Differential Effects of -Tetrahydrocannabinol and Methanandamide in CB1 Knockout and Wild-Type Mice

Mice devoid of CB1 cannabinoid receptors (CB1 / mice) provide a unique opportunity to further investigate the role of CB1 receptors in exocannabinoid and endocannabinoid effects. CB1 / mice (N 18) and their wild-type littermates (CB1 / mice; N 12) were placed in standard mouse operant chambers and trained to lever press under a fixed ratio 10 schedule of reinforcement. When stable lever press responding under the fixed ratio 10 schedule had been established, cannabinoids and noncannabinoids were administered to both groups. CB1 / mice acquired the lever press response more readily than CB1 / mice. -Tetrahydrocannabinol ( -THC) decreased lever press responding in CB1 / mice only, whereas methanandamide, a metabolically stable endocannabinoid analog, produced similar response rate decreases in both genotypic groups. Similar to -THC, another endocannabinoid analog, (R)-(20cyano-16,16-dimethyl docosa-cis-5,8,11,14-tetraeno)-1 -hydroxy-2 -propylamine (O-1812), decreased responding in CB1 / mice, but not in CB1 / mice. The CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) blocked the effects of -THC, but not those of methanandamide. Because methanandamide binds poorly to CB2 receptors, these results suggest possible non-CB1, non-CB2 mechanisms of action for methanandamide-induced behavioral disruption of lever press responding. Ethanol and morphine elicited greater response decreases in CB1 / mice than in CB1 / mice, suggesting a possible role of CB1 receptors in the rate disruptive effects of these drugs. In contrast, diazepam did not produce between group differences, suggesting that CB1 receptors are not involved in diazepam-induced disruption of lever press responding. -Tetrahydrocannabinol ( -THC), the principle psychoactive constituent of marijuana and other natural, synthetic, and endocannabinoids, elicits a well characterized syndrome of pharmacological effects in laboratory animals (Dewey, 1986; Martin et al., 1991). These effects, including hypothermia, hypoactivity, antinociception, and catalepsy, are attributed mainly to the actions of cannabinoids at CB1 cannabinoid receptors in the brain (Martin et al., 1991). Recently, recombinant DNA techniques have made possible the development of CB1 receptor knockout mice (CB1 / ) (Ledent et al., 1999; Zimmer et al., 1999). These mice lack CB1 receptors and provide a unique opportunity to further investigate the role of these receptors in the pharmacological effects of cannabinoids. Research with CB1 / mice demonstrates that the characteristic antinociceptive, hypoactive, and cataleptic effects of -THC are absent in these mice (Zimmer et al., 1999; Di Marzo et al., 2000). Moreover, -THC stimulates [S]GTP S binding in brain membranes of CB1 / mice, but not in those of CB1 / mice (Di Marzo et al., 2000). Thus far, the measures examining the pharmacological effects of cannabinoids in CB1 / mice, albeit important, represent unlearned responses. Previous research has demonstrated that cannabinoids, including -THC, WIN 55,212-2, and methanandamide, also disrupt performance in the Morris water maze, a working memory task (Varvel and Lichtman, 2002). The effects of cannabinoids on schedulecontrolled operant behavior have not been investigated in these mice. In laboratory animals, cannabinoids dose dependently decrease rates of responding from operant baseline in schedule-controlled lever-pressing tasks (Carriero et al., 1998). Moreover, cannabinoid suppression of responding is extensively documented in animal drug discrimination paradigms (Wiley, 1999). CB1 receptors are the proposed mediators of cannabinoid-induced suppression of operant lever This research was supported by National Institute on Drug Abuse Grants DA-03672, DA-09789, and predoctoral award DA-14823. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.055376. ABBREVIATIONS: -THC, -tetrahydrocannabinol; GTP S, guanosine 5 -O-(3-thio)triphosphate; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; WIN 55212-2, (R)-( )-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone; ANOVA, analysis of variance; O-1812, (R)-(20-cyano-16,16-dimethyl docosa-cis-5,8,11,14-tetraeno)-1 -hydroxy-2 -propylamine. 0022-3565/04/3091-86–91$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 309, No. 1 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 55376/1135330 JPET 309:86–91, 2004 Printed in U.S.A. 86 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from responding (Carriero et al., 1998). Thus, this study is the first examination of a learned lever pressing task in CB / mice. In this study, -THC and endocannabinoid analogs methanandamide and O-1812 (Fig. 1) were used to assess the effects of CB1 receptor deletion on cannabinoid disruption of lever press responding. Whereas -THC binds with similar affinity to cannabinoid CB1 receptors (Ki 40.7 1.7 nM) and CB2 receptors (Ki 36.4 10 nM) (Showalter et al., 1996), methanandamide and O-1812 bind with considerably higher affinity to CB1 receptors (Ki 137 20 and Ki 3.4 0.5 nM, respectively) (Adams et al., 1995; Di Marzo et al., 2001a) than to CB2 receptors (Ki 3024 705 and Ki 3870 235 nM, respectively) (our unpublished data; Di Marzo et al., 2001a). To evaluate the pharmacological specificity of effects in this model, we also tested three drugs (morphine, ethanol, and diazepam) that share pharmacological effects with cannabinoids in other assays, albeit these effects are mediated via different (noncannabinoid) mechanisms (Wiley and Martin, 2003). Materials and Methods Subjects. Heterozygotes (CB1 / mice) (obtained from A. Zimmer, National Institute of Mental Health, Bethesda, MD) were bred at Virginia Commonwealth University to provide CB1 / mice, CB1 / mice, and additional CB1 / mice. Experimentally naive, male C57BL/6J CB1 / and CB1 / genotype mice (20–25 g) from this colony were housed individually in clear plastic cages (18 29 13 cm) with steel wire fitted tops and wood-chip bedding. Mice were transported daily (Monday–Friday) from an animal colony room (12-h light/dark cycle, 22–24°C) to the laboratory where experimental training and testing sessions occurred. Sessions were conducted during the light cycle. To initiate