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Abstract: The endocannabinoid anandamide (AEA) activates also transient receptor potential vanilloid-1 (TRPV1) channels. However, no data exist on the potential role of spinal TRPV1 activation by AEA in neuropathic pain. We tested the effect of: 1) AEA (5–100 μg), alone or in the presence of an inhibitor of its hydrolysis, and 2) elevated levels of endogenous AEA (following inhibition of AEA hydrolysis), in CCI rats, and the involvement of TRPV1 or cannabinoid CB1 receptors in the observed effects. Levels of AEA in the spinal cord of CCI rats were measured following all treatments. AEA (50 μg) displayed anti-allodynic and anti-hyperalgesic effects which were abolished by previous antagonism of TRPV1, but not CB1, receptors. Depending on the administered dose, the selective inhibitor of AEA enzymatic hydrolysis, URB597 (10–100 μg), reduced thermal and tactile nociception via CB1 or CB1/TRPV1 receptors. The anti-nociceptive effects of co-administered per se ineffective doses of AEA (5 μg) and URB597 (5 μg) was abolished by antagonism of CB1, but not TRPV1, receptors. Spinal AEA levels were increased after CCI, slightly increased further by URB597, 10 μg i.t., and strongly elevated by URB597, 100 μg. Injection of AEA (50 μg) into the lumbar spinal cord led to its dramatic elevation in this tissue, whereas, when a lower dose was used (5 μg) AEA endogenous levels were elevated only in the presence of URB597 (5 μg). We suggest that spinal AEA reduces neuropathic pain via CB1 or TRPV1, depending on its local concentration. [Copyright &y& Elsevier]

Cannabidiol (CBD), a non-euphorigenic compound derived from Cannabis, shows promise for improving recovery following cerebral ischemia and has recently been shown effective for the treatment of childhood seizures caused by Dravet and Lennox-Gastaut syndromes. Given evidence for activity to mitigate effects of CNS insult and dysfunction, we considered the possibility that CBD may also protect and improve functional recovery of a complex learned behavior. To test this hypothesis, we have applied a songbird, the adult male zebra finch, as a novel pre-clinical animal model. Their learned vocalizations were temporarily disrupted with bilateral microlesions of HVC (used as a proper name) a pre-vocal motor cortical-like brain region that drives song. These microlesions destroy about 10% of HVC, and temporarily impair song production, syntax and phonology for about seven days. Recovery requires sensorimotor learning as it depends upon auditory feedback. Four CBD doses (0, 1, 10 and 100 mg/kg) within three surgery conditions (microlesion, no-microlesion, sham-microlesion) were evaluated (n = 5-6). Birds were recorded over 20 days: three baseline; six pre-microlesion drug treatment days and; 11 post-microlesion treatment and recovery days. Results indicate 10 and 100 mg/kg CBD effectively reduced the time required to recover vocal phonology and syntax. In the case of phonology, the magnitude of microlesion-related disruptions were also reduced. These results suggest CBD holds promise to improve functional recovery of complex learned behaviors following brain injury, and represent establishment of an important new animal model to screen drugs for efficacy to improve vocal recovery., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking, suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI elicited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha (PPAR-α) and the PPAR-α antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in mice suggests that this lipid acts as a functional PPAR-α agonist to attenuate nicotine reward. These findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating nicotine addiction., (Copyright © 2018. Published by Elsevier Ltd.)

