Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in several brain regions of adult male rats treated daily with delta9-tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,<em>2</em>1<em>2</em>-<em>2</em>-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,<em>2</em>1<em>2</em>-<em>2</em>-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of <em>2</em>-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.

In the adult brain, neural stem cells proliferate within the subventricular zone before differentiating into migratory neuroblasts that travel along the rostral migratory stream (RMS) to populate the olfactory bulb with new neurons. Because neuroblasts have been shown to migrate to areas of brain injury, understanding the cues regulating this migration could be important for brain repair. Recent studies have highlighted an important role for endocannabinoid (eCB) signaling in the proliferation of the stem cell population, but it remained to be determined whether this pathway also played a role in cell migration. We now show that mouse migratory neuroblasts express cannabinoid receptors, diacylglycerol lipase α (DAGLα), the enzyme that synthesizes the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and monoacylglycerol lipase, the enzyme responsible for its degradation. Using a scratch wound assay for a neural stem cell line and RMS explant cultures, we show that inhibition of DAGL activity or CB(1)/CB(<em>2</em>) receptors substantially decreases migration. In contrast, direct activation of cannabinoid receptors or preventing the breakdown of <em>2</em>-AG increases migration. Detailed analysis of primary neuroblast migration by time-lapse imaging reveals that nucleokinesis, as well as the length and branching of the migratory processes are under dynamic control of the eCB system. Finally, similar effects are observed in vivo by analyzing the morphology of green fluorescent protein-labeled neuroblasts in brain slices from mice treated with CB(1) or CB(<em>2</em>) antagonists. These results describe a novel role for the endocannabinoid system in neuroblast migration in vivo, highlighting its importance in regulating an additional essential step in adult neurogenesis.

OBJECTIVE

The endocannabinoids, N-arachidonoylethanolamide (anandamide) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are rapidly degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Whilst these lipid mediators are known to modulate vascular tone, the extent to which they are inactivated via local metabolism in the vasculature remains unclear.

METHODS

In rat isolated small mesenteric arteries, the regulatory role of FAAH, MGL and cyclooxygenase (COX) in relaxant responses to anandamide and <em>2</em>-AG was evaluated by using inhibitors of these enzymes. Relaxations to non-hydrolysable analogues of endocannabinoids and arachidonic acid were also examined.

RESULTS

Relaxation to anandamide but not <em>2</em>-AG was potentiated by the selective FAAH inhibitor, URB597 (1 microM). In contrast, MAFP (10 microM; an inhibitor of FAAH and MGL) enhanced responses to both anandamide and <em>2</em>-AG. Inhibition of COX-1 by indomethacin (10 microM) potentiated relaxations to <em>2</em>-AG, whereas inhibition of COX-<em>2</em> by nimesulide (10 microM) potentiated anandamide-induced relaxation. With the exception of MAFP, effects of FAAH and COX inhibitors were dependent on the endothelium. Relaxation to methanandamide and noladin ether, the non-hydrolysable analogues of anandamide and <em>2</em>-AG respectively, were insensitive to the enzyme inhibitors.

CONCLUSIONS

This study shows that local activity of FAAH, MGL and COX, which is present largely in the endothelium, limits the vasodilator action of endocannabinoids in rat small mesenteric arteries. Despite the differential roles played by these enzymes on relaxation to anandamide versus <em>2</em>-AG, our results suggest that inhibitors of these enzymes enhance the vascular impact of endocannabinoids.

OBJECTIVE

<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is an endocannabinoid whose hydrolysis is predominantly catalysed by the enzyme monoacylglycerol lipase (MAGL). The development of MAGL inhibitors could offer an opportunity to investigate the anti-inflammatory and anti-nociceptive role of <em>2</em>-AG, which have not yet been elucidated. On these bases, URB60<em>2</em>, a MAGL inhibitor, was tested in a murine model of inflammation/inflammatory pain.

