Cannabinoid CB1 Receptor Antagonist AM251 Research Articles

Beneficial Changes in Rat Vascular Endocannabinoid System in Primary Hypertension and under Treatment with Chronic Inhibition of Fatty Acid Amide Hydrolase by URB597.

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB1 receptor (CB1R) expression were elevated. The CB1R antagonist AM251 diminished the methanandamide-evoked relaxation only in the sMA of SHR and enhanced the vasoconstriction induced by phenylephrine and the thromboxane analog U46619 in sMA in SHR and WKY. In the sMA of SHR, URB597 elevated anandamide levels, improved the endothelium-dependent vasorelaxation to acetylcholine, and in the presence of AM251 reduced the vasoconstriction to phenylephrine and enhanced the vasodilatation to methanandamide, and tended to reduce hypertrophy. In the aortas, URB597 elevated endocannabinoid levels improved the endothelium-dependent vasorelaxation to acetylcholine and decreased CB1R expression. Our study showed that hypertension and chronic administration of URB597 caused local, resistance artery-specific beneficial alterations in the vascular endocannabinoid system, which may bring further advantages for therapeutic application of pharmacological inhibition of FAAH.

Inhibition of fatty acid amide hydrolase in the CNS prevents and reverses morphine tolerance in male and female mice.

Fatty acid amide hydrolase (FAAH) is an intracellular serine amidase that terminates the signalling of various lipid messengers involved in pain regulation, including anandamide and palmitoylethanolamide. Here, we investigated the effects of pharmacological or genetic FAAH removal on tolerance to the anti-nociceptive effects of morphine. We induced tolerance in male and female mice by administering twice-daily morphine for 7 days while monitoring nociceptive thresholds by the tail immersion test. The globally active FAAH inhibitor URB597 (1 and 3 mg·kg-1 , i.p.) or the peripherally restricted FAAH inhibitor URB937 (3 mg·kg-1 , i.p.) were administered daily 30 min prior to morphine, alone or in combination with the cannabinoid CB1 receptor antagonist AM251 (3 mg·kg-1 , i.p.), the CB2 receptor antagonist AM630 (3 mg·kg-1 , i.p.), or the PPAR-α antagonist GW6471 (4 mg·kg-1 , i.p.). Spinal levels of FAAH-regulated lipids were quantified by LC/MS-MS. Gene transcription was assessed by RT-qPCR. URB597 prevented and reversed morphine tolerance in both male and female mice. This effect was mimicked by genetic FAAH deletion, but not by URB937. Treatment with AM630 suppressed, whereas treatment with AM251 or GW6471, attenuated the effects of URB597. Anandamide mobilization was enhanced in the spinal cord of morphine-tolerant mice. mRNA levels of the anandamide-producing enzyme N-acyl-phosphatidylethanolamine PLD (NAPE-PLD) and the palmitoylethanolamide receptor PPAR-α, but not those for CB2 , CB1 receptors or FAAH, were elevated in spinal cord CONCLUSION AND IMPLICATIONS: FAAH-regulated lipid signalling in the CNS modulated opiate tolerance, suggesting FAAH as a potential target for opiate-sparing medications.

CANNABINOID RECEPTORS ARE NOT INVOLVED IN ANTINOCICEPTION INDUCED BY SYSTEMIC DICLOFENAC IN MICE

Aims: It has been long suspected that the cannabinoid system participates in the antinociceptive effects of nonsteroidal anti-inflammatory drugs. We studied the possible effects of cannabinoid receptor antagonism on diclofenac-induced antinociception in the writhing test in mice. Methods: In our study, male BALB/c mice, weighing 20-30 g, were used. Writhing responses were produced by intraperitoneal injection of 0.6% acetic acid. Different doses of diclofenac (3, 10, 30 mg/kg, i.p.) were tested, then the influence of AM-251 (1 mg/kg, i.p.), a cannabinoid CB1 receptor antagonist and AM-630 (3 mg/kg, i.p.), a cannabinoid CB2 receptor antagonist on the antinociceptive effects of diclofenac was studied. Results: Diclofenac administration elicited a significant, dose-dependent antinociceptive response; however, neither the cannabinoid CB1 receptor antagonist AM-251 nor the cannabinoid CB2 receptor antagonist AM-630 had any influence on the antinociceptive effect of diclofenac. Conclusion: Iinhibition of cannabinoid receptors does not contribute to the antinociceptive action of systemic diclofenac. Further studies are needed to explain the antinociceptive mechanism of diclofenac. Keywords: AM-251, AM-630, antinociception, cannabinoid receptors, diclofenac

