Effects of fatty acid amide hydrolase (FAAH) inhibitors on working memory in rats

Introduction

Cannabis and synthetic cannabinoid agonists can produce certain therapeutic effects, but they can also produce adverse side effects including dependence and memory impairment. They produce these effects by activating cannabinoid CB₁ receptors, mimicking the effects of endogenous cannabinoid substances (endocannabinoids). The two main endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), are produced on demand and are rapidly degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), respectively. Since CB₁ receptors have two separate endogenous ligands, it is likely that the brain circuits involving anandamide and 2-AG underlie distinct sets of neurobehavioral processes that can be selectively _targeted for therapeutic purposes. This can be accomplished by administering inhibitors of FAAH or MGL, thereby increasing the effects of anandamide or 2-AG when and where they are released. This amplification of natural endocannabinoid signaling could potentially produce beneficial effects without the adverse side effects associated with exogenous cannabinoid agonists, which directly activate CB₁ receptors throughout the brain (see reviews by Blankman and Cravatt 2013; Clapper et al. 2009a; Hwang et al. 2010; Panlilio et al. 2013; Pertwee 2014; Schlosburg et al. 2009; Zanettini et al. 2011).

The FAAH inhibitor that has been studied most intensively is URB597 (Piomelli et al. 2006). In preclinical testing, URB597 does not produce classical THC-like effects such as catalepsy, hypothermia, and hyperphagia (Kathuria et al. 2003). URB597 also shows no signs of abuse potential in animal models of cannabis abuse; it does not have THC-like in rats trained to detect the interoceptive effects of THC (Gobbi et al. 2005), and it is not self-administered by squirrel monkeys that have extensive experience self-administering anandamide and other cannabinoid agonists (Justinova et al. 2008). However, other FAAH inhibitors, including URB694 (Justinova et al. 2015), PF-04457845 (Justinova et al. 2014) and AM3506 (Bergman et al. 2011), have shown moderate to strong reinforcing effects when offered as an intravenous solution to squirrel monkeys. These findings indicate that FAAH inhibitors can vary considerably in their effect profiles and should be evaluated individually for specific therapeutic and adverse effects.

Delta-9-tetrahydracannabinol (THC) impairs learning and memory in humans (Ranganathan and D'Souza 2006) and animals (Zanettini et al. 2011), with working memory being particularly sensitive. In rodents, memory has also been shown to be impaired by administration of exogenous anandamide, but only when its degradation by FAAH is prevented (Goonawardena et al. 2011; Lichtman et al. 1995; Mallet and Beninger 1996; 1998; Varvel et al. 2006). Surprisingly, inhibition or genetic deletion of FAAH, which substantially increases endogenous levels of anandamide, has been found to enhance rather than impair memory in rodents trained with procedures involving aversively-motivated behavior (i.e., water maze: Varvel et al., 2006, 2007; or passive-avoidance of a context associated with footshock: Hasanein and Teimuri Far 2015; Mazzola et al. 2009; Morena et al. 2014). However, memory-related studies with appetitively-motivated procedures have mostly shown impairment rather than enhancement after treatment with a FAAH inhibitor (Basavarajappa et al. 2014; Busquets-Garcia et al. 2011; Goonawardena et al. 2011; Seillier et al. 2010; these studies all used URB597). There have been fewer studies involving MGL inhibition. The MGL inhibitor JZL184 did not affect memory in an object-recognition procedure (Busquets-Garcia et al. 2011), but JZL184 and a dual FAAH-MGL inhibitor (JZL195) both impaired memory in a repeated-acquisition water-maze procedure in mice (Wise et al. 2012).