METHODS

Acute inflammation was induced by intraplantar injection of lambda-carrageenan into mice. The highest dose to be employed has been selected performing the tetrad assays for cannabimimetic activity in mice. URB60<em>2</em> anti-inflammatory and anti-nociceptive efficacy (assessed by plethysmometer and plantar test, respectively) was evaluated both in a preventive regimen (drug administered 30 min before carrageenan) and in a therapeutic regimen (URB60<em>2</em> administered 30 min after carrageenan). To elucidate the cannabinoid receptor involvement, rimonabant and SR1445<em>2</em>8, CB1 and CB<em>2</em> selective antagonists, respectively, were given 15 min before URB60<em>2</em>.

RESULTS

Systemic administration of URB60<em>2</em> elicited a dose-dependent anti-oedemigen and anti-nociceptive effect that was reversed exclusively by the CB<em>2</em> receptor antagonist. The efficacy of URB60<em>2</em> persisted also when the compound was administered in a therapeutic regimen, suggesting the ability of URB60<em>2</em> to improve established disease.

CONCLUSIONS

The present report highlighted the ability of the selective MAGL inhibitor, URB60<em>2</em>, to prevent and treat an acute inflammatory disease without producing adverse psychoactive effects. The data presented herein also contributed to clarify the physiological role of <em>2</em>-AG in respect to inflammatory reactions, suggesting its protective role in the body.

In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (C<em>2</em>0:4) (<em>2</em>-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main <em>2</em>-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD1<em>2</em> remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD1<em>2</em> in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD1<em>2</em> preferred the 1 (3)- and <em>2</em>-isomers of <em>arachidonoylglycerol</em>. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D<em>2</em>78-H306, ABHD1<em>2</em>: S<em>2</em>46-D333-H37<em>2</em>) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D<em>2</em>78 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD1<em>2</em>, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD1<em>2</em>, which currently lacks such inhibitors.

The cytoplasm of neural cells contain millimolar amounts of ATP, which flood the extracellular space after injury, activating purinergic receptors expressed by glial cells and increasing gliotransmitter production. These gliotransmitters, which are thought to orchestrate neuroinflammation, remain widely uncharacterized. Recently, we showed that microglial cells produce <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endocannabinoid known to prevent the propagation of harmful neuroinflammation, and that ATP increases this production by threefold at <em>2</em>.5 min (Witting et al., <em>2</em>004). Here we show that ATP increases <em>2</em>-AG production from mouse astrocytes in culture, a response that is more rapid (i.e., significant within 10 sec) and pronounced (i.e., 60-fold increase at <em>2</em>.5 min) than any stimulus-induced increase in endocannabinoid production reported thus far. Increased <em>2</em>-AG production from astrocytes requires millimolar amounts of ATP, activation of purinergic P<em>2</em>X7 receptors, sustained rise in intracellular calcium, and diacylglycerol lipase activity. Furthermore, we show that astrocytes express monoacylglycerol lipase (MGL), the main hydrolyzing enzyme of <em>2</em>-AG, the pharmacological inhibition of which potentiates the ATP-induced <em>2</em>-AG production (up to 113-fold of basal <em>2</em>-AG production at <em>2</em>.5 min). Our results show that ATP greatly increases, and MGL limits, <em>2</em>-AG production from astrocytes. We propose that <em>2</em>-AG may function as a gliotransmitter, with MGL inhibitors potentiating this production and possibly restraining the propagation of harmful neuroinflammation.

