Receptor Antagonist AM251 Research Articles

Cannabidiol-induced panicolytic-like effects and fear-induced antinociception impairment: the role of the CB1 receptor in the ventromedial hypothalamus.

The behavioural effects elicited by chemical constituents of Cannabis sativa, such as cannabidiol (CBD), on the ventromedial hypothalamus (VMH) are not well understood. There is evidence that VMH neurons play a relevant role in the modulation of unconditioned fear-related defensive behavioural reactions displayed by laboratory animals. This study was designed to explore the specific pattern of distribution of the CB1 receptors in the VMH and to investigate the role played by this cannabinoid receptor in the effect of CBD on the control of defensive behaviours and unconditioned fear-induced antinociception. A panic attack-like state was triggered in Wistar rats by intra-VMH microinjections of N-methyl-D-aspartate (NMDA). One of three different doses of CBD was microinjected into the VMH prior to local administration of NMDA. In addition, the most effective dose of CBD was used after pre-treatment with the CB1 receptor selective antagonist AM251, followed by NMDA microinjections in the VMH. The morphological procedures demonstrated distribution of labelled CB1 receptors on neuronal perikarya situated in dorsomedial, central and ventrolateral divisions of the VMH. The neuropharmacological approaches showed that both panic attack-like behaviours and unconditioned fear-induced antinociception decreased after intra-hypothalamic microinjections of CBD at the highest dose (100nmol). These effects, however, were blocked by the administration of the CB1 receptor antagonist AM251 (100pmol) in the VMH. These findings suggest that CBD causes panicolytic-like effects and reduces unconditioned fear-induced antinociception when administered in the VMH, and these effects are mediated by the CB1 receptor-endocannabinoid signalling mechanism in VMH.

Cannabinoid receptor type 1 in the bed nucleus of the stria terminalis modulates cardiovascular responses to stress via local N-methyl-D-aspartate receptor/neuronal nitric oxide synthase/soluble guanylate cyclase/protein kinase G signaling.

Endocannabinoid neurotransmission in the bed nucleus of the stria terminalis is involved in the control of cardiovascular responses to stress. However, the local mechanisms involved is this regulation are not known. The purpose of this study was to assess an interaction of bed nucleus of the stria terminalis endocannabinoid neurotransmission with local nitrergic signaling, as well as to investigate the involvement of local N-methyl-D-aspartate glutamate receptor and nitric oxide signaling in the control of cardiovascular responses to acute restraint stress by bed nucleus of the stria terminalis endocannabinoid neurotransmission in rats. The first protocol evaluated the effect of intra-bed nucleus of the stria terminalis microinjection of the selective cannabinoid receptor type 1 receptor antagonist AM251 in nitrite/nitrate content in the bed nucleus of the stria terminalis following restraint stress. The other protocols evaluated the impact of local pretreatment with the selective N-methyl-D-aspartate glutamate receptor antagonist LY235959, the selective neuronal nitric oxide synthase inhibitor Nω-propyl-L-arginine, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or the protein kinase G inhibitor KT5823 in restraint-evoked cardiovascular changes following bed nucleus of the stria terminalis treatment with AM251. Bilateral microinjection of AM251 into the bed nucleus of the stria terminalis increased local nitric oxide release during restraint stress. Bed nucleus of the stria terminalis treatment with the cannabinoid receptor type 1 receptor antagonist also enhanced the tachycardia caused by restraint stress, but without affecting arterial pressure increase and sympathetic-mediated cutaneous vasoconstriction. The facilitation of restraint-evoked tachycardia following bed nucleus of the stria terminalis treatment with the cannabinoid receptor type 1 receptor antagonist was completely inhibited by local pretreatment with LY235959, Nω-propyl-L-arginine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or KT5823. Our results provide evidence that bed nucleus of the stria terminalis endocannabinoid neurotransmission inhibits local N-methyl-D-aspartate/neuronal nitric oxide synthase/soluble guanylate cyclase/protein kinase G signaling, and this mechanism is involved in the control of the cardiovascular responses to stress.

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.

