What may be the synaptic function, which necessitates principal neurons to target DGL-α so precisely into dendritic spine heads? DGL-α synthesizes 2-AG from diacylglycerol, the common second messenger produced upon Gq/11-coupled receptor activation and phospholipase C-β activity. The most abundant Gq/11-coupled receptor and PLC-β-type enzyme in hippocampal dendritic spine heads is the metabotropic glutamate receptor type 5 and PLC-β1, respectively , and indeed, activation of mGluR5 leads to endocannabinoid-mediated retrograde synaptic suppression , and the elevation of 2-AG levels through PLC-β1 and DGL-α activity. Because 2-AG inhibits glutamate release from excitatory nerve terminals inhippocampal neurons , this negative feed-back pathway operating as a “synaptic circuit-breaker” may have a pivotal functional significance in controlling network excitability during neuronal insults leading to excitotoxicity. Although the medical importance of such a protective messenger system is obvious, it is not yet clear if the same molecular machinery is functional at excitatory synapses of human neurons as well. The postsynaptic accumulation of DGL-α at excitatory synapses in human hippocampal samples described in the present study along with evidence that mGluR5 is also present postsynaptically at excitatory synapses both in the human hippocampus and in primate cortical areas underlies this notion, though similar data are not yet available for PLC-βs in humans. Interestingly, an independent structural support derives from the striking similarity in the density of DGL-α and mGluR5- immunostaining in relation to given hippocampal layers. For example, in the dentate gyrus, higher concentration of DGL-α was found in the inner third of the molecular layer than in the outer two-thirds of stratum moleculare both in rodents and in humans ,cannabis dry rack underlying the observation that excitatory inputs of granule cells received from mossy cells may be more tightly controlled by endocannabinoids than afferents from the entorhinal cortex. 

Similarly to DGL-α distribution, the density of mGluR5-immunostaining is more pronounced in the inner molecular layer both in rodents and in humans. Finally, among glutamatergic terminal types, the concentration of CB1 receptors is also highest in those arborizing in the inner molecular layer. Whether this intensity difference reflects the higher density of excitatory synapses in the inner molecular layer both in rodents , and in humans or it is due to synapse-specific variations in the regulation of synaptic 2-AG signaling needs to be established in further experiments. Nevertheless, the weaker density of MGL-immunostaining in the inner molecular layer indicating reduced capacity for 2-AG inactivation by MGL at mossy cell synapses gives some indirect support for the latter possibility and emphasizes that the termination of 2-AG signaling may be specifically regulated in the human hippocampus as well.We provide anatomical evidence that MGL, the main degrading enzyme of 2-AG has a widespread distribution in the human hippocampal formation. The nature of MGLimmunoreactivity was comparable to DGL-α-immunostaining at the light microscopic level, with the profuse punctate labeling covering the neuropil and outlining the laminar structure of the hippocampus. Electron microscopic analysis uncovered that this compartmentalized staining pattern is due to the accumulation of immunolabeling at excitatory synapses. However, in striking contrast to the postsynaptically localized DGL-α enzyme, MGL was present presynaptically in glutamatergic axon terminals. This anatomical observation is in agreement with previous findings obtained in the rodent hippocampus , and it is also supported by recent physiological experiments demonstrating that MGL limits the duration of synaptic depression at hippocampal excitatory synapses. In addition, although further immunohistochemical studies using antibodies with higher sensitivity may reveal that MGL is not fully restricted to glutamatergic synapses, the present findings indicate that the highest concentration of MGL protein is likely located in excitatory boutons in the human hippocampus.It is estimated that approximately 85% of the brain’s 2-AG hydrolysis activity is accounted for this serine hydrolase. Its widespread distribution in excitatory axon terminals in the human hippocampus suggests that MGL may also play a similarly important role in 2- AG hydrolysis in the human brain.

Together with our previous findings demonstrating the ubiquitous presence of CB1 cannabinoid receptors on the same excitatory terminals , these data collectively corroborate that MGL is the key enzyme terminating synaptic 2-AG signaling after activation of presynaptic CB1 receptors in the human hippocampal formation. Given that acute in vivo administration of JLZ184, the most potent selective inhibitor of MGL currently available, replicates nearly all of the characteristic behavioral effects of Δ9 – tetrahydrocannabinol by protecting endogenously released 2-AG from degradation , it is conceivable to hypothesize that JLZ184 may have a similar effect on the human brain based on the similar neuroanatomical localization of MGL in rodents and humans. Therefore, although MGL inhibitors hold great therapeutic potential in several medical applications , their predicted psychoactive side effects based on their cannabimimetic properties in animals , should be taken into consideration when pondering the use of these compounds in humans. Cannabis and alcohol are two of the oldest drugs used by humans. Together with nicotine, they represent a relevant health problem because of the clinical consequences of their abuse. Their psychotropic effects are well known and recent research has shown that there is a close link between cannabis and alcohol. The endogenous cannabinoid system [a functional set of lipid transmitters and receptors that is the target of both natural and synthetic cannabinoids ] has been shown to mediate some of the pharmacological and behavioral aspects of alcohol. Both cannabinoids and alcohol activate the same reward pathways and the cannabinoid CB1 receptor plays an important role in regulating the positive reinforcing effects of alcohol as well as alcohol relapse. Several studies have documented that endocannabinoid transmission becomes hyperactive in reward-related areas during chronic ethanol administration. This hypothesis is based on two findings. First, the increase in the levels of both anandamide and 2-arachidonylglicerol, the two main endocannabinoids,planting racks observed in animals chronically exposed to ethanol. Second, the down-regulation of CB1 receptors induced by endocannabinoid-mediated over-stimulation. Following this rationale, cannabinoid CB1 receptor knockout mice show reduced alcohol preference and self-administration. In this experiment, rats were tested under a progressive ratio schedule of reinforcement to measure the break point for ethanol.

For this purpose, animals were first trained to self-administer 10% alcohol under a fixed ratio 1 schedule of reinforcement. Following the acquisition of a stable baseline of responding for 10% ethanol, rats were tested under the progressive ratio condition, in which the response requirement was increased as follows. For each of the first four ethanol deliveries the ratio was increased by 1; for the next four deliveries the ratio was increased by 2 and for all of the following deliveries the ratio was increased by 4. Each ethanol-reinforced response resulted in a 1.0 s illumination of the house light, whereas sessions were terminated when more than 30 min had elapsed since the last reinforced response. Drug testing was carried out once a week as follows. The progressive ratio baseline was established on days 1 and 2, whereas progressive ratio drug testing took place on day 3. For the next 2 days, animals were placed in the chambers under fixed ratio 1 condition to re-establish the ethanol self-administration baseline, whereas on days 6 and 7 they remained confined to their home cages. AM404 or its vehicle was given 30 min before the progressive ratio session. The experiment was repeated for the following 2 weeks, counterbalancing the treatment.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, 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 bedding of the chamber was changed and bedding trays were cleaned between 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.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 reducesethanol 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.