Endogenous cannabinoid signalling is widespread throughout the body, and considerable evidence supports its modulatory role in many fundamental physiological processes. The daily and seasonal cycles of the relationship of the earth and sun profoundly affect the terrestrial environment. Terrestrial species have adapted to these cycles in many ways, most well studied are circadian rhythms and hibernation. The purpose of this review was to examine literature support for three hypotheses: (i) endocannabinoid signalling exhibits brain region-specific circadian rhythms; (ii) endocannabinoid signalling modulates the rhythm of circadian processes in mammals; and (iii) changes in endocannabinoid signalling contribute to the state of hibernation. The results of two novel studies are presented. First, we report the results of a study of healthy humans demonstrating that plasma concentrations of the endocannabinoid, N-arachidonylethanolamine (anandamide), exhibit a circadian rhythm. Concentrations of anandamide are threefold higher at wakening than immediately before sleep, a relationship that is dysregulated by sleep deprivation. Second, we investigated differences in endocannabinoids and congeners in plasma from Marmota monax obtained in the summer and during the torpor state of hibernation. We report that <em>2</em>-<em>arachidonoylglycerol</em> is below detection in M. monax plasma and that concentrations of anandamide are not different. However, plasma concentrations of the anorexigenic lipid oleoylethanolamide were significantly lower in hibernation, while the concentrations of palmitoylethanolamide and <em>2</em>-oleoylglycerol were significantly greater in hibernation. We conclude that available data support a bidirectional relationship between endocannabinoid signalling and circadian processes, and investigation of the contribution of endocannabinoid signalling to the dramatic physiological changes that occur during hibernation is warranted.

We have recently reported that in bone the cannabinoid CB1 receptor is present in sympathetic terminals. Here we show that traumatic brain injury (TBI), which in humans enhances peripheral osteogenesis and fracture healing, acutely stimulates bone formation in a distant skeletal site. At this site we demonstrate i) a high level of the main endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and expression of diacylglycerol lipases, enzymes essential for <em>2</em>-AG synthesis; ii) that the TBI-induced increase in bone formation is preceded by elevation of the <em>2</em>-AG and a decrease in norepinephrine (NE) levels. The TBI stimulation of bone formation was absent in CB1-null mice. In wild-type animals it could be mimicked, including the suppression of NE levels, by <em>2</em>-AG administration. The TBI- and <em>2</em>-AG-induced stimulation of osteogenesis was restrained by the beta-adrenergic receptor agonist isoproterenol. NE from sympathetic terminals is known to tonically inhibit bone formation by activating osteoblastic beta<em>2</em>-adrenergic receptors. The present findings further demonstrate that the sympathetic control of bone formation is regulated through <em>2</em>-AG activation of prejunctional CB1. Elevation of bone <em>2</em>-AG apparently suppresses NE release from bone sympathetic terminals, thus alleviating the inhibition of bone formation. The involvement of osteoblastic CB<em>2</em> signaling in this process is minimal, if any.

While endocannabinoid modulation of both GABAergic and glutamatergic synaptic transmission and plasticity has been extensively investigated, our understanding of the role of endocannabinoids in protecting neurons from harmful insults remains limited. <em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), the most abundant endogenous ligand and a full agonist for cannabinoid receptors, exhibits anti-inflammatory and neuroprotective effects via a CB1 receptor (CB1R)-mediated mechanism. However, it is still not clear whether <em>2</em>-AG is also able to protect neurons from β-amyloid (Aβ)-induced neurodegeneration. Here, we demonstrate that exogenous application of <em>2</em>-AG significantly protected hippocampal neurons in culture against Aβ-induced neurodegeneration and apoptosis. This neuroprotective effect was blocked by SR141716 (SR-1), a selective CB1R antagonist, but not by SR1445<em>2</em>8 (SR-<em>2</em>), a selective CB<em>2</em>R antagonist, or capsazepine (CAP), a selective transient receptor potential cation channels, subfamily V, member 1 (TRPV1) receptor antagonist. To determine whether endogenous <em>2</em>-AG is capable of protecting neurons from Aβ insults, hippocampal neurons in culture were treated with URB60<em>2</em> or JZL184, selective inhibitors of monoacylglycerol lipase (MAGL), the enzyme hydrolyzing <em>2</em>-AG. MAGL inhibition that elevates endogenous levels of <em>2</em>-AG also significantly reduced Aβ-induced neurodegeneration and apoptosis. The <em>2</em>-AG-produced neuroprotective effects appear to be mediated via CB1R-dependent suppression of extracellular signal-regulated kinases 1 and <em>2</em> (ERK1/<em>2</em>) and nuclear factor-κB (NF-κB) phosphorylation and cyclooxygenase-<em>2</em> (COX-<em>2</em>) expression. Our results suggest that elevation of endogenous <em>2</em>-AG by inhibiting its hydrolysis has potential as a novel efficacious therapeutic approach for preventing, ameliorating or treating Alzheimer's disease.

Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in <em>2</em>-<em>arachidonoylglycerol</em> and a strong upregulation of the synthesizing enzyme DAGL-alpha, that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(<em>2</em>) receptors. We observed that CB(1) was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(<em>2</em>) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury.

Low concentrations of <em>2</em>-<em>arachidonoylglycerol</em> were found to induce rapid, transient elevation of intracellular free Ca<em>2</em>+ in NG108-15 cells (EC50 was 150 nM). Free arachidonic acid, <em>2</em>-palmitoylglycerol, <em>2</em>-oleoylglycerol, <em>2</em>-linoleoylglycerol and <em>2</em>-docosahexaenoylglycerol were inactive. Anandamide acted as a partial agonist. Importantly, desensitization was observed upon sequential challenge with <em>2</em>-<em>arachidonoylglycerol</em>. Furthermore, cross-desensitization was observed between <em>2</em>-<em>arachidonoylglycerol</em> and WIN 55<em>2</em>1<em>2</em>-<em>2</em>, a cannabinoid receptor agonist. Pretreatment of the cells with SR141716A, a cannabinoid receptor antagonist, abolished the activities of both <em>2</em>-<em>arachidonoylglycerol</em> and WIN 55<em>2</em>1<em>2</em>-<em>2</em>. These results strongly suggest that <em>2</em>-<em>arachidonoylglycerol</em> and WIN 55<em>2</em>1<em>2</em>-<em>2</em> bind to a common cannabinoid receptor to elicit cellular responses and that <em>2</em>-<em>arachidonoylglycerol</em> has some physiological role in nervous tissues.

G protein-coupled receptor (GPR) 55 is sensitive to certain cannabinoids, it is expressed in the brain and, in cell cultures, it triggers mobilization of intracellular Ca(<em>2</em>+). However, the adaptive neurobiological significance of GPR55 remains unknown. Here, we use acute hippocampal slices and combine two-photon excitation Ca(<em>2</em>+) imaging in presynaptic axonal boutons with optical quantal analysis in postsynaptic dendritic spines to find that GPR55 activation transiently increases release probability at individual CA3-CA1 synapses. The underlying mechanism involves Ca(<em>2</em>+) release from presynaptic Ca(<em>2</em>+) stores, whereas postsynaptic stores (activated by spot-uncaging of inositol 1,4,5-trisphosphate) remain unaffected by GPR55 agonists. These effects are abolished by genetic deletion of GPR55 or by the GPR55 antagonist cannabidiol, a constituent of Cannabis sativa. GPR55 shows colocalization with synaptic vesicle protein vesicular glutamate transporter 1 in stratum radiatum. Short-term potentiation of CA3-CA1 transmission after a short train of stimuli reveals a presynaptic, Ca(<em>2</em>+) store-dependent component sensitive to cannabidiol. The underlying cascade involves synthesis of phospholipids, likely in the presynaptic cell, but not the endocannabinoids <em>2</em>-<em>arachidonoylglycerol</em> or anandamide. Our results thus unveil a signaling role for GPR55 in synaptic circuits of the brain.

Endocannabinoids are lipid mediators able to bind to and activate cannabinoid receptors, the primary molecular targets responsible for the pharmacological effects of the Δ9-tetrahydrocannabinol. These bioactive lipids belong mainly to two classes of compounds: N-acylethanolamines and acylesters, being N-arachidonoylethanolamine (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), respectively, their main representatives. During the last twenty years, an ever growing number of fatty acid derivatives (endocannabinoids and endocannabinoid-like compounds) have been discovered and their activities biological is the subject of intense investigations. Here, the most recent advances, from a therapeutic point of view, on endocannabinoids, related compounds, and their metabolic routes will be reviewed.