Cannabichromene is a cannabinoid CB2 receptor agonist.

Cannabichromene (CBC) is one of the most abundant phytocannabinoids in Cannabis spp. It has modest antinociceptive and anti-inflammatory effects and potentiates some effects of Δ9 -tetrahydrocannabinol in vivo. How CBC exerts these effects is poorly defined and there is little information about its efficacy at cannabinoid receptors. We sought to determine the functional activity of CBC at cannabinoid CB1 and CB2 receptors. AtT20 cells stably expressing haemagglutinin-tagged human CB1 and CB2 receptors were used. Assays of cellular membrane potential and loss of cell surface receptors were performed. CBC activated CB2 but not CB1 receptors to produce hyperpolarization of AtT20 cells. This activation was inhibited by a CB2 receptor antagonist AM630, and sensitive to Pertussis toxin. Application of CBC reduced activation of CB2 , but not CB1 , receptors by subsequent co-application of CP55,940, an efficacious CB1 and CB2 receptor agonist. Continuous CBC application induced loss of cell surface CB2 receptors and desensitization of the CB2 receptor-induced hyperpolarization. CBC is a selective CB2 receptor agonist displaying higher efficacy than tetrahydrocannabinol in hyperpolarizing AtT20 cells. CBC can also recruit CB2 receptor regulatory mechanisms. CBC may contribute to the potential therapeutic effectiveness of some cannabis preparations, potentially through CB2 receptor-mediated modulation of inflammation.

Effects of cannabinoids on the development of chick embryos in ovo

We have examined the effects of the synthetic cannabinoids HU 210 and HU 211, the plant-derived cannabidiol and the endogenous cannabinoid anandamide on the viability and development of chick embryos. Fertilized White Leghorn chicken eggs were injected with the test compounds or carrier vehicle, via a drilled small hole in the egg, directly into the egg yolk. After nine days of exposure, the embryonal viability, length and wet weight of embryos, and wet weight of brains were measured, and the development stages were assessed according to the Hamburger and Hamilton (HH) scale. The potent synthetic cannabinoid receptor agonist HU 210 and the non-psychotropic cannabidiol were embryotoxic at the highest concentrations examined (10 µM and 50 µM, respectively), with no viable embryos after the HU 210 injection, and 20% viability after the cannabidiol injections. The effects of HU 210 on the chick embryo were attenuated by α-tocopherol and the cannabinoid receptor antagonist AM251, whereas only α-tocopherol gave a statistically significant protection against the embryotoxic effects of cannabidiol. This study shows that exposure to plant-derived or synthetic cannabinoids during early embryonal development decreases embryonal viability. Extrapolation of data across species is of course difficult, but the data would argue against the use of cannabinoids, be it recreationally or therapeutically, during pregnancy.

N-Linoleyltyrosine Protects against Transient Cerebral Ischemia in Gerbil via CB2 Receptor Involvement in PI3K/Akt Signaling Pathway.

Anandamide (AEA) played potent neuroprotective activities via cannabinoid type 1 (CB1) and 2 (CB2) receptor. N-Linoleyltyrosine (NITyr), as an AEA analogue, was synthesized in our laboratory and evaluated the neuroprotective effects and mechanisms for the first time. NITyr was synthesized via substitution reaction. The neuroprotective effects of NITyr were evaluated in a gerbil model of transient cerebral ischemia. Each gerbil was subjected to open field test (OFT), Rotard rod test (RRT), Morris water maze (MWM) successively and executed after animal behaviors. Part of the brain was stained with hematoxylin and eosin (HE) and Nissl staining, and the rest for biochemical analysis. NITyr could not increase spontaneous locomotor activity and ameliorate the anxiety behavior in the OFT but could improve the motor coordination in the RRT and the spatial memory impairment in the MWM. Immunohistochemically, NITyr could attenuate the ischemia-induced neural loss in the hippocampus. The Enzyme-linked immunosorbent assay (ELISA) suggested that NITyr ameliorated the inflammation and oxidative stress. Consistently, NITyr could up-regulate the expressions of p-phosphadylinositol 3-kinase (PI3K) and p-Akt but not PI3K and Akt in the hippocampus. In addition to oxidative stress, CB2 receptor antagonist AM630 but not CB1 receptor antagonist AM251 could reverse the above phenomena. However, CB1 receptor antagonist AM251 could reverse oxidative stress. Accordingly, NITyr could up-regulate the expressions of CB2 but not CB1. NITyr could improve the motor coordination, learning and memory impairments, neural loss in the hippocampus and the inflammation of the mice via CB2 receptor involvement of PI3K/Akt signaling pathway.

