Facilitation of contextual fear memory extinction and anti-anxiogenic effects of AM404 and cannabidiol in conditioned rats

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Abstract

The present study investigated the central effects of the eCB uptake/metabolism inhibitor AM404 and the phytocannabinoid cannabidiol (CBD) on the extinction of contextual fear memories in rats. Rats were conditioned and 24 h later subjected to three consecutive 9-min non-reinforced exposures to the conditioning context (extinction sessions, 24 h intervals). AM404 or CBD was injected i.c.v. 5 min before each extinction session and a 3-min drug-free test of contextual memory was performed 24 h after the last extinction session. AM404 (1.0 µg/µl, i.c.v.) and CBD (2.0 µg/µl, i.c.v.) facilitated extinction of contextual fear memory, with persistent effects. These responses were antagonized by the CB1-selective antagonist SR141716A (0.2 mg/kg, i.p.), but not by the TRPV1-selective antagonist capsazepine (5.0 µg/µl, i.c.v.). The effect of the anxiolytic drug Diazepam (DZP) on the extinction of contextual fear memory was also investigated. In contrast with the CBD and AM404 results, DZP induced a general reduction in the expression of conditioned freezing. Both AM404 and CBD induced anti-anxiogenic effect in the fear-potentiated plus-maze test, whereas DZP was anxiolytic in conditioned and unconditioned rats. In conclusion, CBD, a non-psychoactive phytocannabinoid could be an interesting pharmacological approach to reduce the anxiogenic effects of stress and promote the extinction of fear memories.

Introduction

Cannabinoid compounds were first identified in extracts of the plant Cannabis sativa, which contains at least 66 compounds of this class. Among these compounds, Δ9-tetrahydrocannabinol (Δ9-THC) is the most widely studied and is considered to be responsible for the majority of Cannabis psychoactive effects (reviewed in Pertwee, 2006). The chemical isolation of Δ9-THC and of other major cannabinoid compounds such as cannabinol and cannabidiol (CBD) was promptly followed by its synthesis, which boosted the researches in the cannabinoid field (Gaoni and Mechoulam, 1964); for a historical review, see (Mechoulam and Hanus, 2000). Although Δ9-THC was readily recognized to be psychoactive, CBD has been considered to be a non-psychoactive cannabinoid (Bisogno et al., 2001, Di Marzo and Petrocellis, 2006, Mechoulam et al., 2002, Mechoulam et al., 1970). However, there is compelling experimental evidence suggesting that CBD induce effects in the central nervous system, being anxiolytic (Crippa et al., 2004) and antipsychotic (Zuardi et al., 1995) in humans and anxiolytic (Moreira et al., 2006, Resstel et al., 2006) and anticonvulsivant (Carlini et al., 1973, Consroe and Wolkin, 1977, Izquierdo et al., 1973) in laboratory animals. The pharmacology of CBD is not completely understood and several mechanisms of action have been proposed, including diffuse targets on the endocannabinoid (eCB) system (Bisogno et al., 2001), enhancement of adenosinergic signaling (Carrier et al., 2006), agonism of 5HT1A serotoninergic receptors (Mishima et al., 2005) and TRPV1 vanilloid receptors (Bisogno et al., 2001) (for recent reviews about the CBD pharmacology, see Mechoulam et al., 2007, Pertwee, 2008). Within the eCB system, CBD weakly binds to CB1 and CB2 receptors and inhibits the uptake and hydrolysis of anandamide, an endocannabinoid ligand (Bisogno et al., 2001). The pharmacological profile of CBD somewhat resembles that of AM404, a synthetic drug known to inhibit anandamide uptake/metabolism (Beltramo et al., 1997, De Petrocellis et al., 2000, Fegley et al., 2004, Fowler et al., 2004, Jarrahian et al., 2000) and to activate TRPV1 receptors (De Petrocellis et al., 2000, Zygmunt et al., 2000). Some of the in vivo effects of AM404 seem to involve enhanced anandamide levels and therefore indirect activation of CB1 cannabinoid receptors (Beltramo et al., 2000, Bortolato et al., 2006, Freund et al., 2003).

