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BEHAVIORAL PHARMACOLOGY

Differential Effects of ⁹-Tetrahydrocannabinol and Methanandamide in CB1 Knockout and Wild-Type Mice

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia

Received for publication June 5, 2003
Accepted December 17, 2003.

	Abstract

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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. ⁹-Tetrahydrocannabinol (⁹-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 ⁹-THC, another endocannabinoid analog, (R)-(20-cyano-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 ⁹-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.

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 ⁹-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 schedule-controlled 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 responding (Carriero et al., 1998). Thus, this study is the first examination of a learned lever pressing task in CB-/- mice.

In this study, ⁹-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 ⁹-THC binds with similar affinity to cannabinoid CB1 receptors (K_i = 40.7 ± 1.7 nM) and CB2 receptors (K_i = 36.4 ± 10 nM) (Showalter et al., 1996), methanandamide and O-1812 bind with considerably higher affinity to CB1 receptors (K_i = 137 ± 20 and K_i = 3.4 ± 0.5 nM, respectively) (Adams et al., 1995; Di Marzo et al., 2001a) than to CB2 receptors (K_i = 3024 ± 705 and K_i = 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).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Chemical structure of 9-THC, anandamide, methanandamide, and O-1812.

	Materials and Methods

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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 x 29 x 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 x 11.5-cm deep x 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 sound- and light-attenuating cubicles.

Drugs. ⁹-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 (Rhône-Poulenc, 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. ⁹-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 Corporation, 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.

	Results

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The results of initial acquisition of the fixed ratio criterion are shown in Fig. 2. Acquisition of the lever press response differed in the two groups with CB1+/+ mice (N = 12) acquiring the lever press criterion more readily than CB1-/- mice (N = 14). The entire group of 12 CB1+/+ mice successfully responded until the required fixed ratio 10 criterion was met. In contrast, seven of the initial 14 CB1-/- mice failed to reach criterion and were removed from the study due to lack of responding (two after session 19, three after session 22, and two after session 27). Four additional CB1-/- mice were added to the study later as replacements. Table 1 shows mean operant responding in CB1-/- and CB1+/+ mice after administration of the vehicle for cannabinoid and noncannabinoid compounds. Overall rates of lever press responding under vehicle controls were less in CB1-/- mice than in CB1+/+ mice. Therefore, data were converted to mean percentage of control responding.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Acquisition of fixed ratio 10 criterion in CB1+/+ () and CB1-/- mice (). Seven of the initial 14 CB1-/- mice failed to reach criterion and were removed from the study (two after session 19, three after session 22, and two after session 27). Values reflect mean (± S.E.M.) of the mean fixed ratios at the end of each session.

Figure 3 shows the effects of cannabinoids on operant responding in CB1-/- and CB1+/+ mice. ⁹-THC produced significant dose-dependent decreases in responding in CB1+/+ mice (Fig. 3A), whereas ⁹-THC did not elicit significant decreases in responding in CB1-/- mice at any dose up to 56 mg/kg. In contrast to results with ⁹-THC, methanandamide produced similar decreases in responding between groups (Fig. 3B). Compared with vehicle control, both CB1-/- and CB1+/+ mice displayed significant decreases in responding at the 100 mg/kg methanandamide dose only. Although ANOVA revealed no significant decreases in responding of CB1-/- and CB1+/+ mice after administration of O-1812, the curvilinear nature of the dose-effect curves may have obscured the ability of ANOVA to detect any differences (Fig. 3C). All mice in both groups responded after vehicle injection. Results of a ² test of homogeneity, however, showed that a significantly greater number of CB1+/+ mice failed to respond (nonresponders, 7 of 9) after injection with 30 mg/kg O-1812 compared with CB1-/- mice (nonresponders, 1 of 7). Hence, when data were considered qualitatively, 30 mg/kg O-1812 decreased responding in CB1+/+ mice, but not in CB1-/- mice.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Mean responding (±S.E.M.) in CB1+/+ () and CB1-/- mice () after administration of 9-THC (A), methanandamide (B), and O-1812 (C). Only CB1-/- mice received 56 mg/kg 9-THC. Therefore, these data were not included in the statistical analysis. ANOVA results: *, P < 0.05 versus vehicle control within groups; #, P < 0.05 across groups at a given dose; and +, P < 0.05 main effect of dose (compared with vehicle). In addition, due to curvilinear nature of dose-effect functions for O-1812, these data were also analyzed qualitatively with 2 test of homogeneity (i.e., responders versus nonresponders). Results showed that 30 mg/kg O-1812 significantly suppressed response rates in CB1+/+ mice, but not in CB1-/- mice.

