CB1 Antagonist SR141716A Research Articles

CB1-RECEPTOR ANTAGONIST SR-141716A MODULATES LOCOMOTOR ACTIVITY IN OBX-RATS

Dysfunction of the endocannabinoid system has been related to depressive-like behavior. In our study, bilateral olfactory bulbectomy (OBX) was used as an animal model of depression. We evaluated the effect of the selective CB1-antagonist SR-141716A (Rimonabant) on the exploratory and locomotor activity of OBX-rats. Rimonabant was administered intragastrically for 14 days to OBX-rats, divided into 3 experimental groups, where the drug was given before; immediately (1-14 days) after; or 14 days (14-28 day) after OBX. Exploratory and locomotor activity of OBX-rats was tested in an Opto-Varimex apparatus. SR-141716A, administered subchronically, intragastrically exerted locomotor stimulating effects in OBX- and sham-operated rats, while the exploratory activity was not affected. The time interval for the drug administration is of significance for the manifestation of the effects on locomotor activity in OBX-rats. SR-141716A applied 14 days before OBX or after the development of a depressive-like state (14-28 days after OBX), but not immediately after OBX (1-14 days) aggravated the OBX-induced hyperlocomotor state.

The Synthetic Cannabinoid URB447 Reduces Brain Injury and the Associated White Matter Demyelination after Hypoxia-Ischemia in Neonatal Rats.

The number of functions controlled by the endocannabinoid system in health and disease continues growing over the years. In the brain, these include the modulation of harmful events such as glutamate excitotoxicity, oxidative stress, and inflammation, mainly regulated by activation/blockade of CB1/CB2 cannabinoid receptors. In the present work, we evaluated the capacity of the CB1 antagonist/CB2 agonist synthetic cannabinoid URB447 on reducing neurodegeneration after brain injury. By using a model of hypoxia-ischemia (HI) in neonatal rats, we found that URB447 strongly reduced brain injury when administered before HI. A comparable effect was observed with the CB1 antagonist SR141716A, whereas the CB1 agonist WIN-55,212-2 reduced the effect of URB447. When administered 3 h after HI, which is considered a clinically feasible therapeutic window to treat perinatal brain injury in humans, URB447 reduced neurodegeneration and white matter damage. Markers of astrogliosis and microglial activation also appeared reduced. These results confirm the important role played by the endocannabinoid system in the neurodegenerative process and strongly encourage further research into the mechanisms of URB447-induced neuroprotection.

Different temporal windows for CB1 receptor involvement in contextual fear memory destabilisation in the amygdala and hippocampus.

Reconsolidation is a process in which re-exposure to a reminder causes a previously acquired memory to undergo a process of destabilisation followed by subsequent restabilisation. Different molecular mechanisms have been postulated for destabilisation in the amygdala and hippocampus, including CB1 receptor activation, protein degradation and AMPA receptor exchange; however, most of the amygdala studies have used pre-reexposure interventions, while those in the hippocampus have usually performed them after reexposure. To test whether the temporal window for destabilisation is similar across both structures, we trained Lister Hooded rats in a contextual fear conditioning task, and 1 day later performed memory reexposure followed by injection of either the NMDA antagonist MK-801 (0.1 mg/kg) or saline in order to block reconsolidation. In parallel, we also performed local injections of either the CB1 antagonist SR141716A or its vehicle in the hippocampus or in the amygdala, either immediately before or immediately after reactivation. Infusion of SR141716A in the hippocampus prevented the reconsolidation-blocking effect of MK-801 when performed after reexposure, but not before it. In the amygdala, meanwhile, pre-reexposure infusions of SR141716A impaired reconsolidation blockade by MK-801, although the time-dependency of this effect was not as clear as in the hippocampus. Our results suggest the temporal windows for CB1-receptor-mediated memory destabilisation during reconsolidation vary between brain structures. Whether this reflects different time windows for engagement of these structures or different roles played by CB1 receptors in destabilisation across structures remains an open question for future studies.

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals, birds, and amphibians

Purpose: Previous studies have found non-CB1 non-CB2 G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians.Materials and methods: Antagonism of the stimulation of [35S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors.Results: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [35S]GTPγS binding by WIN55212-2 was not blocked by the CB1 antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian.Conclusions: The portion of WIN55212-2-stimulated [35S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB1 receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.

