Endocannabinoids modulate a diverse array of functions including progenitor cell proliferation in the central nervous system, and odorant detection and food intake in the mammalian central olfactory system and larval Xenopus laevis peripheral olfactory system. However, the presence and role of endocannabinoids in the peripheral olfactory epithelium have not been examined in mammals. We found the presence of cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptor protein and mRNA in the olfactory epithelium. Using either immunohistochemistry or calcium imaging we localized CB1 receptors on neurons, glia-like sustentacular cells, microvillous cells and progenitor-like basal cells. To examine the role of endocannabinoids, CB1- and CB2- receptor-deficient (CB1-/-/CB2-/-) mice were used. The endocannabinoid 2-arachidonylglycerol (2-AG) was present at high levels in both C57BL/6 wildtype and CB1-/-/CB2-/- mice. 2-AG synthetic and degradative enzymes are expressed in wildtype mice. A small but significant decrease in basal cell and olfactory sensory neuron numbers was observed in CB1-/-/CB2-/- mice compared to wildtype mice. The decrease in olfactory sensory neurons did not translate to impairment in olfactory-mediated behaviors assessed by the buried food test and habituation/dishabituation test. Collectively, these data indicate the presence of an endocannabinoid system in the mouse olfactory epithelium. However, unlike in tadpoles, endocannabinoids do not modulate olfaction. Further investigation on the role of endocannabinoids in progenitor cell function in the olfactory epithelium is warranted.

While there is currently active debate over possible therapeutic applications of marijuana and cannabis-based compounds, consistently their primary drawbacks have been the psychoactive properties, dependence, and abuse potential. Prolonged administration of ∆9-tetrahydrocannabinol (THC), the primary psychoactive constituent in marijuana, demonstrates both tolerance and physical withdrawal in both preclinical and clinical studies. Repeated THC administration also produces CB1 receptor adaptations in the form of reduced activation of receptors, along with a downregulation of membrane surface receptors, in many brain regions involved in THC-associated behaviors. The increased need for drug to maintain therapeutic effects, and a withdrawal syndrome following discontinuation of use, are common risk factors in drugs of abuse. Recently, compounds have been developed that prolong the availability of the major naturally occurring endogenous cannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), through inhibition of their catabolic breakdown by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The overall objectives of this research are to elucidate the physiologic roles of these two endogenous ligands and to determine if either can produce beneficial therapeutic effects without negative cannabis-like CNS effects. Therefore, we tested the impact of acute and prolonged blockade of FAAH and MAGL on a variety of cannabinoid-mediated behaviors and on precipitated cannabinoid withdrawal. Despite that acute blockade of FAAH and MAGL produce similar efficacy in reducing nociceptive responses, and both can reduce THC-induced somatic withdrawal, sustained blockade of these enzymes leads to remarkably different adaptations in CB1 receptor functioning. Namely, prolonged elevations in brain 2-AG leads to marked antinociceptive tolerance, cross-tolerance to exogenous cannabinoid agonists, and physical dependence. In contrast, sustained elevations in brain anandamide continues to dampen pain responses without apparent signs of physical withdrawal, loss of CB1 receptor activation as measured by [35S]GTPγS, or receptor downregulation as measured by [3H]CP,55940. These results suggest that chronic 2-AG elicits greater compensatory changes in CB1 receptor functions than anandamide. With similar efficacy in most therapeutic endpoints tested, and evidence of reduced impact on long-term function of the endocannabinoid system, these results distinguish FAAH as a more promising therapeutic target to treat pain and other conditions than MAGL.