The influence of the cannabinoid receptor CB1 on the periaqueductal gray in mice treated with photobiomodulation after chronic constriction injury of the sciatic nerve: a placebo-controlled trial

BACKGROUND AND OBJECTIVES: Studies have demonstrated that the cannabinoid CB1 receptor is involved in the modulation of pain, mainly by activating the descending pain control pathway. However, the role of photobiomodulation in this process is not well elucidated. Thus, the present study aimed to investigate the involvement of the CB1 receptor in the supraspinal photobiomodulation-induced antinociception. METHODS: Male albino swiss mice were submitted to chronic constriction injury and treated with photobiomodulation. To evaluate the supraspinal involvement of the CB1 receptor in the photobiomodulation-induced antinociception, the cannabinoid CB1 receptor antagonist AM251 (0.1µg/vol 0.2µL) was injected 5 minutes before the photobiomodulation treatment. The photobiomodulation treatment was performed on the fifth day after the stereotactic surgery and chronic constriction injury at a dose of 50J/cm2 in acute condition. The hot plate and von Frey monofilaments tests were performed to evaluate the thermal and mechanical pain sensitivity, respectively. RESULTS: The thermal and mechanical nociceptive threshold was higher in mice with chronic constriction injury, injected with saline and treated with photobiomodulation at the dose of 50J/cm2 in both the hot plate (p<0.001) and von Frey (p>0.001) tests. These antinociceptive effects were not detected in mice with chronic constriction injury pre-treated with AM251. CONCLUSION: The present study suggests that CB1 receptors located in Supraspinal structures, participate in the control of neuropathic pain following photobiomodulation treatment in animals undergoing chronic constriction injury.

Mechanisms of l-alpha-lysophosphatidylinositol-induced relaxation in human pulmonary arteries

Aimsl-Alpha-lysophosphatidylinositol (LPI) is an endogenous agonist of G protein-coupled receptor 55 (GPR55) which relaxes mesenteric arteries on activation. The aim of the present study was to determine the influence and underlying mechanisms of LPI-induced relaxation in human pulmonary arteries (hPAs). Main methodsFunctional studies were performed in isolated hPAs using organ bath technique. The expression of GPR55 in hPAs and bronchioles was determined by real-time qPCR, Western blot analysis, and immunohistochemistry. Key findingsLPI induced a concentration-dependent vasorelaxation in endothelium-intact hPAs. This effect was attenuated by the GPR55 antagonist CID16020046, the peroxisome proliferator-activated receptor-γ (PPARγ) antagonist GW9662, the putative endothelial cannabinoid receptor (CBe) antagonist O-1918 and the inhibitor of nitric oxide (NO) synthase (L-NAME). In addition, vasorelaxation was also attenuated by the presence of a high KCl concentration, selective blockers of small (KCa2.3; UCL1684), intermediate (KCa3.1; TRAM-34) and large conductance (KCa1.1; iberiotoxin) calcium-activated potassium channels and by endothelium denudation. However, vasorelaxation was not attenuated by the cannabinoid CB1 receptor antagonist AM251 or by the cyclooxygenase inhibitor indomethacin. SignificanceThe study showed that the LPI-induced vasorelaxation was endothelium-dependent and mediated by GPR55, PPARγ and CBe receptors, occurred in a NO- and calcium-activated potassium channel-dependent manner in isolated hPAs. LPI seems to possess positive, hypotensive properties in pulmonary vascular bed.

