treated rats.

Figure 2
Under conditions of high environmental aversiveness, both JZL184 and diazepam produced anxiolytic-like responding in the elevated plus maze

3.1.3. The CB1 receptor antagonist rimonabant blocks the anxiolytic-like effects of JZL184 under high environmental aversiveness in the elevated plus maze

A priori analyses revealed that JZL184 (8 mg/kg)-treated rats spent a greater percentage of time on the open arms (t26 = −2.525, p = 0.009; η2 = 0.197; Fig. 3a) and exhibited more open arm entries (t26 = −2.186, p = 0.019; η2 = 0.155; Fig. 3b), relative to vehicle-treated rats. Rimonabant (1 mg/kg) also blocked the JZL184-induced increase in both open arm time and open arm entries; rats receiving JZL184 (8 mg/kg) exhibited a greater percentage of time spent on the open arms (t32 = 1.813, p = 0.040; η2 = 0.096; Fig. 3a) and a greater number of open arm entries (t32 = 2.955, p = 0.003; η2 = 0.220; Fig. 3b) compared to rats treated with rimonabant (1 mg/kg) in either the presence or absence of JZL184 (8 mg/kg). Percentages of open arm time and numbers of open arm entries did not differ between rats receiving vehicle or rimonabant (1 mg/kg) (p > 0.05; Fig. 3a–b). Moreover, percentages of open arm time and the numbers of open arm entries did not differ between rats receiving rimonabant in the absence or presence of JZL184 (8 mg/kg) (p > 0.05; Fig. 3a–b). No drug altered the number of closed arm entries (p > 0.05; Fig. 3c).

Figure 3
The CB1 receptor antagonist rimonabant blocked the anxiolytic-like effects of JZL184 under conditions of high environmental aversiveness in the elevated plus maze

3.1.4. JZL184 produces context-dependent anxiolytic-like effects in the elevated plus maze whereas high environmental aversiveness produces anxiogenic-like effects

Drug treatment altered both the percentage of open arm time (F2,58 = 5.376, p = 0.007; η2 = 0.162; Fig. 4a) and frequency of open arm entries (F 2,58 = 7.684, p = 0.001; η2 = 0.176; Fig. 4b). Both JZL184 (8 mg/kg) (p < 0.05; η2 = 0.162; Fig. 4a) and diazepam (1 mg/kg) (p < 0.05; η2 = 0.085; Fig. 4a) increased the percentage open arm time relative to vehicle. Diazepam (1 mg/kg) also increased the number of open arm entries relative to vehicle (p < 0.05; η2 = 0.095; Fig. 4b). However, JZL184 (8 mg/kg)-treated rats exhibited a lower percentage of open arm time (p < 0.05; η2 = 0.085; Fig. 4a) and fewer open arm entries (p < 0.05; η2 = 0.095; Fig. 4b) relative to diazepam (1 mg/kg). Rats tested under high light exhibited fewer open arm entries (F 1,58 = 3.814, p = 0.056; η2 = 0.046; Fig. 4b) relative to rats tested under low light, but did not reliably differ in the percentage of time spent on the open arms of the elevated plus maze (p > 0.05; Fig. 4a). Drug treatment differentially altered the frequency of open arm entries depending on lighting condition during testing (F 2,58 = 3.859, p = 0.027; η2 = 0.092; Fig. 4b). When tested under low light, diazepam (1mg/kg) elicited a greater number of open arm entries relative to rats treated with the same dose of diazepam (1 mg/kg) under high light (p < 0.05; η2 = 0.219; Fig. 4b), vehicle under either low (p < 0.05; η2 = 0.232; Fig. 4b) or high (p < 0.05; η2 = 0.179; Fig. 4b) light, or JZL184 (8 mg/kg) under either low (p < 0.05; η2 = 0.232; Fig. 4b) or high (p < 0.05; η2 = 0.179; Fig. 4b) light. When tested under high light, rats treated with either JZL184 (8 mg/kg) (p < 0.05; η2 = 0.140; Fig. 4b) or diazepam (1 mg/kg) (p < 0.05; η2 = 0.164; Fig. 4b) exhibited a greater number of open arm entries relative to vehicle. No drug or lighting treatment altered the frequency of closed arm entries (p > 0.05; Fig. 4c).