Cannabinoid receptors (CB1-R) are the target of a novel class of neuromodulators, the endocannabinoids. Yet, their signalling mechanisms in adult brain are poorly understood. We report that, in rat and mouse hippocampal slices, anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, synthetic cannabinoids, and delta(9)-tetrahydrocannabinol activated p38 mitogen-activated protein kinases (MAPK), but not c-Jun N-terminal kinase (JNK). In contrast, lysophosphatidic acid (LPA), a lipid messenger acting on different receptors, increased both p38-MAPK and JNK phosphorylation. The effects of cannabinoids on p38-MAPK were mediated through activation of CB1-R because they were blocked in the presence of SR 141716 A and absent in CB1-R knockout mice, two conditions that did not alter the effects of LPA. The activation of p38-MAPK by cannabinoids was insensitive to inhibitors of SRC: These results provide new insights into the cellular mechanisms by which cannabinoids exert their effects in hippocampus.

The nucleus accumbens (NAc) is a critical component of the reward circuitry, and dysfunction of the NAc may account for anhedonia and other symptoms of depression. Here, we investigated whether alterations in endocannabinoid (eCB) signaling in the NAc contribute to depression-like behaviors induced by chronic unpredictable stress (CUS) in mice. We compared three types of eCB/CB1 receptor-mediated synaptic plasticity in slices prepared from the NAc core of control and stress-exposed mice: depolarization-induced suppression of excitation, long-term depression, and the depression of field excitatory postsynaptic potentials (fEPSPs) induced by group I metabotropic glutamate receptor agonist DHPG. CUS (5-6-week exposure to stressors), but not sub-CUS (1 week exposure to stressors), induces depression-like behaviors and impairs these forms of eCB/CB1 receptor-mediated plasticity examined in the NAc core. Neither sub-CUS nor CUS altered the tissue contents of the eCBs, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> in the striatum. However, exposure to CUS, but not to sub-CUS, attenuated the depression of fEPSPs induced by the CB1 receptor agonist WIN 55 <em>2</em>1<em>2</em>-<em>2</em>. CUS exposure reduced the maximal effect without affecting the EC(50) of WIN 55 <em>2</em>1<em>2</em>-<em>2</em> to induce fEPSP depression. Thus, impaired CB1 receptor function could account for CUS-induced deficiency in eCB signaling in the NAc. Both CUS-induced deficiency in eCB signaling and depression-like behaviors were reversed by in vivo administration of antidepressant fluoxetine. These results suggest that downregulation of eCB signaling in the NAc occurs after CUS and contributes to the pathophysiology of depression.

Neuroinflammation has been implicated in the pathogenesis of neurodegenerative diseases. Cyclooxygenase-<em>2</em> (COX-<em>2</em>), an inducible enzyme converting arachidonic acid (AA) to prostaglandins, is the key player in neuroinflammation. It has been long thought that the COX-<em>2</em>-mediated neuronal injury/degeneration is attributed to the increased production of AA-derived prostaglandins. Recent studies show that endogenous cannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is a natural substrate for COX-<em>2</em>, and it can be oxygenated by COX-<em>2</em> to form prostaglandin glyceryl esters. In this study, we demonstrate that prostaglandin E(<em>2</em>) glyceryl ester (PGE(<em>2</em>)-G), a major COX-<em>2</em> oxidative metabolite of <em>2</em>-<em>arachidonoylglycerol</em>, enhanced hippocampal glutamatergic synaptic transmission indicated by the increased frequency of miniature excitatory post-synaptic currents, and induced neuronal injury/death revealed by the terminal transferase dUTP nick end labeling staining and caspase 3 activation. The actions of PGE(<em>2</em>)-G are not mediated via a cannabinoid receptor 1, but mediated through ERK, p38 mitogen-activated protein kinase, IP(3), and NF-kappaB signal transduction pathways. In addition, the PGE(<em>2</em>)-G-induced neurotoxicity is attenuated by blockade of the NMDA receptors. Our results suggest that the COX-<em>2</em> oxidative metabolism of endocannabinoids is an important mechanism contributing to the inflammation-induced neurodegeneration.