Endocannabinoid-dependent protection against kainic acid-induced long-term alteration of brain oscillations in guinea pigs

Changes in rhythmic activity can serve as early biomarkers of pathological alterations, but it remains unclear how different types of rhythmic activity are altered during neurodegenerative processes. Glutamatergic neurotoxicity, evoked by kainic acid (KA), causes hyperexcitation and acute seizures that result in delayed brain damage. We employed wide frequency range (0.1–300Hz) local field potential recordings in guinea pigs to study the oscillatory activity of the hippocampus, entorhinal cortex, medial septum, and amygdala in healthy animals for three months after KA introduction. To clarify whether the activation of endocannabinoid (eCB) system can influence toxic KA action, AM404, an eCB reuptake inhibitor, and URB597, an inhibitor of fatty acid amide hydrolase, were applied. The cannabinoid CB1 receptor antagonist AM251 was also tested. Coadministration of AM404 or URB597 with KA reduced acute behavioral seizures, but electrographic seizures were still registered. During the three months following KA injection, various trends in the oscillatory activities were observed, including an increase in activity power at all frequency bands in the hippocampus and a progressive long-term decrease in the medial septum. In the KA- and KA/AM251-treated animals, disturbances of the oscillatory activities were accompanied by cell loss in the dorsal hippocampus and mossy fiber sprouting in the dentate gyrus. Injections of AM404 or URB597 softened alterations in electrical activity of the brain and prevented hippocampal neuron loss and synaptic reorganization. Our results demonstrate the protective potential of the eCB system during excitotoxic influences.

P.1.g.072 - Striatonigral-nigrotectal circuits regulate the expression of innate fear responses via cannabinoid receptor type 1 on GABAergic neurons

Cannabinoid CB1 receptor (CB1R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with 13C NMR isotopomer analysis whether CB1Rs could have a local metabolic role in brain areas having high CB1R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-13C]acetate, which is oxidized in glial cells, and [U–13C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB1R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-13C]acetate (−11.6 ± 2.0%) and [U–13C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U–13C]glucose (−11.7 ± 4.0%) but not that of [2-13C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB1R antagonist AM251 (500 nM). These results thus suggest that CB1Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB1R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB1Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB1Rs control cell physiology and afford neuroprotection.

Differential Pharmacological Regulation of Sensorimotor Gating Deficit in CB1 Knockout Mice and Associated Neurochemical and Histological Alterations.

The endocannabinoid system has been widely involved in the pathophysiology of sensorimotor gating deficits. This study aimed to evaluate the pharmacological modulation of the sensorimotor gating impairment induced by cannabinoid CB1 receptor (CB1r) deletion. For this purpose, the prepulse inhibition (PPI) paradigm was used to evaluate the effect of two antipsychotics drugs (risperidone and haloperidol) and a psychostimulant (methylphenidate) on the preattentional deficit presented by CB1KO mice. Furthermore, the effects of the CB1r antagonist AM251 on PPI were evaluated in WT mice. Real-time PCR and immunohistochemical studies were carried out to analyze dopamine transporter (DAT) and α-2C adrenergic receptor (ADRA2C) gene expressions and the distribution of parvalbumin (PV) and cholecystokinin-8 (CCK) immunoreactive (ir) cortical neurons, respectively. Neither risperidone nor haloperidol significantly modified the PPI of WT and CB1KO mice, whereas methylphenidate improved the preattentional deficit of CB1KO mice. In addition, treatment with AM251 (3 mg/kg; i.p.) significantly decreased the PPI of WT animals. The administration of methylphenidate increased DAT and ADRA2C gene expressions in CB1KO mice without producing any effect in WT animals. Immunohistochemical studies revealed that there were no significant changes in CCK immunolabeling between WT and CB1KO mice, whereas the radial distribution of PV-ir neurons was abnormal in CB1KO mice. These data further support the important role of CB1r in sensorimotor gating regulation and the therapeutic usefulness of methylphenidate for the treatment of psychiatric disorders with associated preattentional deficits.

Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress

Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus–pituitary–adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

Cannabinoid CB1 receptors mediate the effects of dipyrone.

