The travel distance was also increasing over time. An interesting exploratory observation indicated that, compared to RMDs located further away from schools, a larger proportion of RMDs in reachable distance to schools had interior child-appealing marketing. It is possible that RMDs intentionally targeted children if they were in closer proximity of schools. Unfortunately, our study was not able to test this hypothesis directly. Almost all the audited RMDs followed California rules on age verification. If continuous monitoring and enforcements are not in place, however, children might get access to abundant child-appealing marketing practices inside of the dispensaries, the consequences of which could be grave. Furthermore, exterior signs of age limit were absent in over 80% RMDs and security personnel were only observed in 40% RMDs. These might increase the risks of accidental or even intentional attempts of children to enter RMD premises, who would be then exposed to interior marketing in waiting area. Compared to laws in other states, California regulations on child-appealing marketing seem to be vague and less comprehensive during the study period. Because content restrictions are inherently subjective, it might be challenging for California RMDs to comply and for regulators to enforce without objective, operationalizable measures of “child-appealing”. Fortunately, after this study was completed, California released new regulations in January 2019 on child-relevant products and marketing. Specifically, marijuana products and packages “shall not use any depictions or images of minors” and “shall not contain the use of objects, such as toys, inflatables, movie characters, cartoon characters, or include any other display, depiction, or image designed in any manner likely to be appealing to minors”. These texts are expected to provide clearer guidance to law compliance and enforcements. In addition to prohibitions in laws, California could also consider screening content materials such as packages before they are available in RMDs. For instance, Massachusetts allows manufacturers to submit artwork to a regulatory board for review to ensure non-child-appealing packaging.

Standardized packaging might be another alternative,cannabis drying trays which has shown effectiveness in tobacco control outside of the US. This study has limitations. First, this study used a cross-sectional design to capture a snapshot in summer 2018, approximately half a year after California’s commercialization of marijuana. This unique transition period was characterized with a lack of law enforcement, delay of dispensary licensing, and inadequate understanding of laws. As the legal market matures and government makes endeavors on law interpretation and enforcement, we might expect a stronger compliance with laws and possibly a reduction in marketing practices. The findings may not be generalizable to other time points in California. Second, our observations were largely constrained within the regulatory regime in California and may not be generalizable to other states where different regulatory measures are in place. Third, frequency or quantity measures in each marketing category would be more informative than simple binary indicators for availability. Unfortunately, a dispensary often displays hundreds or even thousands of products, packages, paraphernalia, and advertisements. Obtaining frequency or quantity information requires the field workers to spend a considerably longer time evaluating the RMD environment, which is infeasible in practice. Fourth, California laws lacked specific details related to children during the study period. The classification of child-appealing was informed by laws in other states and constructed with authors’ own understanding, which may not reflect California lawmakers’ intention or completely align with recently released new regulations. Further, there might be inevitable measurement errors even after two field workers discussed and resolved discrepancies between them. Lastly, this study only gathered data on RMDs in closest proximity to public schools. To improve representativeness, future research is encouraged to audit a random sample of RMDs. The increasing spread of marijuana use, especially among adolescents and young adults , has heightened societal awareness of the risks associated with this drug and has highlighted the need to fully understand its mechanism of action. Basic research has shown that D9 -tetrahydrocannabinol , the main active constituent of marijuana, produces its effects by combining with selective receptors present on the membrane of cells in the brain, the vasculature and the immune system .

Research has also revealed that a group of lipid-derived substances produced by the body engages these receptors and participates in biological processes as diverse as pain perception, memory formation and blood pressure regulation. This knowledge has allowed researchers to interpret the pharmacological properties of marijuana, but remains inadequate to the task of developing strategies for the medicinal management of marijuana dependence. No such strategies exist at present , despite the fact that pharmacotherapy—alone or in combination with behavioral therapy—is considered a primary treatment option for drug dependence when abuse prevention fails . Several basic questions, which are relevant to the pharmacotherapy of marijuana dependence, remain unanswered. For example, while it is clear that D9 -THC acts by hijacking the brain endocannabinoid system, its impact on the various components of this system—synthetic and catabolic enzymes, transporters, and receptors—is still largely undefined. Does D9 -THC produce rapid adaptive changes in neuronal endocannabinoid signaling, as recent evidence indicates ? And, if so, do such changes contribute to the pharmacological actions of the drug? Does prolonged exposure to D9 -THC cause stable alterations in endocannabinoid signaling? And, if so, do such alterations contribute to marijuana dependence and, most importantly, can they be safely reversed to restore normality? Answering these questions may not only help develop effective therapeutic strategies for marijuana dependence, but in light of the broad roles played by the endocannabinoid system in the control of brain reward processes , might also shed new light on fundamental mechanisms of drug addiction. To accomplish this task, it seems important to move forward in two convergent directions: the molecular characterization of endocannabinoid signaling, much of which is still uncharted; and the development of pharmacological agents that interfere with specific components of this system. In the present review, I outline recent progress made in these directions, specifically focusing on endocannabinoid deactivation, and discuss some of the challenges lying ahead.Anandamide was the first endocannabinoid substance to be isolated and structurally characterized . Its formation inneural cells is thought to require two enzymatic steps, which are illustrated in Fig. 1. The first is the activity-dependent cleavage of the phospholipid precursor N-arachidonoyl-PE . This reaction, which is mediated by a unique D-type phospholipase , produces anandamide and phosphatidic acid, which is recycled to produce other glycerol-containing phospholipids.