Ghrelin-Induced Orexigenic Effect in Rats Depends on the Metabolic Status and Is Counteracted by Peripheral CB1 Receptor Antagonism

Ghrelin is an endogenous regulator of energy homeostasis synthesized by the stomach to stimulate appetite and positive energy balance. Similarly, the endocannabinoid system is part of our internal machinery controlling food intake and energy expenditure. Both peripheral and central mechanisms regulate CB1-mediated control of food intake and a functional relationship between hypothalamic ghrelin and cannabinoid CB1 receptor has been proposed. First of all, we investigated brain ghrelin actions on food intake in rats with different metabolic status (negative or equilibrate energy balance). Secondly, we tested a sub-anxiogenic ultra-low dose of the CB1 antagonist SR141716A (Rimonabant) and the peripheral-acting CB1 antagonist LH-21 on ghrelin orexigenic actions. We found that: 1) central administration of ghrelin promotes food intake in free feeding animals but not in 24 h food-deprived or chronically food-restricted animals; 2) an ultra-low dose of SR141716A (a subthreshold dose 75 folds lower than the EC50 for induction of anxiety) completely counteracts the orexigenic actions of central ghrelin in free feeding animals; 3) the peripheral-restricted CB1 antagonist LH-21 blocks ghrelin-induced hyperphagia in free feeding animals. Our study highlights the importance of the animaĺs metabolic status for the effectiveness of ghrelin in promoting feeding, and suggests that the peripheral endocannabinoid system may interact with ghrelińs signal in the control of food intake under equilibrate energy balance conditions.

Involvement of a non-CB1/CB2 cannabinoid receptor in the aqueous humor outflow-enhancing effects of abnormal-cannabidiol

The purpose of this study was to investigate the effects of abnormal-cannabidiol (abn-cbd), a non-psychoactive cannabinoid agonist, on aqueous humor outflow via the trabecular meshwork (TM) of porcine eye, and to examine the involvement of a non-CB1/CB2 cannabinoid receptor and the p42/44 mitogen-activated protein kinase (p42/44 MAPK) pathway. The effects of abn-cbd on aqueous humor outflow were measured using a porcine anterior segment perfused organ culture model. The activation of p42/44 MAPK by abn-cbd was determined in cultured TM cells with western blot analysis using an anti-phospho-p42/44 MAPK antibody. Administration of abn-cbd caused a concentration-dependent enhancement of aqueous humor outflow facility with a maximum effect (155.0 ± 11.7% of basal outflow facility) after administration of 30 nM abn-cbd. Pretreatment with 1 μM of O-1918, a cannabidiol analog that acts as a selective antagonist at the non-CB1/CB2 receptor, produced a full antagonism of 30 nM abn-cbd induced increase of aqueous humor outflow facility. Pretreatment with 1 μM of CB1 antagonist SR141716A partially blocked, whereas pretreatment with either 1 μM of CB1 antagonist AM251 or 1 μM of CB2 antagonist SR144528 had no effect on abn-cbd induced enhancement of outflow facility. Treatment of TM cells with 30 nM of abn-cbd activated p42/44 MAPK, which was blocked completely by pretreatment with O-1918, and partially by pretreatment with SR141716A, but not by either AM251 or SR144528. In addition, PD98059, an inhibitor of p42/44 MAPK pathway, blocked completely the abn-cbd induced p42/44 MAPK activation and blocked partially the abn-cbd induced enhancement of outflow facility. In conclusion, the results from this study demonstrate that abn-cbd increases aqueous humor outflow through the TM pathway of the eye, and this effect is mediated by a non-CB1/CB2 cannabinoid receptor, with an involvement of p42/44 MAPK signaling pathway.