Dipyrone is a non-steroidal anti-inflammatory drug used primarily as an analgesic and antipyretic. Some hypothesize that dipyrone activity can modulate other pathways, including endocannabinoid signalling. Thus, the aim of the present study was to evaluate the possible role of endocannabinoids in mediating dipyrone activity. This study is based on the tetrad effects of cannabinoids, namely an antinociceptive and cataleptic state, hypolocomotion and hypothermia. Dipyrone (500mg/kg, i.p.) treatment decreased locomotor activity, increased the latency to a thermal analgesic response and induced a cataleptic and hypothermic state. These reactions are similar to the tetrad effects caused by the cannabinoid agonist WIN55,212-2 (3mg/kg, i.p.). The cannabinoid CB1 receptor antagonist AM251 (10mg/kg, i.p.) reversed the effects of dipyrone on locomotor activity, the cataleptic response and thermal analgesia. Both AM251 (10mg/kg, i.p.) and the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (10mg/kg, i.p.) accentuated the reduction in body temperature caused by dipyrone. However, the CB2 receptor antagonist AM630 did not alter the hypothermic response to dipyrone. These results indicate involvement of the endocannabinoid system, especially CB1 receptors, in the analgesic and cataleptic effects of dipyrone, as well as hypolocomotion. However, cannabinoid receptors and TRPV1 were not involved in the hypothermic effects of dipyrone. We hypothesize that the mechanism of action of dipyrone may involve inhibition of cyclo-oxygenase and fatty acid amide hydrolase, which together provide additional arachidonic acid as substrate for endocannabinoid synthesis or other related molecules. This increase in endocannabinoid availability enhances CB1 receptor stimulation, contributing to the observed effects.

Repeated morphine treatment alters cannabinoid modulation of GABAergic synaptic transmission within the rat periaqueductal grey.

Cannabinoids and opioids produce antinociception by modulating GABAergic synaptic transmission in a descending analgesic pathway from the midbrain periaqueductal grey (PAG). While chronic opioid treatment produces opioid tolerance, it has recently been shown to enhance cannabinoid-induced antinociception within the PAG. This study examined the effect of repeated opioid treatment on opioid and cannabinoid presynaptic modulation of GABAergic synaptic transmission in PAG. Midbrain PAG slices were prepared from untreated rats, and rats that had undergone repeated morphine or saline pretreatment. Whole-cell voltage-clamp recordings were made from neurons within the ventrolateral PAG. In slices from untreated animals, the cannabinoid receptor agonist WIN55212 and the μ receptor agonist DAMGO inhibited electrically evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) IPSCs in PAG neurons, with IC50 s of 30 and 100 nM respectively. The inhibition of evoked IPSCs produced by WIN55212 (30 nM) and DAMGO (100 nM) was similar in PAG neurons from morphine- and saline-treated animals. The cannabinoid CB1 receptor antagonist AM251 increased the frequency of spontaneous miniature IPSCs in PAG neurons from repeated morphine-, but not saline-treated animals. DAMGO inhibition of evoked IPSCs was enhanced in the presence of AM251 in morphine-, but not saline-treated animals. These results indicate that the efficiency of agonist-induced inhibition of GABAergic synaptic transmission is enhanced by morphine treatment, although this is dampened by endocannabinoid-mediated tonic inhibition. Thus, endocannabinoid modulation of synaptic transmission could provide an alternative analgesic approach in a morphine-tolerant state. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

P-47 * HARMFUL EFFECTS OF LAW-EVADING HERBS AS A NEW TREND IN JAPAN: BEHAVIORAL AND CYTOTOXICOLOGICAL PROPERTIES OF SYNTHETIC CANNABINOIDS

Introduction. Law-evading herbal products consist of finely chopped dry vegetative matter mixed with chemical substances, and the drugs are injurious to health. Analysis of chemical substances in herbal products clarified that they contain synthetic cannabinoids. Method. The present study investigates the behavioral and cytotoxicological properties of synthetic cannabinoid CP-55, 940 in mice. In the current behavioral analysis study, we investigated the effect that CP-55, 940 has on place conditioning in ICR mice. The cytotoxicological effects of CP-55, 940 were characterized in mouse striatal neurons of primary culture in vitro. Results. In a place-conditioning study, CP-55, 940 produced a significant conditioned-place preference. The rewarding effects of CP-55, 940 were completely suppressed by cannabinoid CB1 receptor antagonist AM251. Administration of CP-55, 940 in striatal primary culture caused cell death in a dose-dependent manner. Conclusion. Our findings demonstrated that the CB1 receptor might be involved in the expression of CP-55, 940-induced rewarding effect. Furthermore, our data on striatal primary culture indicate that CP-55, 940 has strong neurotoxicity. These behavioral and neurochemical data indicate that CP-55, 940 might have strong adverse effects and a psychic-dependence liability. We established a psychic-dependence liability and cytotoxicity screening system for synthetic cannabinoids using animals (behavioral analysis in vivo) and cell culture (cytotoxicity analysis in vitro).

