Administration Of AM251 Research Articles

Blockade of Cannabinoid CB1 Receptors in the central amygdala attenuates morphine reward in rats

Event Abstract Back to Event Blockade of Cannabinoid CB1 Receptors in the central amygdala attenuates morphine reward in rats Maryam Sardari1*, Ameneh Rezayof1 and Mohammad Reza-Zarrindast2 1 University of Tehran, School of Biology, College of Sciences, Iran 2 Tehran Univ. of Medical Science, Dept. of Pharmacology, School of Medicine, Iran Our previous studies suggest that the central amygdala (CeA) may play an important role in morphine reward. Considering the anatomy and functions of the CeA, it may influence the mesocorticolimbic dopaminergic system that mediates morphine reward. On the other hand, it has been shown that cannabinoids produce some pharmacological effects similar to morphine, because there are functional interactions between the cannabinoid and opioid systems. In the present study, the effects of bilateral injections of a selective cannabinoids receptor (CB1) antagonist, AM251 into the CeA on morphine-induced conditioned place preference (CPP) were investigated in male wistar rats. In these experiments, the animals weighting 220-250g at the time of surgery were used. The animals were cannulated in the CeA by stereotaxic instrument, and were allowed to recover 1- week before training. All procedures were carried out in accordance with institutional guide lines for animal care and use. Using a 3-day schedule of conditioning, it was found that the conditioning treatments with subcuntaneous (s.c) injections of different doses of morphine (0-6 mg/kg) induce a conditional place preference (CPP) in a dose-dependent manner. Intra-CeA administration of AM251 (0-120 ng/rat) inhibited the morphine-induced conditioned place preference. Furthermore, intra-CeA administration of AM251 alone did not induce a significant place preference or place aversion. These findings suggest that the cannabinoid CB1 Receptors of the central amygdala are involved in mediating morphine reward. Conference: 41st European Brain and Behaviour Society Meeting, Rhodes Island, Greece, 13 Sep - 18 Sep, 2009. Presentation Type: Poster Presentation Topic: Poster presentations Citation: Sardari M, Rezayof A and Reza-Zarrindast M (2009). Blockade of Cannabinoid CB1 Receptors in the central amygdala attenuates morphine reward in rats. Conference Abstract: 41st European Brain and Behaviour Society Meeting. doi: 10.3389/conf.neuro.08.2009.09.292 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Jun 2009; Published Online: 15 Jun 2009. * Correspondence: Maryam Sardari, University of Tehran, School of Biology, College of Sciences, Tehran, Iran, Jandark96@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Maryam Sardari Ameneh Rezayof Mohammad Reza-Zarrindast Google Maryam Sardari Ameneh Rezayof Mohammad Reza-Zarrindast Google Scholar Maryam Sardari Ameneh Rezayof Mohammad Reza-Zarrindast PubMed Maryam Sardari Ameneh Rezayof Mohammad Reza-Zarrindast Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Serotonergic System in the Central Nucleus of Amygdala Mediates Cannabidiol- Induced Sleep Alteration

Cannabidiol (CBD) is one of the psycho-inactive constituents of marijuana, the Cannabis sativa. The pharma- cological property of CBD, especially the anxiolytic effect, is significant in the therapeutic purposes. The central nucleus of amygdala (CeA) plays a key role in the anxiety and its related behavioral responses (e.g. sleep-wake activity), and sero- tonin is one of the major mediators. However, the sleep-wake effect of CBD remains unclear. This study was designed to elucidate the effects of CBD on sleep-wake alteration and the involvement of serotonin in the CeA. Administrations of 5- hydroxytryptamine (5-HT), 5-HT1A receptor partial agonist (buspirone), 5-HT2 antagonist (ritanserin), cannabinoid CB1 receptor agonist (ACEA), or CB1 antagonist (AM-251) were employed to elucidate the action of CBD on CeA presynaptic CB1 receptors, serotonergic activity and the subsequent sleep alteration. We found that microinjection of CBD into the CeA prior to the beginning of the light period dose-dependently decreased slow wave sleep (SWS) with limited effect on rapid eye movement sleep (REMS). CBD-induced SWS suppression during the light period could be mimicked by admin- istering serotonin into the CeA. Buspirone and ritanserin dose-dependently blocked CBD-induced SWS decrease. Fur- thermore, administration of AM-251 exhibits similar effect as that of CBD on sleep-wake activity, and the CBD-induced SWS decrease was partially blocked by ACEA. These observations suggest that CBD acting on the CeA neurons de- creases SWS during the light phase, which is at least partially mediated by the consequence of antagonizing presynaptic CB1 receptors, enhancing serotonin release from the presynaptic terminals and subsequently acting on the postsynaptic 5- HT2 receptors.