Effects of Cannabinoid Agonists on Sheep Sphincter of Oddi in vitro

Background/Aims: According to recent studies, the endocannabinoid system plays an important role in both physiological and pathophysiological situations. The purpose of the present studywas to investigate the effects of cannabinoid (CB) agonists on isolated sheep sphincter of Oddi (SO) in vitro.Methods: The isolated sheep SO tissues were mounted in organ baths and tested for isometric tension and cyclic GMP levels (cGMP) in response to the non-selective CB receptor agonistWIN55,212-2 and the potent C131 receptor agonist methanandamide in the presence and absence of the selective CB1 antagonist SR 141716A, the selective CB2 antagonist SR 144528 and the nonspecific inhibitor of nitric oxide (NO) synthase I-NAME. Results: CB agonists relaxed SO in a concentration-dependent manner. These relaxations did not reduce in the presence of SR 144528 but were significantly reduced by SR 141716Aand I-NAME. Carbachol significantly increased the cGMP levels compared with the control group and both of the CB receptor agonists significantly increased the cGMP levels compared with the control and carbachol groups.On the other hand, I-NAME prevented the increase in cGMP levels caused by CB agonists. Conclusion: These results show that the relaxation by the agonists maybe through CB1 receptors.The decrease of CB relaxation responses by I-NAME, a nonspecific inhibitor of NO synthase, and the increase of cGMP levels in the SO tissues by CB agonists which decreased by I-NAME show that the relaxation effects of these agonists may also partially bevia increasing the NO synthesis or release.

Effects of morphine on pain-elicited and pain-suppressed behavior in CB1 knockout and wildtype mice

Pharmacological manipulations of the type 1 cannabinoid receptor (CB1) suggest a role for CB1 in morphine-induced antinociception, but studies utilizing CB1 knockout (KO) mice do not support this conclusion. Since studies using CB1 KO mice to study morphine's antinociceptive effects have only examined thermal nociception, this study examines these interactions in models that employ a chemical stimulus. To determine whether the findings obtained with thermal pain models extend to other models, the effects of morphine on acetic acid-induced writhing were examined in CB1 KO and wildtype (WT) mice. Behaviors that decrease in response to acid injection, feeding and wheel running, were also examined, and investigations were carried out in the thermal hotplate assay. The CB1 antagonist SR141716A was also examined in these assays. Morphine completely blocked acid-induced writhing (1.0-10.0 mg/kg) and increased response latencies in the hotplate (10.0-32.0 mg/kg) in both genotypes. Morphine (3.2 mg/kg) significantly attenuated the suppression of wheel running but did not completely prevent this effect in either genotype. Morphine did not alter pain-suppressed feeding. In each of these assays, morphine's effects were not altered in CB1 KO mice compared with WT mice; however, SR141716A attenuated morphine's effects in C57BL/6 mice. The effects of morphine do not differ in CB1 KO and WT mice in preclinical pain models using thermal and chemical stimuli. Since SR141716A did attenuate the effects of morphine, it is possible that CB1 KO mice undergo developmental changes that mask the role of CB1 receptors in morphine's antinociceptive effects.

Effects of cannabinoid CB1 receptor agonism and antagonism on SKF81297-induced dyskinesia and haloperidol-induced dystonia in Cebus apella monkeys

Antipsychotic drugs may cause extrapyramidal symptoms (EPS), such as dyskinesia and dystonia. These effects are believed to involve dysfunctional striatal dopamine transmission. Patients with schizophrenia show increased prevalence of cannabis abuse and this has been linked to severity of EPS. Endocannabinoids modulate striatal dopamine activity via type 1 cannabinoid (CB1) receptors, and studies in rats and humans suggest beneficial effects of CB1 ligands on EPS. The present study explored the effects of CB1 receptor ligands on oral dyskinesia induced by the dopamine D1 receptor agonist SKF81297 (SKF) and acute dystonia induced by the dopamine D2 receptor antagonist haloperidol in Cebus apella monkeys. The monkeys were sensitised to EPS by prior exposure to D2 receptor antagonists.SKF (0.3 mg/kg) was administered alone and in combination with the CB1 agonist CP55,940 (0.0025–0.01 mg/kg) or the CB1 antagonist SR141716A (0.25–0.75 mg/kg). Haloperidol (individual doses at 0.01–0.02 mg/kg) was administered alone and in combination with CP55,940 (0.005 or 0.01 mg/kg) or SR141716A (0.5 or 0.75 mg/kg). Subsequently, the monkeys were videotaped, and the recordings were rated for oral dyskinesia or dystonia.SKF-induced oral dyskinesia was dose-dependently reduced by CP55,940, with no effect of SR141716A. Haloperidol-induced dystonia was not affected by either CP55,940 or SR141716A.