In the present study, we focused on the effects of FAAH inhibitors on working memory in rats, using a food-based procedure known to be sensitive to impairment by THC (Justinova et al. 2013; Panlilio et al. 2012; Panlilio et al. 2011). We tested five different FAAH inhibitors (and one MGL inhibitor) at doses sufficient to substantially increase levels of anandamide (or 2-AG). We found that only one of these compounds, the FAAH inhibitor AM3506, impaired working memory at the doses tested. Since pharmacological doses of anandamide may activate alpha-type peroxisome proliferator-activated receptors (PPAR-alpha) and vanilloid transient receptor potential cation channels (TRPV1), and since FAAH inhibition increases endogenous levels of not only anandamide but also other fatty acid amides that are ligands for PPAR-alpha and TRPV1, we explored the mechanism of AM3506's effects by giving AM3506 in combination with a CB₁ antagonist (rimonabant), a PPAR-alpha antagonist (MK886), or a TRPV1 antagonist (capsazepine). These tests indicated that the memory impairment induced by AM3506 was mediated by CB₁ receptors.

Materials and methods

Results

Accuracy (i.e., percentage of trials with a correct response) under the nonmatching-to-sample task was high at the 0-second delay and decreased monotonically as a function of delay under baseline conditions (not shown) and after treatment with vehicle (see Figure 2). Even at the longest delay, accuracy was well above chance level (50%) after treatment with vehicle. During drug testing (Figure 2), the accuracy curves continued to show a general downward slope. The data in each frame of Figure 2 were analyzed separately, and in each case the main effect of delay was highly significant [F(4,44) ranging from 24.0 to 46.0, all p's<.0001]. The main effect of AM3506 on accuracy was significant [F(2,22)=30.2, p<.0001], but the other treatment drugs had no significant main effects or interaction effects (Figure 2). The dose-effect functions for all treatments are summarized in Figure 3, collapsing across delays; only AM3506 had a significant effect, and this was only at the highest dose.

Responding in the side holes is not explicitly required during the delay period, but it appears to serve a rehearsal-like mediating function, enhancing the accuracy of the nonmatching response (Panlilio et al. 2011, 2012). Rats show individual differences in whether their mediating behavior during the delay occurs in the matching or nonmatching hole. Logistic regression indicated that six of the rats in the present study were more likely to choose the correct hole if at least one response had occurred in the to-be-correct hole (i.e., the nonmatching hole) during the delay, while the other five rats were more likely to choose the correct hole if at least one response occurred in the other side hole (i.e., the matching hole) during the delay; this analysis identified the “appropriate” side hole for each rat. Neither AM3506 nor any of the other treatments had a significant effect on 1) the rate of responding in the side holes or center hole during the delay (data shown for AM3506; Figure 4a); or 2) the relative frequency of the four types of trials (i.e., trials with responding in only the appropriate side hole, only the inappropriate side hole, neither side hole, or both side holes; Figure 4b). However, AM3506 treatment [F(1,11)=33.6, p<.0001] and trial type [F(3,33)=12.96, p<.0001] had significant effects on accuracy (Figure 4c), and paired comparisons revealed that the 3 mg/kg dose of AM3506 significantly decreased accuracy of the nonmatching response specifically in trials where at least one response occurred in the appropriate hole (i.e., in “Appropriate Only” trials and “Both” trials).

To explore the mechanism of the AM3506 effect, we attempted to block it with: 1) the CB₁-receptor antagonist, rimonabant; 2) the PPAR-alpha antagonist, MK886; and 3) the TRPV1 antagonist, capsazepine. As in the single-injection experiments (Figure 2), the 3 mg/kg dose of AM3506 was again found to impair accuracy during the antagonist experiments (Figure 5). This replication of the AM3506-induced impairment —obtained after all six inhibitors had been tested under the single-injection procedure— indicates that the effect was reliable and not dependent on the order in which the inhibitors were tested. Accuracy decreased as a function of delay, and the main effect of delay was significant in each frame of Figure 5 [F(4,44) ranging from 34.7 to 68.1, all p's<.0001]. Rimonabant produced a statistically significantly blockade of the AM3506 effect, but the other antagonists did not. For the data in the rimonabant frame of Figure 5, the interaction of pretreatment and treatment was significant [F(1,11)=12.1, p<.005]. In contrast, while the main effect of AM3506 treatment was significant for the data in the MK886 frame [F(1,11)=44.4, p<.0001] and the capsazepine frame [F(1,11)=62.3, p<.0001] of Figure 5, all main effects and interactions involving pretreatment with MK886 or capsazepine were nonsignificant. The results of the antagonist tests are summarized in Figure 6, with the accuracy data collapsed across delays. The 3 mg/kg dose of AM3506 significantly impaired accuracy when it was given in combination with an injection of vehicle, MK886 or capsazepine. However, when AM3506 was combined with rimonabant, accuracy was significantly improved compared to when AM3506 was combined with vehicle. Accuracy was not significantly affected by any of the antagonists when given alone (p's>.3).

