This suggests that it may be preferable to assess the role of anandamide in drug reward processes by measuring changes in anandamide levels directly produced by administration of the drug rather than changes in anandamide levels produced after chronic drug exposure, when alterations may be opposite to those after acute administration and may reflect biochemical alterations associated with withdrawal. This is supported by a recent study in which Caille et al. employed micro-dialysis techniques to monitor extracellular endocannabinoid levels in the brain during active self administration of drugs and found that anandamide levels were elevated during heroin self-administration, consistent with findings by Vigano et al. . Interestingly, extracellular levels of anandamide were not altered during cocaine or ethanol self-administration . Differences in the molecular target and mechanism of actions of drugs of abuse could account for the different effects on accumbal levels of endocannabinoids. There is only limited information on the effects of food reward on brain levels of anandamide. In one study, anandamide levels in the NAcc, but not in the hypothalamus, increased after 24-h food deprivation but not during active feeding . This suggests that appetitive aspects of food reward involve release of anandamide, but consummatory aspects of reward do not. However,growing racks it is also possible that, as in the case of dopamine , natural rewards produce much lower increases in anandamide release than pharmacological rewards and that increases in anandamide occur with food reward but are lower than the limit of detection with current techniques.
Administration of addictive drugs or exposure to food increases dopamine levels in the NAcc and activation of dopamine D2 receptors in this region increases extracellular levels of anandamide . This suggests that increases in anandamide levels in the NAcc after administration of abused drugs or after eating may be secondary to increases in dopamine levels. However, Caille et al. have recently shown that drugs such as cocaine produce large increases in dopamine levels but do not alter anandamide levels in the NAcc. Finally, to the best of our knowledge, the release of CBs during electrical brain self-stimulation remains to be investigated. However, indirect pharmacological evidence indicates that, at least in the VTA, 2-AG and not anandamide is released after application of brain stimulation that would sustain self-administration in rats .As anandamide is quickly degraded, studying its normal roles can be facilitated by blocking the mechanisms of degradation and, thus, magnifying and prolonging its actions. Importantly, increases in anandamide levels would presumably be obtained only in those brain regions where anandamide is released. As previously described, the intracellular step of anandamide degradation is mediated mainly by FAAH activity . Thus, the specific contribution of anandamide to reward functions can be more readily investigated when FAAH is inhibited by drugs such as URB597 . It should be noted that URB597 has no rewarding effects by itself in conditioned place preference paradigms, does not produce THC-like discriminative effects and does not alter dopamine levels in the shell of NAcc in rats. One study found that the directly acting CB receptor agonists such as THC and WIN 55,212-2 increased the reinforcing effects of heroin, but FAAH inhibition by URB597 had no significant effect, suggesting that heroin-induced release of anandamide is not necessary for opiate reward .
Another study found that administration of URB597 increased ethanol preference and intake in a two-bottle free-choice procedure . In a recent study, we found that nicotine can produce THC-like discriminative effects when given in combination with URB597, indicating that nicotine does release anandamide and that, under conditions of FAAH inhibition, can actually produce THC-like behavioural effects . However, it has to be established whether the reinforcing effects of nicotine itself, rather than its ability to produce THC-like discriminative effects, are altered by stimulation of the endocannabinoid system, as another study found that the discriminative effects of nicotine are not altered by administration of URB597 or combinations of URB597 plus anandamide . Finally, one study has found that the effects of ethanol on the mesolimbic dopaminergic system are reduced by URB597 in mutant FAAH-null mice ; however, the behavioural relevance of these neurochemical alterations remains to be established as mutant FAAH-null mice show a higher preference for ethanol . The effects of URB597 on food reward have not yet been systematically studied, but one study investigated the effects of administration of URB597 on brain stimulation reward. FAAH inhibition increased the threshold for intracranial self stimulation, indicating a decrease in the reinforcing effects of brain stimulation . However, these effects were obtained at doses of URB597 3–10 times higher than those required to produce significant inhibition of FAAH and dramatic potentiation of the effects of exogenously administered anandamide and, thus, the interpretation of these results is difficult and may be related to loss of selectivity at high doses.In parallel with results obtained with URB597, the availability of FAAH-null mice has further expanded possibilities of assessing anandamide’s role in brain reward processes. As with CB1-null mice, FAAH-null mice do not show dramatic changes in body weight, food consumption or fertility , suggesting, again, that the endocannabinoid system plays only a modulatory role in basic reward functions. There are only a few studies on the effects of genetic ablation of FAAH on reward processes. Among drugs of abuse, information is available only regarding ethanol. In one study, it was found that ethanol consumption and preference increase in female, but not male, FAAH-null mice , whereas in another study it was found that ethanol consumption and preference increase in both female and male FAAH-null mice . Interestingly, conditioned place preferences were similarly obtained in males, but not in females, when ethanol was injected intraperitoneally , indicating that the consequences of FAAH deletion on ethanol’s rewarding effects may be complex. To date, no study has investigated the effects of FAAH deletion on food or brain stimulation reward.