Figure 4
High environmental aversiveness produces anxiogenic-like responding in the elevated plus maze and unmasks anxiolytic-like effects of JZL184

3.2. Experiment 2: Chronic and acute administration of JZL184 produces comparable anxiolytic-like responding in the elevated plus maze under high environmental aversiveness

A priori analyses revealed that JZL184, administrated either chronically (8 mg/kg i.p. per day for 6 days) or acutely (once daily injections of vehicle for 5 days followed by a terminal injection of 8 mg/kg i.p. on day 6) elicited greater percentage of open arm time (t33 = 2.063, p = 0.024; η2 = 0. 114; Fig. 5a) and number of open arm entries (t27 = 3.031, p = 0.003; η2 = 0.254; Fig. 5b) than vehicle-treated rats. Neither treatment altered the number of closed arm entries (p > 0.05; Fig. 5c). Percentages of open arm time and the number of open arm entries did not differ between rats receiving chronic or acute treatment with JZL184 (8 mg/kg) (p > 0.05; Fig. 5a–b).

Figure 5
Chronic and acute administration of JZL184 produces anxiolytic-like responding in the elevated plus maze under conditions of high environmental aversiveness

3.3. Experiment 3: Diazepam but not JZL184 produces catalepsy

Acute drug treatments differentially altered (F6,62 = 6.119, p = 0.000; η2 = 0.372; Fig. 5a) time spent immobile in the bar test. The high dose of diazepam (3 mg/kg) increased immobility time relative to either vehicle, the low dose of diazepam (1 mg/kg), or a range of doses (1, 4, 8, 16 mg/kg) of JZL184 (p = 0.000; Fig. 6a). JZL184 did not alter descent latencies in the bar test relative to vehicle-treated rats at any dose (p > 0.05; Fig. 6a). Immobility times were similar in groups receiving either the middle (2 mg/kg i.p.) or high (3 mg/kg i.p.) doses of diazepam (p > 0.05; Fig. 6a). Neither chronic (8mg/kg i.p. × 6 days) nor acute (8mg/kg i.p. on day 6 only) administration of JZL184 altered immobility times in the bar test relative to vehicle-treated rats (F2, 19 = 2.796, p = 0.088; Fig. 6b). Immobility times did not differ between groups (p >0.05, Fig. 6b).

Figure 6
Diazepam, but not JZL184, produces catalepsy in the bar test

3.4. Experiment 4: Chronic and acute administration of JZL184 enhance behavioral sensitivity to WIN 55, 212-2, an exogenous cannabinoid

Behavioral sensitivity to WIN55,212-2, a synthetic cannabinoid, was evaluated in the tail-flick test 24 or 72 h after the terminal JZL184/vehicle injection in the same rats used in the elevated plus maze test (see Fig. 6). The maximal possible antinociceptive effect of WIN55, 212-2 (2.5 mg/kg i.p.) was increased (F2,30 = 5.180, p = 0.012; η2 = 0.257; Fig. 7) in rats treated either chronically (8 mg/kg i.p. × 6 days; p = 0.014; η2 = 0.235; Fig. 7) or acutely (8 mg/kg i.p. on day 6; p = 0.006; η2 = 0.282; Fig. 7) with JZL184, relative to vehicle treatment. WIN55, 212-2-induced antinociception did not differ between chronic and acute JZL184-treated groups at either timepoint.

Figure 7
Chronic and acute treatment with JZL184 produces behavioral sensitization to exogenous cannabinoid administration

4. Discussion

The present studies identified context-dependent anxiolytic effects of JZL184, an inhibitor of the 2-AG hydrolyzing enzyme MGL, that were dependent upon the level of environmental aversiveness. Under baseline conditions (low environmental aversiveness), preventing deactivation of 2-AG with JZL184 did not alter behavioral responding in a well characterized test of anxiety, the elevated plus maze. However, under more stressful circumstances (high environmental aversiveness), JZL184 reduced anxiety-like behavior in the elevated plus maze in a CB1-dependent manner. Furthermore, the anxiolytic effects of JZL184 were preserved following chronic administration, suggesting that they did not undergo tolerance in our study. JZL184 produced a distinct anxiolytic profile from standard treatment with the benzodiazepine diazepam. The anxiolytic ability of JZL184 is comparable to diazepam in the magnitude of effectiveness, but, unlike diazepam, emerges only under more stressful circumstances. We attribute the effects of JZL184 to alterations in emotion, and not to locomotor activity, as JZL184, administered chronically or acutely, did not produce motor impairment in the elevated plus maze or catalepsy in the bar test. Our results demonstrate that JZL184 has beneficial and environmentally-dependent effects on anxiety in rats, presumably via inhibition of MGL-mediated hydrolysis of 2-AG.