Discussion

We tested several compounds that enhance endogenous levels of endocannabinoids and found that one compound, AM3506, produced THC-like impairments in a rodent model of working memory. The level of accuracy in the memory task after treatment with a 3-mg/kg dose of AM3506 in this study was comparable to the levels we observed in earlier studies after treatment with a 3-mg/kg dose of THC (Panlilio et al. 2011, 2012; Justinova et al. 2013). Further testing with receptor-specific antagonists indicated that these impairments were mediated by cannabinoid CB₁ receptors, but not by PPAR-alpha or TRPV1, which are known to be affected by anandamide and other fatty acid amides. These findings indicate that the impairments induced by AM3506 were due to the activation of CB₁ receptors by anandamide, possibly in combination with slightly increased levels of 2-AG.

A feature of the nonmatching-to-position procedure used here is that it allows automated recording of the rehearsal-like mediating behavior that occurs during the delay period (Panlilio et al. 2011, 2012). Like THC in earlier studies with this procedure, AM3506 did not change the rate or distribution of behavior during the delay period, but it seemed to make the mediating response less effective, impairing accuracy even when the most propitious behavior occurred during the delay. That is, AM3506 had significant effects on accuracy specifically in the trials in which the appropriate mediating response did occur. In this respect, THC and AM3506 clearly differ from scopolamine, which disrupts performance of the mediating response (specifically decreasing the proportion of trials in which the rat responds only in the appropriate side hole during the delay; Panlilio et al. 2011).

FAAH and MGL are members of the serine hydrolase family, which includes over 200 members, many of which are not well characterized (Bachovchin and Cravatt 2012). AM3506 is a sulfonylfluoride inhibitor and might show substantial differences in cross-reactivity with serine hydrolases compared to the more typical carbamate/urea inhibitors of FAAH like URB597 and PF-04457845. AM3506 mainly affects FAAH and to a lesser extent MGL, and did not have “off _target” effects when tested against a large number of serine hydrolases using activity-based proteomic methods (Godlewski et al. 2010). However, the differences in the effects of AM3506 and the other test compounds on memory might reflect inhibition of other serine hydrolases in addition to FAAH. Alternatively, in vivo AM3506 might irreversibly deactivate a significantly larger FAAH population compared to the other inhibitors.

Squirrel monkeys are sensitive to the reinforcing effects of CB₁ agonists and will intravenously self-administer solutions of THC (Justinova et al. 2003), anandamide (Justinova et al. 2005) or 2-AG (Justinova et al. 2011). The FAAH and MGL inhibitors tested in the present study have all been assessed for reinforcing effects in squirrel monkeys (Bergman et al. 2011; Justinova et al. 2008; Justinova et al. 2014; Justinova et al. 2015), and it is interesting to note that there is only a partial correspondence between their effect in the memory and reinforcement models. That is: 1) URB597 was not self-administered and did not impair memory; 2) AM3506 was self-administered and did impair memory; but 3) the other inhibitors were all self-administered at moderate to high levels, and did not affect memory.