Methamphetamine-seeking behavior is due to inhibition of nicotinic cholinergic transmission by activation of cannabinoid CB1 receptors

We previously reported the involvement of cannabinoid CB1 receptors (CB1Rs) and nicotinic acetylcholine receptors (nAChRs) in the reinstatement of methamphetamine (MAP)-seeking behavior (lever-pressing response for MAP reinforcement under saline infusion). The present study examined whether the reinstatement involves interactions between these receptors. Rats were trained to self-administer MAP with a light and tone (MAP-associated cues). Then, extinction sessions under saline infusion without cues were conducted. After that, a reinstatement tests were conducted by either presenting the cues or a MAP-priming injection. Systemic and intracranial administration of HU210, a cannabinoid CB1R agonist, into the nucleus accumbens core (NAC) and prelimbic cortex (PrC) reinstated MAP-seeking behavior. The reinstatement caused by the systemic HU210 treatment was attenuated by intracranial administration of AM251, a cannabinoid CB1R antagonist, into each region mentioned above. Meanwhile, reinstatement induced by the MAP-associated cues and MAP-priming injection was also attenuated by intracranial administration of AM251 in each region. In these regions, the attenuating effects of AM251 on the reinstatement induced by each stimulus were blocked by the intracranial administration of mecamylamine, a non-selective nAChR antagonist, but not by scopolamine, a muscarinic ACh receptor (mAChR) antagonist. Furthermore, the intracranial administration of DHβE, an α4β2 nAChR antagonist, but not MLA, an α7 nAChR antagonist, into each region blocked the AM251-induced attenuation of the reinstatement. These findings suggest that relapses to MAP-seeking behavior may be due to two steps, first inhibition of ACh transmission by the activation of cannabinoid CB1Rs and then the inactivation of α4β2 nAChRs.

Effects of CB1 receptor antagonist within the nucleus accumbens on the acquisition and expression of morphine-induced conditioned place preference in morphine-sensitized rats

It has been shown that cannabinoids interact with the opiate system in reward-related behaviors and in animal models of addiction. In the present study, the effects of bilateral intra-accumbal administration of AM251, a CB1 receptor antagonist, on the acquisition and expression of ineffective dose of morphine-induced conditioned place preference (CPP) in morphine-sensitized rats were investigated. 158 adult male albino Wistar rats were used in these experiments. Subcutaneous (s.c.) administration of morphine (0.25, 0.5, 0.75, 1, 2.5 and 5 mg/kg) induced CPP only at the dose of 5 mg/kg. In addition, repeated administration of morphine (5 mg/kg; s.c.), once daily for 3 days followed by 5 days free of the opioid (sensitization period), increased conditioning response induced by ineffective doses of morphine (0.25, 0.5 and 0.75 mg/kg). Bilateral intra-accumbal administration of AM251 (5, 25 and 125 ng/0.5 μl per side) dose-dependently reduced the acquisition and expression of morphine-induced CPP in morphine-sensitized rats, while bilateral intra-accumbal administration of neither saline nor DMSO (0.5 μl/side) had effects on the acquisition and expression of morphine-induced CPP in sensitized rats. The results indicated that CB1 receptors within the nucleus accumbens are involved in the acquisition and expression of morphine-induced CPP in sensitized rats. Our findings also suggest the existence of cross-talk between cannabinoids and opiates on the sensitization to morphine and the implication of endocannabinoid system in the process of sensitization to opiates.

Local enhancement of cannabinoid CB1 receptor signalling in the dorsal hippocampus elicits an antidepressant-like effect