In agreement with the neurodevelopmental hypothesis of schizophrenia, prenatal exposure of rats to the antimitotic agent methylazoxymethanol acetate (MAM) at gestational day 17 produced long-lasting behavioral alterations such as social withdrawal and cognitive impairment in the social interaction test and in the novel object recognition test, respectively. At the molecular level, an increased cannabinoid receptor type-1 (CB1) mRNA and protein expression, which might be due to reduction in DNA methylation at the gene promoter in the prefrontal cortex (PFC), coincided with deficits in the social interaction test and in the novel object recognition test in MAM rats. Both the schizophrenia-like phenotype and altered transcriptional regulation of CB1 receptors were reversed by peripubertal treatment (from PND 19 to PND 39) with the non-psychotropic phytocannabinoid cannabidiol (30 mg/kg/day), or, in part, by treatment with the cannabinoid CB1 receptor antagonist/inverse agonist AM251 (0.5 mg/kg/day), but not with haloperidol (0.6 mg/kg/day). These results suggest that early treatment with cannabidiol may prevent both the appearance of schizophrenia-like deficits as well as CB1 alterations in the PFC at adulthood, supporting that peripubertal cannabidiol treatment might be protective against MAM insult., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Endocannabinoids (eCBs) modulate synaptic transmission in the brain, but little is known of their regulatory role in nigral dopaminergic neurons, and whether transmission to these neurons is tonically inhibited by eCBs as seen in some other brain regions. Using whole-cell recording in midbrain slices, we observed potentiation of evoked IPSCs (eIPSCs) in these neurons after blocking CB1 receptors with rimonabant or LY-320,135, indicating the presence of an eCB tone reducing inhibitory synaptic transmission. Increased postsynaptic calcium buffering and block of mGluR1 or postsynaptic G-protein coupled receptors prevented this potentiation. Increasing spillover of endogenous glutamate by inhibiting uptake attenuated eIPSC amplitude, while enhancing the potentiation by rimonabant. Group I mGluR activation transiently inhibited eIPSCs, which could be prevented by GDP-β-S, increased calcium buffering or rimonabant. We explored the possibility that the dopamine-derived eCB N-arachidonoyl dopamine (NADA) is involved. The eCB tone was abolished by preventing dopamine synthesis, and enhanced by l-DOPA. It was not detected in adjacent non-dopaminergic neurons. Preventing 2-AG synthesis did not affect the tone, while inhibition of NADA production abolished it. Quantification of ventral midbrain NADA suggested a basal level that increased following prolonged depolarization or mGluR activation. Since block of the tone was not always accompanied by attenuation of depolarization-induced suppression of inhibition (DSI) and vice versa, our results indicate DSI and the eCB tone are mediated by distinct eCBs. This study provides evidence that dopamine modulates the activity of SNc neurons not only by conventional dopamine receptors, but also by CB1 receptors, potentially via NADA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Exercise-induced antinociception is widely described in the literature, but the mechanisms involved in this phenomenon are poorly understood. Systemic (s.c.) and central (i.t., i.c.v.) pretreatment with CB₁ and CB₂ cannabinoid receptor antagonists (AM251 and AM630) blocked the antinociception induced by an aerobic exercise (AE) protocol in both mechanical and thermal nociceptive tests. Western blot analysis revealed an increase and activation of CB₁ receptors in the rat brain, and immunofluorescence analysis demonstrated an increase of activation and expression of CB₁ receptors in neurons of the periaqueductal gray matter (PAG) after exercise. Additionally, pretreatment (s.c., i.t. and i.c.v.) with endocannabinoid metabolizing enzyme inhibitors (MAFP and JZL184) and an anandamide reuptake inhibitor (VDM11) prolonged and intensified this antinociceptive effect. These results indicate that exercise could activate the endocannabinoid system, producing antinociception. Supporting this hypothesis, liquid-chromatography/mass-spectrometry measurements demonstrated that plasma levels of endocannabinoids (anandamide and 2-arachidonoylglycerol) and of anandamide-related mediators (palmitoylethanolamide and oleoylethanolamide) were increased after AE. Therefore, these results suggest that the endocannabinoid system mediates aerobic exercise-induced antinociception at peripheral and central levels., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