It is possible that the inhibitors that did not disrupt memory in these tests might do so at higher doses. Although it is not clear how doses compare across species, the lowest doses of AM3506, URB 694 and ARN14633 that maintained significant rates of self-administration in squirrel monkeys were the same across drugs (1 g/kg), and these were three times more potent than PF-04457845; thus, there was no indication that AM3506 was more potent than the other inhibitors. The doses selected for the present study were intended to be high enough to produce near-maximal inhibition of FAAH while maintaining selectivity for FAAH versus MGL. However, there has been no study in which the selectivity of all or even most of these drugs have been measured under the same conditions in any species, and it is most parsimonious at this point to assume that they all have the potential to inhibit both FAAH and MGL to some extent. For example, Seillier et al. (2014) found that doses of JZL184 ranging from 5-30 mg/kg increased 2-AG levels 3-5 fold in rat hippocampus, but doses of 15 or 30 mg/kg also increased anandamide levels more than 2 fold. Godlewski et al. (2010) found that a 1 mg/kg i.p. dose of AM3506 fully inhibited FAAH and increased anandamide in rat brain, but did not increase 2-AG; higher doses were not tested in rats, but a 3 mg/kg dose produced a moderate (-39%) inhibition of MGL in mice. One reason that selectivity is important is that treatment with a dual inhibitor of FAAH and MGL or with a selective FAAH inhibitor combined with a selective MGL inhibitor can produce THC-like effects in drug discrimination (Hruba et al. 2015) and short-term memory tests (Wise et al. 2012). Interestingly, Hruba et al. (2015) found that combined treatment with the FAAH inhibitor PF3845 plus the MGL inhibitor JZL184 produced THC-like discriminative effects in mice, but combined treatment with the FAAH inhibitor URB597 plus JZL184 did not. They suggest that this lack of interaction between URB597 and JZL184 might be due to URB597 decreasing levels of 2-AG.

Goonawardena et al. (2011) used a delayed nonmatching-to-position procedure that was similar to the one used in the present study, but used retractable levers instead of lighted nosepoke holes to present the samples and record the responses. Their behavioral procedure is sensitive to impairment by THC, and these impairments correlate with suppressed hippocampal cell firing around the time of the sample response, presumably representing disrupted encoding of the sample (Hampson and Deadwyler 2000). Goonawardena et al. (2011) found that treatment with methanandamide or URB597 (3 mg/kg) produced THC-like effects on memory and hippocampal cell firing. It is unclear why URB597 produced impairments in their procedure but not ours, but one possibility is the use of Long-Evans rats in their study versus Sprague-Dawley rats in ours. They did not test other FAAH inhibitors, but they did find that the anandamide transport inhibitor AM404 only decreased accuracy of nonmatching at the longest delay and did not affect hippocampal firing.

As noted above, FAAH inhibition and FAAH deletion have produced learning enhancements rather than impairments in some previous studies. It appears that aversively-motivated learning is most sensitive to enhancement by FAAH manipulations, possibly due to effects of FAAH inhibition on anxiety (Haller et al. 2009) or coping behavior (Haller et al. 2013). FAAH inhibition also increases endogenous levels of PPAR-alpha ligands that might play a role in these memory enhancements (Mazzola et al. 2009; Panlilio et al. 2013). With respect to the present study, it is worth noting that our delayed nonmatching-to-position procedure is not sensitive to enhancement effects. In past experiments, we did not observe memory enhancement with nicotine, caffeine, galantamine, or rimonabant, all of which have produced cognitive-enhancing effects in other procedures. We also did not observe either enhancement or impairment of memory in an earlier study (unpublished) when —in the same nonmatching task used in the present study— URB597 was tested in Long-Evans rats at doses (0.1 and 0.3 mg/kg) that enhance passive-avoidance learning (Mazzola et al. 2009); the accuracy curves obtained at these lower doses closely resemble the curves for higher doses of URB597 shown in Figure 2 of the present study. The insensitivity of our delayed nonmatching to position procedure to enhancement might be due to the fact that the rats are highly trained prior to drug testing, producing a ceiling effect. Consistent with this possibility, rimonabant produced a more robust enhancement in a delayed nonmatching procedure when extra-long delays were included during the test (up to 80 s; Deadwyler and Hampson 2008) than when the tests used the same delay values as the baseline training schedule (up to 30 s; Deadwyler et al. 2007; Hampson and Deadwyler 2000).

In conclusion, we find that FAAH inhibitors vary in whether they produce THC-like amnestic effects in a rodent model of working memory. In other experiments, we have found that these same FAAH inhibitors vary in whether they produce THC-like reinforcing effects in the squirrel monkey model of cannabinoid self-administration. Puzzlingly, the profiles exhibited by these drugs in the memory-impairment and abuse-potential models do not fully match. These findings strongly suggest that FAAH inhibitors need to be evaluated on a case-by-case basis for different kinds of adverse effects.