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Introduction: Parkinson's disease is a neurodegenerative disorder characterised by balance problems, muscle rigidity, and slow movement due to low dopamine levels and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The endocannabinoid system is known to modulate the nigrostriatal pathway through endogenous ligands such as anandamide (AEA), which is hydrolysed by fatty acid amide hydrolase (FAAH). The purpose of this study was to increase AEA levels using FAAH inhibitor URB597 to evaluate the modulatory effect of AEA on dopaminergic neuronal death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Methods: Our study included 4 experimental groups (n = 6 mice per group): a control group receiving no treatment, a group receiving URB597 (0.2 mg/kg) every 3 days for 30 days, a group treated with MPTP (30 mg/kg) for 5 days, and a group receiving URB597 and subsequently MPTP injections. Three days after the last dose, we conducted a series of behavioural tests (beam test, pole test, and stride length test) to compare motor coordination between groups. We subsequently analysed immunoreactivity of dopaminergic cells and microglia in the SNpc and striatum. Results: Mice treated with URB597 plus MPTP were found to perform better on behavioural tests than those receiving MPTP only. According to the immunohistochemistry study, mice receiving MPTP showed fewer dopaminergic cells and fibres in the SNpc and striatum. Animals treated with URB597 plus MPTP displayed increased tyrosine hydroxylase immunoreactivity compared to those treated with MPTP only. Regarding microglial immunoreactivity, the group receiving MPTP showed higher Iba1 immunoreactivity in the striatum and SNpc than did the group treated with URB597 plus MPTP. Conclusion: Our results show that URB597 exerts a protective effect since it inhibits dopaminergic neuronal death, decreases microglial immunoreactivity, and improves MPTP-induced motor alterations. Resumen: Introducción: La enfermedad de Parkinson es un desorden neurodegenerativo caracterizado por problemas de equilibro, rigidez muscular y lentitud para realizar movimiento, debido a la pérdida de neuronas dopaminérgicas de la sustancia nigra pars compacta (SNpc) y la reducción de los niveles de dopamina. Se sabe que el sistema endocannabinoide modula el funcionamiento de la vía nigroestriatal, a través de ligandos endógenos como anandamida (AEA), que es hidrolizado por la hidrolasa amida de ácidos grasos (FAAH). El objetivo de este trabajo consiste en aumentar los niveles de AEA, a través de la inhibición de FAAH por URB-597 y evaluar la modulación que ejerce AEA en la muerte neuronal dopaminérgica inducida por 1-metil-4-fenil-1,2,3,6-tetrahidropiridina (MPTP). Métodos: Se incluyeron 4 grupos experimentales con una n = 6, el primero fue el grupo control sin tratamiento, un grupo al cual se le administró (0,2 mg/kg) URB-597 cada 3.er día durante 30 días, un grupo tratado con MPTP (30 mg/kg) por 5 días y un grupo inyectado con URB-597 + MPTP. Tres días después de la última administración de los grupos experimentales, se llevó a cabo la aplicación de los siguientes paradigmas conductuales: prueba de la barra vertical, barra inclinada y longitud de la zancada para comparar la coordinación motriz. Posteriormente, se analizó la inmunorreactividad de las células dopaminérgicas y de microglía en SNpc y cuerpo estriado (CE). Resultados: Los resultados muestran que el grupo tratado con URB-597 previo a la administración de MPTP, presentó un mejor desempeño en la realización de las pruebas conductuales comparados con los ratones que recibieron la administración de MPTP. Los hallazgos del análisis inmunohistoquímico de las células dopaminérgicas muestran que el grupo tratado con MPTP presenta una disminución en el número de células y fibras dopaminérgicas tanto en la SNpc y CE. En los animales tratados con URB-597 previo a la administración de MPTP se observó un aumento en la inmunorreactividad de tirosina hidroxilasa en comparación al grupo tratado con MPTP. Con respecto a la inmunorreactividad de las células de microglía, el grupo tratado con MPTP presentó una mayor inmunorreactividad a Iba-1 en el CE y la SNpc comparado con el grupo tratado con URB-597 previo a la administración de MPTP. Conclusión: Los resultados obtenidos muestran que el URB-597 genera un efecto protector, al inhibir la muerte neuronal dopaminérgica, y disminución en la inmunorreactividad microglial y una mejora en las alteraciones motoras causadas por MPTP. Keywords: Cannabinoids, Striatum, Parkinson's disease, Neuroprotective, Substantia nigra, Palabras clave: Cannabinoides, Cuerpo estriado, Enfermedad de Parkinson, Neuroprotector, Sustancia nigra

Recurrent panic attacks, comprising emotional and cardiovascular aversive responses, are common features in panic disorder, a subtype of anxiety disorder. The underlying brain circuitry includes nuclei of the hypothalamus, such as the dorsomedial hypothalamus (DMH). The endocannabinoid system has been proposed to modulate several biological processes in the hypothalamus. Thus, we tested the hypothesis that hypothalamic endocannabinoid signalling controls aversive responses in an animal model of panic attacks. Local infusion of NMDA into the DMH of rats induced panic-like behaviour. This effect was prevented by local, but not intraperitoneal, injection of a 2-arachidonoylglycerol (2-AG) hydrolysis inhibitor (MAGL inhibitor, URB602). The anandamide hydrolysis inhibitor (FAAH inhibitor), URB597, was ineffective. The anti-aversive action of URB602 was reversed by CB1 and CB2 antagonists (AM251 and AM630, respectively), and mimicked by CB1 and CB2 agonists (ACEA and JWH133, respectively). URB602 also prevented the cardiovascular effects of DMH-stimulation in anaesthetised animals. None of the treatments modified blood corticosterone levels. In conclusion, facilitation of 2-AG-signalling in the DMH modulates panic-like responses. The possible mechanisms comprise activation of both CB1 and CB2 receptors in this brain region.

Endocannabinoids (eCBs) anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were shown to be involved in the basis of trauma-induced behavioral changes, particularly contextual conditioned fear, however, their ligand-specific effects and possible interactions are poorly understood. Here we assessed specific eCB effects and interactions on acquisition of contextual conditioned fear employing electric footshocks in a rat model. We selectively increased eCB levels by pharmacological blockade of the degrading enzymes of AEA by URB597 and 2-AG by JZL184 before traumatization either systemically or locally in relevant brain areas, the prelimbic cortex (PrL), ventral hippocampus (vHC) and basolateral amygdala (BLA). Following traumatization, a series of contextual reminders were conducted during which conditioned fear was assessed. While systemic URB597-treatment during traumatization only slightly enhanced the acquisition of contextual conditioned fear, administration of the compound in the PrL and vHC led to the acquisition of stable, lasting conditioned fear, resistant to extinction. These effects of URB597 were blocked by simultaneous administration of JZL184. Similar treatment effects did not occur in the BLA. Treatment effects were not secondary to alterations in locomotor activity or nociception. Our findings suggest that AEA and 2-AG functionally interact in the regulation of acquisition of contextual conditioned fear. AEA signaling in the PrL and vHC is a crucial promoter of fear acquisition while 2-AG potentially modulates this effect. The lack of eCB effects in the BLA suggests functional specificity of eCBs at distinct brain sites.

Purpose The effect of chronic administration of [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597), inhibitor of fatty acid amide hydrolase (FAAH) that hydrolyzes anandamide, on cross-talk between endocannabinoid system, oxidative status and pro-inflammatory factors in the liver of spontaneously hypertensive rats (SHRs) was investigated. Materials/methods Experiments were conducted using SHRs and normotensive control Wistar-Kyoto rats treated by intraperitoneal injection with URB597 for 14 days. The biochemical parameters were assayed in the rat’s livers. Results In the liver of SHRs an increase in endocannabinoids level, the activity of enzymes degrading them and expression of the cannabinoid receptor type 2 (CB2) receptor as well as a decrease in the expression of the CB1 and vanilloid 1 receptor (TRPV1) were shown. These changes were related to inflammatory conditions as well as oxidative stress resulting from increased reactive oxygen species (ROS) generation due to enhanced activity of enzymes generating ROS accompanied by decrease in the effectiveness of transcription activity of nuclear factor erythroid 2 and the activity of antioxidant enzymes, as well as level of glutathione and vitamins. Chronic administration of URB597 to SHRs caused a decrease in FAAH activity and an increase in anandamide and N-arachidonoyl-dopamine level as well as a decrease in CB2 and an increase in TRPV1 receptor expression. The levels/activities of pro- and antioxidant and inflammatory factors tended to normalize, but phospholipid peroxidation and DNA modifications were increased. Conclusion In conclusion, long-term chronic administration of URB597 to SHRs by altering interactions between endocannabinoid and redox systems enhances some liver metabolic disturbances observed in hypertension.