N-palmitoylethanolamine (PEA), an endogenous fatty acid ethanolamide, plays a key role in the regulation of the inflammatory response and pain through, among others, activation of nuclear peroxisome proliferator-activated receptors (PPAR-α). Endogenous cannabinoids play a protective role in several central nervous system (CNS) disorders, particularly those associated with neuronal hyperexcitability. We investigated the effects of PEA and the role of PPAR-α in absence epilepsy using the WAG/Rij rat model. PEA, anandamide (AEA), a PPAR-α antagonist (GW6471) and a synthetic CB1 receptor antagonist/inverse agonist (SR141716) were administered to WAG/Rij rats in order to evaluate the effects on epileptic spike-wave discharges (SWDs) on EEG recordings. We studied also the effects of PEA co-administration with SR141716 and GW6471 and compared these effects with those of AEA to evaluate PEA mechanism of action and focusing on CB1 receptors and PPAR-α. Both PEA and AEA administration significantly decreased SWDs parameters (absence seizures). In contrast, GW6471 was devoid of effects while SR141716 had pro-absence effects. The co-administration of SR141716 with PEA or AEA completely blocked the anti-absence effects of these compounds. GW6471 antagonized PEA's effects whereas it did not modify AEA's effects. Furthermore, we have also measured PEA, AEA and 2-AG (2-arachidonoylglycerol) brain levels identifying significant differences between epileptic and control rats such as decreased PEA levels in both thalamus and cortex that might contribute to absence epilepsy. Our data demonstrate that PEA has anti-absence properties in the WAG/Rij rat model and that such properties depend on PPAR-α and indirect activation of CB1 receptors. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Whether adolescent exposure to chronic delta-9-tetrahydrocannabinol (THC) facilitates progression to opioid consumption is still controversial. In a maternal deprivation model (3 h daily from postnatal day 1-14), we previously reported that adolescent exposure to chronic THC blocks morphine dependence in maternally deprived (D) rats. Owing to the existence of a functional cross-interaction between the opioid and cannabinoid systems in reward, we evaluated if the vulnerability to opiate reward in D rats, may involve an alteration of the endocannabinoid system. Anandamide and 2-arachidonoylglycerol (2-AG), were quantified in the striatum and mesencephalon of adolescent and adult D and non-deprived (animal facility rearing, AFR) rats by isotope dilution liquid chromatography-mass spectrometry. Oral morphine self-administration behavior was analyzed for 14 weeks, 24 days after chronic injection of the cannabinoid CB1 receptor antagonist/inverse agonist, SR141716A (3 mg/kg) for 2 weeks during adolescence (PND 35-48). Adolescent D rats exhibited higher basal levels of anandamide than adolescent AFR rats in the nucleus accumbens (38%), the caudate-putamen nucleus (62%) and the mesencephalon (320%), whereas adult D rats showed an increase of anandamide and 2-AG levels in the nucleus accumbens (50% and 24%, respectively) and of 2-AG in the caudate-putamen nucleus (48%), compared to adult AFR rats. Chronic administration of SR141716A to adolescent D rats blocked the escalation behavior in the morphine consumption test. Our data suggest that altered brain endocannabinoid levels may contribute to the escalation behavior in the morphine consumption test in a maternal deprivation model., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB(1) receptors and "transient receptor potential vanilloid" type 1 (TRPV1) channels, and N-palmitoylethanolamine (PEA) potentiates these effects. In neurons of the rat dorsal root ganglia (DRG), TRPV1 is activated and/or sensitised by AEA as well as upon activation of protein kinases C (PKC) and A (PKA). We investigated here the effect on AEA levels of PKC and PKA activators in DRG neurons. AEA levels were significantly enhanced by both phorbol-miristoyl-acetate (PMA), a typical PKC activator, and forskolin (FSK), an adenylate cyclase stimulant, as well as by thrombin, which also activates PKC by stimulating protease-activated receptors (PARs). The levels of the other endocannabinoid and TRPV1-inactive compound, 2-arachidonoylglycerol (2-AG), were enhanced only by thrombin and to a lesser extent than AEA, whereas PEA was not affected by any of the treatments. Importantly, FSK- and PMA-induced elevation of AEA levels was not sensitive to intracellular Ca2+ chelation with BAPTA-acetoxymethyl (AM) ester. In human embryonic kidney (HEK-293) cells, which constitutively express PARs, thrombin, PMA and FSK elevated AEA levels, and the effects of the two former compounds were counteracted by the PKC inhibitor, RO318220, whereas the effect of FSK was reduced by the PKA inhibitor RpcAMPs. In conclusion, we report that AEA levels are stimulated by both PKC, either directly or after thrombin receptor activation, and PKA, possibly in a way independent from intracellular calcium. Since AEA activates TRPV1, these findings may suggest the existence of an amplificatory cascades on this receptor in sensory neurons.

We evaluated the effects of intra-periaqueductal grey (PAG) N-arachidonoyl-serotonin (AA-5-HT), a compound with a "dual" ability to inhibit the fatty acid amide hydrolase (FAAH) and to antagonize transient receptor vanilloid type 1 (TRPV1) receptors, on endocannabinoid levels, rostral ventromedial medulla (RVM) ON and OFF cell activities, thermal nociception (tail flick in anaesthetized rats) and formalin-induced nocifensive responses in awake rats. AA-5-HT increased endocannabinoid levels in the PAG and induced analgesia. Paradoxically, it also depressed the RVM OFF cell, as well as the ON cell activities. The effect of AA-5-HT was mimicked by co-injecting the selective FAAH inhibitor URB597 and the selective TRPV1 antagonist I-RTX into the PAG, which also induced analgesia and inhibition of ON and OFF cell ongoing activities. The recruitment of "alternative" pathways, such as PAG-locus coeruleus (LC)-spinal cord might be responsible for AA-5-HT effect since we found evidence that (i) intra-PAG AA-5-HT increased LC neuron firing activities, and (ii) intrathecal phentolamine or ketanserin prevented the analgesic effect of AA-5-HT. Moreover, intra-PAG AA-5-HT prevented the changes in the ON and OFF cells firing activity induced by intra-paw formalin, and it inverted the formalin-induced increase in LC adrenergic cell activity. All AA-5-HT effects were antagonized by cannabinoid CB1 and TRPV1 receptor antagonists thus suggesting that co-localization of these receptors in the PAG can be an appropriate neural substrate for AA-5-HT-induced analgesia.