Systemic administration of direct cannabinoid CB1 receptor agonists and inhibitors of the hydrolytic enzyme fatty acid amide hydrolase have been shown to elicit antidepressant effects. Moreover, the endocannabinoid system in the hippocampus is sensitive to both chronic stress and antidepressant administration, suggesting a potential role of this system in emotional changes associated with these regimens. The aim of this study was to determine if cannabinoid CB1 receptors in the hippocampus modulate emotionality in rats as assessed via the forced swim test. Male Sprague-Dawley rats were bilaterally implanted with cannulae directed at the dentate gyrus of the dorsal hippocampus and subsequently received three infusions of either the cannabinoid CB1 receptor agonist HU-210 (1 and 2.5 microg), the fatty acid amide hydrolase inhibitor URB597 (0.5 and 1 microg), the cannabinoid CB1 receptor antagonist AM251 (1 and 2.5 microg), or vehicle (dimethyl sulfoxide) and were assessed in the forced swim test. Infusion of both doses of HU-210 resulted in a dramatic reduction in immobility and increase in swimming behaviour, indicative of an antidepressant response, which was partially reversed by coadministration of AM251. No effect of URB597 administration or any effect following the administration of AM251 alone was, however, observed. These data indicate that activation of CB1 receptors in the dentate gyrus of the hippocampus results in an antidepressant-like response. Collectively, these data highlight the potential importance of changes in the hippocampal endocannabinoid system following stress or antidepressant treatment with respect to the manifestation and/or treatment of depression.

Endocannabinoid modulation of male rat sexual behavior

Synthetic and plant-derived cannabinoid CB(1) receptor agonists have consistently been shown to impair sexual behavior in male rodents; however, the role of the endocannabinoid system in regulating copulatory processes is largely unknown. The aim of this experiment was to determine the effect of pharmacological facilitation or antagonism of endocannabinoid signaling on male rat sexual behavior. Male Long-Evans rats were administered a single injection of either the cannabinoid CB(1) receptor antagonist AM251 (1, 2, or 5 mg/kg), the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.1, 0.3, or 0.5 mg/kg), or the anandamide uptake inhibitor/FAAH inhibitor AM404 (1, 2, and 5 mg/kg), or their respective vehicles, and examined on parameters of appetitive and consummatory sexual behavior. Inhibition of anandamide metabolism through URB597 had no effect on any parameter of sexual behavior. However, the highest dose of AM404 increased the latency to engage in intromitting behavior, but had no other effect on sexual behavior, suggesting that this effect may be due to the sedative-suppressive effects of this drug. AM251 produced a dose-dependent facilitation of ejaculation, such that the number of intromissions required to achieve ejaculation and the ejaculation latency were reduced by AM251 administration. These data suggest that antagonism of the CB(1) receptor facilitates ejaculatory processes, an effect which may be due to interactions with neuropeptidergic systems in the hypothalamus, and further, suggest a novel target for pharmacological agents aimed at treating ejaculatory-based sexual dysfunction.

Effects of IP-751, Ajulemic Acid, on Bladder Overactivity Induced by Bladder Irritation in Rats

Ajulemic acid (IP-751) is a synthetic analog of tetrahydrocannabinol, which is a major ingredient of the plant Cannabis. IP-751 reportedly shows potent anti-inflammatory activity and is a powerful analgesic agent. Thus, we examined whether IP-751 can suppress urinary frequency induced by nociceptive stimuli in the bladder. Continuous cystometry (infusion rate 0.04 mL/min) under urethane anesthesia was performed to evaluate the effect of intravenous injection of IP-751 with or without a cannabinoid-1 receptor antagonist (AM251) or a cannabinoid-2 receptor antagonist (AM630) on bladder function in normal rats and rats with urinary frequency induced by intravesical infusion with 0.25% acetic acid or cyclophosphamide (CYP) (150 mg/kg intraperitoneally, 48 hours before cystometrography). When 10 mg/kg of IP-751 was injected in normal rats, the intercontraction interval (ICI) and pressure threshold increased. A 0.25% acetic acid infusion induced urinary frequency, as evidenced by a reduction in ICIs, which were suppressed by injection of IP-751 (10 mg/kg). Urinary frequency, indicated by significant ICI reductions, was also observed in the CYP-treated rats. Administration of IP-751 (10 mg/kg) significantly suppressed CYP-induced urinary frequency, as evidenced by the increase in the ICI. When AM251, but not AM630, was administered before IP-751, the IP-751-induced increases in the ICI and pressure threshold were prevented in all three groups. In addition, administration of AM251 alone decreased the ICIs in CYP-treated rats. IP-751 can suppress normal bladder activity and urinary frequency induced by bladder nociceptive stimuli, probably by suppression of bladder afferent activity. These inhibitory effects of IP-751 are at least in part mediated by the cannabinoid-1 receptor.

