The Endogenous Cannabinoid Anandamide Produces -9- Tetrahydrocannabinol-Like Discriminative and Neurochemical Effects That Are Enhanced by Inhibition of Fatty Acid Amide Hydrolase but Not by Inhibition of Anandamide Transport

Anandamide is an endogenous ligand for brain cannabinoid CB1 receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that anandamide, given i.v. or i.p., would produce discriminative effects like those of -9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methanandamide (AM-356), a metabolically stable anandamide analog, produced clear dose-related THC-like discriminative effects, but anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3 -carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of anandamide, with 3 mg/kg anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of anandamide after URB-597 and methanandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of anandamide or its dopamine-elevating effects. Thus, anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of anandamide. The endogenous cannabinoid (CB) system, which is targeted by the psychoactive ingredient in cannabis, -9-tetrahydrocannabinol (THC), comprises receptors termed CB1 and CB2 (and others not yet identified) endogenous compounds that activate these receptors and enzymes involved in the synthesis and degradation of these endogenous cannabinoids This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health (NIH), Department of Health and Human Services; by NIH Grants DA09158, DA7215, DA12413, and DA12447; and by the Centre National de la Recherche Scientifique. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.114124. ABBREVIATIONS: CB, cannabinoid; THC, -9-tetrahydrocannabinol; FAAH, fatty acid amide hydrolase; AM-356, methanandamide, R-( )-arachidonyl1 -hydroxy-2 -propylamide; AM-404, N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide; UCM-707, N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide; VDM-11, N-(4-hydroxy-2-methylphenyl) arachidonoyl amide; LY2318912, (5-[(4-azido-3-iodo-benzoylamino)-methyl]-tetrazole-1-carboxylic acid dimethylamide); OL-135, 7-phenyl-1-(5-pyridin-2-yl-oxazol-2-yl)-heptan-1-one; NAc, nucleus accumbens; SR141716, N-piperidino-5-(4-chlorophenyl)-1(2, 4-dichlorophenyl)-4-methylpyrazole-3-carboxamide; VR, vanilloid receptor; Rim, rimonabant; ANOVA, analysis of variance; DA, dopamine. 0022-3565/07/3211-370–380 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 321, No. 1 U.S. Government work not protected by U.S. copyright 114124/3188273 JPET 321:370–380, 2007 Printed in U.S.A. 370 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from (Freund et al., 2003; Piomelli, 2003; Di Marzo et al., 2004). A way to investigate whether exogenous and endogenous cannabinoids produce qualitatively similar effects is to use drugdiscrimination procedures (Solinas et al., 2006b), with THC as the baseline training drug (Browne and Weissman, 1981; Wiley et al., 1995; Burkey and Nation, 1997; Jarbe et al., 2001; Solinas et al., 2003; Alici and Appel, 2004). Among the endogenous ligands for cannabinoid CB1 receptors, anandamide is the best-characterized (Freund et al., 2003; Piomelli, 2003; Di Marzo et al., 2004). Anandamide is synthesized on demand, binds with high affinity to extracellular CB1 receptors, and is rapidly inactivated, presumably by a two-step process thought to involve active transport into neurons by a yet-to-be identified membrane transport mechanism followed by FAAH hydrolysis (Freund et al., 2003; Piomelli, 2003; Di Marzo et al., 2004; Moore et al., 2005), although the existence of a transport process has been a matter of debate (Glaser et al., 2005; Dickason-Chesterfield et al., 2006). It is probably because of this rapid inactivation that anandamide has failed to produce THC-like effects in previous drug-discrimination studies, because methanandamide, a longer-lasting synthetic analog of anandamide that is not metabolized by FAAH, produces complete generalization to THC training stimuli under similar conditions (Burkey and Nation, 1997; Jarbe et al., 2001). Pharmacological tools have been developed that inhibit these two mechanisms of anandamide inactivation (Piomelli, 2003). For example, compounds, such as AM-404 [N-(4-hydroxyphenyl)-eicosa5,8,11,14-tetraenamide], VDM-11, UCM-707 [N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide], and LY2318912, inhibit the transport of anandamide into neurons (Piomelli et al., 1999; De Petrocellis et al., 2000; Lopez-Rodriguez et al., 2003; Glaser et al., 2005; Moore et al., 2005), and compounds, such as cyclohexyl carbamic acid 3 -carbamoyl-biphenyl-3-yl ester (URB-597) and OL-135, inhibit the activity of FAAH (Kathuria et al., 2003; Lichtman et al., 2004). Administration of these compounds or genetic ablation of FAAH results in increases in anandamide concentrations in plasma and some brain areas (Giuffrida et al., 2000; Kathuria et al., 2003; Fegley et al., 2004; Lichtman et al., 2004; Bortolato et al., 2006) and in the potentiation and prolongation of many of the effects of anandamide (Calignano et al., 1997; Cravatt et al., 2001; Kathuria et al., 2003). We recently found that both anandamide and methanandamide increase extracellular dopamine levels in the nucleus accumbens shell when injected intravenously (Solinas et al., 2006a), a characteristic effect of most drugs that have reinforcing or rewarding effects in experimental animals and that are abused by humans (Di Chiara, 2002; Wise, 2002). Anandamide and methanandamide produced similar peak effects, but the effects of methanandamide were more prolonged. Inhibition of FAAH by administration of URB-597 dramatically potentiated both the magnitude of peak elevations in dopamine levels in the nucleus accumbens shell produced by anandamide and the duration of these elevations (Solinas et al., 2006a). These results were obtained after i.v. injections of anandamide, a route of administration that