PCC measures are used to explain changes in alcohol-related mortality and morbidity , for comparison of alcohol use across geographic regions , the study of alcohol policies , the examination of alcohol use over time, the calculation of global alcohol-attributable fractions, and to inform news articles about alcohol use in the U.S. . It is therefore critical that PCC measures are as precise as possible to ensure that conclusions drawn from the applications of these measures are accurate and valid. Through the presentation of estimates based on ABV variation and comparisons to estimates from ABV-invariant methods we suggest that the inclusion of annual estimates of the %ABV of alcoholic beverages sold in the U.S. is necessary to ensure the precision of PCC measures and the accurate detection of changes in alcohol consumption over time and place. Drugs that activate cannabinoid receptors, the molecular target of Δ 9 -tetrahydrocannabinol in marijuana, reduce nausea and emesis produced by chemotherapy , alleviate pain symptoms associated with central and peripheral neuropathies , decrease pain and spasticity in multiple sclerosis , and improve psychomotor deficits in Tourette’s syndrome . Despite such broad therapeutic potential, the clinical usefulness of these agents is limited by their psychotropic and reinforcing effects,grow benches which account for the remarkable prevalence of marijuana as an abused drug . The rewarding properties of plant-derived or synthetic cannabinoid drugs are reasonably well understood.

Mechanistically, they have been linked to the ability of these substances to activate CB1-type cannabinoid receptors in the central nervous system, enhance activity of midbrain dopaminergic neurons, and elicit dopamine release in the reward-controlling shell region of the nucleus accumbens . By contrast, the contribution of endocannabinoid signals to the regulation of normative reward-based behaviors is still unclear, despite indications that pharmacological or genetic interruption of CB1 receptor activity strongly affects such behaviors . One important set of questions that remains unanswered relates to the chemical neuroanatomy of endocannabinoid-mediated reward and, in particular, to the functions served by individual endocannabinoid substances in reward modulation. Two such substances have been characterized: anandamide and 2-arachidonoylglycerol .Both compounds are produced in and released from neuronal and glial cells upon demand, and are eliminated by uptake into cells followed by intracellular hydrolysis. In the brain, anandamide is primarily hydrolyzed by the postsynaptic membrane-associated amidase, fatty acid amide hydrolase , which also cleaves other non-cannabinoid fatty-acid ethanolamides such as oleoylethanolamide . On the other hand, 2-AG is predominantly hydrolyzed by the presynaptic cytosolic lipase, monoacylglycerol lipase and, to a lesser extent, by two additional membrane-associated lipases, ABHD6 and ABHD12 . The existence of distinct biochemical pathways mediating the deactivation of anandamide and 2-AG suggests that selective pharmacological interruption of each of these pathways might help define the contribution of individual endocannabinoid signals to the modulation of reward. The compound URB597 is a potent and selective inhibitor of intracellular FAAH activity . In rodents, URB597 increases brain anandamide levels without changing the levels of 2-AG . Moreover, the drug elicits antinociceptive, anxiolytic-like, and antidepressant-like effects , which are likely mediated by enhanced anandamide activity at CB1 receptors, because they are attenuated by the CB1 antagonists rimonabant and AM251 .

Importantly, URB597 does not cause place preference or substitute for THC in rat drug-discrimination tests, an indication that it may lack hedonic properties . In the present study, we investigated the rewarding properties of URB597 in squirrel monkeys, a primate species that has been extensively used to model human reward-based behavior and has provided precious insights into the reinforcing effects of cannabinoids . We first determined the effects of URB597 on endocannabinoid levels in areas of the brain associated with reward, memory and emotional responses to stress. Next, we tested whether URB597 would either be self-administered by monkeys or would alter their self-administration of THC and cocaine. Finally, to assess the potential of URB597 to precipitate relapse to abuse in abstinent individuals, we examined its ability to reinstate extinguished drug-seeking behavior. Twenty three adult male squirrel monkeys weighing 0.9 to 1.1 kg were housed in individual cages in a temperature- and humidity-controlled room with unrestricted access to water. Monkeys were fed a daily ration consisting of five biscuits of high protein monkey diet and two pieces of Banana Softies that maintained their body weights at a constant level throughout the study. Fresh fruits, vegetables and environmental enrichment were provided daily. One group of five monkeys was used for experiments with the anandamide self-administration baseline: all monkeys had a history of anandamide self-administration . Another group of four monkeys was used for experiments with the THC self administration baseline: three monkeys had a history of THC self-administration and one monkey had history of anandamide self-administration . Another group of four monkeys was used for experiments with the cocaine self-administration baseline; all monkeys had a history of cocaine self-administration . A group of ten monkeys with no prior exposure to cannabinoids was used for neurochemical analyses . Adult male Wistar rats , n = 6–12 per group, were used for evaluation of brain lipid levels in rats. The animals were housed at constant room temperature and humidity under a 12-h light/dark cycle. Food and water were available ad libitum. Monkeys and rats were maintained in facilities fully accredited by AALAC and experiments were conducted in accordance with guidelines of the Institutional Animal Care and Use Committee of the Intramural Research Program, NIDA, NIH, and followed the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research .