Intermedin in Paraventricular Nucleus Attenuates Ang II-Induced Sympathoexcitation through the Inhibition of NADPH Oxidase-Dependent ROS Generation in Obese Rats with Hypertension.

Increased reactive oxygen species (ROS) induced by angiotensin II (Ang II) in the paraventricular nucleus (PVN) play a critical role in sympathetic overdrive in hypertension (OH). Intermedin (IMD), a bioactive peptide, has extensive clinically prospects in preventing and treating cardiovascular diseases. The study was designed to test the hypothesis that IMD in the PVN can inhibit the generation of ROS caused by Ang II for attenuating sympathetic nerve activity (SNA) and blood pressure (BP) in rats with obesity-related hypertension (OH). Male Sprague-Dawley rats (160–180 g) were used to induce OH by feeding of a high-fat diet (42% kcal as fat) for 12 weeks. The dynamic changes of sympathetic outflow were evaluated as the alterations of renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to certain chemicals. The results showed that the protein expressions of Ang II type 1 receptor (AT1R), calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 2 (RAMP2) and RAMP3 were markedly increased, but IMD was much lower in OH rats when compared to control rats. IMD itself microinjection into PVN not only lowered SNA, NADPH oxidase activity and ROS level, but also decreased Ang II-caused sympathetic overdrive, and increased NADPH oxidase activity, ROS levels and mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) activation in OH rats. However, those effects were mostly blocked by the adrenomedullin (AM) receptor antagonist AM22-52 pretreatment. The enhancement of SNA caused by Ang II can be significantly attenuated by the pretreatment of AT1R antagonist lorsatan, superoxide scavenger Tempol and NADPH oxidase inhibitor apocynin (Apo) in OH rats. ERK activation inhibitor U0126 in the PVN reversed Ang II-induced enhancement of SNA, and Apo and IMD pretreatment in the PVN decreased Ang II-induced ERK activation. Chronic IMD administration in the PVN resulted in significant reductions in basal SNA and BP in OH rats. Moreover, IMD lowered NADPH oxidase activity and ROS level in the PVN; reduced the protein expressions of AT1R and NADPH oxidase subunits NOX2 and NOX4, and ERK activation in the PVN; and decreased Ang II levels-inducing sympathetic overactivation. These results indicated that IMD via AM receptors in the PVN attenuates SNA and hypertension, and decreases Ang II-induced enhancement of SNA through the inhibition of NADPH oxidase activity and ERK activation.

Cannabinoid system involves in the analgesic effect of protocatechuic acid.

Protocatechuic acid is an antioxidant which is shown to have analgesic activity in limited studies. However, the mechanisms of action remain unclear. It is aimed to investigate the possible contribution of cannabinoid system that supresses the nociceptive process by the activation of CB1 and CB2 receptors in central and peripheral levels of pain pathways, to the analgesic activity of protocatechuic acid. The analgesic activity of protocatechuic acid was determined at the doses of 75, 150 and 300mg/kg (i.p.) by acetic acid-induced writhing and tail-immersion tests in mice. The results were compared to the analgesic effect of 300mg/kg (i.p.) dipyrone and non-specific CB receptor agonist 5mg/kg (i.p.) WIN 55,212-2. For investigating the contribution of cannabinoid system to protocatechuic acid analgesia; pre-treatment with 8mg/kg (i.p.) CB1 antagonist AM251 and 8mg/kg (i.p.) CB2 antagonist AM630 were performed separately before 300mg/kg protocatechuic acid administration. It was determined that protocatechuic acid has dose-dependent analgesic effect independently from locomotor activity and is comparable with effects of dipyrone and WIN 55,212-2. Pre-treatment with CB1 receptor antagonist AM251 significantly antagonized the protocatechuic acid-induced analgesia in the tail-immersion and writhing tests, whereas pre-treatment of CB2 receptor antagonist AM630 was found to be effective only in the tail-immersion test. It is concluded that cannabinoid modulation contributes to the analgesic effect of protocatechuic acid in spinal level rather than peripheral. CB1 receptor stimulation rather than CB2 receptor stimulation mediates the analgesic effect of protocatechuic acid in both levels, especially peripheral. Graphical abstract Protocatechuic acid inhibits pain response via cannabinoidergic system.