A striking observation of our study was that animals treated chronically with JZL184 (8 mg/kg per day for 6 days) showed no loss in anxiolytic efficacy in the elevated plus maze. Chronic (8 mg/kg i.p. × 6 days) and acute (8 mg/kg i.p. on day 6) treatment with JZL184 produced equally efficacious anxiolytic profiles (i.e. similar magnitude increases in percentage open arm time and number of open arm entries) and also produced similar levels of behavioral sensitization to the antinociceptive effects of WIN55,212-2 (2.5 mg/kg i.p.). Our results suggest that JZL184, administrated either chronically or acutely in rats, produces a prolonged blockade of endocannabinoid deactivation without producing tolerance, at least with the current dosing schedule. By contrast, tolerance and receptor desensitization are observed in mice following repeated administration of higher doses of JZL184 (40 mg/kg i.p. for 6 days) (16), a treatment regimen that produces approximately 85% inhibition of MGL activity after a single injection (42, 43). Differences in species selectivity (rats vs. mice) and dose (8 mg/kg per day for 6 days vs. 40 mg/kg per day for 6 days) of JZl184 employed may account for differences between the present study and that by Schlosburg et al. (2010). Our results suggest that a sustained elevation of endocannabinoid tone was still present 72 h following acute or chronic injection of JZL184 in the rat. However, stimulation-contingent 2-AG mobilization under conditions of high environmental aversiveness was required to unmask anxiolytic effects of JZL184 in the elevated plus maze. Nonetheless, the fact that the MGL inhibitor JZL184 remained effective in suppressing anxiety-like behavior after repeated administration implies that MGL inhibitors may show therapeutic potential as anxiolytics in humans.

Our findings are congruent with other data which suggest that benzodiazepines such as chlordiazepoxide produce anxiolytic-like responding in the elevated plus maze regardless of the degree of environmental aversiveness present during testing (29). Previous studies substantiate that increasing illumination during the elevated plus maze test is aversive (29, 58) and that effects of the endocannabinoid system or endocannabinoid modulators on anxiety are uncovered by altering environmental aversiveness (25, 29, 58). Similar to inhibition of 2-AG hydrolysis with JZL184 (present report), inhibition of anandamide hydrolysis with URB597 produced anxiolytic-like effects in the elevated plus maze only when tested under high light (29). Likely brain regions for which JZL184 exerts its effects on anxiety-like behavior in our study may be analogous to those implicated in studies employing site-specific modulation of anandamide hydrolysis. For example, anxiety-like responding was reduced by local inhibition of FAAH in the basolateral amygdala complex (59), dorsolateral periaqueductal grey area (60), hippocampus (61), infralimbic region (62), and prefrontal cortex (63). Future microinjection studies are needed to determine whether these regions mediate the effects of systemic JZL184 on anxiety described here. Together, these data suggest that both anandamide and 2-AG regulate the behavioral expression of emotion, especially under high stress circumstances.

Evidence supports both overlapping and separate roles for 2-AG and anandamide signaling in the brain to regulate stress. For example, both 2-AG and anandamide are recruited to mimic and occlude glucocorticoid-induced suppression of glutamate release in the hypothalamus (64) and attenuate anxiety-related behaviors induced by repeated drug exposure (65). Similarly, acute inhibition of either 2-AG or anandamide hydrolysis attenuated both defensive burying (44) and rimonabant-precipitated somatic withdrawal in Δ9-tetrahydrocannabinol-dependent mice (66). However, several lines of evidence support a unique role for each endocannabinoid ligand in brain circuitry regulating emotion after footshock exposure (i.e., during stress-induced analgesia) (37), acute corticosterone treatment (67), chronic restraint stress (6871), chronic unpredictable stress (72), and maternal deprivation (73). Selective targeting of these chemical pathways using tools that prevent the synthesis or hydrolysis of endocannabinoids are needed to extend these findings and determine the relative contribution and potential synergism of endocannabinoids in emotion regulation.

Our results, along with those of others (6, 13, 15, 24, 28, 60), suggest that inhibition of endocannabinoid-degrading enzymes prolongs endocannabinoid signaling to produce monophasic, treatment-like effects on anxiety. In our study, similar levels of behavioral sensitization to exogenous cannabinoids were observed in animals receiving either chronic or acute treatment with JZL184. This observation further underscores the ability of JZL184 to enhance endocannabinoid tone. These data stand in contrast to those generated using exogenous cannabinoid CB1 receptor agonists or antagonists in preclinical models, wherein biphasic effects or both anxiogenic and anxiolytic effects are dose-dependently produced (for review see 74, 75). Similarly, clinical studies employing cannabinoid CB1 receptor antagonist/inverse agonists suggest that blocking the function of endocannabinoids may produce anxiety, depression, and suicidal ideation (7678). However, these effects may be due to excessive cannabinoid CB1 receptor blockade and limited to patients at high risk for mental illness (75). Pro-cannabinoid agents, such as MGL inhibitors, that enhance endocannabinoid tone without acting directly on CB1 receptors, may exhibit better clinical utility relative to CB1 receptor blockers. Indirect methods that elevate endocannabinoid tone may offer a more circumscribed and beneficial spectrum of physiological effects compared to direct activation of CB1 receptors. MGL inhibitors may, consequently, offer lasting efficacy in reducing anxiety with minimal tolerance or untoward psychogenic side effects (i.e. compared to either direct cannabinoid agonists or benzodiazepines). More work is necessary to determine whether pharmacological inhibitors of MGL may be used to exploit the therapeutic potential of the endocannabinoid system to suppress anxiety in humans.