Role of intra-accumbal cannabinoid CB1 receptors in the potentiation, acquisition and expression of morphine-induced conditioned place preference

Recent studies demonstrate a functional interaction between opioid and endogenous cannabinoid system. These two systems possess similar pharmacological effects on drug addiction and reward. The present study was designed to investigate the role of intra-accumbal cannabinoid CB1 receptors in the acquisition and expression of morphine-induced conditioned place preference (CPP). Two-hundred forty eight adult male albino Wistar rats were used in these experiments. Using a 3-day schedule of conditioning, it was found that subcutaneous administration of morphine (0.2–10mg/kg) induced CPP at the doses of 5 and 10mg/kg. Solely intra-accumbal administration of WIN55,212-2 (1, 2 and 4mmol/0.5μl DMSO) as CB1 receptor agonist could induce CPP. Also, our results showed that ineffective dose of WIN55,212-2 (1mmol) when administered before the ineffective dose of morphine (2mg/kg) could induce the CPP and potentiate the rewarding effect of morphine. On the other hand, intra-accumbal injection of the cannabinoid CB1 receptor antagonist AM251 (90μmol/0.5μl DMSO) alone induced a significant conditioned place aversion. Moreover, intra-NAc injection of AM251 (45 and 90μmol/0.5μl DMSO) inhibited morphine-induced CPP. Interestingly, injection of WIN55,212-2 (1, 2 and 4mmol) or AM251 (15, 45 and 90μmol) into the NAc had no effect on the expression of morphine (5mg/kg)-induced CPP. These observations provide evidence that cannabinoid CB1 receptors in the NAc are involved in development of reward-related behaviors and they can potentiate the rewarding effects of morphine. It seems that these receptors can affect the reward modulatory system at the level of nucleus accumbens in rats.

A role for endocannabinoids in acute stress-induced suppression of the hypothalamic-pituitary-gonadal axis in male rats

ObjectiveStress is known to be an inhibitor of the reproductive hypothalamic-pituitary-gonadal (HPG) axis. However, the neural and molecular connections between stress and reproduction are not yet understood. It is well established that in both humans and rodents, kisspeptin (encoded by the kiss1 gene) is a strong stimulator of the HPG axis. In the present study we hypothesized that endocannabinoids, an important neuromodulatory system in the brain, can act on the HPG axis at the level of kiss1 expression to inhibit reproductive function under stress.MethodsAdult male Wistar rats were unilaterally implanted with an intracerebroventricular cannula. Afterwards, the animals were exposed to immobilization stress, with or without the presence of the cannabinoid CB1 receptor antagonist AM251 (1 µg/rat). Blood samples were collected through a retro-orbital plexus puncture before and after stress. Five hours after the stress, brain tissue was collected for reverse transcriptase-quantitative polymerase chain reaction measurements of kiss1 mRNA.ResultsImmobilization stress (1 hour) resulted in a decrease in the serum luteinizing hormone concentration. Additionally, kiss1 gene expression was decreased in key hypothalamic nuclei that regulate gonadotrophin secretion, the medial preoptic area (mPOA), and to some extent the arcuate nucleus (ARC). A single central administration of AM251 was effective in blocking these inhibitory responses.ConclusionThese findings suggest that endocannabinoids mediate, at least in part, immobilization stress-induced inhibition of the reproductive system. Our data suggest that the connection between immobilization stress and the HPG axis is kiss1 expression in the mPOA rather than the ARC.

State-Dependent, Bidirectional Modulation of Neural Network Activity by Endocannabinoids

The endocannabinoid (eCB) system and the cannabinoid CB1 receptor (CB1R) play key roles in the modulation of brain functions. Although actions of eCBs and CB1Rs are well described at the synaptic level, little is known of their modulation of neural activity at the network level. Using microelectrode arrays, we have examined the role of CB1R activation in the modulation of the electrical activity of rat and mice cortical neural networks in vitro. We find that exogenous activation of CB1Rs expressed on glutamatergic neurons decreases the spontaneous activity of cortical neural networks. Moreover, we observe that the net effect of the CB1R antagonist AM251 inversely correlates with the initial level of activity in the network: blocking CB1Rs increases network activity when basal network activity is low, whereas it depresses spontaneous activity when its initial level is high. Our results reveal a complex role of CB1Rs in shaping spontaneous network activity, and suggest that the outcome of endogenous neuromodulation on network function might be state dependent.