PRECLINICAL STUDY: FULL ARTICLE: Altered architecture and functional consequences of the mesolimbic dopamine system in cannabis dependence

Cannabinoid withdrawal produces a hypofunction of mesencephalic dopamine neurons that impinge upon medium spiny neurons (MSN) of the forebrain. After chronic treatment with two structurally different cannabinoid agonists, Delta(9)-tetrahydrocannabinol and CP55 940 (CP) rats were withdrawn spontaneously and pharmacologically with the CB1 antagonist SR141716A (SR). In these two conditions, evaluation of tyrosine hydroxylase (TH)-positive neurons revealed significant morphometrical reductions in the ventrotegmental area but not substantia nigra pars compacta of withdrawn rats. Similarly, confocal analysis of Golgi-Cox-stained sections of the nucleus accumbens revealed a decrease in the shell, but not the core, of the spines' density of withdrawn rats. Administration of the CB1 antagonist SR to control rats, provoked structural abnormalities reminiscent of those observed in withdrawal conditions and support the regulatory role of cannabinoids in neurogenesis, axonal growth and synaptogenesis by acting as eu-proliferative signals through the CB1 receptors. Further, these measures were incorporated into a realistic computational model that predicts a strong reduction in the excitability of morphologically altered MSN, yielding a significant reduction in action potential output. These pieces of evidence support the tenet that withdrawal from addictive compounds alters functioning of the mesolimbic system and provide direct morphological evidence for functional abnormalities associated with cannabinoid dependence at the level of dopaminergic neurons and their postsynaptic counterpart and are coherent with recent hypothesis underscoring a hypodopaminergic state as a distinctive feature of the 'addicted brain'.

Statistical Parametric Mapping reveals ligand and region-specific activation of G-proteins by CB1 receptors and non-CB1 sites in the 3D reconstructed mouse brain

CB1 receptors mediate the CNS effects of Δ9-tetrahydrocannabinol and synthetic cannabinoids. Previous studies have investigated cannabinoid-mediated G-protein activity in a subset of brain regions thought to mediate the behavioral effects of cannabinoids, but a detailed regional comparison of the effects of multiple ligands has not been conducted. This study used a novel approach, Statistical Parametric Mapping (SPM), to analyze 3D reconstructed brain images derived from agonist-stimulated [35S]GTPγS autoradiography in a whole-brain unbiased manner. SPM analysis demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP) and WIN55,212-2 (WIN) in CB1+/+ mouse brains. To assess the potential contribution of novel cannabinoid binding sites, experiments were performed in CB1−/− mouse brains. SPM analysis revealed that the aminoalkylindole WIN, but not the bicyclic cannabinoid CP or the endocannabinoid analogue M-AEA, stimulated [35S]GTPγS binding in cortex, hippocampus, hypothalamus, amygdala, cerebellum and certain brainstem areas (dorsal tegmental complex and locus coeruleus). No differences between WIN-stimulated G-protein activity and basal activity were found in basal ganglia. Pharmacological experiments using the CB1 antagonist SR141716A in CB1+/+ mice showed that SR141716A blocked WIN-stimulated G-protein activity in all brain regions, suggesting that it binds to both CB1 and putative non-CB1 sites. These studies show ligand and region-specific cannabinoid-mediated G-protein activity at both CB1 and non-CB1 sites and demonstrate that SPM is a powerful approach for the analysis of reconstructed brain imaging data derived from agonist-stimulated [35S]GTPγS autoradiography.

Age-dependent effects of the cannabinoid CB1 antagonist SR141716A on food intake, body weight change, and pruritus in rats

The cannabinoid CB1 selective antagonist SR141716A (Rimonabant) has been shown to decrease body weight in laboratory animals and humans. Furthermore, SR141716A can elicit scratching behavior in rodents, a behavior that has been hypothesized to contribute to SR141716A-induced decrease in food intake. Although childhood obesity is a rising health issue, it is unknown whether SR141716A is equipotent at modulating food intake and other CB1-mediated behaviors in younger subjects. To determine whether CB1 receptor blockade is equipotent at modulating food and water intake, body weight, and scratching behavior, the effect of a range of SR141716A doses on these behaviors in food-restricted postnatal day (P) 18, 28, and 60 male rats was investigated. Brain concentrations of SR141716A were determined in each age group. SR141716A dose- and age-dependently suppressed food and water intake and body weight gain and elicited head scratching, with the most potent effects observed in P18 and P28 rats. Brain concentrations of SR141716A were significantly elevated in P18 rats relative to P28 and P60 rats. SR141716A-elicited head scratching was attenuated by the 5-HT(2A/2C) antagonist ketanserin. SR141716A is more potent at modulating food intake and head scratching in very young animals; these differences can be attributed to an increase in brain penetration of SR141716A for P18 but not for P28 and P60 rats. In addition, SR141716-elicited head scratching is modulated by 5HT receptor antagonism and is not a contributing factor to SR141716A's anorectic effects.