Role of Endocannabinoid System in the Peripheral Antinociceptive Action of Aripiprazole.

Recently, we demonstrated that the antipsychotic dopaminergic and serotoninergic agonist aripiprazole induced peripheral antinociception. However, the mechanism underlying this effect has not been fully established. Here, our aim was to identify possible relationships between this action of aripiprazole and the endocannabinoid system. All drugs were given locally into the right hind paw of male Swiss mice weighing 30-35 g in a volume of 20 µL. The hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2 μg). Aripiprazole was injected 10 minutes before the measurement, and an irreversible inhibitor of anandamide hydrolase (MAFP), an inhibitor for monoacylglycerol lipase (JZL184), and an anandamide reuptake inhibitor (VDM11) were given 10 minutes before the aripiprazole. Nociceptive thresholds were measured using an algesimetric apparatus in the third hour after prostaglandin E2 injection. Data were analyzed by ANOVA and Bonferroni tests. The antinociceptive effect induced by aripiprazole (100 μg) was blocked by cannabinoid 1 or 2 receptor antagonists AM251 (40 μg [P < .01], 80 μg [P < .0001], and 160 μg [P < .0001]) and AM630 (100 μg [P < .0001], 200 μg [P < .0001], and 400 μg [P < .0001]), respectively. The peripheral antinociception induced by aripiprazole (25 μg) was enhanced by administration of the inhibitor of fatty acid amide hydrolase (MAFP, 0.5 μg [P < .0001]) or monoacylglycerol lipase (JZL184, 4 μg [P < .0001]). Moreover, a similar enhancement was observed with the anandamide reuptake inhibitor (VDM11, 2.5 μg [P < .0001]). These results provide evidence for the involvement of the endocannabinoid system in peripheral antinociception induced by aripiprazole treatment.

CUMYL-4CN-BINACA Is an Efficacious and Potent Pro-Convulsant Synthetic Cannabinoid Receptor Agonist.

Synthetic cannabinoid receptor agonists (SCRAs) are the largest class of new psychoactive substances (NPS). New examples are detected constantly, and some are associated with a series of adverse effects, including seizures. CUMYL-4CN-BINACA (1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)indazole-3-carboxamide) is structurally related to potent, cumylamine-derived SCRAs such as 5F-CUMYL-PINACA, but is unusual due to a terminal aliphatic nitrile group not frequently encountered in SCRAs or pharmaceuticals. We report here that CUMYL-4CN-BINACA is a potent CB1 receptor agonist (K i = 2.6 nM; EC50 = 0.58 nM) that produces pro-convulsant effects in mice at a lower dose than reported for any SCRA to date (0.3 mg/kg, i.p). Hypothermic and pro-convulsant effects in mice could be reduced or blocked, respectively, by pretreatment with CB1 receptor antagonist SR141716, pointing to at least partial involvement of CB1 receptors in vivo. Pretreatment with CB2 receptor antagonist AM-630 had no effect on pro-convulsant activity. The pro-convulsant properties and potency of CUMYL-4CN-BINACA may underpin the toxicity associated with this compound in humans.

Cannabinoids differentially modulate cortical information transmission through the sensorimotor or medial prefrontal basal ganglia circuits.