Figure 4 shows the effects of SR141716A on operant responding in CB1-/- and CB1+/+ mice. SR141716A (3.0 mg/kg) administered alone did not decrease responding significantly from vehicle in CB1-/- and CB1+/+ mice (Fig. 4A). When administered before 30 mg/kg ⁹-THC, however, SR141716A antagonized its rate decreasing effects in CB1+/+ mice (Fig. 4B). Because ⁹-THC did not produce effects in CB1-/- mice, SR141716A was not used to antagonize any dose of ⁹-THC in CB1-/- mice. In contrast, SR141716A administered before 100 mg/kg methanandamide did not antagonize the rate decreasing effects of this drug in either group of mice (Fig. 4C).

View larger version (16K): [in this window] [in a new window]   Fig. 4. A, mean responding (±S.E.M.) after administration of vehicle (white column) and 3 mg/kg SR141716A (black column). B, mean responding (±S.E.M.) after administration of vehicle (white column), 9-THC (black column: 30 mg/kg in CB1+/+ mice; 56 mg/kg in CB1-/- mice), and 30 mg/kg 9-THC antagonized by 3 mg/kg SR141716A (striped column) in CB1+/+ mice. C, mean responding (±S.E.M.) after administration of vehicle (white column), 100 mg/kg methanandamide (black column), and lack of antagonism of 100 mg/kg methanandamide by 3 mg/kg SR141716A (striped column). *, P < 0.05 versus vehicle control within groups.

Figure 5 shows the effects of ethanol, morphine, and diazepam on operant responding in CB1-/- and CB1+/+ mice. Ethanol doses of 0.5, 1, and 2 g/kg decreased responding to a significantly greater extent in CB1-/- mice than in CB1+/+ mice (Fig. 5A). Furthermore, whereas 0.25, 0.5, and 2 g/kg ethanol significantly reduced responding from vehicle levels in CB1-/- mice, only 2 g/kg ethanol produced significant decreases in CB1+/+ mice. In contrast to ethanol, morphine produced different effects in CB1-/- and CB1+/+ mice only at the 3 mg/kg dose (Fig. 5B). Whereas 3 mg/kg morphine significantly decreased responding in CB1-/- mice, it did not do so in their CB1+/+ littermates. At doses of 5.6 and 10 mg/kg, morphine produced significant decreases in responding in both CB1-/- and CB1+/+ mice. As shown in Fig. 5C, diazepam doses of 1.25, 2.5, 5, and 10 mg/kg produced similar reductions in responding from vehicle in CB1-/- and CB1+/+ mice.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Mean fixed ratio responding (±S.E.M.) in CB1+/+ () and CB1-/- mice () after administration of ethanol (A), morphine (B), and diazepam (C). *, P < 0.05 versus vehicle control within groups. #, P < 0.05 across groups at a given dose. +, P < 0.05 main effect of dose (compared with vehicle).