Cannabinoids Ameliorate Pain and Reduce Disease Pathology in Cerulein-Induced Acute Pancreatitis

The functional involvement of the endocannabinoid system in modulation of pancreatic inflammation, such as acute pancreatitis, has not been studied to date. Moreover, the therapeutic potential of cannabinoids in pancreatitis has not been addressed. We quantified endocannabinoid levels and expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in pancreas biopsies from patients and mice with acute pancreatitis. Functional studies were performed in mice using pharmacological interventions. Histological examination, serological, and molecular analyses (lipase, myeloperoxidase, cytokines, and chemokines) were performed to assess disease pathology and inflammation. Pain resulting from pancreatitis was studied as abdominal hypersensitivity to punctate von Frey stimuli. Behavioral analyses in the open-field, light-dark, and catalepsy tests were performed to judge cannabinoid-induced central side effects. Patients with acute pancreatitis showed an up-regulation of cannabinoid receptors and elevated levels of endocannabinoids in the pancreas. HU210, a synthetic agonist at CB1 and CB2, abolished abdominal pain associated with pancreatitis and also reduced inflammation and decreased tissue pathology in mice without producing central, adverse effects. Antagonists at CB1- and CB2-receptors were effective in reversing HU210-induced antinociception, whereas a combination of CB1- and CB2-antagonists was required to block the anti-inflammatory effects of HU210 in pancreatitis. In humans, acute pancreatitis is associated with up-regulation of ligands as well as receptors of the endocannabinoid system in the pancreas. Furthermore, our results suggest a therapeutic potential for cannabinoids in abolishing pain associated with acute pancreatitis and in partially reducing inflammation and disease pathology in the absence of adverse side effects.

ROLE OF DORSAL HIPPOCAMPAL CANNABINOID RECEPTORS AND NITRIC OXIDE IN ANXIETY LIKE BEHAVIOURS IN RATS USING THE ELEVATED PLUS‐MAZE TEST

1. The important role of the cannabinoid system in the modulation of anxiety like behaviours in clinical and experimental studies has been proposed. However, investigations into this effect of cannabinoids has produced contradictory results. It has been reported that different neurotransmitters, such as nitric oxide (NO), are involved in the behavioural effects of cannabinoids. The hippocampus is also an important brain region in the modulation of anxiety in which CB1 receptors are densely expressed. The present study was designed to evaluate the interactions between cannabinoid and NO systems in the CA1 brain region of the rats using the plus-maze test. 2. Rats were anaesthetized with ketamine and xylazine and special cannulas were inserted stereotaxically into the CA1 region of the dorsal hippocampus. After 1 week recovery, the effects of intra-CA1 administration of WIN55212-2 (1, 2.5 and 5 microg/rat), AM251 (2, 10 and 50 ng/rat), L-arginine (0.01, 0.1 and 1 microg/rat) and N(G)-nitro-L-arginine methyl ester (L-NAME; 1, 10 and 100 ng/rat) on percentage open arm time (%OAT) and percentage open arm entries (%OAE) were determined. Moreover, the effects of pretreatment with AM251 (2 ng/rat), L-arginine (0.01 microg/rat) and L-NAME (1 ng/rat) on the response induced by intra-CA1 administration of WIN55212-2 were also assessed. 3. The administration of either L-arginine or L-NAME into the CA1 region produced significant anxiogenic-like responses, whereas administration of AM251 induced anxiolytic effects. Intra-CA1 injection of WIN55212-2 produced a significant anxiogenic-like effect that was reversed by AM251 and was also altered by L-NAME, but not by L-arginine. 4. These data imply that cannabinoids may have anxiogenic-like effects in the CA1 region of the hippocampus in which CB1 receptors and NO may be involved.

CB1‐independent inhibition of dopamine transporter activity by cannabinoids in mouse dorsal striatum

Cannabinoid drugs are known to affect dopaminergic neurotransmission in the basal ganglia circuitry. In this study, we used in vitro and in vivo techniques to investigate whether cannabinoid agonists and antagonist could affect dopaminergic transmission in the striatum by acting at the dopamine transporter. Incubation of striatal synaptosomes with the cannabinoid agonists WIN55,212-2 or methanandamide decreased dopamine uptake (IC(50) = 2.0 micromol/L and 3.1 micromol/L, respectively). A similar inhibitory effect was observed after application of the inactive WIN55,212-2 isomer, S(-)WIN55,212-3. The CB(1) antagonist AM251 did not reverse WIN55,212-2 effect but rather mimicked it. WIN55,212-2 and AM251 partially displaced the binding of the cocaine analog [(3)H]WIN35,428, thus acting as dopamine transporter pseudo-substrates in the high micromolar range. High-speed chronoamperometry measurements showed that WIN55,212-2 (4 mg/kg, i.p.) caused significant release of endogenous dopamine via activation of CB(1) receptors, followed by a reduction of dopamine clearance. This reduction was CB(1)-independent, as it was mimicked by S(-)WIN55,212-3. Administration of AM251 (1 and 4 mg/kg, i.p.) increased the signal amplitude and reduced the clearance of dopamine pressure ejected into the striatum. These results indicate that both cannabinoid agonists and antagonists inhibit dopamine transporter activity via molecular targets other than CB(1) receptors.