Effects of Chronic Exposure to the CB1 Agonist AM 411 in Squirrel Monkeys

Cannabinoid CB1 agonists produce tolerance that may be associated with dependence and increased sensitivity to the behavioral effects of the CB1 antagonist SR141716A, perhaps indicative of antagonist‐precipitated withdrawal. In the present studies, the effects of the long‐acting CB1 agonist AM 411 and the CB1 antagonist SR141716A on operant responding were examined in squirrel monkeys before and during chronic AM 411 treatment (1.0 mg/kg/day, im). Prechronically, AM 411 (0.01‐1 mg/kg) produced dose‐related decreases in responding; the highest dose decreased behavior to approximately 20% of control values. In contrast, SR 141716A (1‐10 mg/kg) had little effect on behavior; the highest dose decreased responding only to >80% of control values. Re‐determination of drug effects during the chronic regimen revealed a >10‐fold rightward shift in the dose‐response function for AM 411, indicative of tolerance. On the other hand, SR 141716A (0.32‐3.2 mg/kg) produced dose‐related decreases in behavior; the highest dose decreased responding to <50% of control values. These data are consistent with the view that chronic CB1 receptor activation produces tolerance to the effects of CB1 agonists and enhances sensitivity to the behaviorally disruptive effects of CB1 antagonists. The increased sensitivity to the effects of SR 141716A may be indicative of antagonist‐precipitated withdrawal (supported by DA19205).

Modulation of cannabinoid receptor activation as a neuroprotective strategy for EAE and stroke.

Recognition of the importance of the endocannabinoid system in both homeostasis and pathologic responses raised interest recently in the development of therapeutic agents based on this system. The CB(2) receptor, a component of the endocannabinoid system, has significant influence on immune function and inflammatory responses. Inflammatory responses are major contributors to central nervous system (CNS) injury in a variety of diseases. In this report, we present evidence that activation of CB(2) receptors, by selective CB(2) agonists, reduces inflammatory responses that contribute to CNS injury. The studies demonstrate neuroprotective effects in experimental autoimmune encephalomyelitis, a model of multiple sclerosis, and in a murine model of cerebral ischemia/reperfusion injury. In both cases, CB(2) receptor activation results in reduced white cell rolling and adhesion to cerebral microvessels, a reduction in immune cell invasion, and improved neurologic function after insult. In addition, administration of the CB(1) antagonist SR141716A reduces infarct size following ischemia/reperfusion injury. Administration of both a selective CB(2) agonist and a CB(1) antagonist has the unique property of increasing blood flow to the brain during the occlusion period, suggesting an effect on collateral blood flow. In summary, selective CB(2) receptor agonists and CB(1) receptor antagonists have significant potential for neuroprotection in animal models of two devastating diseases that currently lack effective treatment options.

Cannabinoid-1 (CB1) receptors regulate colonic propulsion by acting at motor neurons within the ascending motor pathways in mouse colon

Cannabinoid-1 (CB(1)) receptors on myenteric neurons are involved in the regulation of intestinal motility. Our aim was to investigate CB(1) receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Presence of the CB(1) receptor was investigated by RT-PCR, and immunohistochemistry was used for colabeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances, and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in an unpartitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and CB(1)(-/-) mice. Immunostaining with the cytoskeletal marker peripherin showed CB(1) immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed CB(1) on choline acetyltransferase-, calretinin-, and 5-HT-immunopositive myenteric neurons, but nitrergic neurons appeared immunonegative for CB(1) immunostaining. Solitary spindle-shaped CB(1)-immunoreactive cells in between smooth muscle cells lacked specific markers for interstitial cells of Cajal or glial cells. ES elicited neuronally mediated excitatory JP (EJP) and inhibitory JP. Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode 6 and a maximal EJP projection distance of approximately 18 mm. The CB(1) receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber, WIN 55,212-2 reduced EJP at the separated oral sites, proving that CB(1) activation inhibits interneuron-mediated neurotransmission. These effects were absent in the presence of the CB(1) antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in SR141716A-pretreated wild-type or CB(1)(-/-) mice. Activation of the CB(1) receptor modulates excitatory cholinergic neurotransmission in mouse colon by reducing amplitude and spatial spreading of the ascending electrophysiological impulses. This effect on electrophysiological spreading involves CB(1)-mediated effects on motor neurons and ascending interneurons and is likely to underlie the here reported in vivo reduction in colonic propulsion.