Hypertension is a civilization disease leading to remodeling and damage of blood vessels, impaired renal function and premature death. The aim of this study was to compare the effect of chronic administration of URB597, the FAAH (fatty acid amide hydrolase) inhibitor, to rats with primary (SHRs) and secondary (DOCA-salt hypertensive rats) hypertension on electrical and physicochemical properties of kidney cells membranes. Changes in the electrical charge of the membrane may affect the cell functions. The electrical properties of the kidney cells (surface charge density, zeta potential) were measured by electrophoresis. Qualitative and quantitative composition of the membrane (phospholipids and proteins) was determined by HPLC and lipid peroxidation product (4-hydroxy-2E-hexenal; 4-HHE) level was examined by GCMSMS, while the sialic acid content was measured by resorcinol method. In rats with primary hypertension (SHR) and secondary hypertension (DOCA-salt), changes in electrical properties (increase of electric charge and zeta potential) and membrane composition (increase in sialic acid and protein concentration and decrease in phospholipid level) of kidney cells are observed in comparison to control animals. Greater changes were observed in DOCA-salt hypertensive rats. Changes in membrane properties caused by URB597 depend on the type of hypertension. The administration of URB597 to rats with primary hypertension partially prevents changes in the electrical properties (electrical charge, zeta potential) of the membrane caused by hypertension as well as in the sialic acid and proteins content. However, there is no reduction in oxidative stress, assessed by the level of 4-HHE, which may affect the metabolic function of the kidneys. URB597 administered to rats with DOCA salt does not prevent, but rather intensifies, changes caused by hypertension in the kidney. In conclusion, URB597 given to individuals with hypertension, particularly with secondary hypertension, enhancing some disturbances in electric and physicochemical properties of kidney cells observed in hypertension what may lead to additional kidney disorders. Therefore, further researches are necessary.

Background Activation of cannabinoid CB1 receptors suppresses pathological pain but also produces unwanted central side effects. We hypothesized that a positive allosteric modulator of CB1 signaling would suppress inflammatory and neuropathic pain without producing cannabimimetic effects or physical dependence. We also asked whether a CB1 positive allosteric modulator would synergize with inhibitors of endocannabinoid deactivation and/or an orthosteric cannabinoid agonist. Methods GAT211, a novel CB1 positive allosteric modulator, was evaluated for antinociceptive efficacy and tolerance in models of neuropathic and/or inflammatory pain. Cardinal signs of direct CB1-receptor activation were evaluated together with the propensity to induce reward or aversion and physical dependence. Comparisons were made with inhibitors of endocannabinoid deactivation (JZL184, URB597) or an orthosteric cannabinoid agonist (WIN55,212-2). All studies used 4 to 11 subjects per group. Results GAT211 suppressed allodynia induced by complete Freund’s adjuvant and the chemotherapeutic agent paclitaxel in wild-type but not CB1 knockout mice. GAT211 did not impede paclitaxel-induced tumor cell line toxicity. GAT211 did not produce cardinal signs of direct CB1-receptor activation in the presence or absence of pathological pain. GAT211 produced synergistic antiallodynic effects with fatty acid amide hydrolase and monoacylglycerol lipase inhibitors in paclitaxel-treated mice. Therapeutic efficacy was preserved over 19 days of chronic dosing with GAT211, but it was not preserved with the monoacylglycerol lipase inhibitor JZL184. The CB1 antagonist rimonabant precipitated withdrawal in mice treated chronically with WIN55,212-2 but not in mice treated with GAT211. GAT211 did not induce conditioned place preference or aversion. Conclusions Positive allosteric modulation of CB1-receptor signaling shows promise as a safe and effective analgesic strategy that lacks tolerance, dependence, and abuse liability.

An increasing body of evidence shows significant sex differences in the mammalian brain in multiple behaviours and psychiatric and neurological diseases and as well as that the endocannabinoid system may differ between males and females. In this study we investigated sex differences in working, short-term and long-term memory and the expression of β2-adrenergic and D1- and D2-receptors in the mPFC and hippocampus, brain regions that are involved in stress response and memory modulation in rats exposed to the chronic unpredictable stress (CUS) and the potential beneficial effects of the chronic fatty acid amide hydrolase inhibitor URB597 treatment. Chronically stressed male rats had an improvement of working memory, while stressed females showed very low object-recognition abilities. On the other hand, animals of both sexes exhibited long-term memory impairment. Our results showed that CUS decreased the expression of β2-adrenoceptors in the mPFC and D1 receptors in the mPFC and hippocampus of male rats and decreased β2-adrenoceptors and D1- receptors in the hippocampus of female. URB597 treatment had a positive effect on the short-term memory of stressed animals of both sexes whereas failed to restore long-term memory and did not affect the protein levels β2-adrenoceptors and D1 receptors in the hippocampus of CUS female rats. The present results support that endocannabinoids induced long-term memory and neurochemical alternations which are sex dependent, suggesting sex specific treatment strategies of mental disorders.

The interaction between the endocannabinoid and ROS signaling systems has been demonstrated in different organs. Inhibitors of fatty acid amide hydrolase (FAAH), the key enzyme responsible for degradation of the endocannabinoid anandamide, are postulated to possess anti-hypertensive potential. Here, we compared the effects of hypertension and chronic FAAH inhibition by URB597 on the endocannabinoid system and redox balance in spontaneously hypertensive rats (SHR) and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Enhanced oxidative stress and lipid peroxidation were found in both hypertension models. Hypertension affected cardiac and plasma endocannabinoid systems in a model-dependent manner: anandamide and 2-arachidonoylglycerol levels decreased in SHR and increased in DOCA-salt. Cardiac CB1 receptor expression increased in both models while higher CB2 receptor expression was only in DOCA-salt. URB597 increased endocannabinoid levels in both models but produced the partial reduction of oxidative stress in DOCA-salt but not in SHR. Notably, URB597 decreased antioxidant defense and increased lipid peroxidation products in normotension. Therefore, the therapeutic potential of FAAH inhibitors should be interpreted cautiously.

Activating the endocannabinoid system has become a major focus in the search for novel therapeutics for anxiety and deficits in fear extinction, two defining features of PTSD. We examined whether chronic treatment with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.2, 0.3, 0.4 mg/kg, i.p.) or the CB1/2 receptor agonist WIN55,212-2 (0.25, 0.5 mg/kg, i.p.) injected for 3 weeks to rats exposed to the shock and reminders model of PTSD would attenuate post-stress symptoms and affect basolateral amygdala (BLA) and CA1 CB1 receptors. Exposure to shock and reminders enhanced acoustic startle response and impaired extinction. Rats exposed to shock and reminders and chronically treated with URB597 demonstrated normalized startle response and intact extinction kinetics. WIN55,212-2 only affected the startle response. The therapeutic effects of URB597 and WIN55,212-2 were found to be CB1 receptor dependent, as these effects were blocked when a low dose of the CB1 receptor antagonist AM251 (0.3 mg/kg, i.p. for 3 weeks) was co-administered. Moreover, URB597, but not WIN55,212-2, normalized the shock/reminders-induced upregulation in CB1 receptor levels in the BLA and CA1. One hour after the shock, N-arachidonoylethanolamine (AEA) was increased in the BLA and decreased in the CA1. Circulating 2-arachidonoylglycerol (2-AG) concentrations were decreased in shocked rats, with no significant effect in the BLA or CA1. FAAH activity was increased in the CA1 of shocked rats. Chronic cannabinoid treatment with URB597 can ameliorate PTSD-like symptoms suggesting FAAH inhibitors as a potentially effective therapeutic strategy for the treatment of disorders associated with inefficient fear coping.

The aim of this study was to examine the influence of deoxycorticosterone acetate-salt (DOCA-salt) hypertension and chronic treatment with the fatty acid amide hydrolase inhibitor, URB597, on small and intermediate conductance calcium-activated potassium channels and endothelium-dependent hyperpolarization (KCa2.3/KCa3.1-EDH) in rat small mesenteric arteries (sMAs). The EDH-type response was investigated, in endothelium-intact sMAs using a wire myograph, by examining acetylcholine-evoked vasorelaxation in the presence of Nω-nitro-L-arginine methyl ester and indomethacin (inhibitors of nitric oxide synthase and cyclooxygenase, respectively). In normo- and hypertension the efficacy of EDH-type relaxation was similar and inhibition of KCa2.3 and KCa3.1 by UCL1684 and TRAM-34, respectively, given alone or in combination, attenuated EDH-mediated vasorelaxation. KCa3.1 expression and NS309 (KCa2.3/KCa3.1 activator)-induced relaxation was reduced in sMAs of DOCA-salt rats. Endothelium denudation and incubation with UCL1684 and TRAM-34 attenuated the maximal NS309-evoked vasorelaxation in both groups. URB597 had no effect in functional studies, but increased the expression of KCa3.1 in the sMAs. KCa2.3/KCa3.1-EDH-mediated relaxation was maintained in the sMAs of DOCA-salt rats despite endothelial dysfunction and down-regulation of KCa3.1. Furthermore, KCa3.1 played a key role in the EDH-type dilator response of sMAs in normo- and hypertension. The hypotensive effect of URB597 is independent of KCa2.3/KCa3.1-EDH-type relaxation.

Objective Status epilepticus (SE) provokes changes, which lead to neuronal alterations. Endocannabinoids (eCBs) can affect the neuronal survival during excitotoxicity and brain damage. Using a kainic acid (KA)-induced experimental SE model, we investigated whether cellular changes entail damage to endoplasmic reticulum (ER), mitochondria, and nuclei in hippocampal cells (CA1 field), and whether these alterations can be diminished by treatment with URB597, an inhibitor of eCB enzymatic degradation. Material and methods SE was induced in Wistar rats by the microinjection of KA into the lateral ventricle. URB597 or a vehicle (10% DMSO) were injected in the same way into the brain of animals 24 h after the KA infusion and then daily for the next nine days. The behavior of animals was controlled visually and recorded with a video system. The intensity of SE significantly varied in different animals. Convulsive (stages 3–5 according to the Racine scale) and nonconvulsive seizures (mainly stages 1, 2 and rarely 3, 4) were recognized. Results Two weeks after SE, a significant loss of hippocampal cells occurred in animals with KA injections. In survived cells, ultrastructural alterations in ER, mitochondria, and nuclei of hippocampal neurons were observed. The degree of cell injury depended on the severity of SE. Alterations evoked by moderate seizures were prevented or diminished by URB597, but strong seizures induced mostly irreversible damage. Conclusions The beneficial impact of the FAAH inhibitor URB597 can give impetus to the development of novel neuroprotective strategies.