Endocannabinoids modulate stress‐induced suppression of hippocampal cell proliferation and activation of defensive behaviours

The endocannabinoid system has been shown to regulate both the hypothalamic-pituitary-adrenal (HPA) axis and emotionality. The present experiment was designed to examine whether pharmacological modulation of the endocannabinoid system would affect the suppression of hippocampal cell proliferation and increase in defensive behaviours seen following exposure to predator odour (trimethylthiazoline; TMT) stress. Rats were administered either an endocannabinoid uptake inhibitor (AM404; 2 mg/kg) or a cannabinoid CB1 receptor antagonist (AM251; 5 mg/kg) 30 min prior to exposure to TMT. Exposure to TMT reduced cell proliferation in the dentate gyrus and increased the expression of defensive burying. Administration of AM404 significantly inhibited defensive burying, and attenuated the reduction in cell proliferation in response to TMT exposure. Administration of AM251 alone significantly increased cell proliferation; however, pretreatment with AM251 prevented neither the stress-induced suppression of cell proliferation nor the stress-induced increase in behavioural responses. These results support previous research demonstrating that augmentation of endocannabinoid signalling can suppress stress-responsive systems. They also suggest that endocannabinoids may play a complex role in the regulation of neurogenesis via cell proliferation in the hippocampus.

Cannabinoid CB1Receptor Antagonist AM251 Inhibits Cocaine-Primed Relapse in Rats: Role of Glutamate in the Nucleus Accumbens

Blockade of cannabinoid CB1 receptors has been reported to inhibit cocaine- or cocaine cue-induced reinstatement of drug seeking. However, the mechanisms underlying this action are poorly understood. Given the importance of dopamine, glutamate, and GABA in cocaine reward and relapse, we studied the effects of AM251 [N-(piperidin-1-yl)-5-(4-iodophonyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a novel highly selective CB1 receptor antagonist, on cocaine-primed reinstatement of drug-seeking behavior and on cocaine-induced changes in extracellular DA, glutamate, and GABA in the nucleus accumbens (NAc) under reinstatement conditions. We found that systemic administration of AM251 selectively inhibited cocaine-induced, but not sucrose plus sucrose cue-induced, reinstatement of reward-seeking behavior. AM251 alone did not trigger reinstatement. Local perfusion of AM251 into the NAc or the dorsal striatum also inhibited cocaine-triggered reinstatement. AM251 alone dose dependently elevated NAc glutamate in a voltage-dependent Na+ channel-dependent manner. AM251 did not affect NAc DA or GABA. Pretreatment with AM251 dose dependently inhibited cocaine-induced increases in NAc glutamate but not in DA. Blockade of NAc metabotropic glutamate mGluR2/3 receptors by LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] slightly facilitated cocaine-enhanced glutamate release but blocked the antagonism of cocaine-induced reinstatement by AM251. These data suggest the following: (1) CB1 receptors exert tonic inhibition over NAc glutamate release under cocaine-extinction conditions; (2) blockade of CB1 receptors by AM251 inhibits cocaine-enhanced NAc glutamate release and cocaine-triggered reinstatement; and (3) these effects appear to be mediated by activation of presynaptic mGluR2/3 autoreceptors secondary to AM251-induced increase (disinhibition) of NAc glutamate release.

Effects of the CB1 cannabinoid receptor inverse agonist AM251 on food intake and body weight in mice lacking μ-opioid receptors

We used μ-opioid receptor-deficient (MOR−/−) mice to determine the effects of the CB1 cannabinoid receptor inverse agonist AM251 on feeding in the absence of MOR signaling. A single dose of AM251 at 0.6, 2 or 6 mg/kg caused similar dose-dependent suppression of food intake in wild-type and MOR−/− mice. Administration of AM251 at 3 mg/kg/day for 9 consecutive days also led to a similar reduction (∼8%) in body weight in wild-type and MOR−/− mice. Our results suggest that MOR signaling is not necessary for cannabinoid-mediated effects on feeding and that physiological antagonism of opioid receptor tone might be required for the observed synergistic effects of a CB1 inverse agonist and an opioid receptor antagonist on feeding.