5. Conclusions

Here we demonstrate that JZL184 has beneficial and environmentally-dependent effects on anxiety in rats, presumably via inhibition of MGL-mediated hydrolysis of 2-AG. Anxiolytic efficacy of JZL184 was revealed under conditions of high environmental aversiveness. Moreover, the anxiolytic profile of JZL184 showed no evidence of undergoing tolerance following repeated administration of the drug. These data warrant further testing of inhibitors of 2-AG deactivation in animal models of anxiety to elucidate the role of 2-AG in emotion regulation. Our data implicate a pivotal role for 2-AG in controlling anxiety and stress responsiveness and further validate MGL as a therapeutic target.

Acknowledgments

This research was funded by DA021644 (to AGH). NRS was funded by a NIDA Diversity Supplement (DA021644S1 to AGH).

Abbreviations

ANOVA
Analysis of variance
AEA
anandamide
2-AG
2-arachidonoylglycerol
CB1
cannabinoid receptor type 1
C385A
common single nucleotide polymorphism
EPM
elevated plus maze
FAAH
fatty acid amide hydrolase
i.p
intraperitoneal
MAFP
14-eicosatetraenyl-methyl ester phosphonofluoridic acid
MGL
monoacylglycerol lipase
SS
and sums of squares

Footnotes

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References

1. Moreira FA, Grieb M, Lutz B. Central side-effects of therapies based on CB1 cannabinoid receptor agonists and antagonists: focus on anxiety and depression. Best Pract Res Clin Endocrinol Metab. 2009;23:133–44. [PubMed]
2. Hill MN, Gorzalka BB. The endocannabinoid system and the treatment of mood and anxiety disorders. CNS Neurol Disord Drug Targets. 2009;8:451–8. [PubMed]
3. Navarro M, Hernandez E, Munoz RM, del Arco I, Villanua MA, Carrera MR, Rodriguez de Fonseca F. Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in the rat. Neuroreport. 1997;8:491–6. [PubMed]
4. Arevalo C, de Miguel R, Hernandez-Tristan R. Cannabinoid effects on anxiety-related behaviours and hypothalamic neurotransmitters. Pharmacol Biochem Behav. 2001;70:123–31. [PubMed]
5. Haller J, Varga B, Ledent C, Freund TF. CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents. Behav Pharmacol. 2004;15:299–304. [PubMed]
6. Patel S, Hillard CJ. Pharmacological evaluation of cannabinoid receptor ligands in a mouse model of anxiety: further evidence for an anxiolytic role for endogenous cannabinoid signaling. J Pharmacol Exp Ther. 2006;318:304–11. [PubMed]
7. Haller J, Matyas F, Soproni K, Varga B, Barsy B, Nemeth B, Mikics E, Freund TF, Hajos N. Correlated species differences in the effects of cannabinoid ligands on anxiety and on GABAergic and glutamatergic synaptic transmission. Eur J Neurosci. 2007;25:2445–56. [PMC free article] [PubMed]
8. Rodgers RJ, Evans PM, Murphy A. Anxiogenic profile of AM-251, a selective cannabinoid CB1 receptor antagonist, in plus-maze-naive and plus-maze-experienced mice. Behav Pharmacol. 2005;16:405–13. [PubMed]
9. Haller J, Bakos N, Szirmay M, Ledent C, Freund TF. The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. Eur J Neurosci. 2002;16:1395–8. [PubMed]
10. Griebel G, Stemmelin J, Scatton B. Effects of the cannabinoid CB1 receptor antagonist rimonabant in models of emotional reactivity in rodents. Biol Psychiatry. 2005;57:261–7. [PubMed]
11. Petrosino S, Di Marzo V. FAAH and MAGL inhibitors: therapeutic opportunities from regulating endocannabinoid levels. Curr Opin Investig Drugs. 2010;11:51–62. [PubMed]
12. Cippitelli A, Cannella N, Braconi S, Duranti A, Tontini A, Bilbao A, Defonseca FR, Piomelli D, Ciccocioppo R. Increase of brain endocannabinoid anandamide levels by FAAH inhibition and alcohol abuse behaviours in the rat. Psychopharmacology (Berl) 2008;198:449–60. [PubMed]
13. Piomelli D, Tarzia G, Duranti A, Tontini A, Mor M, Compton TR, Dasse O, Monaghan EP, Parrott JA, Putman D. Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597) CNS Drug Rev. 2006;12:21–38. [PubMed]