Endocannabinoids are paracrine/autocrine lipid mediators with several biological functions. One of these, i.e. the capability to stimulate food intake via cannabinoid CB(1) receptors, has been particularly studied, thus leading to the development of the first CB(1) receptor blocker, rimonabant, as a therapeutic tool against obesity and related metabolic disorders. Hypothalamic endocannabinoids stimulate appetite by regulating the expression and release of anorexic and orexigenic neuropeptides via CB(1) receptors. In turn, the tone of the latter receptors is regulated by hormones, including leptin, glucocorticoids and possibly ghrelin and neuropeptide Y, by modulating the biosynthesis of the endocannabinoids in various areas of the hypothalamus. CB(1) receptor stimulation is also known to increase blood glucose during an oral glucose tolerance test in rats. Here we investigated in the rat if insulin, which is known to exert fundamental actions at the level of the mediobasal hypothalamus (MBH), and the melanocortin system, namely alpha-melanocyte stimulating hormone (alpha-MSH) and melanocortin receptor-4 (MCR-4), also regulate hypothalamic endocannabinoid levels, measured by isotope-dilution liquid chromatography coupled to mass spectrometry. No effect on anandamide and 2-arachidonoylglycerol levels was observed after 2h infusion of insulin in the MBH, i.e. under conditions in which the hormone reduces blood glucose, nor with intra-cerebroventricular injection of alpha-MSH, under conditions in which the neuropeptide reduces food intake. Conversely, blockade of MCR-4 receptors with HS014 produced a late (6h after systemic administration) stimulatory effect on endocannabinoid levels as opposed to a rapid and prolonged stimulation of food-intake (observable 2 and 6h after administration). These data suggest that inhibition of endocannabinoid levels does not mediate the effect of insulin on hepatic glucose production nor the food intake-inhibitory effect of alpha-MSH, although stimulation of endocannabinoid levels might underlie part of the late stimulatory effects of MCR-4 blockade on food intake.

Recent studies have shown that activation of the cannabinoid CB(1) receptor by synthetic agonists, and pharmacological elevation of endocannabinoid levels, suppress hyperalgesia and allodynia in animal models of neuropathic pain. However, the concentrations of endocannabinoids in the nervous tissues involved in pain transmission during neuropathic pain have never been measured. Here we have determined the levels of anandamide and 2-arachidonoylglycerol (2-AG), as well as of the analgesic anandamide congener, palmitoylethanolamide (PEA), in three brain areas involved in nociception, i.e. the dorsal raphe (DR), periaqueductal grey (PAG) and rostral ventral medulla (RVM), as well as in the spinal cord (SC), following chronic constriction injury (CCI) of the sciatic nerve in the rat, in comparison with sham-operated rats. After 3 days from CCI, anandamide or 2-AG levels were significantly enhanced only in the SC or PAG, respectively. After 7 days from CCI, when thermal hyperalgesia and mechanical allodynia are maximal, a strong (1.3-3-fold) increase of both anandamide and 2-AG levels was observed in the PAG, RVM and SC. At this time point, anandamide, but not 2-AG, levels were also enhanced in the DR. PEA levels were significantly decreased in the SC after 3 days, and in the DR and RVM after 7 days from CCI. These data indicate that anandamide and 2-AG, operating at both spinal and supra-spinal levels, are up-regulated during CCI of the sciatic nerve, possibly to inhibit pain. Yet to be developed substances that inhibit both endocannabinoid and PEA inactivation might be useful for the treatment of neuropathic pain.

