Elsevier

Neuropharmacology

Volume 54, Issue 1, January 2008, Pages 141-150
Neuropharmacology

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

https://doi.org/10.1016/j.neuropharm.2007.07.005Get rights and content

Abstract

Anandamide and 2-arachidonoyl glycerol, referred to as endocannabinoids (eCBs), are the endogenous agonists for the cannabinoid receptor type 1 (CB1). Several pieces of evidence support a role for eCBs in the attenuation of anxiety-related behaviours, although the precise mechanism has remained uncertain. The fatty acid amid hydrolase (FAAH), an enzyme responsible for the degradation of eCBs, has emerged as a promising target for anxiety-related disorders, since FAAH inhibitors are able to increase the levels of anandamide and thereby induce anxiolytic-like effects in rodents. The present study adopted both genetic and pharmacological approaches and tested the hypothesis that FAAH-deficient (FAAH−/−) mice as well as C57BL/6N mice treated with an FAAH inhibitor (URB597) would express reduced anxiety-like responses. Furthermore, as it is known that anandamide can bind several other targets than CB1 receptors, we investigated whether FAAH inhibition reduces anxiety via CB1 receptors. FAAH−/− mice showed reduced anxiety both in the elevated plus maze and in the light-dark test. These genotype-related differences were prevented by the CB1 receptor antagonist rimonabant (3 mg/kg). Moreover, URB597 (1 mg/kg) induced an anxiolytic-like effect in C57BL/6N mice exposed to the elevated plus maze, which was prevented by rimonabant (3 mg/kg). The present work provides genetic and pharmacological evidence supporting the inhibition of FAAH as an important mechanism for the alleviation of anxiety. In addition, it indicates an increased activation of CB1 receptors as a mechanism underlying the effects of FAAH inhibition in two models of anxiety.

Introduction

The herb Cannabis sativa may induce a diversity of emotional responses ranging from anxiolytic and relaxing effects to the induction of panic attacks (Hall and Solowij, 1998). Divergences have also been observed in both humans and rodents after the administration of Δ9-tetrahydrocannabinol (Δ9-THC), the main active compound of marijuana, or its synthetic counterparts (Berrendero and Maldonado, 2002, Marco et al., 2004, Patel and Hillard, 2006, Zuardi et al., 1982).

The mechanisms underlying these bidirectional effects remain to be determined. In the brain, cannabinoids activate the type 1 cannabinoid (CB1) receptor, which is densely expressed in a number of regions related to the modulation of fear and anxiety (Mackie, 2005, Pacher et al., 2006). However, several variables are likely to interfere with the activity of these compounds on experimental anxiety (for a review, see Viveros et al., 2005). First, the effect may depend on the dose administered. In general, low doses tend to be anxiolytic and high doses tend to be anxiogenic (Marco et al., 2004). Second, the characteristics of the experimental environment, such as the intensity of illumination, may influence the effects of CB1 activation or blockade (Haller et al., 2004a, Naidu et al., 2007). Third, the various protocols employed for measuring anxiety may generate diverse aversive states, with which cannabinoids may interfere in opposite ways (Viveros et al., 2005). Finally, the previous history of the subjects, such as exposure to stresses or drug treatments, may also be of relevance to the response to cannabinoids (Rodgers et al., 2005).