14. Long JZ, Nomura DK, Vann RE, Walentiny DM, Booker L, Jin X, Burston JJ, Sim-Selley LJ, Lichtman AH, Wiley JL, Cravatt BF. Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo. Proc Natl Acad Sci U S A. 2009;106:20270–5. [PMC free article] [PubMed]
15. Scherma M, Medalie J, Fratta W, Vadivel SK, Makriyannis A, Piomelli D, Mikics E, Haller J, Yasar S, Tanda G, Goldberg SR. The endogenous cannabinoid anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase (FAAH) inhibition. Neuropharmacology. 2008;54:129–40. [PMC free article] [PubMed]
16. Schlosburg JE, Blankman JL, Long JZ, Nomura DK, Pan B, Kinsey SG, Nguyen PT, Ramesh D, Booker L, Burston JJ, Thomas EA, Selley DE, Sim-Selley LJ, Liu QS, Lichtman AH, Cravatt BF. Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system. Nat Neurosci. 2010;13:1113–9. [PMC free article] [PubMed]
17. Viveros MP, Marco EM, Llorente R, Lopez-Gallardo M. Endocannabinoid system and synaptic plasticity: implications for emotional responses. Neural Plast. 2007 doi: 10.1155/2007/52908. [PMC free article] [PubMed] [Cross Ref]
18. Cravatt BF, Giang DK, Mayfield SP, Boger DL, Lerner RA, Gilula NB. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature. 1996;384:83–7. [PubMed]
19. McKinney MK, Cravatt BF. Structure and function of fatty acid amide hydrolase. Annu Rev Biochem. 2005;74:411–32. [PubMed]
20. Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR, Lichtman AH. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc Natl Acad Sci USA. 2001;98:9371–6. [PMC free article] [PubMed]
21. Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, Sensi SL, Kathuria S, Piomelli D. Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci USA. 2002;99:10819–24. [PMC free article] [PubMed]
22. Dinh TP, Kathuria S, Piomelli D. RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-arachidonoylglycerol. Mol Pharmacol. 2004;66:1260–4. [PubMed]
23. Blankman JL, Simon GM, Cravatt BF. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol. 2007;14:1347–56. [PMC free article] [PubMed]
24. Moreira FA, Kaiser N, Monory K, Lutz B. Reduced anxiety-like behaviour induced by genetic and pharmacological inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) is mediated by CB1 receptors. Neuropharmacology. 2008;54:141–50. [PubMed]
25. Naidu PS, Varvel SA, Ahn K, Cravatt BF, Martin BR, Lichtman AH. Evaluation of fatty acid amide hydrolase inhibition in murine models of emotionality. Psychopharmacology (Berl) 2007;192:61–70. [PubMed]
26. Kathuria S, Gaetani S, Fegley D, Valino F, Duranti A, Tontini A, Mor M, Tarzia G, La Rana G, Calignano A, Giustino A, Tattoli M, Palmery M, Cuomo V, Piomelli D. Modulation of anxiety through blockade of anandamide hydrolysis. Nat Med. 2003;9:76–81. [PubMed]
27. Rossi S, De Chiara V, Musella A, Sacchetti L, Cantarella C, Castelli M, Cavasinni F, Motta C, Studer V, Bernardi G, Cravatt BF, Maccarrone M, Usiello A, Centonze D. Preservation of striatal cannabinoid CB1 receptor function correlates with the antianxiety effects of fatty acid amide hydrolase inhibition. Mol Pharmacol. 2010;78:260–8. [PubMed]
28. Micale V, Cristino L, Tamburella A, Petrosino S, Leggio GM, Drago F, Di Marzo V. Anxiolytic effects in mice of a dual blocker of fatty acid amide hydrolase and transient receptor potential vanilloid type-1 channels. Neuropsychopharmacology. 2009;34:593–606. [PubMed]
29. Haller J, Barna I, Barsvari B, Gyimesi Pelczer K, Yasar S, Panlilio LV, Goldberg S. Interactions between environmental aversiveness and the anxiolytic effects of enhanced cannabinoid signaling by FAAH inhibition in rats. Psychopharmacology (Berl) 2009;204:607–16. [PMC free article] [PubMed]