Antinociceptive effects induced through the stimulation of spinal cannabinoid type 2 receptors in chronically inflamed mice

The stimulation of spinal cannabinoid type 2 (CB2) receptors is a suitable strategy for the alleviation of experimental pain symptoms. Several reports have described the up-regulation of spinal cannabinoid CB2 receptors in neuropathic settings together with the analgesic effects derived from their activation. Besides, we have recently reported in two murine bone cancer models that the intrathecal administration of cannabinoid CB2 receptor agonists completely abolishes hyperalgesia and allodynia, whereas spinal cannabinoid CB2 receptor expression remains unaltered. The present experiments were designed to measure the expression of spinal cannabinoid CB2 receptors as well as the analgesic efficacy derived from their stimulation in mice chronically inflamed by the intraplantar injection of complete Freund's adjuvant 1week before. Both spinal cannabinoid CB2 receptors mRNA measured by real-time PCR and cannabinoid CB2 receptor protein levels measured by western blot remained unaltered in inflamed mice. Besides, the intrathecal (i.t.) administration of the cannabinoid CB2 receptor agonists AM1241, (R,S)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole, (0.03-1μg) and JWH 133, (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran, (3–30μg) dose-dependently blocked inflammatory thermal hyperalgesia and mechanical allodynia. The analgesic effects induced by both agonists were counteracted by the coadministration of the selective cannabinoid CB2 receptor antagonist SR144528, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide, (5μg) but not by the cannabinoid CB1 receptor antagonist AM251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, (10μg). The effects induced by AM1241 were also inhibited by the coadministration of the opioid receptor antagonist, naloxone (1μg). These results demonstrate that effective analgesia can be achieved in chronic inflammatory settings through the stimulation of spinal cannabinoid CB2 receptors even if this receptor population is not up-regulated.

Cholecystokinin Exerts an Effect via the Endocannabinoid System to Inhibit GABAergic Transmission in Midbrain Periaqueductal Gray

Cholecystokinin modulates pain and anxiety via its functions within brain regions such as the midbrain periaqueductal gray (PAG). The aim of this study was to examine the cellular actions of cholecystokinin on PAG neurons. Whole-cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of cholecystokinin and its effects on synaptic transmission. Sulfated cholecystokinin-(26-33) (CCK-S, 100-300 nM), but not non-sulfated cholecystokinin-(26-33) (CCK-NS, 100-300 nM) produced an inward current in a sub-population of opioid sensitive and insensitive PAG neurons, which did not reverse over a range of membrane potentials. The CCK-S-induced current was abolished by the CCK1 selective antagonist devazepide (100 nM), but not by the CCK2 selective antagonists CI988 (100 nM, 1 μM) and LY225910 (1 μM). CCK-S, but not CCK-NS produced a reduction in the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) and an increase in the evoked IPSC paired-pulse ratio. By contrast, CCK-S had little effect on the rate and amplitude of TTX-resistant miniature IPSCs under basal conditions and when external K(+) was elevated. The CCK-S-induced inhibition of evoked IPSCs was abolished by the cannabinoid CB1 receptor antagonist AM251 (3 μM), the mGluR5 antagonist MPEP (10 μM) and the 1, 2-diacylglycerol lipase (DAGLα) inhibitor tetrahydrolipstatin (10 μM). In addition, CCK-S produced an increase in the rate of spontaneous non-NMDA-mediated, TTX-dependent excitatory postsynaptic currents (EPSCs). These results suggest that cholecystokinin produces direct neuronal depolarisation via CCK1 receptors and inhibits GABAergic synaptic transmission via action potential-dependent release of glutamate and mGluR5-induced endocannabinoid signaling. Thus, cholecystokinin has cellular actions within the PAG that can both oppose and reinforce opioid and cannabinoid modulation of pain and anxiety within this brain structure.