might have reduced its hepatic first-passage metabolism, thus increasing concentrations of anandamide in the brain. Because of previous failures to demonstrate significant THC-like discriminative effects of anandamide when given intraperitoneally or intramuscularly, we tested the hypothesis that anandamide would produce THC-like discriminative effects when administered intravenously. We first trained rats to discriminate i.p. injections of 3 mg/kg THC from saline with a single daily session procedure (Solinas et al., 2003) followed by training with a multiple sessions per day procedure until a baseline was reestablished and THC doseresponse curves were determined by testing different i.p. and i.v. doses of THC. We determined whether i.v. or i.p. injections of anandamide alone or in combination with FAAH inhibition by URB-597 or transport inhibition by AM-404 or UCM-707 produced THC-like discriminative effects and compared these effects with those obtained with i.p. anandamide. We also compared effects of i.v. anandamide and methanandamide and assessed the receptors involved in the effects of anandamide by treating rats with specific cannabinoid CB1 and vanilloid VR1 receptor antagonists before administering anandamide. Finally, we performed parallel in vivo microdialysis experiments to compare these behavioral effects with the effects of i.p. and i.v. anandamide, given alone or in combination with FAAH inhibition by URB597 or transport inhibition by AM-404, on dopamine levels in the shell of the nucleus accumbens. Materials and Methods Subjects. For drug-discrimination studies, male Sprague-Dawley rats initially weighing 350 to 380 g (Charles River, Wilmington, MA) were housed individually. Weights of rats were gradually reduced to approximately 85% of free feeding by limiting daily access to food before the start of drug-discrimination training sessions. Once drugdiscrimination sessions were started, weight was maintained at approximately 85% of free feeding by giving 15 g of food pellets shortly after the end of each daily session. Water was available ad libitum. For microdialysis studies, male Sprague-Dawley rats weighing 300 to 350 g (Charles River) were used. They were housed two per group with food and water available ad libitum. All rats were housed in temperatureand humidity-controlled rooms and were maintained on 12-h light/dark cycles. The lights were on from 6:45 AM to 6:45 PM, and experiments were conducted during the light phase. Animals used in these studies were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC), and all experiments were conducted in accordance with the guidelines of the Institutional Care and Use Committee of the Intramural Research Program, National Institute on Drug Abuse (NIDA), National Institutes of Health and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003). Drugs. THC and rimonabant (SR141716, N-piperidino-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methylpyrazole-3-carboxamide) were obtained from the National Institute on Drug Abuse, NIH (Rockville, MD). Anandamide and capsazepine were purchased from Sigma/RBI (St. Louis, MO). UCM-707 was purchased from Tocris Cookson (Ellisville, MO). Methanandamide [AM-356, R-( )-arachidonyl-1 -hydroxy-2 -propylamide] and AM-404 were provided by Dr. Alex Makriyannis (Center for Drug Discovery, Northeastern University, Boston, MA) and University of Connecticut, Center for Drug Discovery and Departments of Pharmaceutical Sciences and Molecular Cell Biology (Storrs, CT). URB-597 was provided by Dr. Daniele Piomelli (Department of Pharmacology, University of California, Irvine, CA). THC, 50 mg/ml in ethanol, was dissolved in a solution, 40% w/v of -hydroxy-cyclodextrine (Sigma/RBI) for i.p. administration and in vehicle containing 2% Tween 80, 2% ethanol, and 96% saline for i.v. administration. All the other drugs were dissolved in Anandamide Produces THC-Like Central Effects 371 at A PE T Jornals on N ovem er 4, 2017 jpet.asjournals.org D ow nladed from vehicle containing 2% Tween 80, 2% ethanol, and 96% saline and were injected in a volume of 1.0 ml/kg i.p. or i.v., with the exception of URB-597, which was dissolved in a vehicle containing 20% dimethyl sulfoxide in saline and injected in a volume of 2.0 ml/kg i.p. Drug-Discrimination Apparatus. Standard operant-conditioning chambers (Coulbourn Instruments, Lehigh Valley, PA) were used. Each chamber contained two levers separated by a recessed tray into which a pellet dispenser could deliver 45 mg of food pellets (F0021; Bioserv, Frenchtown, NJ). Each press of a lever with a force of 0.4 N through 1 mm was recorded as a response and was accompanied by an audible click. The operant-conditioning chambers were controlled by computers using a MED-PC software package (MED Associates, East Fairfield, VT). Single Session per Day Training Procedure. Rats were trained under a discrete-trial schedule of food-pellet delivery to respond on one lever after an injection of a training dose of 3 mg/kg THC and on the other lever after an injection of 1 ml/kg THC vehicle. Injections of THC or vehicle were given i.p. 30 min before the start of the session. At the start of the session, a white house light was turned on and, in its presence, the rats were required to make 10 consecutive responses (fixed-ratio 10 schedule of food delivery) on the lever appropriate to the presession treatment. The completion of 10 consecutive responses on the injection-appropriate lever produced delivery of a 45-mg food pellet and initiated a 45-s time-out during which lever-press responses had no programmed consequences and the chamber was dark. Responses on the injection-inappropriate lever reset the fixed-ratio requirement on the injection-appropriate lever. After each time-out, the white house light was again turned on and the next trial began. Each session ended after completion of 20 fixed-ratio trials or after 30 min had elapsed, whichever occurred