Orexin A and B are hypothalamic peptides known to modulate arousal, feeding, and reward via OX1 and OX<em>2</em> receptors. Orexins are also antinociceptive in the brain, but their mechanism(s) of action remain unclear. Here, we investigated the antinociceptive mechanism of orexin A in the rat ventrolateral periaqueductal gray (vlPAG), a midbrain region crucial for initiating descending pain inhibition. In vlPAG slices, orexin A (30-300 nm) depressed GABAergic evoked IPSCs. This effect was blocked by an OX1 [1-(<em>2</em>-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea (SB 334867)], but not OX<em>2</em> [N-acyl 6,7-dimethoxy-1,<em>2</em>,3,4-tetrahydroisoquinoline hydrochloride (compound <em>2</em>9)], antagonist. Orexin A increased the paired-pulse ratio of paired IPSCs and decreased the frequency, but not amplitude, of miniature IPSCs. Orexin A-induced IPSC depression was mimicked by (R)-(+)-[<em>2</em>,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,<em>2</em>,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,<em>2</em>1<em>2</em>-<em>2</em>), a cannabinoid 1 (CB1) receptor agonist. 1-(<em>2</em>,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM <em>2</em>51), a CB1 antagonist, reversed depressant effects by both agonists. Orexin A-induced IPSC depression was prevented by 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-<em>2</em>,5-dione (U731<em>2</em><em>2</em>) and tetrahydrolipstatin, inhibitors of phospholipase C (PLC) and diacylglycerol lipase (DAGL), respectively, and enhanced by cyclohexyl[1,1'-biphenyl]-3-ylcarbamate (URB60<em>2</em>), which inhibits enzymatic degradation of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Moderate DAGLα, but not DAGLβ, immunoreactivity was observed in the vlPAG. Orexin A produced an overall excitatory effect on evoked postsynaptic potentials and hence increased vlPAG neuronal activity. Intra-vlPAG microinjection of orexin A reduced hot-plate nociceptive responses in rats in a manner blocked by SB 334867 and AM <em>2</em>51. Therefore, orexin A may produce antinociception by activating postsynaptic OX1 receptors, stimulating synthesis of <em>2</em>-AG, an endocannabinoid, through a Gq-protein-mediated PLC-DAGLα enzymatic cascade culminating in retrograde inhibition of GABA release (disinhibition) in the vlPAG.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type <em>2</em> (CB(<em>2</em>)), and enzymes that regulate the eCB ligands <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of <em>2</em>-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of <em>2</em>-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(<em>2</em>) and D(<em>2</em>). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(<em>2</em>) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing <em>2</em>-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.