Experimental chambers and other apparatus used in this study were the same as previously described . Monkeys were surgically prepared with chronic indwelling venous catheters . The catheters were connected to polyethylene tubing, which passed out of the isolation chamber where it was attached to a motor-driven syringe pump. Before the start of each session, monkeys were placed into Plexiglas chairs and restrained in the seated position by waist locks. Before each session, catheters were flushed with 1 ml of saline and one priming injection was delivered . At the start of the session, the white house light was turned off and green stimulus lights were turned on. In the presence of the green lights,grow racks with lights monkeys were required to make 10 responses on a lever to produce an injection of anandamide, THC or cocaine. Completion of 10 responses on the lever turned off the green lights and produced an intravenous injection of 40 µg/kg of anandamide, 4 µg/kg of THC or 30 µg/kg of cocaine paired with a 2-s illumination of amber stimulus lights . Duration of each injection was 0.2 s and injection volume was 0.2 ml. Each injection was followed by a 60-s timeout period, during which the chamber was dark and lever presses had no programmed consequences. One-hour sessions were conducted five days per week . All monkeys had learned to respond under the FR10 schedule for the particular training drug prior to beginning this study. After completing the previous experiments, monkeys self administered the training dose of each drug for at least five sessions until responding was stable . Self administration behavior was then extinguished by substituting vehicle for THC, anandamide, or cocaine, but maintaining the presentation of the brief-stimulus associated with each injection. Then we tested reinstatement of extinguished drug-taking behavior by priming injections of THC or URB597 in all three groups of monkeys. Reinstatement effects of each pretreatment were studied for three consecutive sessions starting after at least three days of stable vehicle extinction. Monkeys received injections of vehicle or URB597 in their home cage. THC was injected in the chair immediately before the session.Food was withheld 12 h prior to this procedure. Anaesthesia was induced and maintained with isoflurane . Monkeys were weighed, prepared with a venous line , placed on a surgery table and kept warm by heat lamps. Body temperature and ECG were monitored throughout anaesthesia. After animals were stabilized , URB597 or its vehicle was intravenously injected 1 h prior to euthanasia and the venous line was flushed with 0.5 ml of saline. Body temperature, SPO2, pulse and rate of respiration were recorded every 10 min. After 1 h, monkeys were euthanized with Euthasol .Brains were quickly removed, the cerebella were separated and forebrains were dissected.

Each hemisphere was cut into 3 parts by two coronal sections made at approximately AP +12.5 and −5 . Brain fragments were snap-frozen in isopentane , wrapped in aluminium foil, and placed in dry ice. Samples were stored in the freezer for 2 days and then shipped on dry ice to the University of California Irvine for analyses. In one set of experiments, rats were sacrificed by rapid decapitation under light anaesthesia 2 h after injection of URB597 or vehicle. In other experiments, rats were food deprived for 12 h and sacrificed by decapitation 1 h after injection of URB597 or vehicle, while maintained under isoflurane anaesthesia for the duration of the experiment. In either case, brains were rapidly removed, and the hippocampus and prefrontal cortex was dissected from the fresh tissue over ice. Brain regions were frozen in dry ice, and stored at −80°C until lipid and enzymatic analyses. Cumulative-response records were obtained during all sessions to assess within-session patterns of responding. Rates of responding during self-administration sessions are expressed as responses per second averaged over the one-hour session, with responding during time-outs not included in calculations. Injections per session represent total number of injections delivered per session. Data for dose-effect curves are expressed as mean response rates and numbers of injections per session ± SEM over the last three sessions. In addition, total intake of anandamide, THC or cocaine for each session was calculated. Reinstatement data and effects of pretreatment with URB597 on drug self-administration are expressed as mean ± SEM of total numbers of injections per session over three sessions. Statistical analysis was done using single-factor repeated measures ANOVA to assess differences between vehicle and test-drug pretreatment conditions or between different doses of anandamide, THC, cocaine or URB597 and vehicle. Significant main effects were analyzed further by subsequent paired comparisons to control values using Dunnett’s test . Bonferroni t-test was used when the number of observations did not allow for the use of the Dunnett’s test. Differences between effects of vehicle and URB597 pretreatment on lipid levels and FAAH activity were analyzed using single-factor ANOVA. Differences were considered statistically significant when p < 0.05.We found that the selective FAAH inhibitor URB597 suppresses FAAH activity and increases anandamide levels in regions of the squirrel monkey brain that participate in motivational, cognitive and emotional functions. This effect is accompanied by a marked decrease in the levels of 2-AG, a major endocannabinoid substance in the brain, even though URB597 does not affect activities of 2-AG-metabolizing enzymes such as DGL and MGL. We further observed that URB597 does not display overt reinforcing property in monkeys over a broad range of experimental conditions. Indeed, the drug did not reinforce self-administration behavior even when its cumulative intake exceeded by several folds a fully effective dose for FAAH inhibition. Furthermore, neither previous cocaine nor THC exposure predisposed monkeys to self-administer URB597: even monkeys that had previously self-administered anandamide at very high rates failed to respond to the FAAH inhibitor. Lastly, URB597 did not affect the reinforcing effects of THC or cocaine, and did not reinstate extinguished drug seeking behavior in monkeys that had previously self-administered THC or cocaine. We interpret these results to indicate that URB597, by enhancing anandamide signaling, causes a compensatory down-regulation in 2-AG mobilization; and the potentiation of anandamide-mediated transmission produced by URB597 is insufficient per se to produce reinforcing effects. Our findings further imply that FAAH inhibitors such as URB597 – which have demonstrated analgesic, anxiolytic, antidepressant and antihypertensive properties in rodents – may be used in humans without anticipated risk of inducing abuse or provoking relapse to drug use in abstinent individuals. The pharmacological profile of URB597 is strikingly different from that of THC and other direct-acting CB1 receptor agonists. Studies in rodents have shown that URB597 does notproduce THC-like effects such as catalepsy, hypothermia or hyperphagia . Further, URB597 does not mimic the discriminative-stimulus actions of THC . Even further, URB597 does not increase dopamine levels in the nucleus accumbens shell of rats, a defining neurochemical feature of reinforcing drugs . Finally, URB597 does not elicit conditioned place preferences indicative of rewarding properties in rats .