The finding that FAAH-null mice show a slightly antidepressant-like phenotype is consistent with a role for anandamide in mood regulation .The best evidence for a role of 2-AG in brain reward processes comes from studies that measured changes in brain levels of 2-AG produced by drugs, food or brain stimulation . Chronic administration of many abused drugs alters levels of 2-AG in brain areas involved in reward. For example, it has been shown that chronic administration of THC decreases tissue levels of 2-AG in the striatum , ethanol decreases tissue levels of 2-AG in the midbrain but not in the nucleus accumbens , nicotine or cocaine do not alter 2-AG levels either in the midbrain or the striatum and morphine decreases 2-AG levels in the striatum without altering 2-AG levels in the striatum . Moreover, Vigano et al. found that acute injection of morphine decreased tissue levels of 2-AG in the striatum more than chronic administration and that a challenge injection of morphine, after a 2-week period of withdrawal, in rats chronically treated with morphine,vertical growing racks had effects similar to those of an acute injection. On the other hand, no changes in extracellular levels of 2-AG were found in the NAcc of rats self-administering heroin, but there was an increase in 2-AG levels in rats self-administering ethanol . The general picture that can be drawn from these data is that 2-AG is involved in some consequences of repeated administration of abused drugs but it may be more important for the rewarding effects of ethanol than for the rewarding effects of other abused drugs, such as heroin, cocaine or nicotine. One study investigated the effects of manipulation of food intake and food deprivation on brain levels of 2-AG and found that tissue levels of 2-AG were increased by fasting but were decreased by feeding , indicating that 2-AG would be more important for appetitive aspects than consummatory aspects of food reward. Finally, no study has investigated brain levels of 2-AG during electrical brain self-stimulation reward. However, one study has shown that electrical stimulation of the medial prefrontal cortex produces a decrease in presynaptic glutamate release that might be explained by release of 2-AG . Future studies are needed to verify that these neurochemical results have behavioural relevance.As noted in section ‘Synthesis and degradation of endocannabinoids and endocannabinoid transport’, endocannabinoid transport remains one of the less understood features of the endocannabinoid system. Although endocannabinoid transport was proposed long ago and drugs thought to inhibit the transport of endocannabinoids through the cell membrane have been available for a decade , the molecular entity mediating this transport is still unknown and the nature of endocannabinoid transport is debated .
However, drugs such as AM404, VDM11, UCM707 and AM1172 , which are thought to block endocannabinoid uptake into the cell, can be important tools for investigating the role of the endocannabinoid system in brain reward processes. Unlike the FAAH inhibitor URB597, the uptake inhibitor AM404 has some rewarding effects by itself using a conditioned place preference procedure, although its rewarding effects measured with this procedure are clearly smaller than those of the direct CB receptor agonist WIN 55,212-2 . Interestingly, AM404 induced conditioned place preferences in rats at a dose that did not significantly increase tissue levels of anandamide or 2-AG in the brain areas investigated . On the other hand, AM404 does not produce THC-like discriminative effects and does not alter dopamine levels in the shell of NAcc in rats . It is, thus, surprising that the effects of AM404 or other uptake inhibitors on drug, food or brain stimulation reward have seldom been studied. To our knowledge, only one study has investigated the effects of uptake inhibition on drug reward and in that study AM404 did not increase heroin self-administration under a progressive-ratio schedule and, instead, produced a small decrease . In that study, CB1 receptor agonists such as THC and WIN 55,212-2 potentiated heroin self administration and URB597 had no effect on heroin self administration. One interpretation of these results is that enhancement of endocannabinoid tone has either an inhibitory or a neutral effect on opioid reward, whereas broad activation of CB1 receptors has a facilitatory effect on opioid reward. The effects of blockade of anandamide transport on food intake and electrical brain stimulation reward have been investigated using the uptake inhibitors VDM-11 and OMDM-2. It was found that VDM-11 does not alter food intake in rats and that high doses of OMDM-2 increase the threshold for electrical brain self-stimulation reward . Obviously, more research is needed to interpret the effects of inhibition of endocannabinoid uptake on brain reward processes and to understand its role in the regulation of the activity of the endocannabinoid system. For example, we have recently found that some effects of anandamide, such as its ability to produce THC-like discriminative effects and to increase dopamine levels in the NAcc, are potentiated by URB597 but not by AM404 , suggesting that regional differences may exist in the relative roles of uptake inhibition and FAAH inhibition.The importance of the endocannabinoid system in drug reward varies greatly depending on the drug studied, with a pronounced role for the endocannabinoid system in opioid and ethanol reward and more subtle roles in nicotine and psychostimulant reward. It is clear that activity of CB1 receptors is important for opioids to produce maximal reinforcing effects . Although opioids release anandamide in the NAcc , the importance of this release remains unclear given that compounds that increase brain concentrations of anandamide and prolong anandamide’s actions do not alter heroin self-administration . Thus, interactions between CB1 receptors and mu-opioid receptors may be more important than endocannabinoid release in the modulation of opioid effects.It is also clear that activity of CB1 receptors is important for ethanol to produce maximal reinforcing effects . Although it has been reported that ethanol self-administration increases 2-AG but not anandamide levels in the NAcc , FAAH-null mice show higher preferences for ethanol but no higher ethanol-induced conditioned place preferences . Thus, although release of endocannabinoids appears to participate in ethanol reward, the relative importance of 2-AG and anandamide needs to be established. CB1 receptors may be critically involved in the rewarding effects of nicotine .