Cannabinoid agonists, including marijuana containing delta-9-tetrahydrocannabinol (THC), are found rewarding by humans. In addition to human self-reports and experimental studies that show marijuana is rewarding, contributions from preclinical studies also have implicated cannabinoid receptors in reward-motivated behavior. One way to assess these preclinical effects of cannabinoids is intracranial self-stimulation (ICSS), where an animal performs a response to receive electrical stimulation of a specific brain area or circuit known to be involved in reward. Drugs of abuse, such as psychomotor stimulants, facilitate responding for ICSS. While a few studies have shown facilitating effects of cannabinoids in rats, several have shown the opposite effect, and no studies so far have evaluated cannabinoids in mouse ICSS. Furthermore there are no studies evaluating specific inhibitors of endocannabinoid catabolic enzymes in ICSS in any species. In these studies we assessed the cannabinoid agonist THC, as well as the fatty acid amide hydrolase (FAAH) inhibitor, PF-3845, the monoacylglycerol lipase (MAGL) inhibitor JZL184, and the combined FAAH/MAGL inhibitor SA-57 in ICSS of the medial forebrain bundle in C57BL/6 mice. Additionally, we assessed the psychomotor stimulant cocaine as a positive control to facilitate ICSS. These studies were complimented with spontaneous locomotor activity and food-maintained operant experiments to assess the sensitivity of ICSS to cannabinoids. Additionally, brain endocannabinoid levels were measured in brain regions associated with the mesolimbic system after enzyme inhibitor treatments. THC, JZL184, and SA-57 all produced time-dependent reductions in ICSS that were mediated through CB1 receptors, as they were blocked by pre-treatment with the CB1 antagonist rimonabant, but not with the CB2 antagonist SR144528. PF-3845 also reduced ICSS, but did so independent of CB1 and CB2 receptors, and only with one dose (30.0 mg/kg) that has not been assessed previously in vivo. We showed that ICSS was more sensitive to the rate-reducing effects of cannabinoids than other measures of behavior with motor components including spontaneous locomotor activity and operant nose-poking for food, and that the reduction of ICSS produced by both JZL184 and SA-57 is accompanied by increases in 2-AG in mesolimbic brain areas. Thus, cannabinoids do not facilitate ICSS in C57BL/6 mice over a range of doses and pre-treatment times, similar to most studies with rats. These data suggest that cannabinoids may produce rewarding effects through non-mesolimbic areas of the brain.

Decades of research has provided evidence for the role of the endocannabinoid system in human health and disease. This versatile system, consisting of two receptors (CB1 and CB2), their endogenous ligands (endocannabinoids), and metabolic enzymes has been implicated in a wide variety of disease states, ranging from neurological disorders to cancer. CB2 has gained much interest for its beneficial immunomodulatory role that can be obtained without eliciting psychotropic effects through CB1. Recent studies have shed light on a protective role of CB2 in cardiovascular disease, an ailment which currently takes more lives each year in Western countries than any other disease or injury. By use of CB2 knockout mice and CB2-selective ligands, knowledge of how CB2 signaling affects atherosclerosis and ischemia has been acquired, providing a major stepping stone between basic science and translational clinical research. Here, we summarize the current understanding of the endocannabinoid system in human pathologies and provide a review of the results from preclinical studies examining its function in cardiovascular disease, with a particular emphasis on possible CB2-targeted therapeutic interventions to alleviate atherosclerosis.

Cardiovascular disease leads in morbidity and mortality in Western societies with no known cure. NADPH oxidase (Nox) contributes to atherosclerosis through the indirect activation of macrophages leading to the internalization of oxidized low density lipoproteins (oxLDL). Chronic inflammation in activated macrophages contributes to atherosclerosis. Because macrophages are positioned at the cross-roads of lipid metabolism in vessel walls, they are important in the cellular pathology of atherosclerosis. Components of the endocannabinoid (eCB) system are vital to atherosclerotic development, since the eCB system has been found to play an important role in the amelioration of atherosclerosis. The eCB system has several components, including the G-protein-coupled cannabinoid receptors (CB1 and CB2); their endogenous ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); and biosynthetic enzymes that produce and degrading these compounds. CB2 signaling has been shown to upregulate immunoprotective and anti-oxidative pathways, whereas CB1 signaling has opposite effects. We hypothesized a mechanistic link between scavenger receptor activation and Nox activity, which leads to enhanced 2-AG biosynthesis via a signaling pathway that activates diacylglycerol lipase beta (DAGLB). Activation of CB2-mediated signaling by enhanced “eCB tone” can potentially reduce oxidative stress in macrophages. The released 2-AG is subsequently catabolized hydrolytic enzymes, leading to enhanced 2-AGbiosynthesis via activated DAGLB. We first proved that macrophage treated with oxLDL can activate Nox and increase reactive oxygen species production. We used human and mouse macrophages to demonstrate cause and effect. Secondly, we demonstrated that increased levels of superoxide causes enhanced 2-AG biosynthesis within the macrophage, and that upregulation in eCB production is an adaptive response to oxidative stress. Finally, we identified and quantified the serine hydrolases found in smooth muscle cells (SMCs) using an activity-based protein profiling (ABPP)-MudPIT approach that our laboratory has previously done using human macrophages. Additionally, the catabolism of 2-AG by primary SMCs was explored to demonstrate SMCs can hydrolyze 2-AG to its metabolites arachidonic acid and glycerol by the known hydrolytic enzymes. We demonstrated that enhancing endocannabinoid tone within the vessel wall is a valuable strategy to reduce the occurrence of inflammation that leads to atherosclerosis.