Our current knowledge of the implications of endocannabinoids in fear and anxiety is largely based on fear conditioning paradigms and approach-avoidance conflicts. Here we establish the ethobehavioral beetle mania task (BMT), which confronts mice with an erratically moving robo-beetle. With the help of this task we demonstrate decreased tolerance yet increased avoidance responses to an approaching beetle in high-anxiety behavior (HAB) and BALBc mice compared to C57BL/6N, CD1 and normal-anxiety behavior (NAB) mice. Also DBA/2N mice showed decreased passive and increased active behavior, but followed the robo-beetle more often than HAB and BALBc mice. Treatment with diazepam (1 mg/kg) increased tolerance without affecting avoidance behavior in HAB mice. Treatment with the MAGL inhibitor JZL184 (8 mg/kg) increased flight behavior, but did not affect tolerance. The FAAH inhibitor URB597 (0.3 mg/kg), however, reduced flight behavior and enhanced tolerance to the robo-beetle. The latter effects were blocked by co-treatment with the CB1 receptor antagonist SR141716A (3 mg/kg), which failed to affect the behavior by itself. Taken together, we validate the BMT as a novel test for studying endocannabinoids beyond traditional paradigms and for assessing active fear responses in mice. Furthermore, we demonstrate panicolytic consequences of pharmacological enhancement of anandamide, but not 2-AG signaling.

Tardive dyskinesia (TD) is a side effect associated with the long-term use of certain antipsychotics. Considering the modulatory role of the endocannabinoid system upon dopaminergic neurotransmission, the present study tested the hypothesis that increasing endocannabinoid (anandamide and 2-arachidonoylglycerol) levels attenuates haloperidol-induced TD (vacuous chewing movements, VCMs) in male Wistar rats. The animals received administration of chronic haloperidol (38 mg/kg; 29 days) followed by acute FAAH (URB597, 0.1–0.5 mg/kg) or MAGL (JZL184, 1–10 mg/kg) inhibitors before VCM quantification. The underlying mechanisms were evaluated by pre-treatments with a CB1 receptor antagonist (AM251, 1 mg/kg) or a TRPV1 channel blocker (SB366791, 1 mg/kg). Moreover, CB1 receptor expression was evaluated in the striatum of high-VCM animals. As expected, haloperidol induced VCMs only in a subset of rats. Either FAAH or MAGL inhibition reduced VCMs. These effects were prevented by CB1 receptor antagonism, but not by TRPV1 blockage. Remarkably, CB1 receptor expression was increased high-VCM rats, with a positive correlation between the levels of CB1 expression and the number of VCMs. In conclusion, increasing endocannabinoid levels results in CB1 receptor-mediated protection against haloperidol-induced TD in rats. The increased CB1 receptor expression after chronic haloperidol treatment suggests a counter-regulatory protective mechanism.

Current clinical and pre-clinical data suggest that both cannabinoid agents and blockage of CRF through corticotrophin releasing factor receptor type 1 (CRFr1) may offer therapeutic benefits for post-traumatic stress disorder (PTSD). Here we aim to determine whether they are more effective when combined when microinjected into the basolateral amygdala (BLA) or CA1 area of the hippocampus after exposure to a stressful event in the shock/reminders rat model for PTSD. Injection of the fatty acid amide hydrolase (FAAH) inhibitor URB597 after the shock into either the BLA or CA1 facilitated extinction, and attenuated startle response and anxiety-like behavior. These preventive effects of URB597 were found to be mediated by the CB1 receptor. Intra-BLA and intra-CA1 microinjection of the CRFr1 antagonist, CP-154,526 attenuated startle response. When microinjected into the BLA, CP-154,526 also attenuated freezing behavior during exposure to the first reminder and decreased anxiety-like behavior. The combined treatment of URB597 and CP-154,526 was not more effective than the separate treatments. Finally, mRNA levels of CRF, CRFr1 and CB1r were significantly higher in the BLA of rats exposed to shock and reminders compared to non-shocked rats almost one month after the shock. Taken together, the results show that enhancing endocannabinoid signaling in the amygdala and hippocampus produced a more favorable spectrum of effects than those caused by the CRFr1 antagonist. The findings suggest that FAAH inhibitors may be used as a novel treatment for stress-related anxiety disorders.

Neurogenic inflammation is a local inflammatory response that is driven by the peripheral release of neuropeptides from small diameter afferents which occurs in many organs including joints. The knee joint has a rich endocannabinoid system which has been shown to decrease acute synovitis. The aim of this study was to investigate the influence of joint afferents on leukocyte-endothelial interactions within the synovial microcirculation of mice and determine the role of endocannabinoids on this inflammatory response. Electrical, antidromic stimulation of the saphenous nerve decreased leukocyte rolling at the lowest frequency tested (0.5Hz), while increasing leukocyte rolling at higher frequencies (2.0 and 5.0Hz). The leukocyte rolling effect of nerve stimulation was completely abolished by pre-treating the knee with the vasoactive intestinal peptide antagonist VIP6-28; however, neither calcitonin gene related peptide nor substance P antagonism had an effect on this neurogenic inflammatory response. Treating knees with the endocannabinoid breakdown inhibitor URB597 completely blocked leukocyte rolling and this effect could be reversed with the non-canonical cannabinoid antagonist O-1918. These results provide evidence that antidromic stimulation of the mouse saphenous nerve promotes leukocyte rolling within the synovial microcirculation, and that endocannabinoids can attenuate this neurogenic inflammatory response.

Disruption of the neurovascular unit (NVU), induced by chronic cerebral hypoperfusion (CCH), has been broadly found in various neurological disorders. SUMO-specific protease 3 (SENP3) is expressed in neurons, astrocytes, and microglia, and regulates a variety of cell events. However, whether SENP3 is involved in neurovascular injury under the condition of CCH is still elusive. To address this issue, we investigated the effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on NVU and the role of SENP3 in this process, as well as the underling mechanisms. The expression of SENP3 was detected by immunochemistry. The function and structure of the NVU was assessed by Western blot analysis and transmission electron microscopy. CCH caused the upregulation of SENP3, the disruption of cell and non-cell components at the protein level within the NVU, and ultrastructural deterioration. The NVU impairment as well as overexpression of SENP3 were reversed by treatment with URB597. These results reveal a novel neuroprotective role in URB597, which implicates URB597 in the amelioration of CCH-induced NVU impairment by inhibiting SENP3.

Background URB597 is a compound largely linked to the inhibition of fatty acid amide hydrolase (FAAH), an enzyme responsible for the metabolic degradation of the endocannabinoid anandamide (AEA). Despite this pharmacological property accounts for its modulatory profile demonstrated in some neurotoxic paradigms, the possible protective properties of this agent have been poorly investigated, and deserve exploration in different neurotoxic models. In this study, we explored the effects of URB597 on oxidative damage to lipids and other major endpoints of toxicity in two neurotoxic models in vivo in rats (the first one produced by the mitochondrial neurotoxin 3-nitropropionic acid [3-NP], and the other generated by the striatal injection of the pro-oxidant toxin 6-hydroxidopamine [6-OHDA]) in order to provide further supporting evidence of its modulatory profile. Methods Male Wistar adult rats were treated for 5 or 7 consecutive days with URB597 (0.3 mg/kg, i.p.) and simultaneously exposed to three injections of 3-NP (30 mg/kg, i.p.) or a single intrastriatal infusion of 6-OHDA (0.02 mg/2 μl), respectively. Twenty four hours after all treatments were administered, lipid peroxidation was measured in the striatum of 3-NP-treated rats, and in the midbrain of 6-OHDA-treated rats. Motor skills and histological assessment in the striatum were also evaluated in 3-NP-treated rats 6 and 7 days after the last drug administration, respectively; whereas apomorphine-induced circling behavior and tyrosine hydroxylase immunolocalization in the striatum and substantia nigra were investigated 21 and 22 days after the last drug infusion, respectively. Results URB597 prevented the oxidative damage to lipids induced by 3-NP in the striatum, and this effect could account for the attenuation of motor deficits in this model. Attenuation of motor disturbances induced by URB597 in both models was associated with the morphological preservation of the striatum in the 3-NP model and the partial preservation of tyrosine hydroxylase in the 6-OHDA model in the SNpc and striatum. Conclusion The modulatory actions exerted by URB597 in both toxic models support its potential against toxic conditions implying motor and neurochemical alterations linked to energy depletion, excitotoxicity and oxidative stress. Although most of these effects could be attributable to its action on FAAH and further AEA accumulation, in light of our present findings other properties are suggested.

