Effects of URB597 on FAAH activity and brain endocannabinoid levels
Systemic administration of URB597 (0.3 mg/kg, intravenous, i.v.) resulted in a marked inhibition of FAAH activity in all brain areas examined () (midbrain: F(1,6)
= 56.88, p
< 0.001; putamen: F(1,6)
= 126.93, p
< 0.001; nucleus accumbens: F(1,6)
= 8.52, p
= 0.027; prefrontal cortex: F(1,6)
= 53.04, p
< 0.001; thalamus: F(1,6)
= 153.84, p
< 0.001; amygdala: F(1,5)
= 162.93, p
< 0.001; hippocampus: F(1,6)
= 107.27, p
< 0.001). As previously observed in rodents (17
), FAAH inhibition was accompanied by an increase in the levels of anandamide () and OEA (), a non-endocannabinoid FAAH substrate (31
). Surprisingly, because in contrast with prior results obtained in rats (32
), URB597 treatment did not significantly increase OEA levels in putamen, nucleus accumbens and amygdala, and only marginally increased them in prefrontal cortex, suggesting that enzyme activities other than FAAH catalyze OEA hydrolysis in these regions of the monkey brain. Moreover, and again in contrast with previous rodent data (17
), URB597 administration significantly decreased 2-AG levels in monkey brain (). Parallel experiments confirmed that URB597 (1 mg/kg, intraperitoneal) does not affect 2-AG levels in rat hippocampus, even when experimental conditions closely matched those used in monkeys (12 h food deprivation, ongoing isoflurane anaesthesia) (). Similar results were obtained in the prefrontal cortex (data not shown). The ability of URB597 to reduce 2-AG levels in the monkey brain was not attributable to a direct effect of the drug on 2-AG-metabolizing enzymes, as incubation with URB597 (30 min) only altered DGL or MGL activities in monkey brain homogenates at concentrations significantly higher than those required to inhibit FAAH (residual DGL activity in putamen, as percent of control: 1 µM URB597, 106.6 ± 0.4; 10 µM URB597, 114.8 ± 6.2; residual MGL activity in putamen: 1 µM URB597, 78.0 ± 0.9, 10 µM URB597, 50.8 ± 0.3; mean ± SEM; n = 3) (17
Figure 1 Fatty-acid amide hydrolase (FAAH) activity in squirrel monkey brain regions 1 hour after injection of URB597 (0.3 mg/kg, i.v.). (a) Midbrain, (b) putamen, (c) nucleus accumbens, (d) prefrontal cortex, (e) thalamus, (f) amygdala, and (g) hippocampus. Activity (more ...)
Figure 2 Anandamide tissue levels in squirrel monkey brain regions 1 hour after injection of URB597 (0.3 mg/kg, i.v.). (a) Midbrain, (b) putamen, (c) nucleus accumbens, (d) prefrontal cortex, (e) thalamus, (f) amygdala, and (g) hippocampus. Data are mean ± (more ...)
Figure 3 Oleoylethanolamide (OEA) tissue levels in squirrel monkey brain regions 1 hour after injection of URB597 (0.3 mg/kg, i.v.). (a) Midbrain, (b) putamen, (c) nucleus accumbens, (d) prefrontal cortex, (e) thalamus, (f) amygdala, and (g) hippocampus. Data (more ...)
Figure 4 2-Arachidonoylglycerol (2-AG) tissue levels in squirrel monkey brain regions 1 hour after injection of URB597 (0.3 mg/kg, i.v.). (a) Midbrain, (b) putamen, (c) nucleus accumbens, (d) prefrontal cortex, (e) thalamus, (f) amygdala, and (g) hippocampus. (more ...)
Endocannabinoid levels in rat hippocampus after URB597 administration.
