A microcomputer controlled the delivery of fluids, presentation of auditory and visual stimuli, and recording of the behavioral data. Rats were trained to self-administer 10% ethanol , 0.2% saccharin or water in 30 min daily sessions on a fixed-ratio 1 schedule of reinforcement, where each response resulted in delivery of 0.1 mL of fluid as previously described Briefly, for the first 3 days of training, water availability in the home cage was restricted to 2 h ⁄ day in order to facilitate acquisition of operant responding for a liquid reinforcer. During this time, rats were permitted to lever-press for a 0.2% saccharin solution. At this point, water was made freely available and saccharin self-administration training continued for another 3 days. The rats were then trained to self-administer ethanol by using a modification of the sucrose-fading procedure that used saccharin instead of sucrose . During the first 6 days of training rats were allowed to lever-press for a 5.0% ethanol solution containing 0.2% saccharin . Starting on day 7, the concentration of ethanol was gradually increased from 5.0 to 8.0% and finally to 10.0% , while the concentration of saccharin was correspondingly decreased to 0%. At the beginning of the saccharin-fading procedure a second but inactive lever was introduced. Responses at this lever were recorded during all training and testing phases as a measure of non-specific behavioral activation but they had no programmed consequences.At completion of the fading procedure, animals were trained to discriminate between 10% ethanol and water in 30 min daily sessions. Beginning with self-administration training at the 10% ethanol concentration, discriminative stimuli predictive of ethanol vs. water availability were presented during the ethanol and water self administration sessions, respectively. The discriminative stimulus for ethanol consisted of the odour of an orange extract , whereas water availability was signaled by an anize extract . The olfactory stimuli were generated by depositing six to eight drops of the respective extract into the bedding of the operant chamber. In addition,hydroponic grow table each lever-press resulting in delivery of ethanol was paired with illumination of the chamber’s house light for 5 s . The corresponding cue during water sessions was a 5 s tone .

Concurrently with the presentation of these stimuli, a 5 s time-out period was in effect, during which responses were recorded but not reinforced. The olfactory stimuli serving as S+ or S– for ethanol availability were introduced 1 min before extension of the levers and remained present throughout the 30 min sessions.The rats were only given ethanol sessions during the first 3 days of the conditioning phase. Subsequently ethanol and water sessions were conducted in random order across training days, with the constraint that all rats received a total of 10 ethanol and 10 water sessions.Reinstatement tests began the day after the last extinction session. These tests lasted 30 min under conditions identical to those during the conditioning phase, except that alcohol and water were not made available. Sessions were initiated by the extension of both levers and presentation of either the ethanol S+ or water S– paired stimuli. The respective discriminative stimulus remained present during the entire session and responses at the previously active lever were followed by activation of the delivery mechanism and a 5 s presentation of the CS+ in the S+ condition or the CS– in the S– condition. Animals were tested under the S+ ⁄ CS+ condition on day 1 and under the S– ⁄ CS– condition on day 2. Subsequently, reinstatement experiments were conducted every fourth day , in which AM404 was administered 30 min prior to the sessions. Responding at the inactive lever was constantly recorded to monitor possible non-specific behavioral effects.Pre-treatment with the anandamide transport inhibitor AM404 30 min prior to the ethanol self-administration session significantly reduced the operant response for ethanol in a dose-dependent manner . This effect was not due to a decrease in the reinforcing value of ethanol because progressive ratio experiments resulted in similar break points for animals treated with vehicle or AM404 . They were not derived or a motor inhibition induced by AM404 as the 2 mg ⁄ kg dose did not affect locomotion at the time of operant behavior testing . The effects were selective for ethanol because pre-treatment with AM404 did not modify operant responding for saccharin .

In addition, administration of AM404 did not alter food motivation and thus, food intake in rats deprived of food for 24 h . These results suggest that the pharmacological effects of the anandamide transport inhibitor are not related to a devaluation of the motivational state or a devaluation of motivational properties of natural reinforcers.In a subsequent experiment, we tested the efficacy of AM404 as a modulator of not only the operant responses for ethanol but also the operant responses elicited by the contextual stimuli associated with alcohol. As the highest dose tested resulted in significant inhibition of locomotion, we did not administer it in this context. Once a stable extinction baseline was observed, we induced relapse by presenting cues associated with ethanol delivery during training. Ethanol-related contextual stimuli elicited ethanol-seeking behavior, as operant responses induced by ethanol-associated stimuli were more intense and significantly higher than those observed on the last day of extinction . When AM404 was injected 30 min prior to cue presentation, it failed to alter the responses for ethanol seeking , indicating that anandamide uptake inhibition was not effective in preventing cue-induced relapse.The major finding of the present study is the demonstration that acute administration of the anandamide transport inhibitor AM404 reduce sethanol self-administration under an operant conditioning schedule. This compound does not affect the relapse induced by contextual cues associated with ethanol. The effects of AM404 seem to be selective for ethanol, as it was unable to suppress responding for other reinforcers, such as saccharin or food intake, suggesting that this effect is not related to a decrease in a general motivational state. This is confirmed by the lack of action of AM404 on the motivational properties of ethanol, as measured in the progressive ratio paradigm. This suppressive effect of AM404 on ethanol self-administration seems to be independent of the already known anandamide-induced motor impairment, as the lowest effective dose tested did not alter motor behavior in the open field. Moreover, the actions of AM404 were found to be independent of a potentiation of the sedative effects of ethanol.

