Main Research Findings

1) Treatment with URB-597 has been shown to enhance endocannabinoid signalling related to improved auditory discrimination sensitivity.

2) Administration of URB-597 has the potential to facilitate the extinction of floor aversion in animals trained to associate a floor cue with naloxone-precipitated morphine withdrawal.

 

Selected Data
1) The research team of Sokolic et al investigated the effects of delta-9-THC and fatty acid amide hydrolysis inhibitor URB-597 on the performance of auditory and olfactory discrimination tasks in rats. For the purpose of this study, a total of 32 male Australian Hooded Wistar rats, bred in-house at the University of Sydney, were used in this study. The rats, weighing between 116 and 229 grams and aged 40–50 days at the start, were housed in groups of eight in a temperature-controlled colony room set to 21°C. The room followed a reverse light–dark cycle and the rats were placed on a water deprivation schedule, receiving 10 ml of water per day but having free access to water on most weekends [1].

Five different experiments took place in 12-channel air-dilution olfactometers, two of which were modified for auditory discrimination tasks. The test chamber measured 17 cm × 24 cm × 25 cm and contained a stainless steel grid floor, a ventilation fan, a sniff port for odor detection, and a lick tube for water rewards. The olfactometers generated olfactory stimuli by diverting an air stream through saturator bottles containing odorants, mixing the odorized air with a main airflow before delivering it through the sniff port. Rats initiated trials by nose poking into the port, which triggered a 2-second stimulus presentation period [1].

For odor discrimination tasks, rats sampled one of 16 different odorants, including common food-related scents (e.g., lemon, vanilla, banana) and aromatic oils (e.g., jasmine, honeysuckle). Aldehydes were diluted, while other odorants remained undiluted. Auditory discrimination trials followed the same response protocols but presented sound stimuli instead of odors, using either a beeper or white noise. A custom-written software program controlled the olfactometers and data recording.
Initially, rats learned to associate licking the lick tube with a water reward, followed by nose-poking training to ensure adequate sampling of stimuli. The final task was a go/no-go successive discrimination paradigm. The olfactory group was trained to distinguish between lemon (S+) and strawberry (S−), while the auditory group learned to differentiate between a beeper (S+) and white noise (S−). A correct response on an S+ trial was defined as licking the tube and resulted in a water reward, while incorrect responses on S− trials, defined as licking when they shouldn’t, led to no reward and a longer intertrial interval. A performance criterion of 17 correct responses in 20 consecutive trials was set for all tasks [1].

Once rats reached criterion performance, the first experiment began with dividing the animals into treatment subgroups that received either delta-9-THC or rimonabant at increasing doses of 0.3, 1, and 3 mg/kg. Drug administration occurred 15 minutes before testing, with washout days between drug trials to ensure baseline recovery. Higher doses of delta-9-THC impaired auditory discrimination performance significantly, prompting the use of additional washout days. To assess whether rimonabant could counteract delta-9-THC effects, a final test combined both drugs at 3 mg/kg. The second experiment examined the potential long-term effects of delta-9-THC on auditory discrimination with rats previously tested in Experiment 1 receiving a single high dose of 10 mg/kg of delta-9-THC. Testing occurred 24 hours later and performance was compared between the delta-9-THC-treated group and a vehicle-treated control group [1].

Three weeks after Experiment 2, the third experiment was initiated using the same rats that were tested with URB-597 and increased endocannabinoid levels. On the first test day, all rats received a vehicle injection, while on the second day, they were split into two groups receiving either 0.1 or 0.3 mg/kg of URB-597. Two washout days followed before reversing the dosage groups and repeating the process. A final test examined whether 3 mg/kg of rimonabant could block the effects of URB-597 when coadministered.
Experiment 4 evaluated whether delta-9-THC affects the ability to acquire and reverse novel olfactory discriminations. The olfactory group from Experiment 1 was used, while the auditory group was excluded due to severe impairment under delta-9-THC. Rats were assigned to either drug or control groups for five different odor discriminations. Doses of delta-9-THC at 0.3, 1, and 3 mg/kg were tested, followed by a combined dose of 3 mg/kg of delta-9-THC and 3 mg/kg of rimonabant trial, as well as a rimonabant-alone trial [1].

Acquisition was measured by the number of errors made before reaching the 17-out-of-20 correct response criterion. If rats did not reach criterion within 400 trials on the first day, they continued on subsequent days. Once acquired, the discrimination was tested over four additional days to establish asymptotic performance. The reversal phase reallocated rats to new treatment groups to assess whether drug treatment affected their ability to reverse the learned discrimination.
Finally, the fifth experiment included rats from previous experiments that were divided into an experimental group treated with URB-597 at 0.3 mg/kg and a control group to test their ability to acquire a coffee (S+) vs. jasmine (S−) discrimination. Performance was measured similarly to Experiment 4. After reaching criterion, the discrimination was reversed and a final test assessed whether rimonabant blocked URB-597 effects when coadministered before a frangipani (S+) vs. honeysuckle (S−) discrimination task [1].