The role of the endocannabinoid system in nicotine addiction is being increasingly acknowledged. Acute inhibition of anandamide (AEA) degradation efficiently reduces nicotine withdrawal-induced affective symptoms in rats and fatty acid amide hydrolase (FAAH), the degradation enzyme of AEA, has been proposed as a possible treatment against nicotine addiction. However, it is unclear whether chronic inhibition of AEA during nicotine abstinence will have beneficial or deleterious affective side-effects. Using a rat model of nicotine addiction, we found that, during abstinence, rats injected daily with a FAAH inhibitor (URB597) developed a depressive-like phenotype. Our results show that in the nicotine abstinent rats, URB597 induced low saccharin consumption, persistent immobility in the forced swim test and increased corticosterone levels in response to stress. In addition, URB597decreased CB1 receptor binding and activity in the habenula, a key structure in the control of nicotine-related emotional states. In contrast, non-treated abstinent rats showed increased CB1 receptor activity and behaviors comparable to controls. No FAAH inhibition-induced alterations were observed in animals that had a previous history of saline self-administration. Taken together, our results suggest that chronic FAAH inhibition prevents the homeostatic adaptations of habenular CB1 receptor function that are necessary for the recovery from nicotine dependence.

Elements of the endocannabinoid system are strongly expressed in the basal ganglia where they suffer profound rearrangements after dopamine depletion. Modulation of the levels of the endocannabinoid 2-arachidonoyl-glycerol by inhibiting monoacylglycerol lipase alters glial phenotypes and provides neuroprotection in a mouse model of Parkinson's disease. In this study, we assessed whether inhibiting fatty acid amide hydrolase could also provide beneficial effects on the time course of this disease. The fatty acid amide hydrolase inhibitor, URB597, was administered chronically to mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) over 5weeks. URB597 (1mg/kg) prevented MPTPp induced motor impairment but it did not preserve the dopamine levels in the nigrostriatal pathway or regulate glial cell activation. The symptomatic relief of URB597 was confirmed in haloperidol-induced catalepsy assays, where its anti-cataleptic effects were both blocked by antagonists of the two cannabinoid receptors (CB1 and CB2), and abolished in animals deficient in these receptors. Other fatty acid amide hydrolase inhibitors, JNJ1661010 and TCF2, also had anti-cataleptic properties. Together, these results demonstrate an effect of fatty acid amide hydrolase inhibition on the motor symptoms of Parkinson's disease in two distinct experimental models that is mediated by cannabinoid receptors. This work was supported by the projects PI14/02070 and SAF2012-39875-C02-01 from the Spanish Government (Plan estatal I+D+I 2013–2016 and ISCIII-FEDER), Fundación Gangoiti and the UTE-project/Foundation for Applied Medical Research (FIMA). Estefanía Rojo is supported by a predoctoral fellowship from Colfuturo. Partial support was also provided by the Research and Education Program of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin

Cannabinoids are part of an endogenous signaling system found throughout the body, including the eye. Hepler and Frank showed in the early 1970s that plant cannabinoids can lower intraocular pressure (IOP), an effect since shown to occur via cannabinoid CB1 and GPR18 receptors. Endocannabinoids are synthesized and metabolized enzymatically. Enzymes implicated in endocannabinoids breakdown include monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), but also ABHD12, NAAA, and COX-2. Inhibition of MAGL activity raises levels of the endocannabinoid 2-arachidonoyl glycerol and substantially lowers IOP. Blocking other cannabinoid metabolizing enzymes or cannabinoid transporters may similarly contribute to lowering IOP and so serve as therapeutic targets for treating glaucoma. We have tested blockers for several cannabinoid-metabolizing enzymes and transporters (FABP5 and membrane reuptake) for their ability to alter ocular pressure in a murine model of IOP. Of FAAH, ABHD12, NAAA, and COX2, only FAAH was seen to play a role in regulation of IOP. Only the FAAH blocker URB597 lowered IOP, but in a temporally, diurnally, and sex-specific manner. We also tested two blockers of cannabinoid transport (SBFI-26 and WOBE437), finding that each lowered IOP in a CB1-dependent manner. Though we see a modest, limited role for FAAH, our results suggest that MAGL is the primary cannabinoid-metabolizing enzyme in regulating ocular pressure, thus pointing towards a role of 2-arachidonoyl glycerol. Interestingly, inhibition of cannabinoid transport mechanisms independent of hydrolysis may prove to be an alternative strategy to lower ocular pressure.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder linked to various converging toxic mechanisms. Evidence suggests that hyperglycemia induces oxidative stress, mitochondrial dysfunction, inflammation and excitotoxicity, all of which play important roles in the onset and progression of AD pathogenesis. The endocannabinoid system (ECS) orchestrates major physiological responses, including neuronal plasticity, neuroprotection, and redox homeostasis, to name a few. The multi-targeted effectiveness of the ECS emerges as a potential approach to treat AD. Here we characterized the protective properties of the endocannabinoids arachidonylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), the synthetic cannabinoids CP 55-940 and WIN 55,212-2, and the fatty acid amide hydrolase (FAAH) inhibitor URB597, on a combined hyperglycemia + oligomeric amyloid beta peptide (A beta(1-42)) neurotoxic model in primary hippocampal neurons which exhibit several AD features. Cells were treated with cannabinoid agents at increased concentrations (1 nM-1 mu M) for 6 h, and then co-treated with 150 mM glucose (GLU, 24 h), followed by incubation with 500 nM A beta(1-42) (24 h). Cell viability/survival, reactive oxygen species (ROS) levels, antioxidant enzyme (SOD, CAT, GPx and GRx) activities, biological products of oxidative damage (AGE and HNE adducts) and nitrosative stress (3-NT), several endpoints of inflammation (iNOS, IL-1 beta and TNF-alpha), amyloid quantification, mitochondrial membrane potential, and the involvement of the Nrf2 pathway, were all evaluated. The combined high glucose + amyloid beta 1-42 (GLU + A beta(1-42)) condition decreased cell viability and mitochondrial membrane potential, while augmenting oxidative damage and inflammation. All agents tested preserved cell viability and stimulated mitochondrial membrane potential, while reducing all the evaluated toxic endpoints in a differential manner, with URB597 showing the highest efficacy. The neuroprotective efficacy of all cannabinoid agents, except for URB597, led to partial recruitment of specific antioxidant activity and Nrf2 pathway regulation. Our results support the neuroprotective potential of these agents at low concentrations against the damaging effects of GLU + A beta(1-42), affording new potential modalities for the design of AD therapies.

Anxiety disorders are the most prevalent mental health disorder worldwide, with a lifetime prevalence of 5-7% of the human population. Although the etiology of anxiety disorders is incompletely understood, one aspect of host health that affects anxiety disorders is the gut-brain axis. We used a mouse model of gastrointestinal (GI) colonization to demonstrate that the commensal fungus Candida albicans affects host health via the gut-brain axis. In mice, bacterial members of the gut microbiota can influence the host gut-brain axis, affecting anxiety-like behavior and the hypothalamus-pituitary-adrenal (HPA) axis which produces the stress hormone corticosterone (CORT). Here we showed that mice colonized with C. albicans demonstrated increased anxiety-like behavior and increased basal production of CORT as well as dysregulation of CORT production following acute stress. The HPA axis and anxiety-like behavior are negatively regulated by the endocannabinoid anandamide (AEA). C. albicans -colonized mice exhibited changes in endocannabinoid levels within the GI tract and a negative correlation between brain AEA levels and serum CORT. Further, increasing AEA levels using the well-characterized fatty acid amide hydrolase (FAAH) inhibitor URB597 was sufficient to reverse both neuroendocrine phenotypes in C. albicans -colonized mice. Thus, a commensal fungus that is a common colonizer of humans had widespread effects on the physiology of its host. To our knowledge, this is the first report of microbial manipulation of the endocannabinoid (eCB) system that resulted in neuroendocrine changes contributing to anxiety-like behavior. Significance Statement Anxiety disorders are the most prevalent mental health disorder worldwide yet their etiology is incompletely understood and treatment options are limited. The gut-brain axis has been shown to influence the development of anxiety disorders. Recent work has established that the bacterial gut microbiota can influence the gut-brain axis and result in changes to neuroendocrine health including anxiety-like behavior in mice. We describe a novel role for a fungal member of the gut microbiota, Candida albicans , that influences the neuroendocrine system through manipulation of the endocannabinoid system. Additionally, this work demonstrates that acute, limited manipulation of endocannabinoids can have a significant impact on host health, emphasizing the potential of this system as a target for the treatment of anxiety disorders.