Finally, neither experiments with cannabinoid CB1 receptor agonists nor with cannabinoid CB1 and CB2 receptor antagonists allowed us to obtain a direct pharmacological confirmation of the role of known cannabinoid receptors on the effects of AM404. The finding of a similar profile of effects using ACEA, a selective cannabinoid CB1 receptor ligand that shares the arachidonoyl moiety with both anandamide and AM404, suggests a common unknown target responsible for the effects of AM404 on ethanol self-administration. The lack of effects of WIN 55,212-2 and HU-210 at doses devoid of motor side-effects suggests that AM404 does not exert its actions through a CB1 receptor-mediated mechanism. AM404 was the first synthetic inhibitor of anandamide uptake and it has been shown to potentiate many effects elicited by anandamide in vitro and in vivo . As AM404 does not activate cannabinoid receptors , the effects of this drug were suggested to result from the elevation of endogenous anandamide levels . However, recent findings suggest that AM404 also directly activates the vanilloid VR1 receptor , complicating the identification of its mechanism of action on ethanol self-administration. However, the effect of AM404 was not reversed or enhanced by pre-treatment with the competitive vanilloid VR1 receptor antagonist capsazepine, indicating that the inhibitory action of AM404 is not mediated through VR1 stimulation and may be derived from other targets in the endocannabinoid system. Following this rationale we studied the involvement of the cannabinoid CB1 receptor, the natural target of anandamide. In order to confirm its participation we first studied whether the cannabinoid receptor antagonist SR141716A reversed the actions of AM404. This pharmacological test was complicated by the inhibitory actions of SR141716A on ethanol self-administration that precluded the observation of a reversal of the actions of AM404. A second strategy was to compare the actions of AM404 with those of selective cannabinoid CB1 receptor agonists belonging to three of the four main classes of cannabinoid agonists: eicosanoids ,flood tray aminoalkylindoles and classical cannabinoids . The effects of these compounds in ethanol self-administration are not similar to those of AM404. ACEA and WIN 55,212-2 reduced ethanol self-administration, although the component of motor inhibition of WIN 55,212-2 might be responsible for this effect. However, the classical cannabinoid receptor agonist HU-210 did not affect ethanol self-administration . We replicated this finding in a separate study in Marchigian Sardinian alcohol-preferring rats . These results indicate that the contribution of the CB1 receptors to AM404 cannot be supported. The similar profile of actions observed after systemic administration of either cannabinoid CB1 receptor agonists or antagonist seems to be challenging. It has been reported that both cannabinoid CB1 receptor agonists, such as tetrahydrocannabinol, CP55 940 and WIN 55,212-2, and cannabinoid receptor antagonist ⁄ inverse agonists, such as SR141716A, suppress operant behavior . These reports stress the pleiotropic spectrum of actions found after the interference with endocannabinoid signaling. The complex roles of the endocannabinoid system on the regulation of GABA and glutamate synapses throughout the brain circuits processing the appetitive ⁄ motivational properties of ethanol might explain these findings .

As an example, we have recently described that intracerebral injections of SR141716A only affect ethanol selfadministration in rats when the CB1 antagonist is infused in the prefrontal cortex but not in the hippocampus or dorsal striatum . Moreover, in this study, local blockade of fatty acid amidohydrolase, the main enzyme that degrades anandamide, enhances ethanol self-administration when injected into the prefrontal cortex. However, we cannot exclude additional targets such as noncloned cannabinoid-like receptors on which anandamide and WIN 55,212-2 may act. Thus, the present study stressed the need to clarify the growing complexity of endocannabinoid pharmacology, especially in the field of motivated behaviors. Although the present results exclude VR1, CB1 and CB2 receptors as the targets of the effects of AM404, we cannot exclude the contribution of endocannabinoids elevated by AM404 to the present actions, especially because the endocannabinoid system has been recently implicated in the neuroadaptations that occur during acute alcohol exposure, alcohol dependence and abstinence. Several studies have documented that endocannabinoid transmission is acutely inhibited by ethanol and becomes hyperactive during chronic ethanol administration, as revealed by the increase in the levels of endocannabinoids and the down-regulation of CB1 receptors . Thus, it is tempting to imagine that those compounds that increase endocannabinoid transmission, such as AM404, might be useful in reducing operant responses for ethanol. With the precautions derived from the non-CB1 profile of the effects of AM404, we propose that the increased levels of endogenous cannabinoids occurring during chronic ethanol administration contribute to facilitate the action of AM404; the neuroadaptations in the central nervous system associated with chronic ethanol intake lead to an increase in anandamide levels and this event could enhance the action of AM404 acting through the increased endogenous anadamide. However, we also demonstrate that the acute administration of AM404 was not able to suppress the relapse response for ethanol, i.e. the reinstatement of ethanol responding induced by the presentation of contextual cues associated with ethanol after a period of extinction. The differential response to AM404 in self-administration and relapse conditions may have a neuropharmacological basis in the recently described changes in endocannabinoid levels after chronic ethanol exposure . A possible explanation for these differences may reside in the probable alterations induced by chronically consumed ethanol in the functionality of the receptor systems mediating the central effects of ethanol that sustain ethanol-drinking behavior in rats. These neuroadaptation processes might result in a decreased potency and efficacy of the ligands. The increased levels of anandamide observed during ethanol consumption may return to basal levels or even disappear and thereby AM404 could not be acting in such a situation.This hypothesis is supported by the results obtained recently by Gonzalez et al. who showed that the levels of endocannabinoids underwent significant changes in reward-related areas during relapse, showing the lowest values in this phase.