2) The research team of Manwell et al conducted various experiments examining the effects of URB-597 treatment on condition aversion responses. Experiments 1 and 2a used Sprague–Dawley rats, whereas Experiments 2b–d and 3 utilized male Long–Evans rats to enhance the robustness of conditioned preference/aversion responses. The animals were housed in shoebox cages under a 12-hour light/dark cycle at 21°C. Food and water were provided ad libitum, except in Experiment 1, where rats were maintained at 85% of their body weight [2].

Morphine was administered in doses of 10 mg/ml for Experiments 2a–d and doses of 20 mg/ml for Experiment 3 while 1 mg/ml of naloxone was administered subcutaneously 10 minutes prior to conditioning. 0.3 mg/kg of URB-597 was administered intraperitoneally 2 hours before extinction testing. Previous studies indicated that URB-597 does not induce conditioned preference or aversion but affects anandamide metabolism and produces anxiolytic, antidepressant, and anti-nausea effects. SR141716 was administered intraperitoneally in a dose of 1 mg/ml, 30 minutes before extinction testing, while AM-251 in doses of 1, 3, or 8 mg/ml was administered in Experiment 2a.

For Experiment 1, operant chambers equipped with levers and sucrose pellet dispensers were used to assess operant behavior. Experiments 2 and 3 used a black Plexiglas rectangular box with a wire-mesh lid. Distinctive floor textures served as conditioning cues, and the rats’ time spent on each floor was recorded via an automated tracking system. Rats previously trained to press a lever for sucrose underwent extinction trials. Groups received URB-597 in doses 0.3 mg/kg, delivered 2 hours before trials, a vehicle treatment, or SR141716 in doses of 2.5 mg/kg, delivered 30 minutes before trials. Extinction sessions were 1 hour long, conducted over three consecutive days, with conditions identical to training but without sucrose reinforcement [2].

During Experiment 2a, rats underwent four conditioning cycles where 10 mg/kg of morphine or saline was paired with distinctive floor textures. Following conditioning, extinction trials began 48 hours after the fourth cycle, and rats received AM-251 in doses of 1, 3, or 8 mg/kg or vehicle 30 minutes before each trial. Trials continued until no significant preference for the morphine-paired floor remained. Similar to Experiment 2a, rats underwent four conditioning cycles. Extinction trials started 72 hours post-conditioning, with URB-597 administered in doses of 0.03, 0.1, or 0.3 mg/kg, or a vehicle administered 2 hours before each trial. Extinction sessions occurred every 48 hours until conditioned preference disappeared [2].

Experiment 2c began following six conditioning cycles where the rats underwent extinction with forced exposure to floor cues. Rats were divided into no extinction, a vehicle, 0.1 mg/kg URB-597, or 0.3 mg/kg URB-597 groups. Extinction included counterbalanced 30-minute exposures to both floor types, separated by 48 hours. Preference tests followed every 48 hours until extinction was complete. To generate a more robust conditioned preference, rats underwent eight conditioning cycles for Experiment 2d. Only those showing a preference by spending at least 45 seconds more on the morphine-paired floor continued. Extinction trials began 96 hours post-conditioning and occurred every 72–96 hours over 10 sessions, and 0.3 mg/kg of URB-597 or vehicle was administered 2 hours before each 10-minute extinction test. One week after extinction, reinstatement tests were conducted with saline and morphine at doses of 2.5 mg/kg to assess the effects of prior URB-597 treatment on relapse [2].

The third experiment that was conducted investigated the effects on morphine withdrawal-induced conditioned aversion. The rats included in this experiment underwent two conditioning cycles over three-day schedules defined as follows: day 1, saline injection and 30-minute exposure to one floor; day 2, 20 mg/kg of morphine injected in the home cage; and day 3, 1 mg/kg of naloxone injected with 30 minutes of exposure to the opposite floor. Extinction trials began 72 hours after conditioning. Groups received URB-597 at a dose of 0.3 mg/kg, delivered 2 hours prior to a 20-minute test trial, a vehicle, or SR141716 at a dose of 2.5 mg/kg, 30 minutes prior to a 20-minute test trial. Seventy-two hours after extinction, drug-free conditioned aversion tests were conducted. One week later, reinstatement tests assessed the effect of naloxone-precipitated withdrawal on aversion recovery [2].
This study examined the effects of cannabinoid-related compounds on extinction learning and morphine-induced conditioned preference and aversion. URB-597 and AM-251 influenced extinction and reinstatement, with URB-597 extending the duration of conditioned responses. The findings contribute to understanding the role of endocannabinoid modulation in addiction-related behaviors.