The cellular stores of NAPE are small, but can be refilled by an N-acyltransferase activity, which catalyzes the intermolecular passage of anarachidonic acid group from the sn-1 positionof phosphatidylcholine to the head group of phosphatidylethanolamine . Incultures of rat cortical neurons, NAT activity is controlled by two intracellular second messengers: Ca2+, which is required to activate the enzyme, and cyclic 30 , 50 -adenosine monophosphate , which stimulates protein kinase A-dependent protein phosphorylation and, via an unknown mechanism, enhances NAT activity . Although separate enzymes catalyze the syntheses of anandamide and NAPE,heavy duty propagation trays the two events are likely to occur simultaneously because Ca2+- stimulated anandamide production is often accompanied by de novo formation of NAPE . Anandamide synthesis can be elicited in vitro by a variety of agents that elevate intracellular Ca2+ levels. For example, the Ca2+ ionophore ionomycin stimulates [ 3 H]anandamide formation in cultures of rat striatal and cortical neurons labeled by incubation with [ 3 H]ethanolamine . In the same neurons, Ca2+-dependent [3 H]anandamide production may be elicited by the glutamate receptor agonist, kainate, by the K+ channel blocker 4- aminopyridine, and by membrane-depolarizing concentrations of K+ ions . Depolarizationof neural cells was also shownto evoke Ca2+-dependent anandamide release in vivo . Along with Ca2+ entry, activation of certain G protein-coupled receptors can also initiate anandamide generation. Administration of the dopamine D2-receptor agonist quinpirole causes a profound stimulation of anandamide synthesis in the rat basal ganglia, which is prevented by the D2 antagonist raclopride . Importantly, cocaine elicits a similar response , suggesting a role for anandamide in the actions of these psychostimulant drugs. The ability of the anandamide transport inhibitor AM404 to reduce D2 agonist induced hyperactivity, discussed below, further supports this possibility .The prototype of this class of drugs, the arachidonate derivative AM404 , has provided important information on the properties of anandamide transport, not only aiding the in vitro characterization of this process, but also helping to reveal its possible functions in animals. Importantly, the partial cannabimetic profile exhibited by this agent in vivo suggests that anandamide transport might provide a useful target in disease conditions in which the endocannabinoid system is hypofunctional . Evidence indicates that one such condition could be opiate withdrawal, which is markedly reduced in rodents by administering AM404 . These theories have been hindered by the fact that the putative transport system responsible for anandamide internalization is still uncharacterized at the molecular level. In fact, the presence of such a system has been recently questioned, based onthe observation that [ 3 H]anandamide uptake in certain cell lines is saturable at longer , but not at shorter incubationtimes . This finding has been interpreted to suggest that fatty-acid amide hydrolase —a key enzyme of intracellular anandamide degradation, described in a subsequent section—may be responsible for the saturation of uptake noted at longer incubation times . However, the result may also be explained on purely technical grounds, as the high concentration of serum albumin used inthe experiments of Glaser and collaborators was previously shownto prevent [3 H]anandamide internalization . Consistent with this interpretation, recent studies have provided additional evidence for the existence of an anandamide transport system independent of FAAH . In particular, one of these studies has shown that cultures of cortical neurons isolated from the brain of FAAHnull mice internalize anandamide as efficiently as do neurons that express normal levels of the enzyme. The same study also demonstrated that the transport inhibitor AM404 is equally effective at reducing anandamide internalization in neurons of FAAH-null and wild-type mice. These results indicate that FAAH does not provide the driving force for anandamide uptake or serve as a target for AM404. Invivo experiments further support this conclusion, showing that AM404 not only enhances the actions of exogenous anandamide in FAAH-null mice, but acts more effectively in this mutant strain than it does in control animals. This implies that AM404 is not in fact a FAAH inhibitor, as it has been proposed , but a FAAH substrate. In support of this idea, it was found that membranes prepared from the brains of normal mice rapidly hydrolyze AM404, whereas those prepared from mice that lack FAAH are unable to carry out this reaction.The fact that FAAH is not directly involved in anandamide internalization raises the question of what mechanism provides the driving force for this process. One possibility is that an intracellular protein may sequester anandamide at the membrane, driving its internalization and facilitating its movement to the mitochondria and the endoplasmic reticulum, where FAAH is primarily localized . If selective for anandamide, such a protein might participate in the transport process as well as serve as a target for transport inhibitors. This hypothetical model is consistent with fattyacid transport into cells, which is also thought to require the cooperation of membrane transporters and intracellular fatty-acid binding proteins .AM404 increases endogenous anandamide levels in brain tissue and peripheral blood of rats and mice . This effect is accompanied by a series of behavioral responses that, though blocked by the CB1 antagonist rimonabant , are clearly distinguishable from those of direct cannabinoid agonists. For example, administration of AM404 into the cerebral ventricles of rats decreases exploratory activity without producing catalepsy and analgesia, two hallmarks of direct CB1 receptor activation. Inaddition, AM404 reduces two characteristic effects caused by activation of D2 family receptors: the yawning response elicited in mice by low doses of the D1/D2-receptor agonist apomorphine; and the stimulationof locomotor activity evoked in rats by the D2- receptor agonist quinpirole . These effects are observed at doses of AM404 that selectively target anandamide transport and produce only mild hypokinesia when the drug is administered alone .