30. Hariri AR, Gorka A, Hyde LW, Kimak M, Halder I, Ducci F, Ferrell RE, Goldman D, Manuck SB. Divergent effects of genetic variation in endocannabinoid signaling on human threat- and reward-related brain function. Biol Psychiatry. 2009;66:9–16. [PMC free article] [PubMed]
31. Hill MN, Miller GE, Carrier EJ, Gorzalka BB, Hillard CJ. Circulating endocannabinoids and N-acyl ethanolamines are differentially regulated in major depression and following exposure to social stress. Psychoneuroendocrinology. 2009;34:1257–62. [PMC free article] [PubMed]
32. Chouker A, Kaufmann I, Kreth S, Hauer D, Feuerecker M, Thieme D, Vogeser M, Thiel M, Schelling G. Motion sickness, stress and the endocannabinoid system. PLoS One. 2010;5:e10752. [PMC free article] [PubMed]
33. Schroeder C, Batkai S, Engeli S, Tank J, Diedrich A, Luft FC, Jordan J. Circulating endocannabinoid concentrations during orthostatic stress. Clin Auton Res. 2009;19:343–6. [PubMed]
34. Mangieri RA, Hong KI, Piomelli D, Sinha R. An endocannabinoid signal associated with desire for alcohol is suppressed in recently abstinent alcoholics. Psychopharmacology (Berl) 2009;205:63–72. [PMC free article] [PubMed]
35. Sutt S, Raud S, Areda T, Reimets A, Koks S, Vasar E. Cat odour-induced anxiety--a study of the involvement of the endocannabinoid system. Psychopharmacology (Berl) 2008;198:509–20. [PubMed]
36. Suarez J, Rivera P, Llorente R, Romero-Zerbo SY, Bermudez-Silva FJ, de Fonseca FR, Viveros MP. Early maternal deprivation induces changes on the expression of 2-AG biosynthesis and degradation enzymes in neonatal rat hippocampus. Brain Res. 2010;1349:162–73. [PubMed]
37. Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R, Krey JF, Walker JM, Holmes PV, Crystal JD, Duranti A, Tontini A, Mor M, Tarzia G, Piomelli D. An endocannabinoid mechanism for stress-induced analgesia. Nature. 2005;435:1108–12. [PubMed]
38. Fone KC, Porkess MV. Behavioural and neurochemical effects of post-weaning social isolation in rodents-relevance to developmental neuropsychiatric disorders. Neurosci Biobehav Rev. 2008;32:1087–102. [PubMed]
39. Cirulli F, Berry A, Bonsignore LT, Capone F, D’Andrea I, Aloe L, Branchi I, Alleva E. Early life influences on emotional reactivity: evidence that social enrichment has greater effects than handling on anxiety-like behaviors, neuroendocrine responses to stress and central BDNF levels. Neurosci Biobehav Rev. 2010;34:808–20. [PubMed]
40. Sciolino NR, Bortolato M, Eisenstein SA, Fu J, Oveisi F, Hohmann AG, Piomelli D. Social isolation and chronic handling alter endocannabinoid signaling and behavioral reactivity to context in adult rats. Neuroscience. 2010;168:371–86. [PMC free article] [PubMed]
41. Eisenstein SA, Clapper JR, Holmes PV, Piomelli D, Hohmann AG. A role for 2-arachidonoylglycerol and endocannabinoid signaling in the locomotor response to novelty induced by olfactory bulbectomy. Pharmacol Res. 2010;61:419–29. [PMC free article] [PubMed]
42. Long JZ, Li W, Booker L, Burston JJ, Kinsey SG, Schlosburg JE, Pavon FJ, Serrano AM, Selley DE, Parsons LH, Lichtman AH, Cravatt BF. Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat Chem Biol. 2009;5:37–44. [PMC free article] [PubMed]
43. Long JZ, Nomura DK, Cravatt BF. Characterization of monoacylglycerol lipase inhibition reveals differences in central and peripheral endocannabinoid metabolism. Chem Biol. 2009;16:744–53. [PMC free article] [PubMed]
44. Kinsey SG, O’Neal ST, Long JZ, Cravatt BF, Lichtman AH. Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay. Pharmacol Biochem Behav. 2010 doi: 10.1016/j.pbb.2010.12.002. [PMC free article] [PubMed] [Cross Ref]
45. Mechan AO, Moran PM, Elliott M, Young AJ, Joseph MH, Green R. A comparison between Dark Agouti and Sprague-Dawley rats in their behaviour on the elevated plus-maze, open-field apparatus and activity meters, and their response to diazepam. Psychopharmacology (Berl) 2002;159:188–95. [PubMed]