Discussion

1) Experiment 1 out of 5 experiments conducted by Sokolic et al examined how delta-9-THC affects performance on an auditory discrimination task. The results showed that delta-9-THC significantly impaired task performance at doses of 0.3, 1, and 3 mg/kg. The impairment was also observed the day after administration of the highest dose of 3 mg/kg. However, when rimonabant at 3 mg/kg was coadministered with delta-9-THC at 3 mg/kg, the detrimental effects were significantly reduced, suggesting a role of CB1 receptor blockade in mitigating these cognitive deficits. Notably, rimonabant alone had no significant effect on auditory discrimination performance. Additionally, neither delta-9-THC nor rimonabant affected performance on an olfactory discrimination task, indicating that the observed impairment was task-specific rather than a general cognitive deficit [1].

Experiment 2 assessed the longer-term impact of delta-9-THC, rats were tested on a well-learned go/no-go auditory discrimination task 24 and 48 hours after administration of a high dose of 10 mg/kg. Performance was severely impaired 24 hours post-administration but recovered by 48 hours. This suggests that delta-9-THC can cause a temporary but substantial disruption in auditory discrimination abilities. That being said, the third experiment revealed that URB-597 administered at doses of 0.1 and 0.3 mg/kg significantly impaired auditory discrimination performance, and this impairment persisted for 24 hours after administration. However, when URB-597 was coadministered with rimonabant, the cognitive impairment was prevented, suggesting that endocannabinoid-mediated CB1 receptor activation plays a crucial role in auditory discrimination deficits [1].

The fourth experiment evaluated the effects of delta-9-THC on the acquisition of novel olfactory discrimination tasks. Unlike its effect on auditory tasks, delta-9-THC administered at doses of 0.3, 1, and 3 mg/kg did not significantly impair the acquisition of olfactory discrimination learning. The same was true when delta-9-THC was administered with rimonabant. These findings indicate that these treatments did not impact learning in this domain. However, when rimonabant was administered alone there was a slight tendency to enhance learning.
However, in an olfactory discrimination reversal task assessing cognitive flexibility, delta-9-THC at 1 and 3 mg/kg significantly impaired performance, increasing errors during reversals. The lowest dose of 0.3 mg/kg did not significantly affect performance. Notably, the impairment in reversal learning was prevented when delta-9-THC was coadministered with rimonabant, reinforcing the role of CB1 receptor activity in this cognitive function. Again, rimonabant alone had a mild facilitatory effect, potentially improving cognitive flexibility [1].

The final experiment examined the effects of URB-597 on olfactory discrimination learning. Similar to delta-9-THC, URB-597 administered at a dose of 0.3 mg/kg did not impair the acquisition of novel olfactory discrimination tasks. Additionally, coadministration of URB-597 with rimonabant did not affect acquisition. However, URB-597 did significantly impair performance in the reversal task, in a manner similar to delta-9-THC. This impairment was eliminated when URB-597 was administered with rimonabant, further supporting the hypothesis that CB1 receptor activation underlies the observed cognitive inflexibility [1].

2) The study conducted by Manwell et al examined the effects of various pharmacological agents on the extinction of conditioned responses related to sucrose-seeking behavior and morphine-induced conditioned place preference. The primary drugs investigated were URB-597, a fatty acid amide hydrolase inhibitor; SR141716, a cannabinoid receptor antagonist; and AM-251, another cannabinoid receptor antagonist. The research aimed to assess whether these drugs influenced the rate of extinction and the potential for reinstatement following drug priming [2].

The first set of experiments focused on operant responding for a sucrose reward. The results reported that pretreatment with URB-597 during extinction training did not alter the rate at which operant responding decreased. However, SR141716 significantly suppressed operant responding, particularly during the early phase of the extinction session. The results indicated that extinction progressed over time, with significant reductions in responding across days and intervals. A mixed factor ANOVA confirmed significant effects of drug treatment, time, and their interactions, suggesting that while extinction occurred across sessions, SR141716 accelerated the suppression of operant behavior. This effect was most pronounced in the first 10 minutes of responding on Day 1. By Days 2 and 3, SR141716 continued to suppress responding more than the vehicle condition. However, URB-597 did not significantly influence the rate of extinction, indicating that FAAH inhibition did not facilitate extinction learning in the context of sucrose-seeking behavior [2].