Although most commonly associated with actions at cannabinoid CB1 receptors on the extracellular surface of the plasma membrane, the endocannabinoid anandamide (AEA) is also transported into the cell, by the putative anandamide membrane transporter (AMT), and activates the vanilloid receptor 1 (TRPV1) at an intracellular site. AEA is then inactivated by fatty acid amide hydrolase (FAAH). As systemic administration of TRPV1 ligands reduces locomotor activity in normal rodents, we hypothesised that activation of TRPV1 by endocannabinoids could play a role in the control of voluntary movement and that such actions could be regulated by AMT and FAAH. Motor activity was assessed in normal, in reserpine-treated, and in reserpine-treated rats treated with L-DOPA. In normal rats, the TRPV1 agonist capsaicin (1 mg/kg) or the FAAH inhibitor URB597 (10 mg/kg) caused a significant reduction in movement in both the horizontal (locomotion) and vertical (rearing) planes (-45% and -53% respectively with capsaicin; -33% and -37% for URB597). Capsaicin-induced hypolocomotion was attenuated by the TRPV1 antagonist, capsazepine. There was no effect of capsaicin, URB597 or the AMT inhibitor OMDM-2 on motor activity in reserpine-treated rats. L-DOPA treatment of reserpine-treated rats elicited high levels of motor activity in both the horizontal and vertical planes. Horizontal activity was attenuated by capsaicin (1 mg/kg, -60%), but not by URB597 (10 mg/kg) or OMDM-2 (5 mg/kg). Vertical activity was attenuated by capsaicin (1 mg/kg, -61%) and by URB597 (10 mg/kg, -54%), but not by OMDM-2. These data suggest that activation of the TRPV1 system can suppress spontaneous locomotion in normal animals and modulates several L-DOPA-induced behaviours in reserpine-treated rats.

The chemotherapeutic agent cisplatin may produce emesis via release of several neurotransmitters such as serotonin (5-HT), substance P and/or dopamine as well as production of prostaglandins (PGs). Administration of synthetic 2-arachidonoylglycerol (2-AG) but not of anandamide, which are two putative endocannabinoids, causes vomiting via its downstream metabolites such as arachidonic acid (AA) and PGs in the least shrew (Cryptotis parva). We report here that cisplatin (0, 5, 10 and 20 mg/kg, i.p.) causes dose- and time-dependent increases in brain tissue levels of 2-AG but not anandamide in this vomiting species. Concomitantly, intestinal tissue levels of both endocannabinoids are relatively reduced. Selective inhibitors [arachidonoyl-serotonin (AA-5-HT) and URB597, 0-5 and 0-10 mg/kg, i.p.] of one of the major endocannabinoid metabolic enzymes, the intracellular fatty acid amide hydrolase (FAAH), do not significantly prevent vomiting produced by emetic doses of i.p.-administered 2-AG, cisplatin or the dopamine receptor agonist apomorphine. At large doses (10 and 20 mg/kg, respectively), both FAAH inhibitors caused emesis per se. Administration of one selective uptake inhibitor of endocannabinoids, OMDM1 (0-5 mg/kg, i.p.), also did not significantly prevent emesis by the direct and indirect emetic stimuli, and likewise caused emesis by itself at a high (10 mg/kg) dose. However, another selective uptake inhibitor, VDM11, did not produce significant emesis per se and prevented emesis caused by apomorphine. Both the corticosteroid dexamethasone, and the cyclooxygenase inhibitor indomethacin, reduced vomiting produced by cisplatin. These data: (a) provide the first evidence that cisplatin causes a selective increase in 2-AG levels in the brain, and (b) support the established notion that 2-AG may produce some of its effects, including emesis, via downstream metabolites produced independently of FAAH.

The endogenous cannabimimetic compound, and anandamide analogue, N-palmitoyl-ethanolamine (PEA), was shown to exert potent anti-inflammatory and analgesic effects in experimental models of visceral, neuropathic and inflammatory pain by acting via several possible mechanisms. However, only scant data have been reported on the regulation of PEA levels during pathological conditions in animals or, particularly, humans. We review the current literature on PEA and report the results of three separate studies indicating that its concentrations are significantly increased during three different inflammatory and neuropathic conditions, two of which have been assessed in humans, and one in a mouse model. In patients affected with chronic low back pain, blood PEA levels were not significantly different from those of healthy volunteers, but were significantly and differentially increased (1.6-fold, P<0.01, N=10 per group) 30 min following an osteopathic manipulative treatment. In the second study, the paw skin levels of PEA in mice with streptozotocin-induced diabetic neuropathic pain were found to be significantly higher (1.5-fold, P<0.005, N=5) than those of control mice. In the third study, colonic PEA levels in biopsies from patients with ulcerative colitis were found to be 1.8-fold higher (P<0.05, N=8-10) than those in healthy subjects. These heterogeneous data, together with previous findings reviewed here, substantiate the hypothesis that PEA is an endogenous mediator whose levels are increased following neuroinflammatory or neuropathic conditions in both animals and humans, possibly to exert a local anti-inflammatory and analgesic action.