The eCB system is important for a number of physiological brain functions and there is an emerging interest in eCB-mediated modulation of emotionality (Kathuria et al., 2003, Viveros et al., 2005). Most cannabinoid effects in the brain occur through activation of CB1 receptors, which are densely expressed in regions known to play an important role in anxiety and aversive learning, including amygdala and hippocampus (Freund et al., 2003, Herkenham et al., 1990), where eCB-related enzymes, such as FAAH are also found (Egertova et al., 2003). Therefore, not only exogenous cannabinoids can influence anxiety, but also enhancement of eCB neurotransmission modulates it, inducing anxiolytic-like effects (Patel and Hillard, 2006). Since the finding that CB1 receptors play a pivotal role in extinction of conditioned fear (Marsicano et al., 2002), intense efforts have been made to further understand how the eCB system modulates aversive memories extinction and its possible consequences for anxiety pharmacotherapy. Given the similarities between extinction procedures and exposure-based psychotherapy used for the treatment of fear disorders in humans (Myers and Davis, 2007), it is believed that the eCB system represents a novel pharmacological target for anxiety disorders related to inappropriate retention of aversive memories (Chhatwal et al., 2005, Marsicano et al., 2002). So far, there is one report that the inhibition of eCB uptake/metabolism facilitates extinction of tone-cued fear-potentiated startle (Chhatwal et al., 2005), but it remains to be determined if this is true for other behavioral tasks.

In contextual fear conditioning, aversive memories are studied by exposing the animal to a context (e.g., conditioning chamber) where an aversive stimulus (normally a mild foot shock) is delivered (Rudy et al., 2004). Re-exposure to the same context induces conditioned fear responses, such as freezing behavior, defined by the absence of movements except for those necessary for breathing (Blanchard and Blanchard, 1969). Extinction of contextual fear memory is elicited with repeated or prolonged non-reinforced exposures to the context, which tends to decrease the conditioned fear responses (Pavlov, 1927); for a recent view, see (Myers and Davis, 2007). In a broader sense, memory extinction may reflect behavioral flexibility and adaptation to environmental changes (Hill et al., 2006, Kamprath et al., 2006). Bearing in mind that eCBs are released in specific brain areas during fear memory extinction (Marsicano et al., 2002), the aim of the present study was to investigate the effects of i.c.v. injection of the eCB metabolism/uptake inhibitor, AM404, and the phytocannabinoid, CBD, on the extinction of contextual conditioned fear in rats. Experiments of pharmacological antagonism were performed using SR141716A, a selective CB1 antagonist, and capsazepine, an antagonist of TRPV1 vanilloid receptors. The elevated plus-maze (EPM) test was used to investigate whether selected doses of CBD and AM404 induce anxiolytic-like effect in naive and/or conditioned rats.

Section snippets

Animals

Male adult Wistar rats (3 months old) bred and reared at the animal facility of our department were used. The animals were kept in collective plastic cages (4–5 rats/cage) with food and water available ad libitum. The animals were maintained in a room at a controlled temperature (23 ± 2 °C) under a 12:12-h light/dark cycle (lights on at 7:00 A.M.). Each behavioral test was conducted during the light phase of the cycle (9:00 A.M.–5:00 P.M.) using independent experimental groups consisting of 7–16

Experiment 1

Effects of i.c.v. administration of AM404 or CBD on the extinction of contextual fear memory. The timeline of the behavioral procedures of Experiment 1 is shown in Fig. 1A. The effects of AM404 (0.2, 1.0 or 2.0 µg/µl, i.c.v.) on the extinction of contextual fear memory are shown in Fig. 1B and D. Two-way ANOVA revealed significant effects of treatment [F(3,141) = 3.94, p < 0.01] and sessions [F(2,141) = 12.78, p < 0.01], but there was no effect of treatment × session interaction [F(6,141) = 0.25, p = 0.95].

Discussion

The present study demonstrates that the eCB uptake/metabolism inhibitor, AM404, and the relatively unheralded phytocannabinoid, CBD, facilitate the extinction of contextual fear memory in rats. These responses were antagonized by the CB1-selective antagonist SR141716A, but not by the TRPV1-selective antagonist CPZ, thus suggesting the involvement of CB1 cannabinoid receptors in the facilitation of extinction by these drugs. Moreover, animals treated with either AM404 or CBD during the

Role of the funding source

The Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil) supported this study, but had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

Contributors

RMB performed all the experiments and statistical analysis. FAP wrote the protocol and collaborated with the experiments. RNT, RMB and FAP performed the analysis of the data, managed the literature searches and the final manuscript. All authors have approved the final manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors would like to thank Sanofi-Aventis (France) for the donation of SR141716A for the present study. The assistance of Dr. Filipe S. Duarte is gratefully acknowledged. R.M.B. received fellowships from CAPES-Brazil and F.A.P. received fellowships from CNPq-Brazil. R.N.T. is the recipient of a CNPq fellowship.

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