An alternative approach to the direct activation of CB1 receptors is the enhancement of the availability of the endogenous ligands, referred to as endocannabinoids (eCBs), of which anandamide and 2-arachidonoylglycerol (2-AG) are the mostly investigated (Piomelli, 2003). Their actions are terminated by a putative uptake process, followed by degradation by fatty acid amide hydrolase (FAAH) and by monoacylglycerol lipase (McKinney and Cravatt, 2005), respectively. Specific inhibitors of FAAH have been developed that significantly increase the brain levels of anandamide, but not 2-AG, thereby potentiating the effects of anandamide (Kathuria et al., 2003). FAAH inhibitors induce analgesia, enhance memory extinction and attenuate anxiety via an increased activation of CB1 receptors (Kathuria et al., 2003, Patel and Hillard, 2006, Varvel et al., 2007). However, anandamide is a promiscuous neuromodulator that may bind to other sites in the brain, implying that alternative mechanisms, apart from the activation of CB1 receptors, may be involved in its actions (Ross, 2003). For example, the transient receptor potential vanilloid type 1 channel (TRPV1) is activated by anandamide and located in several brain regions related to emotions (Cristino et al., 2006). TRPV1 was shown to have a role on the modulation of both anxiety and conditioned fear (Marsch et al., 2007). Therefore, increasing the endogenous levels of anandamide may induce effects that are not only CB1-mediated, but also dependent on other receptors.

Apart from the pharmacological studies mentioned above, genetic approaches have also proved to be very useful for the study of the endocannabinoid system. Cravatt et al. (2001) generated mouse mutants lacking the FAAH gene (FAAH−/− mice). These animals have a 10–15-fold increase in the levels of anandamide and an enhanced response to the injection of this endocannabinoid, altered nociceptive responses and enhanced memory extinction (Cravatt et al., 2001, Varvel et al., 2007). Despite these results, the response of FAAH−/− mice in models of anxiety has remained uncertain. Furthermore, behavioural changes not related to an increased activation of the CB1 receptor have also been observed after genetic inhibition of FAAH (Wise et al., 2007).

Therefore, the aim of this study was to test the notion that FAAH−/− mice might show a reduced anxiety-like behaviour as compared to their wild-type (WT) littermates. The elevated plus maze (EPM) and the light dark test (LDT) were employed as animal models. Furthermore, in order to mimic these experiments pharmacologically, studies were conducted with injections of an FAAH inhibitor in C57BL/6N mice. Finally, to test whether there was an involvement of CB1 receptors, we investigated the effects of rimonabant in blocking the behavioural changes observed after genetic or pharmacological inhibition of FAAH.

Section snippets

Subjects

The animals used in this study were male C57BL/6N mice, FAAH knock out (FAAH−/−) mice and their WT littermates (FAAH+/+), with a weight of 20–30 g and age of 3–4 months. The generation of FAAH−/− mice was described previously (Cravatt et al., 2001). Mutant mice were backcrossed 6 times into C57BL/6N background and heterozygous breedings were utilized. Tail biopsy and ear clip sampling were performed at an age of 6–10 weeks, and genotyping by PCR was performed as described (Cravatt et al., 2001).

Pharmacological validation of the animal models of anxiety

As positive controls for the EPM and the LDT, wild-type male C57BL/6N mice received an injection of the anxiolytic diazepam (2 mg/kg) or its vehicle 30 min prior to the experiments. The effect of diazepam on locomotion was evaluated in an open-field, where no significant change in the total distance moved was observed (Table 1). In the EPM, diazepam induced an increase in the percentage of entries (vehicle 14.76 ± 6.68% and diazepam 66.75 ± 11.01%; t14 = 4.03, p = 0.0012; n = 8/group) and in the time spent

Discussion

The present study shows that FAAH−/− mice exhibit reduced anxiety-like behaviour in two experimental models. Since this phenotype was reversed after injection of the CB1 antagonist rimonabant, it is likely that elevated levels of eCBs acting via CB1 receptors are responsible for the decreased anxiety in animals lacking FAAH. Furthermore, an anxiolytic-like effect was observed in C57BL/6N mice after injection of the FAAH inhibitor URB597, an effect blocked by rimonabant, and, thus, also mediated

Acknowledgements

F.A.M. is a recipient of a fellowship from the Alexander von Humboldt Foundation (Germany). We thank Benjamin Cravatt for providing the FAAH−/− mice, Michel Steiner for valuable suggestions on the manuscript, Francisco Guimarães for critical comments on the statistical analyses, and Andrea Conrad and Anisa Kosan for the technical support with the breeding and genotyping of mice.

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