The second set of experiments investigated whether AM-251 altered the extinction of a morphine-induced conditioned place preference. The rats displayed a strong initial preference for the morphine-paired floor, but this preference diminished over the course of extinction training. The ANOVA revealed significant effects of trial and trial-by-floor interactions, but no significant effects of drug pretreatment. This suggests that AM-251 did not influence the rate at which the conditioned preference for the morphine-paired floor extinguished. Across conditions, rats showed a significant preference for the morphine-paired floor on the first two extinction trials, but this preference was no longer significant by the third trial. Importantly, the initial preference test showed a robust conditioned preference for the morphine-paired floor, but AM-251 did not alter the extinction of this preference.
Subsequent experiments evaluated whether URB-597 could facilitate extinction of morphine-induced conditioned place preference. Statistical analyses revealed significant effects of the conditioning floor and a significant drug-by-floor-by-trial interaction. However, further analyses showed that the effect of the conditioning floor was significant only in the first two extinction trials, but not after. The extinction drug itself did not significantly alter behavior at any point. Given that conditioned preference extinguished within two trials, the researchers examined whether within-session extinction occurred by analyzing data from the first and second halves of the trials. These analyses confirmed that while extinction occurred, URB-597 did not enhance the rate of extinction [2].

Additional experimentation investigated whether administering URB-597 during confinement in the morphine-paired and saline-paired floors influenced extinction. Results showed significant effects of the conditioning floor after one and two cycles of extinction training, but not after three cycles. No significant interactions were observed, indicating that URB-597 did not facilitate extinction under these conditions. In Experiment 2d, rats underwent an extended conditioning phase with eight cycles to strengthen the conditioned preference. Statistical analyses revealed a significant effect of conditioning floor and a floor-by-trial interaction but no significant effects of extinction treatment. Rats exhibited a significant preference for the morphine-paired floor during the first few extinction trials, but this preference diminished over time. Again, URB-597 did not accelerate the extinction process [2].

The study also examined reinstatement of morphine-induced conditioned place preference following a morphine prime. The ANOVA revealed significant effects of conditioning floor and a floor-by-trial interaction, indicating that a morphine prime reinstated the extinguished preference. However, prior treatment with URB-597 during extinction did not alter the magnitude of reinstatement. This suggests that although URB-597 did not hinder extinction, it also did not enhance the long-term suppression of conditioned preference.
A separate analysis examined extinction across six blocks of four trials each, showing that SR141716 impaired extinction while URB-597 had no significant effect. During the early extinction phase, all groups showed a significant preference for the morphine-paired floor. In subsequent blocks, the vehicle and SR141716 groups retained this preference for longer, whereas the URB-597 group showed a more rapid decline in preference. By the final extinction block, no significant floor preference remained in any group. These findings suggest that while SR141716 may slow extinction, URB-597 does not appear to facilitate it [2].

Finally, a post-extinction, drug-free test 72 hours after the last extinction trial assessed whether extinction was maintained. The results indicated no significant effects of prior drug treatment, suggesting that extinction persisted without the treatment drugs. Additionally, when a naloxone-precipitated withdrawal prime was administered, it reinstated the conditioned aversion to the morphine-paired floor. While rats displayed a significant reactivation of their aversion to the withdrawal-associated environment, prior extinction treatment did not modify the strength of reinstatement. This suggests that neither URB-597 nor SR141716 had long-term effects on the reinstatement of withdrawal-induced aversion [2].

Overall, the study demonstrated that URB-597 did not facilitate the extinction of either sucrose-seeking behavior or morphine-induced CPP. While SR141716 effectively suppressed operant responding for sucrose, it also appeared to impair extinction learning. AM-251, another cannabinoid antagonist, did not significantly impact extinction of morphine-induced conditioned place preference. The findings suggest that FAAH inhibition alone does not enhance extinction learning and that cannabinoid receptor antagonism may have complex effects depending on the behavioral context.

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Sokolic L, Long LE, Hunt GE, Arnold JC, McGregor IS. Disruptive effects of the prototypical cannabinoid Δ⁹-tetrahydrocannabinol and the fatty acid amide inhibitor URB-597 on go/no-go auditory discrimination performance and olfactory reversal learning in rats. Behav Pharmacol. 2011 Jun;22(3):191-202. doi: 10.1097/FBP.0b013e328345c82b. PMID: 21512341.

[2] Manwell LA, Satvat E, Lang ST, Allen CP, Leri F, Parker LA. FAAH inhibitor, URB-597, promotes extinction and CB(1) antagonist, SR141716, inhibits extinction of conditioned aversion produced by naloxone-precipitated morphine withdrawal, but not extinction of conditioned preference produced by morphine in rats. Pharmacol Biochem Behav. 2009 Nov;94(1):154-62. doi: 10.1016/j.pbb.2009.08.002. Epub 2009 Aug 19. PMID: 19698735.

 

 

 

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