Heavy episodic drinking (binging), which is highly prevalent among teenagers, results in oxidative damage. Because the prefrontal cortex (PFC) is not completely mature in adolescents, this brain region may be more vulnerable to the effects of alcohol during adolescence. As endocannabinoids may protect the immature PFC from the harmful effects of high doses of alcohol, this study investigated the effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on oxidative stress induced by acute or chronic binge alcohol intake in adolescent rats. At 40min after intraperitoneal pre-treatment with URB597 (0.3mg/kg) or vehicle (Veh), ethanol (EtOH; 3 or 6g/kg, intragastrically) or distilled water (DW) was administered in 3 consecutive sessions (acute binging) or 3 consecutive sessions over 4 weeks (chronic binging). Oxidative stress in PFC slices in situ was measured by dihydroethidium fluorescence staining. At the higher EtOH dose (6g/kg), pre-treatment with URB597 significantly reduced (p

Hypertension is accompanied by perturbations to the endocannabinoid and antioxidant systems. Thus, potential pharmacological treatments for hypertension should be examined as modulators of these two metabolic systems. The aim of this study was to evaluate the effects of chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl)phenyl]N-cyclohexylcarbamate (URB597) on the endocannabinoid system and on the redox balance in the livers of DOCA-salt hypertensive rats. Hypertension caused an increase in the levels of endocannabinoids [anandamide (AEA), 2-arachidonoyl-glycerol (2-AG) and N-arachidonoyl-dopamine (NADA)] and CB1 receptor and the activities of FAAH and monoacylglycerol lipase (MAGL). These effects were accompanied by an increase in the level of reactive oxygen species (ROS), a decrease in antioxidant activity/level, enhanced expression of transcription factor Nrf2 and changes to Nrf2 activators and inhibitors. Moreover, significant increases in lipid, DNA and protein oxidative modifications, which led to enhanced levels of proapoptotic caspases, were also observed. URB597 administration to the hypertensive rats resulted in additional increases in the levels of AEA, NADA and the CB1 receptor, as well as decreases in vitamin E and C levels, glutathione peroxidase and glutathione reductase activities and Nrf2 expression. Thus, after URB597 administration, oxidative modifications of cellular components were increased, while the inflammatory response was reduced. This study revealed that chronic treatment of hypertensive rats with URB597 disrupts the endocannabinoid system, which causes an imbalance in redox status. This imbalance increases the levels of electrophilic lipid peroxidation products, which later participate in metabolic disturbances in liver homeostasis.

Injury to the bronchial epithelium in respiratory diseases such as asthma and COPD results in the loss of barrier function and an elevated sensitivity to environmental insults. An increased release of the endogenous cannabinoid, anandamide in response to inhalation of allergen in asthmatic patients has been reported. The aim of this study was, therefore, to determine the effects of endocannabinoids on bronchial epithelial cell permeability and to investigate the mechanisms involved. Calu-3 human bronchial epithelial cells were cultured at air-liquid interface to allow development of tight junctions. Changes in Transepithelial Electrical Resistance (TEER), a reflection of epithelial permeability, were measured at various time points post-treatment, and expression of the tight junction proteins, occludin and ZO-1, were determined using Western immunoblotting. Anandamide produced a significant reduction in TEER, which was unaffected by cannabinoid receptor antagonists, but attenuated by URB597, an inhibitor of fatty acid amide hydrolase, and by a combination of cyclooxygenase (COX) and lipoxygenase (LOX) blockade. The anandamide metabolite, arachidonic acid, showed similar TEER decrease that was also prevented in the presence of COX and LOX inhibitor. Expression of occludin and ZO-1 were also reduced by anandamide. These findings indicate a pro-inflammatory-like effect of anandamide on bronchial epithelial permeability, mediated by cyclooxygenase and lipoxygenase metabolites, and suggest that inhibition of anandamide degradation might provide a novel approach to treat airway inflammation.

In the present study, we investigated the effects of acute pharmacological manipulation of the endocannabinoid (EC) system on the valence of cognitive judgement bias of rats in the ambiguous-cue interpretation (ACI) paradigm. To accomplish this goal, after initial behavioural training, different groups of rats received single, systemic injections of the irreversible anandamide (AEA) hydrolysis inhibitor URB597, the cannabinoid receptor type 1 (CB1) inverse agonist AM251, the cannabinoid receptor type 2 (CB2) inverse agonist AM630, the combination of URB597 and AM251, and a combination of URB597 and AM630 and were subsequently tested with the ACI paradigm. We report that URB597 at a dose of 1 mg/kg significantly biased animals towards positive interpretation of the ambiguous cue and that this effect was abolished by pre-treatment with AM251 (1 mg/kg) or AM630 (1 mg/kg). The CB1 and CB2 inverse agonists administered alone (1 mg/kg) had no statistically significant effects on the interpretation of the ambiguous cue by rats. Our findings suggest involvement of the endocannabinoid system in the mediation of optimistic judgement bias.

Aims This study was performed to examine whether hypertension affects the sympathetic transmission to resistance vessels of pithed rats via inhibitory presynaptic cannabinoid CB1 receptors and whether endocannabinoids are involved in this response. Materials and methods We compared uninephrectomised rats rendered hypertensive by high salt diet and deoxycorticosterone acetate (DOCA) injections with normotensive animals (uninephrectomy only). Experiments were performed on vagotomised and pithed animals. Increases in diastolic blood pressure (DBP) were induced four times (S1–S4) by electrical stimulation or phenylephrine injection. Key findings Electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 impulses) of the preganglionic sympathetic nerve fibres innervating the blood vessels more strongly increased DBP in normotensive than in DOCA–salt rats. Phenylephrine (0.01 μmol/kg) induced similar increases in DBP in both groups. The cannabinoid receptor agonist CP55940 (0.01–1 μmol/kg) did not modify the rises in DBP induced by phenylephrine. However, it inhibited the electrically stimulated increases in DBP, more strongly in DOCA–salt than in normotensive animals (maximally by 50 and 30%, respectively). The effect of CP55940 was attenuated by the CB1 antagonist AM251 (3 μmol/kg). AM251 enhanced the neurogenic vasopressor response during S4 by itself in hypertensive rats only. URB597 (3 μmol/kg), which inhibits degradation of the endocannabinoid anandamide, did not modify the electrically stimulated increases in DBP. Significance The function of inhibitory presynaptic CB1 receptors on sympathetic nerves is enhanced in DOCA–salt hypertensive rats. Thus, the CB1 receptor-mediated inhibition of noradrenaline release from the sympathetic nerve fibres innervating the resistance vessels might play a protective role in hypertension.

The changes in sympathetic innervations in lymphoid organs could be a key factor in immune dysregulation. The endocannabinoid system has been shown to exhibit potent immunomodulatory effects that may differ between males and females, representing a potential therapeutic target for peripheral and central inflammatory disorders. Thus, in the present study, an examination was made of the effect of fatty acid amide hydrolase inhibitor URB597 treatment on splenic catecholamine content, synthesis, uptake and degradation in chronically unpredictably stressed (CUS) female and male rats. The results show that CUS increases anxiety-like behaviors and that URB597 had an anxiolytic effect on chronically stressed animals of both sexes. CUS induced the expression of plasma interleukin - 6 (IL-6), interleukin - 10 (IL-10) and IL-6 in the spleen, whereas the expression of IL-10 was reduced in the spleen of both sexes. URB597 treatment did not cause changes in IL-6 in plasma or the spleen, whereas it increased IL-10 in the spleen in CUS animals of both sexes. CUS caused a significant depletion of noradrenaline content in the spleen of female rats and a reduction in noradrenaline uptake in the spleen of female rats, while stressed males had a small but insignificant decrease of splenic noradrenaline levels and an enhanced uptake. The FAAH inhibitor URB597 enhances reduced noradrenaline content, affecting its uptake directly at the level of the spleen. It gives rise to the possibility that endocannabinoids exert a neurorestorative effect on the sympathetic nerve system and cell-mediated immune responses in the spleen of chronically stressed rats.

The endocannabinoid (eCB) system is a potential target for the treatment of symptoms of post-traumatic stress disorder (PTSD). Similar to clinical PTSD, approximately 25–30% of rats that undergo cued fear conditioning exhibit impaired extinction learning. In addition to extinction-resistant fear, these “weak extinction” (WE) rats show persistent anxiety-like behaviors. The goal of the present study was to test the hypothesis that behavioural differences between WE animals and those presenting normal extinction patterns (strong extinction; SE) could be mediated by the eCB system. Rats undergoing fear conditioning/extinction and fear recall sessions were initially segregated in weak and strong-extinction groups. Two weeks later, animals underwent a fear recall session followed by a novelty-suppressed feeding (NSF) test. In acute experiments, WE rats were injected with either the fatty acid amide hydrolase (FAAH) inhibitor URB597 or the CB1 agonist WIN55,212-2 1 h prior to long-term recall and NSF testing. SE animals were injected with the inverse CB1 receptor agonist AM251. In chronic experiments, WE and SE rats were given daily injections of URB597 or AM251 between short and long-term recall sessions. We found that acute administration of WIN55,212-2 but not URB597 reduced anxiety-like behaviour in WE rats. In contrast, AM251 was anxiogenic in SE animals. Neither treatment was effective in altering freezing expression during fear recall. The chronic administration of AM251 to SE or URB597 to WE did not alter fear or anxiety-like behaviour or changed the expression of FAAH and CB1. Together, these results suggest that systemic manipulations of the eCB system may alter anxiety-like behaviour but not the behavioural expression of an extinction-resistant associative fear memory.

Background Fatty-acid amide hydrolase (FAAH) is an intracellular serine hydrolase that catalyzes the cleavage of endogenous fatty-acid amides, including the endocannabinoid anandamide (AEA). We previously reported that the peripherally restricted FAAH inhibitor URB937, which selectively increases AEA levels outside the central nervous system, reduces hyperalgesia and c-Fos expression in the trigeminal nucleus caudalis (TNC) and the locus coeruleus in an animal model of migraine based on nitroglycerin (NTG) administration. Aim To further investigate the relevance of FAAH inhibition in the NTG animal model of migraine by testing the effects of the globally active FAAH inhibitor URB597. Methods Our experimental approach involved mapping neuronal c-Fos protein expression, measurement of AEA levels in brain areas and in trigeminal ganglia, evaluation of pain-related behavior and quantification of molecular mediators in rats that received URB597 (2 mg/kg i.p.) either before or after NTG administration (10 mg/kg, i.p.). Results Pre-treatment with URB597 significantly reduced c-Fos immunoreactivity in the TNC and inhibited NTG-induced hyperalgesia in the orofacial formalin test. This behavioral response was associated with a decrease in neuronal nitric oxide synthase, calcitonin gene-related peptide and cytokine gene expression levels in central and peripheral structures. Administration of URB597 after NTG had no such effect. Conclusions The findings suggest that global FAAH inhibition may offer a therapeutic approach to the prevention, but not the abortive treatment, of migraine attacks. Further studies are needed to elucidate the exact cellular and molecular mechanisms underlying the protective effects of FAAH inhibition.