The endocannabinoid system is a valuable target for drug discovery, because it is involved in the regulation of many cellular and physiological functions. The endocannabinoid system constitutes the endogenous lipids anandamide, <em>2</em>-<em>arachidonoylglycerol</em> and noladin ether, and the cannabinoid CB1 and CB<em>2</em> receptors as well as the proteins for their inactivation. It is thought that (endo)cannabinoid-based drugs may potentially be useful to reduce the effects of neurodegeneration. This paper reviews recent developments in the endocannabinoid system and its involvement in neuroprotection. Exogenous (endo)cannabinoids have been shown to exert neuroprotection in a variety of in vitro and in vivo models of neuronal injury via different mechanisms, such as prevention of excitotoxicity by CB1-mediated inhibition of glutamatergic transmission, reduction of calcium influx, and subsequent inhibition of deleterious cascades, TNF-alpha formation, and anti-oxidant activity. It has been suggested that the release of endogenous endocannabinoids during neuronal injury might be a protective response. However, several observations indicate that the role of the endocannabinoid system as a general endogenous protection system is questionable. The data are critically reviewed and possible explanations are given.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG) is the endocannabinoid that mediates retrograde suppression of neurotransmission in the brain. In the present study, we investigated the <em>2</em>-AG signaling system at mossy cell (MC)-granule cell (GC) synapses in the mouse dentate gyrus, an excitatory recurrent circuit where endocannabinoids are thought to suppress epileptogenesis. First, we showed by electrophysiology that <em>2</em>-AG produced by diacylglycerol lipase α (DGLα) mediated both depolarization-induced suppression of excitation and its enhancement by group I metabotropic glutamate receptor activation at MC-GC synapses, as they were abolished in DGLα-knock-out mice. Immunohistochemistry revealed that DGLα was enriched in the neck portion of GC spines forming synapses with MC terminals, whereas cannabinoid CB(1) receptors accumulated in the terminal portion of MC axons. On the other hand, the major <em>2</em>-AG-degrading enzyme, monoacylglycerol lipase (MGL), was absent at MC-GC synapses but was expressed in astrocytes and some inhibitory terminals. Serial electron microscopy clarified that a given GC spine was innervated by a single MC terminal and also contacted nonsynaptically by other MC terminals making synapses with other GC spines in the neighborhood. MGL-expressing elements, however, poorly covered GC spines, amounting to 17% of the total surface of GC spines by astrocytes and 4% by inhibitory terminals. Our findings provide a basis for <em>2</em>-AG-mediated retrograde suppression of MC-GC synaptic transmission and also suggest that <em>2</em>-AG released from activated GC spines is readily accessible to nearby MC-GC synapses by escaping from enzymatic degradation. This molecular-anatomical configuration will contribute to adjust network activity in the dentate gyrus after enhanced excitation.

BACKGROUND

A substantial number of individuals are at risk for the development of motion sickness induced nausea and vomiting (N&V) during road, air or sea travel. Motion sickness can be extremely stressful but the neurobiologic mechanisms leading to motion sickness are not clear. The endocannabinoid system (ECS) represents an important neuromodulator of stress and N&V. Inhibitory effects of the ECS on N&V are mediated by endocannabinoid-receptor activation.

RESULTS

We studied the activity of the ECS in human volunteers (n = <em>2</em>1) during parabolic flight maneuvers (PFs). During PFs, microgravity conditions (<10(-<em>2</em>) g) are generated for approximately <em>2</em><em>2</em> s which results in a profound kinetic stimulus. Blood endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, <em>2</em>-AG) were measured from blood samples taken in-flight before start of the parabolic maneuvers, after 10, <em>2</em>0, and 30 parabolas, in-flight after termination of PFs and <em>2</em>4 h later. Volunteers who developed acute motion sickness (n = 7) showed significantly higher stress scores but lower endocannabinoid levels during PFs. After <em>2</em>0 parabolas, blood anandamide levels had dropped significantly in volunteers with motion sickness (from 0.39+/-0.40 to 0.<em>2</em><em>2</em>+/-0.<em>2</em>5 ng/ml) but increased in participants without the condition (from 0.43+/-0.<em>2</em>3 to 0.60+/-0.38 ng/ml) resulting in significantly higher anandamide levels in participants without motion sickness (p = 0.0<em>2</em>). <em>2</em>-AG levels in individuals with motion sickness were low and almost unchanged throughout the experiment but showed a robust increase in participants without motion sickness. Cannabinoid-receptor 1 (CB1) but not cannabinoid-receptor <em>2</em> (CB<em>2</em>) mRNA expression in leucocytes 4 h after the experiment was significantly lower in volunteers with motion sickness than in participants without N&V.

CONCLUSIONS

These findings demonstrate that stress and motion sickness in humans are associated with impaired endocannabinoid activity. Enhancing ECS signaling may represent an alternative therapeutic strategy for motion sickness in individuals who do not respond to currently available treatments.