46. Stemmelin J, Cohen C, Terranova JP, Lopez-Grancha M, Pichat P, Bergis O, Decobert M, Santucci V, Francon D, Alonso R, Stahl SM, Keane P, Avenet P, Scatton B, le Fur G, Griebel G. Stimulation of the beta3-Adrenoceptor as a novel treatment strategy for anxiety and depressive disorders. Neuropsychopharmacology. 2008;33:574–87. [PubMed]
47. Griebel G, Perrault G, Simiand J, Cohen C, Granger P, Decobert M, Francon D, Avenet P, Depoortere H, Tan S, Oblin A, Schoemaker H, Evanno Y, Sevrin M, George P, Scatton B. SL651498: an anxioselective compound with functional selectivity for alpha2- and alpha3-containing gamma-aminobutyric acid(A) (GABA(A)) receptors. J Pharmacol Exp Ther. 2001;298:753–68. [PubMed]
48. Kinsey SG, Long JZ, O’Neal ST, Abdullah RA, Poklis JL, Boger DL, Cravatt BF, Lichtman AH. Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain. J Pharmacol Exp Ther. 2009;330:902–10. [PMC free article] [PubMed]
49. Guindon J, Guijarro A, Piomelli D, Hohmann AG. Peripheral antinociceptive effects of inhibitors of monoacylglycerol lipase in a rat model of inflammatory pain. Br J Pharmacol. 2010 doi: 10.1111/j.1476–5381.2010.01192.x. [PMC free article] [PubMed] [Cross Ref]
50. Spradley JM, Guindon J, Hohmann AG. Inhibitors of monoacylglycerol lipase, fatty-acid amide hydrolase and endocannabinoid transport differentially suppress capsaicin-induced behavioral sensitization through peripheral endocannabinoid mechanisms. Pharmacol Res. 2010;62:249–58. [PMC free article] [PubMed]
51. Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods. 1985;14:149–67. [PubMed]
52. Pertwee RG, Ross TM. Drugs which stimulate or facilitate central cholinergic transmission interact synergistically with delta-9-tetrahydrocannabinol to produce marked catalepsy in mice. Neuropharmacology. 1991;30:67–71. [PubMed]
53. Rahn EJ, Makriyannis A, Hohmann AG. Activation of cannabinoid CB1 and CB2 receptors suppresses neuropathic nociception evoked by the chemotherapeutic agent vincristine in rats. Br J Pharmacol. 2007;152:765–77. [PMC free article] [PubMed]
54. Martin WJ, Hohmann AG, Walker JM. Suppression of noxious stimulus-evoked activity in the ventral posterolateral nucleus of the thalamus by a cannabinoid agonist: correlation between electrophysiological and antinociceptive effects. J Neurosci. 1996;16:6601–11. [PubMed]
55. Cohen J. Statistical power analysis for the behavioral sciences. 2. New York: Lawrence Erlbaum Associates; 1998.
56. Levine TR, Hullett CR. Eta squared, partial eta squared, and misreporting of effect size in communication research. Human Communication Research. 2002;28:612–25.
57. Pierce CA, Block RA, Aguinis H. Cautionary Note on Reporting Eta-Squared Values From Multifactor ANOVA Designs. Educational and Psychological Measurement. 2004;64:916–24.
58. Haller J, Varga B, Ledent C, Barna I, Freund TF. Context-dependent effects of CB1 cannabinoid gene disruption on anxiety-like and social behaviour in mice. Eur J Neurosci. 2004;19:1906–12. [PubMed]
59. Hill MN, McLaughlin RJ, Morrish AC, Viau V, Floresco SB, Hillard CJ, Gorzalka BB. Suppression of amygdalar endocannabinoid signaling by stress contributes to activation of the hypothalamic-pituitary-adrenal axis. Neuropsychopharmacology. 2009;34:2733–45. [PMC free article] [PubMed]
60. Lisboa SF, Resstel LB, Aguiar DC, Guimaraes FS. Activation of cannabinoid CB1 receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test. Eur J Pharmacol. 2008;593:73–8. [PubMed]
61. Roohbakhsh A, Keshavarz S, Hasanein P, Rezvani ME, Moghaddam AH. Role of endocannabinoid system in the ventral hippocampus of rats in the modulation of anxiety-like behaviours. Basic Clin Pharmacol Toxicol. 2009;105:333–8. [PubMed]