Cannabinoid modulation of electrically evoked pH and oxygen transients in the nucleus accumbens of awake rats

Cannabinoid receptors have been implicated in the regulation of blood flow in the cerebral vasculature. Because the nucleus accumbens (NAc) shows high levels of central cannabinoid receptor 1 (CB1) expression we examined the effects of cannabinoids on the local transient alkaline shifts and increases in extracellular oxygen induced by electrical stimulation of the medial forebrain bundle (MFB) in conscious animals. These changes result from increases in cerebral blood flow (CBF) and metabolism in the NAc that are evoked by the stimulation. Oxygen and pH changes were monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in the NAc of freely moving rats. Administration of the cannabinoid receptor agonist WIN55,212-2 potently inhibited extracellular oxygen and pH changes, an effect that was reversed and prevented by pre-treatment with the CB1 receptor antagonists SR141716A and AM251. The effects on pH following WIN55,212-2 were similar to those following nimodipine, a recognized vasodilator. When AM251 was injected alone, the amplitude of electrically evoked pH shifts was unaffected. Administration of AM404 and VDM11, endocannabinoid transport inhibitors, partially inhibited pH transients in a CB1 receptor-dependent manner. The present findings suggest that CB1 receptor activation modulates changes in two well-established indices of local blood flow and metabolism resulting from electrically evoked activation of ascending fibers. Although endogenous cannabinoid tone alone is not sufficient to modify these responses, uptake blockade demonstrates that the system has the potential to exert CB1-specific effects similar to those of full agonists.

Modulation of Neuropathic and Inflammatory Pain by the Endocannabinoid Transport Inhibitor AM404 [N-(4-Hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide

The endocannabinoid system may serve important functions in the central and peripheral regulation of pain. In the present study, we investigated the effects of the endocannabinoid transport inhibitor AM404 [N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide] on rodent models of acute and persistent nociception (intraplantar formalin injection in the mouse), neuropathic pain (sciatic nerve ligation in the rat), and inflammatory pain (complete Freund's adjuvant injection in the rat). In the formalin model, administration of AM404 (1-10 mg/kg i.p.) elicited dose-dependent antinociceptive effects, which were prevented by the CB(1) cannabinoid receptor antagonist rimonabant (SR141716A; 1 mg/kg i.p.) but not by the CB2 antagonist SR144528 (1 mg/kg i.p.) or the vanilloid antagonist capsazepine (30 mg/kg i.p.). Comparable effects were observed with UCM707 [N-(3-furylmethyl)-eicosa-5,8,11,14-tetraenamide], another anandamide transport inhibitor. In both the chronic constriction injury and complete Freund's adjuvant model, daily treatment with AM404 (1-10 mg/kg s.c.) for 14 days produced a dose-dependent reduction in nocifensive responses to thermal and mechanical stimuli, which was prevented by a single administration of rimonabant (1 mg/kg i.p.) and was accompanied by decreased expression of cyclooxygenase-2 and inducible nitric-oxide synthase in the sciatic nerve. The results provide new evidence for a role of the endocannabinoid system in pain modulation and point to anandamide transport as a potential target for analgesic drug development.

Inhibition of the cannabinoid-1 receptor enhances the cerebrocortical hyperemic response to hypoxia/hypercapnia