<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is a putative endogenous ligand for cannabinoid receptors and was suggested to play an important role in both physiological and pathological events in the central nervous system (CNS) as well as in peripheral organs. The sequential hydrolysis of arachidonic acid (<em>2</em>0:4n-6, AA)-containing phospholipids has been proposed as a major biosynthetic route of <em>2</em>-AG. On the other hand, the manipulation of the dietary n-3 polyunsaturated fatty acid (PUFA) status changes the AA level in tissue phospholipids. We, therefore, conducted two separate experiments to confirm whether the dietary n-3 PUFA status influences the <em>2</em>-AG level in the mouse brain. In the first experiment, we fed mice with n-3 PUFA-deficient diet, which resulted in a marked decrease in the docosahexaenoic acid (<em>2</em><em>2</em>:6n-3, DHA) levels without a change in the AA level in brain phospholipids as compared with the mice fed with an n-3 PUFA-sufficient diet. The brain <em>2</em>-AG level in the n-3 PUFA-deficient group was significantly higher than in the n-3 PUFA sufficient group. In the second experiment, we found that short-term supplementation of DHA-rich fish oil reduced brain <em>2</em>-AG level as compared with the supplementation with low n-3 PUFA. The decrease in the AA level and the increase in the DHA level in the major phospholipids occurred in the brains of the mice fed the fish oil diet compared with those fed the low n-3 PUFA diet. Our results indicate that the n-3 PUFA deficiency elevates and n-3 PUFA enrichment reduces the brain <em>2</em>-AG level in mice, suggesting that physiological and pathological events mediated by <em>2</em>-AG through cannabinoid receptor in the CNS could be modified by the manipulation of the dietary n-3 PUFA status.

A series of <em>2</em>1 analogues of tetrahydrolipstatin (THL, 1) were synthesized and tested as inhibitors of the formation or hydrolysis of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG). Three of the novel compounds, i.e., 11, 13, and 15, inhibited <em>2</em>-AG formation via the diacylglycerol lipase alpha (DAGLalpha) with IC 50 values lower than 50 nM (IC 50 of THL = 1 microM) and were between <em>2</em>3- and 375-fold selective vs <em>2</em>-AG hydrolysis by monoacylglycerol lipase (MAGL) as well as vs cannabinoid CB 1 and CB <em>2</em> receptors and anandamide hydrolysis by fatty acid amide hydrolase (FAAH). Three other THL analogues, i.e., 14, 16, and 18, were slightly more potent than THL against DAGLalpha and appreciably selective vs MAGL, CB receptors, and FAAH (15-<em>2</em>6-fold). One compound, i.e., 8, was a potent inhibitor of MAGL-like activity (IC 50 = 0.41 microM), and relatively ( approximately 7-fold) selective vs the other targets tested.

Human reproduction is a rather inefficient process, yet the molecular reasons for this inefficiency remain unknown. IVF and embryo transfer (IVF-embryo transfer) also results in a high frequency of implantation failures and early spontaneous abortions. Here we show that the anandamide (AEA)-degrading enzyme, fatty acid amide hydrolase (FAAH), had significantly lower activity (46 +/- 17 versus 161 +/- 74 pmol/min per mg protein) and protein content (0.10 +/- 0.03 versus 0.<em>2</em>3 +/- 0.06 units) in lymphocytes of IVF-embryo transfer patients who failed to achieve an ongoing pregnancy than in those who become pregnant, and this was paralleled by a significant increase in blood AEA (4.0 +/- <em>2</em>.<em>2</em> pmol/ml and 0.9 +/- 1.0 pmol/ml respectively). The blood levels of the other endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, or of the AEA congener, N-palmitoylethanolamine, which are metabolized by enzymes different from FAAH, was not different between the pregnant and nonpregnant women, nor was there any difference in the activity of the AEA membrane transporter or the amounts of cannabinoid receptors in lymphocytes. Taken together with the reported negative effects of AEA on embryo implantation, this study indicates that low FAAH activity and subsequent increased AEA levels in blood might be one of the causes of implantation failure or pregnancy loss, thereby leading to a better understanding of the pathophysiological and therapeutic implications of endocannabinoids in human fertility.