Background and PurposeIn the sensorimotor (SM) and medial prefrontal (mPF) basal ganglia (BG) circuits, the cortical information is transferred to the substantia nigra pars reticulata (SNr) through the hyperdirect trans‐subthalamic pathway and through the direct and indirect trans‐striatal pathways. The cannabinoid CB1 receptor, which is highly expressed in both BG circuits, may participate in the regulation of motor and motivational behaviours. Here, we investigated the modulation of cortico‐nigral information transmission through the BG circuits by cannabinoids.Experimental ApproachWe used single‐unit recordings of SNr neurons along with simultaneous electrical stimulation of motor or mPF cortex in anaesthetized rats.Key ResultsCortical stimulation elicited a triphasic response in the SNr neurons from both SM and mPF‐BG circuits, which consisted of an early excitation (hyperdirect transmission pathway), an inhibition (direct transmission pathway), and a late excitation (indirect transmission pathway). In the SM circuit, after Δ9‐tetrahydrocannabinol or WIN 55,212‐2 administration, the inhibition and the late excitation were decreased or completely lost, whereas the early excitation response remained unaltered. However, cannabinoid administration dramatically decreased all the responses in the mPF circuit. The CB1 receptor antagonist AM251 (2 mg·kg−1, i.v.) did not modify the triphasic response, but blocked the effects induced by cannabinoid agonists.Conclusions and ImplicationsCB1 receptor activation modulates the SM information transmission through the trans‐striatal pathways and profoundly decreases the cortico‐BG transmission through the mPF circuit. These results may be relevant for elucidating the involvement of the cannabinoid system in motor performance and in decision making or goal‐directed behaviour.

Effects of the synthetic cannabinoid 5F-AMB on anxiety and recognition memory in mice.

N-[[1-(5-fluoropentyl)-1H-indazol-3-yl]carbonyl]-L-valine methyl ester (5F-AMB) is a synthetic cannabinoid that has been distributed recently. Although inhalation of 5F-AMB produces adverse effects, such as impaired memory and disturbed consciousness, in humans, the psychopharmacological effects of 5F-AMB in rodents have not been investigated. We first examined the effects of intraperitoneal and intracerebroventricular injections of 5F-AMB on anxiety-like behavior and locomotor activity in the open field (OF) test and recognition memory in the novel object recognition test (NOR) in C57BL/6J mice. We also examined whether a cannabinoid 1 (CB1) receptor antagonist AM251 blocks the effects of 5F-AMB. We next examined the effects of 5F-AMB infusion into the medial prefrontal cortex (mPFC), a brain region associated with anxiety and memory, on these tests. Intraperitoneal injection of 5F-AMB (0.3mg/kg) dramatically decreased locomotor activity in the OF, and this effect was partially reversed by AM251 (3mg/kg). Intracerebroventricular infusion of 5F-AMB (10nmol) produced an anxiolytic effect in the OF and impaired acquisition, but not retrieval, of recognition memory in the NOR, and these effects were blocked by co-infusion of AM251 (1.8nmol). Bilateral intra-mPFC infusion of 5F-AMB (10pmol/side) similarly produced impaired recognition memory acquisition, but no anxiolytic effect. The results demonstrate that centrally administered 5F-AMB produces anxiolytic effect and impaired recognition memory acquisition via activation of CB1 receptors, while systemic 5F-AMB severely impaired locomotor activity. The mPFC is involved in 5F-AMB-induced impairment of recognition memory acquisition. However, other brain region(s) may contribute to the 5F-AMB-induced anxiolytic effect.

Indomethacin plus minocycline coadministration relieves chemotherapy and antiretroviral drug-induced neuropathic pain in a cannabinoid receptors-dependent manner