GABA neurons in the ventral tegmental area of the midbrain are important components in the brain's reward circuit. Long term changes in this circuit occur through the process of synaptic plasticity. It has been shown that high frequency stimulation, as well as treatment with endocannabinoids, can cause GABA neurons in the ventral tegmental area to undergo long term depression, a form of synaptic plasticity that decreases excitability of cells. The present study elaborates on the mechanism whereby high frequency stimulation can result in long term depression of ventral tegmental area GABA neurons. Using the whole cell patch clamp technique in acute brain slices, we recorded excitatory currents from ventral tegmental area GABA neurons in GAD-GFP expressing CD1 mice and observed how the excitatory currents changed in response to different treatments. We confirm that high frequency stimulation causes long term depression, and the cannabinoid type 1 receptor antagonist AM-251 blocks this effect. Long term depression is also elicited by treatment with the cannabinoid type 1 receptor agonist 2-arachidonylglycerol. It is inconclusive whether treatment with 2-arachidonylglycerol occludes further long term depression by high frequency stimulation. We also demonstrate that activation of group I metabotropic glutamate receptors by DHPG produces long term depression. These results support the model that at these excitatory synapses, high frequency stimulation causes the release of glutamate from presynaptic terminals, activating group I metabotropic glutamate receptors, causing production of 2-arachidonylglycerol. 2-arachidonylglycerol in turn acts on presynaptic cannabinoid type 1 receptors to decrease release of glutamate onto GABA neurons. This model can be tested by further research, which should include cannabinoid type 1 receptor knockout mice. This study provides more insight into how drugs of abuse such as tetrahydrocannabinol, the active component of marijuana that activate cannabinoid type I receptors, can corrupt the natural reward mechanisms of the brain.

Il est aujourd'hui bien établi que les amibes libres jouent le rôle de vecteurs à la bactérie pathogène Legionella pneumophila favorisant ainsi son développement et sa propagation dans l'environnement. Ainsi, et jusqu'à maintenant, toutes les espèces d'amibes libres étudiées ont démontré une capacité à soutenir et favoriser la croissance de la bactérie responsable des légionelloses. Toutefois, l'ensemble des études a porté sur un nombre restreint d'espèces amibiennes, et leurs capacités relatives à soutenir la croissance bactérienne n'ont que très peu été abordées. Nous avons comparé la capacité de différentes espèces amibiennes à soutenir la prolifération de différentes souches de L. pneumophila du sérogroupe 1. Ces études ont mis en évidence les propriétés particulières d'une souche d'amibe appartenant à l'espèce Willaertia magna. Cette souche présente, au contraire de toutes les autres espèces, la capacité à inhiber et diminuer la prolifération de certaines souches de L. pneumophila. Nous avons par ailleurs pu démontrer l'existence d'une phagocytose interamibienne entre différentes espèces d'amibes, un phénomène jamais mis en évidence à notre connaissance. Les conséquences de cette phagocytose interamibienne sur la croissance et la prolifération de L. pneumophila sont aussi rapportées. La microscopie électronique suggère fortement que la bactérie L. pneumophila ne peut inhiber la fusion phagolysosomale chez W. magna à l'inverse du phénomène observé chez les espèces amibiennes permissives. Ces observations démontrent l'importance de phénomènes membranaires dans la capacité des bactéries à parasiter leur hôte amibien. L'analyse comparée de la composition lipidique des membranes de différentes espèces amibiennes montre chez W. magna une expression élevée de phosphatidylcholine. L'inhibition de la voie de biosynthèse de ce phospholipide par méthylation de phosphatidyléthanolamine résulte en une forte diminution de la croissance amibienne, suggérant que cette voie métabolique joue un rôle important dans les capacités de résistance de W. magna.