Ethanol Self‐Administration Is Regulated by CB1 Receptors in the Nucleus Accumbens and Ventral Tegmental Area in Alcohol‐Preferring AA Rats

Endogenous cannabinoids and their receptors, CB1 receptors in particular, have been implicated in mediation of ethanol reinforcement. Previously, suppression of ethanol drinking by CB1 antagonists has been demonstrated in many experimental paradigms. However, the exact mechanism by which CB1 antagonists modulate ethanol drinking remains elusive. In the present study, we assessed the role of CB1 receptors within the key regions of the mesolimbic dopamine pathway, the nucleus accumbens (NAcc) and ventral tegmental area (VTA), in regulation of ethanol self-administration. Adult male alcohol-prefer AA rats were trained to self-administer either 10% (w/v) ethanol or 0.1% (w/v) saccharin under an FR1 schedule during daily 30-minute sessions. Following stable baseline responding, rats were tested after systemic administration of the CB1 antagonist SR141716A (0 to 10 mg/kg) and the agonist WIN55,212-2 (0 to 2 mg/kg). Separate groups of rats were implanted with bilateral cannulas aimed at the NAcc or VTA, and tested after microinjections of SR141716A (0 to 3 microg) and WIN55,212-2 (0 to 5 microg) into the NAcc or VTA. The highest intracerebral doses were tested also in rats responding for a 0.1% saccharin solution. SR141617A dose-dependently suppressed ethanol responding after systemic administration. Microinjections of SR141617A both into NAcc and VTA attenuated ethanol responding. In addition, intra-NAcc injections of SR141617A suppressed saccharin intake. Although low doses of systemically given WIN55,212-2 increased ethanol responding, no effects were seen after WIN55,212-2 microinjections into NAcc or VTA. Bidirectional changes in ethanol self-administration by the systematically administered CB1 agonist and antagonist show that ethanol reinforcement is controlled by CB1 receptors in alcohol-preferring AA rats. Replication of the suppressive effects by CB1 antagonism in the NAcc and VTA suggests that endocannabinoids and their receptors mediate ethanol reinforcement through interaction with the mesolimbic dopamine pathway.

Role of cannabinoid receptors in regulation of cardiac tolerance to ischemia and reperfusion

Coronary artery occlusion (45 min) and reperfusion (2 h) were modeled in vivo in anesthetized artificially ventilated Wistar rats. Total ischemia (45 min) and reperfusion (30 min) of the isolated rat heart were performed in vitro. The selective agonist of cannabinoid (CB) receptors HU-210 was injected intravenously at a dose of 0.1 mg/kg 15 min prior to the coronary artery ligation. The selective CB1 antagonist SR141716A and the selective CB2 antagonist SR144528 were injected intravenously 25 min prior to ischemia. In vitro, HU-210 and SR141716A were added to the perfusion solution at the final concentrations of 0.1 microM prior to total ischemia. Preliminary injection of HU-210 reduced the infarct size-to-area at risk (IS/AAR) ratio in vivo. This cardioprotective effect was completely abolished by SR141716A but remained after SR144528 injection. Both antagonists had no effect on the IS/AAR ratio. Preliminary injection of the K(ATP) channel blocker glibenclamide did not abolish the cardioprotective effect of HU-210. The addition of HU-210 prior to ischemia reduced the creatine phosphokinase (CPK) level in the coronary effluent and decreased left ventricular developed pressure. SR141716A alone had no effect on cardiac contractility and CPK levels. These results suggest that cardiac CB1 receptor activation increases cardiac tolerance to ischemia-reperfusion and has a negative effect on the cardiac pump function. Endogenous cannabinoids are not involved in the regulation of cardiac contractility and tolerance to ischemia and reperfusion. ATP-sensitive E+ channels are not involved in the mechanism of the cardioprotective effect of HU-210.