Neuropathic pain sometimes occurs during chemotherapy with paclitaxel or HIV/AIDS antiretroviral therapy with nucleoside reverse transcriptase inhibitors (NRTIs). We previously reported that coadministration of indomethacin plus minocycline (IPM) was antihyperalgesic in a cannabinoid type 1 (CB1) receptor-dependent manner in a mouse model of paclitaxel-induced neuropathic pain. We evaluated if IPM combination has antihyperalgesic and antiallodynic activities in animal models of paclitaxel or NRTI (ddC, zalcitabine)-induced neuropathic pain, and whether antagonists of CB1, CB2 receptors or G protein-coupled receptor 55 (GPR55) can inhibit these activities of IPM. IPM produced antihyperalgesic and antiallodynic effects against paclitaxel and ddC-induced thermal hyperalgesia and mechanical allodynia. WIN 55,212-2, a cannabinoid receptor agonist, also had antihyperalgesic activity. The antihyperalgesic and antiallodynic activities of IPM were antagonized by a CB1 receptor antagonist AM251 and a CB2 receptor antagonist AM630, but not a GPR55 antagonist ML193. IPM had no effects on the mean time spent on the rotarod, whereas WIN 55,212-2 reduced it in a dose-dependent manner. These results show that IPM at a fixed ratio produces antihyperalgesic and antiallodynic effects in mice models of both paclitaxel and NRTI-induced neuropathic pain which is dependent on both CB1 and CB2 receptors, without causing the typical cannabinoid receptor agonist-induced motor impairment.

Layer-specific involvement of endocannabinoid signaling in muscarinic-induced long-term depression in layer 2/3 pyramidal neurons of rat visual cortex

Neuromodulatory facilitation of long-term synaptic plasticity is important in learning, memory, and experience-dependent cortical plasticity. Although muscarinic-induced long-term depression (mLTD) in the visual cortex is well known, its cellular mechanisms are not fully understood yet. Since endocannabinoid signaling mediates presynaptic expression of LTD in various brain areas including the primary visual cortex of rats, we investigated the involvement of endocannabinoids in the induction of mLTD in different dendritic compartments of layer 2/3 pyramidal neurons. With an unloading experiment of FM1-43 as an indicator of synaptic vesicle recycling, we confirmed that layer 1 and layer 4 stimulations mainly activated distal apical (in layer 1) and perisomatic (in layer 2/3) dendritic compartments, respectively. Bath application of muscarine (10 min) induced LTD in synaptic inputs activated by stimulation of layers 1 (L1-mLTD) and 4 (L2/3-mLTD). Both mLTDs were blocked by intracellular Ca2+ chelator BAPTA and bath application of NMDA receptor antagonist d-AP5. However, only L2/3-mLTD exhibited an increase in paired-pulse ratio. In addition, only L2/3-mLTD was blocked by treatment with CB1 receptor antagonist AM251. Both mLTDs were blocked by intracellular NMDA receptor antagonist MK801, but not by glia-specific metabolic inhibitor fluoroacetate, implying that neither presynaptic NMDA receptors nor astrocytes are involved in mLTD. These results suggest that L2/3-mLTD is expressed presynaptically via retrograde endocannabinoid signaling while L1-mLTD is endocannabinoid independent in layer 2/3 pyramidal neurons of the visual cortex. Therefore, layer-specific involvement of endocannabinoids in the induction of mLTD might play an important role in cortical development and information processing in the neocortex.

Ketamine induces central antinociception mediated by endogenous cannabinoids and activation of CB1 receptors

The participation of endocannabinoids in central and peripheral antinociception induced by several compounds has been shown by our group. In this study, we investigated the effect of endocannabinoids on the central antinociception induced by ketamine. The nociceptive threshold for thermal stimulation was measured using the tail-flick test in Swiss mice. The drugs were administered intracerebroventricularly. Probabilities less than 5% (p < 0.05) were considered to be statistically significant (Two-way ANOVA/Bonferroni’s test). The CB1-selective cannabinoid receptor antagonist AM251 (2 and 4 μg) completely reversed the central antinociception induced by ketamine (4 μg) in a dose-dependent manner. In contrast, the CB2-selective cannabinoid receptor antagonist AM630 (2 and 4 μg) did not antagonize this effect. Additionally, the administration of the anandamide amidase inhibitor MAFP (0.2 μg) and anandamide uptake inhibitor VDM11 (4 μg) significantly enhanced the antinociception induced by a low dose of ketamine (2 μg). It was concluded that central antinociception induced by ketamine involves the activation of CB1 cannabinoid receptors. Mobilization of cannabinoids might be required for the activation of those receptors, since inhibitors of the endogenous cannabinoids potentiate the effect of Ketamine.