62. Lin HC, Mao SC, Su CL, Gean PW. The role of prefrontal cortex CB1 receptors in the modulation of fear memory. Cereb Cortex. 2009;19:165–75. [PubMed]
63. Rubino T, Realini N, Castiglioni C, Guidali C, Vigano D, Marras E, Petrosino S, Perletti G, Maccarrone M, Di Marzo V, Parolaro D. Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex. Cereb Cortex. 2008;18:1292–301. [PubMed]
64. Di S, Malcher-Lopes R, Marcheselli VL, Bazan NG, Tasker JG. Rapid glucocorticoid-mediated endocannabinoid release and opposing regulation of glutamate and gamma-aminobutyric acid inputs to hypothalamic magnocellular neurons. Endocrinology. 2005;146:4292–301. [PubMed]
65. Hayase T, Yamamoto Y, Yamamoto K. Persistent anxiogenic effects of a single or repeated doses of cocaine and methamphetamine: interactions with endogenous cannabinoid receptor ligands. Behav Pharmacol. 2005;16:395–404. [PubMed]
66. Schlosburg JE, Carlson BL, Ramesh D, Abdullah RA, Long JZ, Cravatt BF, Lichtman AH. Inhibitors of endocannabinoid-metabolizing enzymes reduce precipitated withdrawal responses in THC-dependent mice. AAPS J. 2009;11:342–52. [PMC free article] [PubMed]
67. Hill MN, Carrier EJ, Ho WS, Shi L, Patel S, Gorzalka BB, Hillard CJ. Prolonged glucocorticoid treatment decreases cannabinoid CB1 receptor density in the hippocampus. Hippocampus. 2008;18:221–6. [PubMed]
68. Rademacher DJ, Meier SE, Shi L, Ho WS, Jarrahian A, Hillard CJ. Effects of acute and repeated restraint stress on endocannabinoid content in the amygdala, ventral striatum, and medial prefrontal cortex in mice. Neuropharmacology. 2008;54:108–16. [PubMed]
69. Hill MN, McLaughlin RJ, Bingham B, Shrestha L, Lee TT, Gray JM, Hillard CJ, Gorzalka BB, Viau V. Endogenous cannabinoid signaling is essential for stress adaptation. Proc Natl Acad Sci USA. 2010;107:9406–11. [PMC free article] [PubMed]
70. Patel S, Roelke CT, Rademacher DJ, Hillard CJ. Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling. Eur J Neurosci. 2005;21:1057–69. [PubMed]
71. Patel S, Roelke CT, Rademacher DJ, Cullinan WE, Hillard CJ. Endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamic-pituitary-adrenal axis. Endocrinology. 2004;145:5431–8. [PubMed]
72. Hill MN, Patel S, Carrier EJ, Rademacher DJ, Ormerod BK, Hillard CJ, Gorzalka BB. Downregulation of endocannabinoid signaling in the hippocampus following chronic unpredictable stress. Neuropsychopharmacology. 2005;30:508–15. [PubMed]
73. Llorente R, Llorente-Berzal A, Petrosino S, Marco EM, Guaza C, Prada C, Lopez-Gallardo M, Di Marzo V, Viveros MP. Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: a possible role for the endocannabinoid system. Dev Neurobiol. 2008;68:1334–47. [PubMed]
74. Moreira FA, Aguiar DC, Campos AC, Lisboa SF, Terzian AL, Resstel LB, Guimaraes FS. Antiaversive effects of cannabinoids: is the periaqueductal gray involved? Neural Plast. 2009;2009:625469. [PMC free article] [PubMed]
75. Le Foll B, Gorelick DA, Goldberg SR. The future of endocannabinoid-oriented clinical research after CB1 antagonists. Psychopharmacology (Berl) 2009;205:171–4. [PMC free article] [PubMed]
76. Christensen R, Kristensen PK, Bartels EM, Bliddal H, Astrup A. Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials. Lancet. 2007;370:1706–13. [PubMed]
77. Topol EJ, Bousser MG, Fox KA, Creager MA, Despres JP, Easton JD, Hamm CW, Montalescot G, Steg PG, Pearson TA, Cohen E, Gaudin C, Job B, Murphy JH, Bhatt DL. Rimonabant for prevention of cardiovascular events (CRESCENDO): a randomised, multicentre, placebo-controlled trial. Lancet. 2010;376:517–23. [PubMed]
78. Rucker D, Padwal R, Li SK, Curioni C, Lau DC. Long term pharmacotherapy for obesity and overweight: updated meta-analysis. BMJ. 2007;335:1194–9. [PMC free article] [PubMed]
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