Endocannabinoid- and mGluR5-Dependent Short-Term Synaptic Depression in an Isolated Neuron/Bouton Preparation From the Hippocampal CA1 Region

Endocannabinoids released from the postsynaptic neuronal membrane can activate presynaptic CB1 receptors and inhibit neurotransmitter release. In hippocampal slices, depolarization of the CA1 pyramidal neurons elicits an endocannabinoid-mediated inhibition of gamma-aminobutyric acid release known as depolarization-induced suppression of inhibition (DSI). Using the highly reduced neuron/synaptic bouton preparation from the CA1 region of hippocampus, we have begun to examine endocannabinoid-dependent short-term depression (STD) of inhibitory synaptic transmission under well-controlled physiological and pharmacological conditions in an environment free of other cells. Application of the CB1 synthetic agonist WIN55212-2 and endogenous cannabinoids 2-AG and anandamide produced a decrease in spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude, indicating the presence of CB1 receptors at synapses in this preparation. Endocannabinoid-dependent STD is different from DSI found in hippocampal slices and the neuron/bouton preparation from basolateral amygdala (BLA) since depolarization alone was not sufficient to induce suppression of sIPSCs. However, concurrent application of the metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) and postsynaptic depolarization resulted in a transient (30-50 s) decrease in sIPSC frequency and amplitude. Application of DHPG alone had no effect on sIPSCs. The depolarization/DHPG-induced STD was blocked by the CB1 antagonist SR141716A and the mGluR5 antagonist MPEP and was sensitive to intracellular calcium concentration. Comparing the present findings with earlier work in hippocampal slices and BLA, it appears that endocannabinoid release is less robust in isolated hippocampal neurons.

Activation of CB1 specifically located on GABAergic interneurons inhibits LTD in the lateral amygdala

Previously, we found that in the lateral amygdala (LA) of the mouse, WIN55,212-2 decreases both glutamatergic and GABAergic synaptic transmission via activation of the cannabinoid receptor type 1 (CB1), yet produces an overall reduction of neuronal excitability. This suggests that the effects on excitatory transmission override those on inhibitory transmission. Here we show that CB1 activation by WIN55,212-2 and Delta(9)-THC inhibits long-term depression (LTD) of basal synaptic transmission in the LA, induced by low-frequency stimulation (LFS; 900 pulses/1 Hz). The CB1 agonist WIN55,212-2 blocked LTD via G(i/o) proteins, activation of inwardly rectifying K+ channels (K(ir)s), inhibition of the adenylate cyclase-protein kinase A (PKA) pathway, and PKA-dependent inhibition of voltage-gated N-type Ca2+ channels (N-type VGCCs). Interestingly, WIN55,212-2 effects on LTD were abolished in CB1 knock-out mice (CB1-KO), and in conditional mutants lacking CB1 expression only in GABAergic interneurons, but were still present in mutants lacking CB1 in principal forebrain neurons. LTD induction per se was unaffected by the CB1 antagonist SR141716A and was normally expressed in CB1-KO as well as in both conditional CB1 mutants. Our data demonstrate that activation of CB1 specifically located on GABAergic interneurons inhibits LTD in the LA. These findings suggest that CB1 expressed on either glutamatergic or GABAergic neurons play a differential role in the control of synaptic transmission and plasticity.

Novel inhibitors of fatty acid amide hydrolase

A class of bisarylimidazole derivatives are identified as potent inhibitors of the enzyme fatty acid amide hydrolase (FAAH). Compound 17 (IC 50 = 2 nM) dose-dependently (0.1–10 mg/kg, iv) potentiates the effects of exogenous anandamide (1 mg/kg, iv) in a rat thermal escape test (Hargreaves test), and shows robust antinociceptive activity in animal models of persistent (formalin test) and neuropathic (Chung model) pain. Compound 17 (20 mg/kg, iv) demonstrates activity in the formalin test that is comparable to morphine (3 mg/kg, iv), and is dose-dependently inhibited by the CB1 antagonist SR141716A. In the Chung model, compound 17 shows antineuropathic effects similar to high-dose (100 mg/kg) gabapentin. FAAH inhibition shows potential utility for the clinical treatment of persistent and neuropathic pain.