Lack of URB597 self-administration in monkeys that previously self-administered anandamide, THC or cocaine
Before the experiments, three groups of squirrel monkeys had learned to self-administer anandamide, THC or cocaine under a ten-response, fixed-ratio schedule of i.v. drug injection (every 10th lever-press response produced a drug injection followed by a one min timeout; FR10, TO 1-min). Varying the injection dose of anandamide, THC or cocaine resulted in inverted, U-shaped, dose-effect curves. Anandamide (; F(5,15) = 11.91, p < 0.001), THC (; F(4,8) = 16.83, p < 0.001) or cocaine (; F(4,8) = 9.41, p < 0.001) maintained significantly higher rates of responding and higher numbers of injections per session (, anandamide: F(5,15) = 47.05, p < 0.001; , THC: F(4,8) = 43.59, p < 0.001; , cocaine: F(4,8) = 22.28, p < 0.001) than vehicle. Maximal responding was maintained by doses of 30 and 56 µg/kg anandamide per injection (, 0.67 ± 0.10 and 0.65 ± 0.14 response per second, respectively), 4 µg/kg THC per injection (, 0.86 ± 0.17 response per second) and 30 µg/kg cocaine per injection (, 0.92 ± 0.25 response per second). When vehicle injections were substituted for any of these drugs, rates of responding and number of injections self-administered per session rapidly decreased to very low levels ().
After establishing dose-response curves for the three groups of monkeys, vehicle was substituted for THC, anandamide or cocaine for 5 to 10 sessions and URB597 was then substituted for vehicle. Each URB597 dose was studied for 4−5 consecutive sessions and each dose-condition was separated by 4–5 consecutive sessions of vehicle extinction. Varying the injection dose of URB597 (1, 3, 30, 100 µg/kg/injection) did not result in inverted U-shaped dose-effect curves in any of the groups of monkeys (). Self-administration behavior was never significantly above vehicle levels at any dose of URB597 or any of the subjects tested. Neither number of injections per session (, anandamide group: F(6,17) = 1.00, p = 0.46; , THC group: F(3,6) = 3.91, p = 0.08; , cocaine group: F(3,6) = 1.20, p = 0.39) nor rates of responding (, anandamide group: F(6,17) = 0.810, p = 0.58; , THC group: F(3,6) = 4.36, p = 0.06; , cocaine group: F(3,6) = 1.00, p = 0.46) differed significantly from vehicle. When URB597 was available at an injection dose of 100 µg/kg, intake over the 1-hour session reached an average of 1.31 ± 0.17 mg/kg (), more than four times greater than the 0.3 mg/kg dose of URB597 that produced a 10-fold increase in brain anandamide levels ().
Reinstatement of extinguished THC-, cocaine- or anandamide-seeking behavior by a priming injection of THC, but not URB597
Next, we examined the effects of a priming injection of THC (40 µg/kg, i.v.) in monkeys experienced with anandamide, THC, or cocaine and then tested the impact of a priming injection of URB597 at a dose that suppresses FAAH activity (0.3 mg/kg, i.v.; ). At this dose, URB597 did not affect responding for food under an FR10, TO 1-min schedule (F(1,8) = 0.12, p = 0.74; data not shown), when given for 5 consecutive sessions 30 min before session inception. Testing of priming injections always started after vehicle extinction had been stable for at least three days and reinstatement tests with priming injections were conducted for three consecutive sessions at each dose. THC produced a significant reinstatement of drug-taking behavior in the three groups of monkeys, while URB597 did not reinstate drug-taking in any of the animals (, anandamide group: F(2,7) = 22.87, p <0.001; , THC group: F(2,6) = 23.19, p = 0.002; , cocaine group: F(2,4) = 131.85, p < 0.001).
Effects of URB597 on self-administration of THC, cocaine, or anandamide
Finally, we investigated whether AAH inhibition influences self-administration of low and peak doses of anandamide, THC, or cocaine. An i.v. dose of 0.3 mg/kg of URB597, which strongly inhibited FAAH activity (), decreased the number of self-administered injections of 56 µg/kg anandamide (, F(1,4) = 19.56, p = 0.011), and increased the number of self-administered injections of 3 µg/kg anandamide (, F(1,4) = 14.25, p = 0.02). This leftward shift in the anandamide dose-response curve confirmed that URB597 was pharmacologically active under our experimental conditions. By contrast, URB597 did not produce significant changes in self-administration behavior for injections of 1 and 4 µg/kg THC (, THC 1: F(1,3) = 0.153, p = 0.72; THC 4: F(1,4) = 3.63, p = 0.15) or 1 and 30 µg/kg cocaine (, cocaine 1: F(1,2) = 7.75, p = 0.11; cocaine 30: F(1,3) = 6.06, p = 0.09)).