The endogenous cannabinoid anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase (FAAH) inhibition
Introduction
Converging evidence indicates that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes (Tanda et al., 1997, Tanda et al., 2000, Ledent et al., 1999, Justinova et al., 2005, Solinas et al., 2006) and emotional responses to stress (for review see: Valverde, 2005). However, little is known about direct effects of endogenous ligands for cannabinoid receptors on brain reward processes and emotional responses to stress. Anandamide (N-arachidonylethanolamide) was the first identified endogenous ligand for brain cannabinoid receptors (Devane et al., 1992). It is produced upon demand through activity-dependent cleavage of membrane lipid precursors, and is released from cells immediately after its production. After release, anandamide is rapidly eliminated through a two-step process involving transport into cells followed by intracellular hydrolysis to arachidonic acid and ethanolamide, catalyzed by fatty acid amide hydrolase (FAAH) (for review see: Piomelli, 2003). This rapid inactivation of anandamide has made study of its in vivo behavioral and neurochemical effects difficult. However, selective inhibitors of FAAH, such as URB597 (cyclohexyl carbamic acid 3′-carbamoyl-3-yl ester) have recently been developed which can be used to magnify and prolong the effects of both endogenously released and exogenously administered anandamide (Kathuria et al., 2003, Lichtman et al., 2004, Mor et al., 2004, Fegley et al., 2005, Solinas et al., 2006, Solinas et al., 2007).
To date, there are only a few studies showing that endogenous cannabinoids such as anandamide interact and participate in the neuronal circuitry that signals rewarding events and controls emotional responses to stress. We recently demonstrated that intravenously administered anandamide is an effective reinforcer of drug-taking behavior in squirrel monkeys (Justinova et al., 2005) and produces THC-like discriminative effects in rats that are potentiated after FAAH inhibition by URB597 (Solinas et al., 2007). These effects of anandamide were blocked by the CB1-receptor antagonist rimonabant (SR141716). They were obtained with intravenous injection of anandamide, a route of administration that results in a more rapid onset and greater magnitude of effects than the more commonly used intraperitoneal route of administration (Smith et al., 1994). Intravenously administered anandamide also produces a rapid, short-lasting increase in extracellular levels of dopamine (DA) in the nucleus accumbens shell of rats, which is magnified and prolonged by URB597 and blocked by rimonabant and is a neurobiological marker of most drugs abused by humans (Solinas et al., 2006).
In this study, we first investigated the effects of intravenously administered anandamide with a place-conditioning paradigm, a procedure widely used in rodents to assess drug effects related to reward or aversion, that can be used to explore motivational brain reward processes related to drugs abused by humans (Mucha et al., 1982, Schechter and Calcagnetti, 1998, Tzschentke, 1998). Since anandamide is rapidly inactivated by FAAH enzymes, we also investigated the effects of intravenous anandamide after FAAH inhibition by URB597. For comparison, we examined the motivational effects of intravenous administration of the synthetic CB1-receptor agonist WIN 55,212-2 with the same place-conditioning procedure. In addition, we attempted to reverse any effects obtained in the place-conditioning experiments with AM251, a selective CB1-receptor antagonist (an analog of rimonabant with greater selectivity for CB1- over CB2-receptors; Gatley et al., 1996, Gatley et al., 1997).
It is known that the endocannabinoid system plays a modulatory role in emotional states such as anxiety and fear (Viveros et al., 2005). Several studies utilizing rodent models of anxiety or depression showed that FAAH inhibition produced anxiolytic-like effects (Kathuria et al., 2003, Patel and Hillard, 2006) and anti-depressant-like effects (Gobbi et al., 2005) in rats and mice, although these effects are not always found (Naidu et al., 2007). Also, the intra-dorsolateral periaqueductal injection of a low dose of anandamide has been reported to produce anxiolytic-like effects when assessed with the elevated plus-maze (Moreira et al., 2007). Consequently, in separate groups of animals, we next investigated the effects of intravenously administered anandamide alone and after FAAH inhibition by URB597 on anxiety-like responses at doses and combinations employed in the place-conditioning experiments. We used a light/dark test, a paradigm based on the innate aversion of rodents to brightly illuminated areas and on the spontaneous exploratory behavior of rodents in response to mild stressors, such as, novel environments and light (Crawley and Goodwin, 1980). Finally, to control for motor disturbances that anandamide or URB597 might induce, which could influence effects in the place-conditioning and light/dark box experiments, we investigated the effects of anandamide and URB597, alone and in combination, on spontaneous locomotor activity in an open-field test.
Section snippets
Animals
Subjects were male Sprague–Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) weighing 300–325 g at the beginning of the experiments. They were individually housed in a temperature- and humidity-controlled room on a 12-h light/dark cycle. Experiments were conducted during the light phase. Food and water were available ad libitum and each animal was adapted to daily handling for 1 week before the start of experiments. Animals used in this study were maintained in facilities fully
Place-conditioning study
No significant baseline differences were found between the time spent by the different groups of rats in the two compartments of the place-conditioning apparatus during the pre-conditioning session before conditioning sessions were started.
Discussion
In the present study with Sprague–Dawley rats, the motivational effects of the endogenous cannabinoid receptor ligand anandamide in a place-conditioning procedure designed to measure rewarding or aversive effects of psychoactive drugs were compared with the emotional effects of anandamide in a light/dark box designed to measure anxiety. Intravenously administered anandamide, by itself, did not produce either conditioned place preferences or conditioned place aversions. However, when rats were
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, National Institute on Drug Abuse, Department of Health and Human Services, by NIH Grants DA09158, DA7215, DA12413, and DA12447, by the Institute of Experimental Medicine, Hungarian Academy of Sciences, and by the Italian Ministry of University and Scientific Research (MIUR) and the Centre of Excellence on ‘Neurobiology of Dependence’.
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