	Discussion

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Acquisition of the lever press response differed in CB1-/- and CB1+/+ mice with CB1+/+ mice acquiring the response more readily than CB1-/- mice. Several alternative explanations of these data are possible. First, differences in motivation for food may have contributed to differences in acquisition. Under drug-free conditions, the exploratory behaviors of CB1-/- mice were reduced in comparison with CB1+/+ mice (Steiner et al., 1999). Similarly, initiation of milk suckling in newborn CB1-/- mice was significantly reduced compared with CB1+/+ mice (Fride et al., 2003). CB1-/- mice also consumed less food overall than CB1+/+ mice and the CB1 receptor antagonist SR141716A has been shown to decrease food intake in mice (Di Marzo et al., 2001b). These data suggest that CB1 receptors may control mechanisms that regulate initiation of goal-directed, motivational behaviors and may play a critical role in food consumption (Steiner et al., 1999; Fride et al., 2003). Thus, it may be that removal of CB1 receptors reduced motivation to press the lever for food reward in CB1-/- mice. Second, consistent with previous research demonstrating reduced locomotor activity in CB1-/- mice compared with CB1+/+ mice (Zimmer et al., 1999), motor suppression may have contributed to the observed decrease in initiation of the lever press response in CB1-/- mice. This alternative is consistent with the fact that overall rates of responding under vehicle control for CB1-/- mice were less than those of CB1+/+ mice (Table 1). A third alternative is that acquisition deficits in CB1-/- mice are the result of deletion of all CB1 receptors in the hippocampus, a central nervous system area highly associated with learning and memory and high in CB1 receptor density (Herkenham et al., 1991). Because cannabinoids impair learning and memory in a variety of mammalian species, including humans, enhanced memory in CB1-/- mice would have been anticipated. Yet, variations in memory effects in CB1-/- mice have been reported (Reibaud et al., 1999; Varvel and Lichtman, 2002). Hence, memory enhancement or suppression may be task specific. Finally, our results are consistent with other "paradoxical effects" observed in CB1-/- mice (Steiner et al., 1999) and may be the result of adaptations in neuronal activity in these mice (Steiner et al., 1999; Fride et al., 2003).

Whereas 30 mg/kg ⁹-THC almost abolished responding in CB1+/+ mice, the effects of ⁹-THC were absent in CB1-/- mice up to doses of 56 mg/kg, producing a more than 10-fold difference in the minimally effective dose between the genetic lines. Thus, it seems that CB1 receptor activation accounts for ⁹-THC-induced disruption of lever press responding. The reversal of these effects in CB1+/+ mice by SR141716A strongly supports this suggestion. These data agree with prior research demonstrating differences between CB1-/- and CB1+/+ mice in other ⁹-THC-induced effects, including hypoactivity, catalepsy, and antinociception (Zimmer et al., 1999; Di Marzo et al., 2000).

In contrast to results with ⁹-THC, methanandamide (Fig. 1), a synthetic anandamide analog, failed to produce significant differences in responding in CB1-/- and CB1+/+ mice. These data concur with earlier reports demonstrating differential effects of ⁹-THC and anandamide in CB1-/- and CB1+/+ mice. For example, whereas the characteristic analgesic, cataleptic, and hypoactive effects of ⁹-THC were produced in CB1+/+ mice only, anandamide precipitated these effects in both genotypic groups (Di Marzo et al., 2000). Similarly, ⁹-THC-induced stimulation of [³⁵S]GTPS binding in brain membranes of CB1+/+ mice only contrasts with anandamide's stimulation of [³⁵S]GTPS binding in both genotypic groups (Di Marzo et al., 2000). Non-CB1, non-CB2 receptor activation may account for these differences (Di Marzo et al., 2000). Potential differences in receptor activation underlying endocannabinoid-induced disruption of operant responding are strongly supported by SR141716A attenuation of ⁹-THC-induced disruption in CB1+/+ mice, but lack of reversal of methanandamide's effects in either group. Likewise, previous research revealed that SR141716A clearly attenuated the hypoactive, antinociceptive, cataleptic, and hypothermic effects of ⁹-THC (Rinaldi-Carmona et al., 1994), whereas those effects of anandamide were not blocked by SR141716A (Adams et al., 1998). Although the CB2 receptor antagonist SR144528 was not used in this study, it is unlikely that differences in the effects of ⁹-THC and the endocannabinoid methanandamide are the result of CB2 receptor activation due to methanandamide's weak affinity for the CB2 receptor. Indeed, if CB2 receptors were involved, one would expect ⁹-THC with its equal affinities for CB1 and CB2 receptors to have been active in both groups of mice.