Activation of cannabinoid-1 (CB1) receptors may evoke diverse vascular effects under physiological conditions: it has been shown to induce vasodilation but also to increase the vascular tone by inhibition of the endothelium-derived hyperpolarizing factor (EDHF). However, the role of CB1 receptors in certain pathophysiological states is obscure. We hypothesized that activation of the CB1 receptors may influence the cerebral hyperemic response to hypoxia/hypercapnia (H/H). Therefore, we investigated the effect of the CB1-antagonist AM-251 on H/H-induced vasodilation in the cerebral cortex. Adult male Wistar rats were anesthetized with intraperitoneal injection of 1.3 g/kg Urethane. Femoral arteries were cannulated for monitoring arterial blood pressure and for measuring arterial blood gas and acid-base parameters. CBF was measured in the parietal cortex simultaneously on both sides by laser-Doppler (LD) flowmetry. H/H was induced by inhalation of different gas mixtures. In the first step, during mild H/H, animals inhaled a gas mixture of 10%O2-10%CO2-80%N2, which produced arterial pO2 of 75-85 mmHg and pCO2 of 50-60 mmHg. In the second step, during moderate H/H, animals inhaled a gas mixture of 5%O2-20%CO2-75%N2, which produced arterial pO2 of 55-65 mmHg and pCO2 of 80-90 mmHg. In the third step, during severe H/H, animals inhaled a gas mixture of 0%O2-20%CO2-80%N2, which produced arterial pO2 of 45-50 mmHg and pCO2 of 90-100 mmHg. In the first experimental group, animals were subjected to these three steps of H/H, then brought back to physiological conditions. After steady state obtained, 10 mg/kg AM-251 was injectedintravenously, followed by repetition of the stepwise H/H. In the second, control group the same protocol was performed, but between the two series of H/H only the vehicle (ethanol and emulphor, dissolved in saline) was administered intravenously. Inhalation of the different gas mixtures induced similar changes of the blood gas tensions and acid-base parameters before and after administration of AM-251 or its vehicle. Application of AM-251 did not alter baseline LD flow or arterial blood pressure. AM-251 has significantly increased, however the H/H-induced hyperemia in each step: by 30% during mild H/H, by 22% during moderate H/H, and by 14% during severe H/H. In contrast, vehicle of AM-251 had no significant effect. H/H did not change significantly the mean arterial pressure of the untreated or vehicle-treated animals, but it has induced slight hypertension (116 mmHg) following AM-251 treatment. The observed enhancement of the cerebrocortical hyperemic response to H/H after blockade of the CB1 receptors indicates that during hypoxia/hypercapnia the activation of CB1 receptors attenuates the vasodilation in the cerebral cortex. A possible explanation of these results is that endothelial CB1 receptors suppress the production of EDHF or other vasodilator mediators during H/H. If this is the case, increased cannabinoid levels during H/H limit the magnitude of reactive hyperemia in the brain tissue. Alternatively, activation of neuronal CB1 receptors may also affect the cerebrocortical blood flow during H/H by influencing the cerebral metabolic rate or the neural regulatory mechanisms of the cerebrovascular tone.

Functional role of the endocannabinoid system and AMPA/kainate receptors in 5-HT 2A receptor-mediated wet dog shakes

These experiments sought to determine the influence of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and the endocannabinoid system in the functional expression of the serotonin (5-HT) type 2A receptor-mediated wet dog shake response. Male Long–Evans rats were pretreated with either 1 mg/kg i.p. of the 5-HT 2A/2C receptor antagonist ketanserin; 1, 10 or 30 mg/kg i.p. of the AMPA/kainate antagonist 6,7-dinitroquinnoxaline-2,3-dione (DNQX); 1, 5 or 10 mg/kg i.p. of the endocannabinoid uptake inhibitor AM404; or 1, 5 or 10 mg/kg i.p. of the cannabinoid CB 1 receptor antagonist AM 251 prior to injection of the 5-HT 2A/2C receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, 1 mg/kg i.p.). Results demonstrated that 10 mg/kg of AM404 significantly reduced the expression of DOI-induced wet dog shakes, but lower doses were ineffective. Administration of AM251 did not induce wet dog shakes behavior when administered alone, but significantly potentiated DOI-induced wet dog shaking behavior at a dose of 10 mg/kg. Pretreatment with DNQX significantly reduced the expression of DOI-induced wet dog shakes at all doses tested. These data suggest that AMPA/kainate receptors play a role in the mediation of 5-HT 2A receptor activity, whereas the endocannabinoid system may act as a regulatory buffer system during periods of elevated activity, but not under basal conditions.

Enhancing Cannabinoid Neurotransmission Augments the Extinction of Conditioned Fear