the lever press response, the mice were maintained at 85% of their free-feeding body weights by restricting their daily food ration of standard rodent chow. When stable rates of responding were achieved, the mice were allowed to gradually gain weight as operant training progressed, culminating in free feed status as long as the mice consistently passed testing criterion. Ad libitum water access in the home cages was permitted. The mice were cared for according to the Guide for the Care and use of Laboratory Animals (National Institutes of Health, 1985). Apparatus. Six computer-interfaced operant chambers, the construction of which has been described previously (Balster and Moser, 1987), were used for behavioral training and testing. In brief, the inner test chambers consisted of a 15-cm long 11.5-cm deep 17.5-cm high area surrounded by an aluminum chassis box with a single Plexiglas side containing a door. Each inner chamber was equipped with two response levers (8 cm apart) that extended 0.8 cm into the chamber from a wall of the box and were positioned 2.5 cm above a floor constructed of parallel stainless steel rods. Above each lever was a house light, which was illuminated when an experimental session was in progress. Midway between the levers was a recessed food trough into which a commercial dipper (model E14-05; Coulbourn Instruments, Lehigh Valley, PA) delivered 0.02 ml of sweetened condensed milk (by volume: 1 part condensed milk, 1 part sugar, and 2 parts water). Sweetened condensed milk has been used successfully as a reinforcer in previous studies (Balster and Moser, 1987). Inner test chambers were enclosed within soundand lightattenuating cubicles. Drugs. -THC (National Institute on Drug Abuse, Rockville, MD), methanandamide (Organix, Inc., Woburn, MA), O-1812 (Organix, Inc.), and SR141716A (Pfizer Inc., Groton, CT) were dissolved in a 1:1:18 vehicle mixture of absolute ethanol, Emulphor-620 (RhônePoulenc, Inc., Princeton, NJ), and saline. Morphine sulfate (National Institute on Drug Abuse) was dissolved in 0.9% saline. Diazepam (Elkins-Sinn, Inc., Cherry Hill, NJ), commercially purchased at a concentration of 5 mg/ml, was diluted with absolute ethanol, propylene glycol and sterile water in a ratio of 1:1:8. Absolute ethanol (Aaper Alcohol and Chemical, Shelbyville, KY) was diluted with sterile distilled water. All injections were administered intraperitoneally in a volume of 10 ml/kg. With the exception of ethanol, vehicles for each dose-effect curve corresponded to drug diluent. The vehicle for ethanol was 0.9% saline. -THC was injected 30 min before the start of a test session. Methanandamide, O-1812, morphine, and diazepam were administered 15 min before the start of a test session. Ethanol was administered 20 min presession. SR141716A (3.0 mg/kg) was injected 10 min before the experimental session when administered alone and 10 min before the administration of drug when used as an antagonist. Mice were tested in a within-subjects design and received every drug dose within a given dose-effect curve. The number of animals tested across drugs differs due to the deaths or illnesses of animals during the long time span of this study, and the addition of four CB1 / knockout mice that were added later in the study to replace mice that did not learn the operant task. Procedure. During daily (Monday–Friday) 15-min training sessions, each mouse was placed in a standard operant chamber and trained to press the right lever for 0.02 ml of sweetened condensed milk according to an fixed ratio 1 schedule of reinforcement, i.e., milk reinforcement was delivered after every right lever press. During acquisition, training, and testing, the house light and the dipper were the only programmed stimuli. Throughout the study, there were no programmed consequences for presses on the left lever. When the lever press response under the fixed ratio 1 reinforcement schedule had been acquired, the ratio value was gradually increased to a final value of fixed ratio 10, in which 10 responses on the right lever were required for delivery of milk reinforcement. Fixed ratio values were increased within sessions subjectively. When stable rates of responding under the fixed ratio 10 schedule of reinforcement were reached (approximately 8–10 training sessions after acquisition of the fixed ratio 10), cannabinoid and noncannabinoid drug testing began. Test sessions were scheduled if response rates (response per second) during the training session preceding test day were within 20% of the average response rate of the five previous training sessions. Tests were usually conducted on Tuesdays and Fridays. Data Analysis. During training and testing, response rates were calculated for the entire session. Test response rates were converted to a percentage of the control response rate during corresponding vehicle test sessions. For each dose-effect determination, responding during vehicle was compared with responding during administration of varying drug doses. Separate split-plot analyses of variance (ANOVA) were conducted for each drug’s dose-effect determination (between subjects factor genotype; within subjects factor dose) using SigmaStat statistical software version 2.0 (Jandel CorporaFig. 1. Chemical structure of -THC, anandamide, methanandamide, and O-1812. Operant Behavior in CB1 Knockout Mice 87 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from tion, San Rafael, CA). Significant ANOVAs were further analyzed with Student-Newman-Keuls post hoc tests ( 0.05) to specify differences between means. Results of the ANOVA for O-1812 data suggested that the curvilinear nature of dose-effect curves for both genotypes may have interfered with our ability to determine significance with ANOVA. To investigate this hypothesis in further detail, we converted response rates for vehicle and the highest 30-mg/kg dose into qualitative data (i.e., rats that pressed the lever at least once were categorized as responders and those that did not press the lever at all were categorized as nonresponders). A 2 test of homogeneity ( 0.05) was performed on the resulting conversions. Because all vehicle-treated mice responded, expected frequencies were calculated based upon 100% of animals in each group as responders.