Results with the synthetic anandamide analog O-1812 were more ambiguous. Although ANOVA did not reveal significant response rate decreases or differences between groups, visual inspection of the graph indicated that differences may have been obscured by the curvilinear nature of the dose-effect curves for each genotype. Follow-up evaluation with a ² test of homogeneity showed that 30 mg/kg O-1812 eliminated responding in a significantly greater proportion of CB1+/+ mice compared with CB1-/- mice. Previous similar anomalies in the patterns of pharmacological effects produced by different anandamide analogs have been observed. First, the results of structure-activity relationship studies have shown that the correlation between affinity for the CB1 receptor and potency in a series of in vivo mouse tests is not nearly as strong for anandamide analogs as it is for traditional cannabinoids such as ⁹-THC (Adams et al., 1995a,b). Second, ⁹-THC discrimination studies have demonstrated that some, but not all, anandamide analogs substitute for ⁹-THC (for review, see Wiley, 1999). Furthermore, when it occurs, substitution is usually accompanied by response rate suppression (which did not occur with traditional cannabinoids) (Wiley et al., 1998). Together, these differences in pharmacological effects among anandamide analogs suggest that alteration of neurotransmission at CB1 receptors may not be the sole mechanism of action for all anandamide analogs. Further research is needed to delineate further other mechanisms that may play a role in the pharmacology of endocannabinoids.

In addition to tests with cannabinoids, three noncannabinoid drugs were evaluated in the CB1-/- and CB1+/+ mice. Response rates were reduced in CB1-/- mice compared with CB1+/+ mice at one or more doses for two of these drugs, ethanol and morphine, but not for the third drug, diazepam. These results were rather unexpected; however, they were not without precedent. Previous research with these mice showed that voluntary ethanol consumption was decreased in CB1-/- mice compared with CB1+/+ mice (Hungund et al., 2003; Poncelet et al., 2003). The larger response rate reductions in CB1-/- mice observed in the present study suggests that these mice may have been more affected behaviorally by ethanol which, in turn, may have resulted in reduced voluntary ethanol drinking. On the other hand, acute ethanol-induced hypothermia and locomotor suppression were similar in both groups (Racz et al., 2003). In addition, the decrease in ethanol consumption in CB1-/- mice was accompanied by a lack of ethanol-induced dopamine release in the nucleus accumbens that was observed in CB1+/+ mice (Hungund et al., 2003). Similarly, Mascia et al. (1999) reported that CB1-/- mice show a similar absence of dopamine release in the nucleus accumbens in response to morphine. In each study, drug-induced release of dopamine in CB1+/+ mice was reversed by SR141716A, further suggesting a role for CB1 receptors in this effect. The results of these studies in combination with those of the present study suggest that the endocannabinoid system may modulate some of the pharmacological effects of ethanol and morphine. Further research in this area is obviously needed.

In sum, CB1-/- and CB1+/+ mice differed in acquisition of the lever press response with CB1+/+ mice acquiring the response more readily than CB1-/- mice. CB1-/- and CB1+/+ mice also differed in response to the rate disruptive effects of the classical cannabinoid ⁹-THC, but not to the rate disruptive effects of the anandamide analog methanandamide. Furthermore, SR141716A blocked the rate disruptive effects of ⁹-THC in CB1+/+ mice, but not the rate disruptive effects of methanandamide in either group. Because methanandamide binds with poor affinity to CB2 receptors, these results suggest that possible non-CB1, non-CB2 mechanisms of action for the rate disruptive effects of methanandamide may exist. In contrast, a second anandamide analog, O-1812, produced response rate suppression in CB1+/+ mice, but not in CB1-/- mice, an effect more similar to that produced by ⁹-THC than to that produced by methanandamide. In addition, CB1-/- and CB1+/+ mice displayed modest differences in response to ethanol and morphine, suggesting modulation of some of the pharmacological effects of these drugs by interaction with CB1 receptors. In contrast, diazepam did not produce differences in learned schedule-controlled operant responding in CB1-/- and CB1+/+ mice, indicating that CB1 receptors do not modulate its rate disruptive effects.

	Footnotes

 
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: ⁹-THC, ⁹-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.

Address correspondence to: Dr. Jenny L. Wiley, Department of Pharmacology and Toxicology, Virginia Commonwealth University, P.O. Box 980613, Richmond, VI 23298-0613. E-mail: jwiley{at}hsc.vcu.edu

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