The endogenous cannabinoid (eCB) system represents a major therapeutic target for the treatment of a variety of anxiety-related disorders. A recent study has demonstrated that pharmacologic or genetic disruption of CB1-receptor-mediated neurotransmission decreases the extinction of conditioned fear in mice. Here, we examined whether CB1 blockade would similarly disrupt extinction in rats, using fear-potentiated startle as a measure of conditioned fear. We also examined whether pharmacologic enhancement of CB1 activation would lead to enhancements in extinction. Our results indicate that systemic administration of the CB1 antagonist rimonabant (SR141716A) prior to extinction training led to significant, dose-dependent decreases in extinction. While the administration of the CB1 agonist WIN 55,212-2 did not appear to affect extinction, administration of AM404, an inhibitor of eCB breakdown and reuptake, led to dose-dependent enhancements in extinction. In addition to showing decreased fear 1 and 24 h after extinction training, AM404-treated animals showed decreased shock-induced reinstatement of fear. Control experiments demonstrated that the effects of AM404 could not be attributed to alterations in the expression of conditioned fear, locomotion, shock reactivity, or baseline startle, as these parameters seemed unchanged by AM404. Furthermore, coadministration of rimonabant with AM404 blocked this enhancement of extinction, suggesting that AM404 was acting to increase CB1 receptor activation during extinction training. These results demonstrate that the eCB system can be modulated to enhance emotional learning, and suggest that eCB modulators may be therapeutically useful as adjuncts for exposure-based psychotherapies such as those used to treat Post-Traumatic Stress Disorder and other anxiety disorders.

Compounds acting at the endocannabinoid and/or endovanilloid systems reduce hyperkinesia in a rat model of Huntington's disease.

We have recently reported that the administration of AM404, an inhibitor of the endocannabinoid re-uptake process, which also has affinity for the vanilloid VR1 receptors, is able to reduce hyperkinesia, and causes recovery from neurochemical deficits, in a rat model of Huntington's disease (HD) generated by bilateral intrastriatal injections of 3-nitropropionic acid (3NP). In the present study, we wanted to explore the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP-lesioned rats by employing several experimental approaches. First, we tried to block the effects of AM404 with selective antagonists for the CB1 or VR1 receptors, i.e. SR141716A and capsazepine, respectively. We found that the reduction caused by AM404 of the increased ambulation exhibited by 3NP-lesioned rats in the open-field test was reversed when the animals had been pre-treated with capsazepine but not with SR141716A, thus suggesting a major role of VR1 receptors in the antihyperkinetic effects of AM404. However, despite the lack of behavioral effects of the CB1 receptor antagonist, the pretreatment with this compound abolished the recovery of neurochemical [gamma-aminobutyric acid (GABA) and dopamine] deficits in the caudate- putamen caused by AM404, as also did capsazepine. In a second group of studies, we wanted to explore the potential antihyperkinetic effects of various compounds which, compared to AM404, exhibit more selectivity for either the endovanilloid or the endocannabinoid systems. First, we tested VDM11 or AM374, two selective inhibitors or the endocannabinoid re-uptake or hydrolysis, respectively. Both compounds were mostly unable to reduce hyperkinesia in 3NP-lesioned rats, although VDM11 produced a certain motor depression, and AM374 exhibited a trend to stimulate ambulation, in control rats. We also tested the effects of selective direct agonists for VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin exhibited a strong antihyperkinetic activity and, moreover, was able to attenuate the reductions in dopamine and GABA transmission provoked by the 3NP lesion, whereas CP55,940 had also antihyperkinetic activity but was unable to cause recovery of either dopamine or GABA deficits in the basal ganglia. In summary, our data indicate a major role for VR1 receptors, as compared to CB1 receptors, in the antihyperkinetic effects and the recovery of neurochemical deficits caused in 3NP-lesioned rats by compounds that activate both CB1 and VR1 receptors, either directly or via manipulation of the levels of endogenous agonists.

Antiobesity effects of chronic cannabinoid CB 1 receptor antagonist treatment in diet-induced obese mice

We determined the effect of a cannabinoid CB1 receptor antagonist (AM-251; N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide) on food intake, body weight and adipose tissue mass in Western diet-induced obese (DIO) mice using a chronic, interrupted, oral dosing paradigm. The dosing paradigm was 2 weeks on treatment (treatment 1), 2 weeks off-treatment, followed by 2 weeks on treatment (treatment 2). During treatment 1 and treatment 2, food intake and body weight were reduced after a single dose. At 30 mg/kg/day, anorectic efficacy was maintained through 12 days (treatment 1) and 7 days (treatment 2). Body weight of AM-251-treated mice remained less than vehicle-treated mice throughout treatment 1 and treatment 2. Administration of AM-251 reduced inguinal subcutaneous, retroperitoneal and mesenteric adipose tissue mass. Antiobesity effects of AM-251 were lost during the off-treatment period, and hyperphagia was observed in treated animals. With re-initiation of AM-251 treatment, mice again responded to the effects of the compound. These results support the hypothesis that chronic treatment of obese individuals with cannabinoid CB1 receptor antagonists is a viable pharmacologic approach to sustained weight loss.