Modulation of cannabinoid and neuropeptide Y (NPY) receptors may offer therapeutic benefits for post-traumatic stress disorder (PTSD). In this study, we aimed to investigate the functional interaction between these systems in the basolateral amygdala (BLA) in a rat model of PTSD. Rats were exposed to the shock and reminders model of PTSD and tested for hyper arousal/PTSD- and depression-like behaviors 3 weeks later. Immediately after shock exposure rats were microinjected into the BLA with URB597, a selective inhibitor of fatty acid amide hydrolase (FAAH) that increases the levels of the endocannabinoid anandamide or with the NPY1 receptor agonist Leu31,Pro34-NPY (Leu). Intra-BLA URB597 prevented the shock/reminders-induced PTSD- behaviors (extinction, startle) and depression-behaviors (despair, social impairments). These preventing effects of URB597 on PTSD- and depression-like behaviors were shown to be mostly mediated by cannabinoid CB1 and NPY1 receptors, as they were blocked when URB597 was co-administered with a low dose of a CB1 or NPY1 receptor antagonist. Similarly, intra-BLA Leu prevented development of all the behaviors. Interestingly, a CB1 antagonist prevented the effects of Leu on despair and social behavior, but not the effects on extinction and startle. Moreover, exposure to shock and reminders upregulated CB1 and NPY1 receptors in the BLA and infralimbic prefrontal cortex and this upregulation was restored to normal with intra-BLA URB597 or Leu. The findings suggest that the functional interaction between the eCB and NPY1 systems is complex and provide a rationale for exploring novel therapeutic strategies that target the cannabinoid and NPY systems for stress-related diseases.

The endocannabinoid system (ECS) has therapeutic potential for treating chronic cerebral hypoperfusion (CCH)-induced cerebral diseases. This study investigated the protective effects of two ECS compounds, cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase inhibitor URB597 (URB) on CCH-induced neuronal apoptosis in vivo. CCH was induced in male Sprague-Dawley rats by bilateral common carotid artery occlusion (BCCAo); the rats were then treated with WIN or URB for 12weeks and their spatial learning and memory abilities were assessed using the Morris water maze. Changes in neuronal number were examined by labeling neurons with an antibody against the neuronal nuclei antigen, and apoptosis of cortical and hippocampal CA1 neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The expression of B cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax), and activated caspase-3 as well as mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, phosphorylated (p-)ERK, p-JNK, and p-P38 was examined by Western blotting. Rats treated with WIN or URB showed better learning and memory performance than controls. The neuroprotective effects of URB were greater than those of WIN, and co-administration of WIN and URB had a synergistic effect. In addition, WIN and URB blocked JNK phosphorylation as well as the decrease in Bcl-2/Bax ratio and caspase-3 activation induced by CCH, implying that these agents modulate neuronal survival. Moreover, the selective JNK inhibitor SP600125 improved mitochondrial membrane dysfunction and blocked neuronal apoptosis induced by JNK-dependent Bcl-2 signaling. WIN and URB enhanced the effects of SP600125, implying that they may exert anti-apoptotic effects in part by inhibiting a non-nuclear JNK pathway. These findings indicate that WIN and URB promote neuronal survival and may potentially be used to protect neurons against chronic ischemic insults.

There is evidence to suggest that a dysregulation of endocannabinoid signaling may contribute to the etiology and pathophysiology of migraine. Thus, patients suffering from chronic migraine or medication overuse headache showed alterations in the activity of the arachidonoylethanolamide (AEA) degrading enzyme fatty acid amide hydrolase (FAAH) and a specific AEA membrane transporter, alongside with changes in AEA levels. The precise role of different endocannabinoid system components is, however, not clear. We have therefore investigated mice with a genetic deletion of the two main cannabinoid receptors CB1 and CB2, or the main endocannabinoid degrading enzymes, FAAH and monoacylglycerol lipase (MAGL), which degrades 2-arachidonoylglycerol (2-AG), in a nitroglycerine-induced animal model of migraine. We found that nitroglycerin-induced mechanical allodynia and neuronal activation of the trigeminal nucleus were completely abolished in FAAH-deficient mice. To validate these results, we used two structurally different FAAH inhibitors, URB597 and PF3945. Both inhibitors also dose-dependently blocked nitroglycerin-induced hyperalgesia and the activation of trigeminal neurons. The effects of the genetic deletion of pharmacological blockade of FAAH are mediated by CB1 receptors, because they were completely disrupted with the CB1 antagonist rimonabant. These results identify FAAH as a target for migraine pharmacotherapy.

Anandamide (AEA) is an endocannabinoid (EC) that modulates multiple functions in the CNS and that is released in areas of injury, exerting putative neuroprotective actions. In the present study, we have used intravital microscopy to analyze the role of the EC system in the glial response against an acute insult. Our data show that AEA modulates astroglial function in vivo by increasing connexin-43 hemichannel (HC) activity. Furthermore, the genetic inactivation of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH), also increased HC activity and enhanced the microglial response against an acute injury to the brain parenchyma, effects that were mediated by cannabinoid CB1 receptors. The contribution of ATP released through an astrocytic HC was critical for the microglial response, as this was prevented by the use of the HC blocker flufenamic acid and by apyrase. As could be expected, brain concentrations of AEA, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were elevated in FAAH-null mice, while 2-arachidonoylglycerol (2-AG) concentrations remained unaltered. In summary, these findings demonstrate that AEA modifies glial functions by promoting an enhanced pro-inflammatory glial response in the brain.

Paracetamol (acetaminophen) is the most commonly used analgesic in the world. Recently, a new view of its action has emerged: that paracetamol would be a pro-drug that should be metabolized by the FAAH enzyme into AM404, its active metabolite. However, this hypothesis has been demonstrated only in naive animals, a far cry from the clinical pathologic context of paracetamol use. Moreover, FAAH is a ubiquitous enzyme expressed both in the central nervous system and in the periphery. Thus, we explored: (i) the involvement of FAAH in the analgesic action of paracetamol in a mouse model of inflammatory pain; and (ii) the contributions of central versus peripheral FAAH in this action. The analgesic effect of paracetamol was evaluated in thermal hyperalgesia, mechanical allodynia and hyperalgesia induced by an intra-plantar injection of carrageenan (3%) in FAAH knock-out mice or their littermates. Moreover, the contribution of the central and peripheral enzymes was explored by comparing the effect of a global FAAH inhibitor (URB597) to that of a peripherally restricted FAAH inhibitor (URB937) on paracetamol action. Here, we show that in a model of inflammatory pain submitted to different stimuli, the analgesic action of paracetamol was abolished when FAAH was genetically or pharmacologically inhibited. Whereas a global FAAH inhibitor, URB597 (0.3 mg/kg), reduced the anti-hyperalgesic action of paracetamol, a brain-impermeant FAAH inhibitor, URB937 (0.3 mg/kg), had no influence. However, administered intracerebroventricularly, URB937 (5 μg/mouse) reduced the action of paracetamol. These results demonstrate that the supra-spinally-located FAAH enzyme is necessary for the analgesic action of paracetamol.

Cannabinoid and endocannabinoid systems have been implicated in several physiological functions including modulation of cognition. In this study we evaluated the effects and interaction between fatty-acid amide hydrolase (FAAH) inhibitor URB597 and CB1 receptor agonist WIN55, 212-2 on memory using object recognition and passive avoidance learning (PAL) tests. Learning and memory impairment was induced by WIN 55, 212-2 administration (1 mg/kg, i.p.) 30 min before the acquisition trial. URB597 (0.1, 0.3 and 1 mg/kg, i.p.) or SR141716A (1 mg/kg, i.p.) was injected to rats 10 min before WIN 55, 212-2 or URB597 respectively. URB597 (0.3 and 1 mg/kg) but not 0.1 mg/kg induced higher discrimination index (DI) in object recognition test and enhanced memory acquisition in PAL test. The cognitive enhancing effect of URB597 was blocked by a CB1 receptor antagonist, SR141716A which at this dose alone had no effect on cognition. WIN55, 212-2 caused cognition deficits in both tests. URB597 (0.3 and 1 mg/kg) treatment could alleviate the negative influence of WIN 55, 212-2 on cognition and memory. These results indicate URB597 potential to protect against memory deficits induced by cannabinoid. Therefore, in combination with URB597 beneficial effects, this study suggests that URB597 has recognition and acquisition memory enhancing effects. It may also constitute a novel approach for the treatment of cannabinoid induced memory deficits and lead to a better understanding of the brain mechanisms underlying cognition.

