Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice

Abstract

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184).

Keywords: 2-arachidonoylglycerol; Anandamide; Drug discrimination; Fatty acid amide hydrolase; Rimonabant; Δ(9)-Tetrahydrocannabinol.

Figure 1

Panels A and B show the effects of THC (filled squares), anandamide (AEA; unfilled squares), and O-1812 (unfilled circles) on % THC-aperture responding in FAAH wild type (+/+; panel A) and knockout (-/-; panel B) mice trained to discriminate 5.6 mg/kg THC vs. vehicle. Response rates for each genotype (responses/min on both apertures) are shown in corresponding bottom panels (panels C and D, respectively). In panels A-D, points above VEH and THC represent the results of control tests with vehicle and THC, respectively, on % THC-aperture responding (panels A and B) and response rates (panels C and D) conducted prior to the dose-effect curve determination for each test compound. The rightmost panels show the effects of 5.6 mg/kg THC in combination with vehicle and with increasing doses of rimonabant (0.1 – 1 mg/kg) on % THC-appropriate responding (panel E) and response rates (panel F) in FAAH knockout and wildtype mice. Also shown in these panels are the effects of 10 mg/kg anandamide in combination with vehicle and with increasing doses of rimonabant (0.1 – 1 mg/kg) on the same measures (right side of panels E and F, respectively). Values represent the mean (±S.E.M.) of data from 13 FAAH knockout mice for THC, THC/rimonabant combination, and anandamide tests and 6 FAAH knockout mice for O-1812 tests. Values represent the mean (±S.E.M.) of data from 9-11 wildtype mice for THC, THC/rimonabant combination, and anandamide tests and 7-8 wildtype mice for O-1812 tests. Number signs (#) designate a significant main effect of dose across genotypes (p< 0.05) and subsequent Tukey post hoc determination of a significant decrease in THC-appropriate responding or response rate compared to mean responding during the vehicle control test.

Figure 2

Panel A shows the effects of MAGL inhibitor JZL184 (administered alone) on % THC-aperture responding in FAAH knockout (unfilled circles) and wildtype (filled circles) mice trained to discriminate 5.6 mg/kg THC vs. vehicle. Panel B shows the response rates for each genotype during these tests. The middle panels show concentrations of 2-AG (panel C) and anandamide (panel D) in the brains of FAAH knockout and wildtype mice following a single injection with vehicle or JZL184 (N=5-6 per group). Panels E and F show the effects of the dual MAGL/FAAH inhibitor JZL195 (administered alone) on % THC-appropriate responding and response rates, respectively, in FAAH knockout (unfilled circles) and wildtype (filled circles) mice. Results of combination tests of 40 mg/kg JZL195 and 3 mg/kg rimonabant on % THC-appropriate responding (panel E) and response rates (panel F) in FAAH knockout (unfilled triangles) and wildtype (filled triangles) are also shown. Points above VEH and THC (panels A, B, E, and F) represent the results of control tests with vehicle and THC, respectively, conducted prior to the dose-effect curve determination for each test compound. Values in the drug discrimination graphs represent the mean (±S.E.M.) of data from 6 FAAH knockout mice and 6-7 wildtype mice. Number signs (#) designate a significant main effect of dose across genotype (p< 0.05) and subsequent Tukey post hoc determination of a significant increase in 2-AG concentrations in the brain. Asterisks (*) designate a significant genotype × dose interaction (p< 0.05) and Tukey post hoc determination of a significant increase in anandamide concentrations in the brain of FAAH knockout mice compared to wildtype mice at the same JZL195 dose.