Functional interaction between morphine and central amygdala cannabinoid CB1 receptors in the acquisition and expression of conditioned place preference

The present study was done to determine whether cannabinoid CB1 receptors of the central amygdala (CeA) are implicated in morphine-induced place preference. Using a 3-day schedule of conditioning, it was found that subcutaneous (s.c.) administration of morphine (2, 4 and 6 mg/kg) caused a significant dose-dependent conditioned place preference (CPP) in male Wistar rats. Intra-CeA microinjection of the cannabinoid CB1 receptor agonist arachidonylcyclopropylamide (ACPA; 0.5, 2.5 and 5 ng/rat) dose-dependently potentiated the morphine (2 mg/kg)-induced CPP. Furthermore, the administration of ACPA (5 ng/rat, intra-CeA) alone induced a significant CPP. It should be considered that the higher dose of ACPA (5 ng/rat, intra-CeA) in combination with morphine decreased locomotor activity on the testing phase. On the other hand, intra-CeA microinjection of the cannabinoid CB1 receptor antagonist AM251 (120 ng/rat) alone induced a significant conditioned place aversion (CPA). Moreover, intra-CeA microinjection of AM251 (90 and 120 ng/rat) inhibited the morphine-induced place preference with a significant interaction. Intra-CeA microinjection of AM251 reversed the effect of ACPA on morphine response. Interestingly, microinjection of ACPA (2.5 and 5 ng/rat) or AM251 (60–120 ng/rat) into the CeA increased or decreased the expression of morphine (6 mg/kg)-induced place preference respectively. These observations provide evidence that cannabinoid CB1 receptors of the CeA are involved in mediating reward and these receptors are also implicated in the acquisition and expression of morphine-induced CPP.

Local pretreatment with the cannabinoid CB1 receptor antagonist AM251 attenuates methamphetamine intra-accumbens self-administration

The endocannabinoid system is a potential target for therapeutic intervention of substance abuse. Cannabinoid CB1 receptor antagonist decreases intravenous methamphetamine self-administration in animal models. This study examined whether the nucleus accumbens (NAcc) is a site of interaction between methamphetamine and the CB1 receptor antagonist AM251. Male Sprague–Dawley rats were trained to lever press and then were surgically implanted with a guide cannula into the right NAcc. Rats were allowed one week to recover and then AM251 (0.1 or 1.0 μg/μL) was reverse dialyzed directly into the NAcc prior to methamphetamine (10 μg/μL) intra-accumbens self-administration. AM251 (1.0 μg/μL) reduced methamphetamine self-administration while AM251 (0.1 μg/μL) had an intermediary effect. The mechanism of self-administration attenuation is not known but could be mediated by AM251 affecting the negative feedback from the NAcc to the ventral tegmental area (VTA). This study provides evidence that the endocannabinoid system is involved with rewarding effects of methamphetamine and suggests a possible therapeutic intervention for methamphetamine abuse.

Fast Feedback Inhibition of the HPA Axis by Glucocorticoids Is Mediated by Endocannabinoid Signaling

Glucocorticoid hormones are secreted in response to stimuli that activate the hypothalamo-pituitary-adrenocortical (HPA) axis and self-regulate through negative feedback. Negative feedback that occurs on a rapid time scale is thought to act through nongenomic mechanisms. In these studies, we investigated fast feedback inhibition of HPA axis stress responses by direct glucocorticoid action at the paraventricular nucleus of the hypothalamus (PVN). Local infusion of dexamethasone or a membrane-impermeant dexamethasone-BSA conjugate into the PVN rapidly inhibits restraint-induced ACTH and corticosterone release in a manner consistent with feedback actions at the cell membrane. The dexamethasone fast feedback response is blocked by the cannabinoid CB1 receptor antagonist AM-251, suggesting that fast feedback requires local release of endocannabinoids. Hypothalamic tissue content of the endocannabinoid 2-arachidonoyl glycerol is elevated by restraint stress, consistent with endocannabinoid action on feedback processes. These data support the hypothesis that glucocorticoid-induced fast feedback inhibition of the HPA axis is mediated by a nongenomic signaling mechanism that involves endocannabinoid signaling at the level of the PVN.