Long-term administration of l-3,4-dihydroxyphenylalanine (levodopa), the mainstay treatment for Parkinson's disease (PD), is accompanied by fluctuations in its duration of action and motor complications (dyskinesia) that dramatically affect the quality of life of patients. Levodopa-induced dyskinesias (LID) can be modeled in rats with unilateral 6-OHDA lesions via chronic administration of levodopa, which causes increasingly severe axial, limb, and orofacial abnormal involuntary movements (AIMs) over time. In previous studies, we showed that the direct activation of CB1 cannabinoid receptors alleviated rat AIMs. Interestingly, elevation of the endocannabinoid anandamide by URB597 (URB), an inhibitor of endocannabinoid catabolism, produced an anti-dyskinetic response that was only partially mediated via CB1 receptors and required the concomitant blockade of transient receptor potential vanilloid type-1 (TRPV1) channels by capsazepine (CPZ) (Morgese et al., 2007). In this study, we showed that the stimulation of peroxisome proliferator-activated receptors (PPAR), a family of transcription factors activated by anandamide, contributes to the anti-dyskinetic effects of URB + CPZ, and that the direct activation of the PPARγ subtype by rosiglitazone (RGZ) alleviates levodopa-induced AIMs in 6-OHDA rats. AIM reduction was associated with an attenuation of levodopa-induced increase of dynorphin, zif-268, and of ERK phosphorylation in the denervated striatum. RGZ treatment did not decrease striatal levodopa and dopamine bioavailability, nor did it affect levodopa anti-parkinsonian activity. Collectively, these data indicate that PPARγ may represent a new pharmacological target for the treatment of LID.

The endocannabinoid system is an important regulator of neuroendocrine and behavioral adaptation in stress related disorders thus representing a novel potential therapeutic target. The aim of this study was to determine the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on stress mediators of HPA axis and to study the role of the basolateral amygdala (BLA) in responses to forced swim stress. Systemic administration of URB597 (0.1 and 0.3mg/kg) reduced the forced swim stress-induced activation of HPA axis. More specifically, URB597 decreased stress-induced corticotropin-releasing hormone (CRH) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus, and pro-opiomelanocortin (POMC) mRNA expression dose-dependently in pituitary gland without affecting plasma corticosterone levels. URB597 treatment also attenuated stress-induced neuronal activation of the amygdala and PVN, and increased neuronal activation in the locus coeruleus (LC) and nucleus of solitary tract (NTS). Injection of the CB1 receptor antagonist AM251 (1ng/side) in the BLA significantly attenuated URB597-mediated effects in the PVN and completely blocked those induced in the BLA. These results suggest that the BLA is a key structure involved in the anti-stress effects of URB597, and support the evidence that enhancement of endogenous cannabinoid signaling by inhibiting FAAH represents a potential therapeutic strategy for the management of stress-related disorders.

The two most studied endocannabinoids are anandamide (AEA), principally catalyzed by fatty-acid amide hydrolase (FAAH), and 2-arachidonoyl glycerol (2-AG), mainly hydrolyzed by monoacylglycerol lipase (MGL). Inhibitors targeting these two enzymes have been described, including URB597 and URB602, respectively. Several recent studies examining the contribution of CB1 and/or CB2 receptors on the peripheral antinociceptive effects of AEA, 2-AG, URB597 and URB602 in neuropathic pain conditions using either pharmacological tools or transgenic mice separately have been reported, but the exact mechanism is still uncertain. Mechanical allodynia and thermal hyperalgesia were evaluated in 436 male C57BL/6, cnr1KO and cnr2KO mice in the presence or absence of cannabinoid CB1 (AM251) or CB2 (AM630) receptor antagonists in a mouse model of neuropathic pain. Peripheral subcutaneous injections of AEA, 2-AG, WIN55,212-2 (WIN; a CB1/CB2 synthetic agonist), URB597 and URB602 significantly decreased mechanical allodynia and thermal hyperalgesia. These effects were inhibited by both cannabinoid antagonists AM251 and AM630 for treatments with 2-AG, WIN and URB602 but only by AM251 for treatments with AEA and URB597 in C57BL/6 mice. Furthermore, the antinociceptive effects for AEA and URB597 were observed in cnr2KO mice but absent in cnr1KO mice, whereas the effects of 2-AG, WIN and URB602 were altered in both of these transgenic mice. Complementary genetic and pharmacological approaches revealed that the anti-hyperalgesic effects of 2-AG and URB602 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-allodynic actions. The antinociceptive properties of AEA and URB597 were mediated only by CB1 receptors.

Studies in male rodents have shown that stress-induced increases in circulating corticosterone are increased by both CB1 receptor (CB1R) antagonist treatment and genetic deletion. The purposes of the current study were to determine whether female mice respond in the same manner as males, and whether indirect CB1R agonists accelerate the return of corticosterone to baseline. In agreement with earlier studies, CB1R null and rimonabant-treated male mice had significantly increased circulating corticosterone 30 minutes following the end of a restraint episode compared to wild type and vehicle-treated, respectively. Females treated with rimonabant had significantly higher circulating corticosterone compared to vehicle. However, corticosterone concentrations were not different between CB1R null and wild type females at 30 minutes recovery, although CB1R null mice had higher corticosterone concentrations at 90 minutes of recovery. Female CB1R null mice exhibited greater serum binding capacity for corticosterone than wild type. The monoacylglycerol lipase inhibitor, JZL184, attenuated corticosterone concentrations at restraint offset in male, and at 30 minutes recovery in female mice compared to vehicle. Male mice treated with JZL184 exhibited greater concentrations of circulating corticosterone at 120 minutes recovery, even in the absence of restraint. JZL184 had no effect on corticosterone concentrations in CB1R null mice. The fatty acid amide hydrolase inhibitor, URB597, did not affect corticosterone responses to restraint in male or female, wild type or CB1R null mice. These data suggest that 2-arachidonoylglycerol is the primary endocannabinoid involved in CB1R regulation of the recovery of the HPA axis from restraint stress. These data support a role for endocannabinoid-CB1R signaling in the regulation of the corticosterone response to restraint stress and suggest that female mice with life-long loss of the CB1R undergo compensatory changes that minimize the impact of loss of endocannabinoid signaling on circulating corticosterone.

The endocannabinoid, N-arachidonoylethanolamine (AEA), is degraded by the enzyme fatty acid amide hydrolase (FAAH). This study examined whether the FAAH inhibitor, URB597, increases retinal ganglion cell (RGC) survival following optic nerve axotomy in young and aged animals. URB597 alone, or together with either a CB1 or CB2 receptor antagonist, was administered daily for 1 or 2 weeks post-axotomy. Histological assessment of retinas indicated that URB597 increased RGC survival in young retina at 1 and 2 weeks post-axotomy. The increase in RGC survival at 2 weeks was accompanied by a reduction in phagocytic microglia. The CB1 antagonist, AM281, but not the CB2 antagonist, AM630, ablated URB597-mediated RGC neuroprotection. CB1 or CB2 antagonism increased phagocytic microglia in URB597 and vehicle-treated animals. In aged animals, URB597 increased RGC survival at 1 week, but not at 2 weeks post-axotomy and had no effect on microglia. Retinal Iba-1 positive microglia were also decreased in URB597-treated axotomized young animals and this decrease was mitigated by CB1 but not CB2 antagonism. As seen with phagocytotic microglia, the CB2 antagonist, AM630, increased Iba-1 positive microglia in the absence of URB597 treatment. Measurement of retinal endocannabinoid levels in URB597-treated animals at 2 weeks post-axotomy revealed a significant increase in AEA levels, accompanied by a decrease in the AEA metabolite, N-arachidonoyl glycine, in young animals but not aged animals. 2-arachidonoylglycerol levels were similar across all experimental groups. These data demonstrate that URB597-mediated retinal neuroprotective effects are mediated primarily through CB1 receptors and that URB597 neuroprotective efficacy declines with age.

Enhancing the effects of endogenously-released cannabinoid ligands in the brain might provide therapeutic effects more safely and effectively than administering drugs that act directly at the cannabinoid receptor. Inhibitors of fatty acid amide hydrolase (FAAH) prevent the breakdown of endogenous ligands for cannabinoid receptors and peroxisome proliferator-activated receptors (PPAR), prolonging and enhancing the effects of these ligands when they are naturally released. This review considers recent research on the effects of FAAH inhibitors and PPAR activators in animal models of addiction and cognition (specifically learning and memory). These studies show that FAAH inhibitors can produce potentially therapeutic effects, some through cannabinoid receptors and some through PPAR. These effects include enhancing certain forms of learning, counteracting the rewarding effects of nicotine and alcohol, relieving symptoms of withdrawal from cannabis and other drugs, and protecting against relapse-like reinstatement of drug self-administration. Since FAAH inhibition might have a wide range of therapeutic actions but might also share some of the adverse effects of cannabis, it is noteworthy that at least one FAAH-inhibiting drug (URB597) has been found to have potentially beneficial effects but no indication of liability for abuse or dependence. Although these areas of research are new, the preliminary evidence indicates that they might lead to improved therapeutic interventions and a better understanding of the brain mechanisms underlying addiction and memory.