Il est aujourd’hui bien établi que les amibes libres jouent le rôle de vecteurs à la bactérie pathogène Legionella pneumophila favorisant ainsi son développement et sa propagation dans l’environnement. Ainsi, et jusqu’à maintenant, toutes les espèces d’amibes libres étudiées ont démontré une capacité à soutenir et favoriser la croissance de la bactérie responsable des légionelloses. Toutefois, l’ensemble des études a porté sur un nombre restreint d’espèces amibiennes, et leurs capacités relatives à soutenir la croissance bactérienne n’ont que très peu été abordées. Nous avons comparé la capacité de différentes espèces amibiennes à soutenir la prolifération de différentes souches de L. pneumophila du sérogroupe 1. Ces études ont mis en évidence les propriétés particulières d’une souche d’amibe appartenant à l’espèce Willaertia magna. Cette souche présente, au contraire de toutes les autres espèces, la capacité à inhiber et diminuer la prolifération de certaines souches de L. pneumophila. Nous avons par ailleurs pu démontrer l’existence d’une phagocytose interamibienne entre différentes espèces d’amibes, un phénomène jamais mis en évidence à notre connaissance. Les conséquences de cette phagocytose interamibienne sur la croissance et la prolifération de L. pneumophila sont aussi rapportées. La microscopie électronique suggère fortement que la bactérie L. pneumophila ne peut inhiber la fusion phagolysosomale chez W. magna à l’inverse du phénomène observé chez les espèces amibiennes permissives. Ces observations démontrent l’importance de phénomènes membranaires dans la capacité des bactéries à parasiter leur hôte amibien. L’analyse comparée de la composition lipidique des membranes de différentes espèces amibiennes montre chez W. magna une expression élevée de phosphatidylcholine. L’inhibition de la voie de biosynthèse de ce phospholipide par méthylation de phosphatidyléthanolamine résulte en une forte diminution de la croissance amibienne, suggérant que cette voie métabolique joue un rôle important dans les capacités de résistance de W. magna / Free living amoeba is a known vector of L. pneumophila in the environment and it has been shown to favour bacterial growth. Until now, all studied amoeba species showed a capacity to support the growth of the bacterium responsible for the Legionnaire’s disorder. However, these studies were related to a restricted number of amoebic species, and their relative capacity to support the bacterial growth. We compared the capacity of various amoebic species to support the proliferation of various strains of L. pneumophila. These studies highlighted the particular properties of a strain belonging to the Willaertia magna species. This amoeba has, contrary to all the other species, the capacity to inhibit and decrease L. pneumophila proliferation. We also demonstrate the existence of an inter-amoebic phagocytosis between various species of amoebas, a phenomenon never studied to our knowledge. The consequences of this inter-amoebic phagocytosis on the growth and proliferation of L. pneumophila are also reported. Electron microscopy strongly suggests that the bacterium cannot inhibit the phagolysosomal fusion in W. magna contrary to permissive amoebic species. These observations suggest the importance of membrane phenomena in the capacity of the bacteria to infest their amoebic host. Compared analysis of the lipidic composition of various amoebic species shows in W. magna a high expression of phosphatidylcholine the major phospholipid. The inhibition of the PE N-methyltransferase biosynthesis pathway of this phospholipid results in a strong reduction of the amoebic growth, suggesting that this metabolic pathway plays an important role in the resistance capacity of W. magna to L.pneumophila.

The endogenous cannabinoids (endocannabinoids) anandamide (AEA) and 2-arachidonylglycerol (2-AG) exert their effects predominantly through cannabinoid CB1 and CB2 receptors, but these actions are short-lived because of rapid hydrolysis by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective inhibition of either enzyme elevates CNS levels of the appropriate endocannabinoid and produces analgesic effects with fewer psychomimetic side effects than Δ9-tetrahydrocannabinol (THC), the primary active constituent of marijuana. While cannabinoid receptor agonists and FAAH inhibitors reliably produce anti-inflammatory and anti-hyperalgesic effects in the carrageenan test and other inflammatory pain models, much less is known about the consequences of inhibiting MAGL in these assays. Here, we tested whether the selective MAGL inhibitor JZL184 would reduce nociceptive behavior in the carrageenan test. JZL184 significantly attenuated carrageenan-induced paw edema and mechanical allodynia, whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Interestingly, the anti-allodynic effects of the combination of low dose of JZL184 (4mg/kg) and high dose of the selective and long-acting FAAH inhibitor PF-3845 (10 mg/kg) was augmented compared with each drug alone. On the contrary, the combination treatment did not reduce edema more than either JZL184 or PR-3845 given alone. These results suggest that low doses of MAGL inhibitors alone or in combination with FAAH inhibitors, reduce inflammatory nociception through the activation of both CB1 and CB2 receptors with no evidence of tolerance following repeated administration.

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