The endogenous cannabinoid anandamide (AEA) exerts the majority of its effects at CB1 and CB2 receptors and is degraded by fatty acid amide hydrolase (FAAH). FAAH KO mice and animals treated with FAAH inhibitors are impaired in their ability to hydrolyze AEA and other non-cannabinoid lipid signaling molecules, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). AEA and these other substrates activate non- cannabinoid receptor systems, including TRPV1 and PPAR-α receptors. In this mini review, we describe the functional consequences of FAAH inhibition on nicotine reward and dependence as well as the underlying endocannabinoid and non-cannabinoid receptor systems mediating these effects. Interestingly, FAAH inhibition seems to mediate nicotine dependence differently in mice and rats. Indeed, pharmacological and genetic FAAH disruption in mice enhances nicotine reward and withdrawal. However, in rats, pharmacological blockade of FAAH significantly inhibits nicotine reward and has no effect in nicotine withdrawal. Studies suggest that non-cannabinoid mechanisms may play a role in these species differences.

Recent studies suggest that endocannabinoid signaling is modulated by 17β-estradiol (17Eβ) however it is unclear if this applies to the cardiovascular actions of anandamide, a major endocannabinoid. This study examined the in vitro effects of 17Eβ on vasorelaxation to anandamide in myograph-mounted small mesenteric arteries obtained from Wistar rats and Spontaneously Hypertensive Rats (SHRs) of both sexes. Treatment with 1 μM 17Eβ but not its enantiomer 17Eα significantly enhanced relaxation to anandamide in male Wistar rats. This effect was independent of a functional endothelium but was blocked by the Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor antagonist SB366791 (2 μM) or prolonged treatment with the TRPV1 agonist capsaicin (10 μM). A TRPV1-dependent potentiation by 17Eβ was also observed in male SHRs, but not in female Wistar rats or female SHRs. Whilst inhibition of anandamide hydrolysis by 1 μM URB597 (an inhibitor of fatty acid amide hydrolase; FAAH) similarly augmented anandamide relaxation in male, but not female, Wistar rats and SHRs, URB597 did not affect the 17Eβ-induced potentiation. Female SHRs displayed a larger maximal relaxation to anandamide; however sex difference was not found in Wistar rats. We conclude that pharmacological levels of 17Eβ potentiate mesenteric relaxation to anandamide through mechanisms dependent on TRPV1 receptors but not FAAH-mediated hydrolysis in male Wistar rats and male SHRs. Sexual dimorphism was observed in the modulatory effects of 17Eβ and URB597, which does not necessarily lead to a greater anandamide response in female rats.

Endocannabinoids exhibit vasodilatory properties and reduce blood pressure in vivo. However, the influence and mechanism of action of the prominent endocannabinoid, anandamide (AEA), in pulmonary arteries are not known. The present study determined the vascular response to AEA in isolated rat pulmonary arteries. AEA relaxed rat pulmonary arteries that were pre-constricted with U-46619. This relaxation was reduced by the following conditions:removal of the endothelium; in KCl pre-constricted preparations; in the presence of the potassium channel (K(Ca)) blockers, tetraethylammonium and the combination of charybdotoxin and apamin, and the prostacyclin receptor antagonist, RO1138452. Inhibitors of cyclooxygenase (indomethacin), nitric oxide (NO) synthase (N(G)-nitro-l-arginine methyl ester) and fatty acid amide hydrolase (URB597) alone or in combination diminished AEA-induced relaxation in endothelium-intact vessels. The remaining experiments were performed in the presence of URB597 to eliminate the influence of AEA metabolites. Antagonists of the endothelial cannabinoid receptor (CB(x)), O-1918 and cannabidiol, attenuated the AEA-induced response. Antagonists of CB(1), CB(2) and TRPV1 receptors, AM251, AM630 and capsazepine, respectively, did not modify the AEA-induced response. A reference activator of CB(x) receptors, abnormal cannabidiol, mimicked the receptor-mediated AEA effects. The present study demonstrated that AEA relaxed rat pulmonary arteries in an endothelium-dependent fashion via the activation of the O-1918-sensitive CB(x) receptor and/or prostacyclin-like vasoactive products of AEA. One or both of these mechanisms may involve K(Ca) or the NO pathway.

CB1 receptors in the amygdala have been shown to mediate learned and unlearned anxiety states, however, the role of amygdalar TRPV1 receptors remains unclear. In the present study we investigated the potential anxiolytic action of intra-basolateral amygdala (BLA) infusion of N-arachidonoyl-serotonin (AA-5-HT), a dual blocker of the endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), and a TRPV1 antagonist. Varying doses of AA-5-HT (0–0.5 nmol) were administered into the BLA prior to elevated plus maze testing. AA-5-HT significantly increased both time spent and number of entries into the open arms. Next, to determine whether the anxiolytic effects were the result of blocking FAAH, TRPV1, or whether a combined action was required, rats were given intra-BLA infusions of either 0.25 nmol AA-5-HT, 1.0 nmol capsazepine (CZP, a TRPV1 antagonist), 0.01 μg URB597 (a selective FAAH inhibitor), or vehicle. Again, AA-5-HT increased the time spent in the open arms as well as the number of open arm entries. In contrast, CZP and URB597 did not reliably alter plus maze performance. We then investigated the effects of co-administration of CZP (1.0 or 10.0 nmol) and URB597 (0.01 or 0.1 μg). At lower doses, co-injections significantly increased both open arm entries as well as the time spent in the open arms, compared to vehicle or either compound alone. While co-administration of the higher doses had no significant effect when compared to either vehicle or CZP treatment, we did observe that open arm activity was elevated in rats receiving combined CZP-URB597 treatment compared to URB597 alone. Overall, our findings indicate that simultaneous FAAH activity and TRPV1 activation are important with respect to the expression of unconditioned fear as mediated within the BLA.

Many motivated and addiction-related behaviors are sustained by activity of both dopamine D1- and D2-type receptors (D1Rs and D2Rs) as well as CB1 receptors (CB1Rs) in the nucleus accumbens (NAc). Here, we use in vitro whole-cell patch-clamp electrophysiology to describe an endocannabinoid (eCB)–dopamine receptor interaction in adult rat NAc core neurons. D1R and D2R agonists in combination enhanced firing, with no effect of a D1R or D2R agonist alone. This D1R+D2R-mediated firing increase required CB1Rs, since it was prevented by the CB1R antagonists AM251 and Rimonabant. The D1R+D2R firing increase also required phospholipase C (PLC), the major synthesis pathway for the eCB 2-arachidonoylglycerol (2-AG) and one of several pathways for anandamide. Further, inhibition of 2-AG hydrolysis with the monoglyceride lipase (MGL) inhibitor JZL184 allowed subthreshold levels of D1R+D2R receptor agonists to enhance firing, while inhibition of anandamide hydrolysis with the fatty acid amide hydrolase (FAAH) inhibitors URB597 or AM3506 did not. Filling the postsynaptic neuron with 2-AG enabled subthreshold D1R+D2R agonists to increase firing, and the 2AG+D1R+D2R increase in firing was prevented by a CB1R antagonist. Also, the metabotropic glutamate receptor 5 (mGluR5) blocker MPEP prevented the ability of JZL184 to promote subthreshold D1R+D2R enhancement of firing, while the 2-AG+D1R+D2R increase in firing was not prevented by the mGluR5 blocker, suggesting that mGluR5s acted upstream of 2-AG production. Thus, our results taken together are consistent with the hypothesis that NAc core eCBs mediate dopamine receptor (DAR) enhancement of firing, perhaps providing a cellular mechanism underlying the central role of NAc core D1Rs, D2Rs, CB1Rs, and mGluR5s during many drug-seeking behaviors.

Chronic morphine exposure causes tolerance and dependence. The cessation of morphine consumption induces a withdrawal syndrome that may involve cannabinoids and is characterized by undesirable psychological and physical signs. The present study examined whether augmentation of the endocannabinoid system by inhibition of fatty acid amide hydrolase could suppress the morphine withdrawal syndrome in morphine-addicted rats. Morphine dependency was induced by 7 consecutive days of morphine injection. The morphine-addicted rats received URB597 (1, 0.5, 0.3, 0.1, 0.03 mg/kg), a fatty acid amide hydrolase inhibitor, before the precipitation of morphine withdrawal syndromes by naloxone. Withdrawal symptoms including jumping, teeth chattering, paw tremor, wet dog shakes, face grooming, penis licking, standing, rearing, sniffing and percent of weight loss were recorded during 30 min after naloxone injection. The results showed that the morphine withdrawal precipitated rats had significantly more withdrawal symptoms than naive control rats and the administration of URB597 (all doses except 0.03 mg/kg) reduced most of the morphine withdrawal symptoms. We conclude that the administration of URB597 modulated morphine withdrawal symptoms. This finding shows that endocannabinoids interact with the opioid system during the morphine withdrawal period and that potentiation of the endogenous cannabinoid system by URB597 may be a new target strategy for the management of morphine addiction.

Sub-chronic administration of PCP produces a social interaction deficit that is reversed by URB597, an inhibitor of the catabolic enzyme of the endocannabinoid anandamide. Since increased anxiety may contribute to social withdrawal and URB597 has been shown to have an anxiolytic action, we studied whether this drug affected saline- and PCP-treated rats' performance in the Elevated-Plus-Maze task, which has been used to assess anxiety-like effects. Sub-chronic PCP produced a CB(1)-dependent decrease in anxiety-like behavior that was reversed by URB597 in a CB(1)-independent fashion, as it was not blocked by the CB(1) antagonist AM251. These findings suggest that PCP-treated rats have altered endocannabinoid transmission and that anxiety does not contribute to the PCP-induced social withdrawal.