Opposing roles of dorsomedial hypothalamic CB1 and TRPV1 receptors in anandamide signaling during the panic-like response elicited in mice by Brazilian rainbow Boidae snakes.

The endocannabinoid system plays an important role in the organization of panic-like defensive behavior. Threatening situations stimulate brain areas, such as the dorsomedial hypothalamus (DMH). However, there is a lack of studies addressing the role of the DMH endocannabinoid system in panic-like responses. We aimed to verify which mechanisms underlie anandamide-mediated responses in the DMH. To test the hypothesis that the anandamide produces panicolytic-like effects, we treated mice with intra-DMH microinjections of vehicle or increasing doses of anandamide (0.5, 5, or 50pmol) and then performed confrontation with the South American snake Epicrates cenchria assisi. Intra-DMH anandamide treatment yielded a U-shaped dose-response curve with no effect of the lowest (0.5pmol) or the highest (50pmol) dose and significant inhibition of panic-like responses at the intermediate (5pmol) dose. In addition, this panicolytic-like effect was prevented by pretreatment of the DMH with the CB1 receptor antagonist AM251 (100pmol). However, pretreatment of the DMH with the TRPV1 receptor antagonist 6-iodo-nordihydrocapsaicin (3nmol) restored the panicolytic-like effect of the highest dose of anandamide. Immunohistochemistry revealed that CB1 receptors were present primarily on axonal fibers, while TRPV1 receptors were found almost exclusively surrounding the perikarya in DMH. The present results suggest that anandamide exerts a panicolytic-like effect in the DMH by activation of CB1 receptors and that TRPV1 receptors are related to the lack of effect of the highest dose of anandamide.

Blockade of Serotonin 2C Receptors with SB-242084 Moderates Reduced Locomotor Activity and Rearing by Cannabinoid 1 Receptor Antagonist AM-251

The endocannabinoid and serotonin (5-HT) systems have key roles in the regulation of several physiological functions such as motor activity and food intake but also in the development of psychiatric disorders. Here we tested the hypothesis, whether blockade of serotonin 2C (5-HT2C) receptors prevents the reduced locomotor activity and other behavioral effects caused by a cannabinoid 1 (CB1) receptor antagonist. As a pretreatment, we administered SB-242084 (1 mg/kg, ip.), a 5-HT2C receptor antagonist or vehicle (VEH) followed by the treatment with AM-251 (5 or 10 mg/kg, ip.), a CB1 receptor antagonist or VEH. The effects of the two drugs alone or in co-administration were investigated in social interaction (SI) and elevated plus maze (EPM) tests in male Wistar rats. Our results show that AM-251 decreased the time spent with rearing in the SI test and decreased locomotor activity in EPM test. In contrast, SB-242084 produced increased locomotor activity in SI test and evoked anxiolytic-like effect in both SI and EPM tests. When applied the drugs in combination, these behavioral effects of AM-251 were moderated by SB-242084. Based on these findings, we conclude that certain unwanted behavioral effects of CB1 receptor antagonists could be prevented by pretreatment with 5-HT2C receptor antagonists.

Intrathecal administration of adrenomedullin induces mechanical allodynia and neurochemical changes in spinal cord and DRG

This study investigated the effect of adrenomedullin (AM) on mechanical pain sensitivity and its possible mechanisms. Intrathecal injection of AM receptor agonist AM1-50 (20 μg) once per day briefly reduced mechanical pain threshold on days 1 and 2 but induced prolonged mechanical allodynia on day 3. However, AM1-50 did not change mechanical pain sensation when the AM receptor antagonist AM22-52 (20 μg) was intrathecally co-administered. Daily administration of AM1-50 (20 μg) for 3 days increased expression of phosphorylated extracellular signal-regulated protein kinase (pERK) and neuronal nitric oxide synthase (nNOS) in the spinal dorsal horn. The AM-induced increase in pERK and nNOS was inhibited by the co-administration of AM22-52. The chronic administration of AM1-50 also increased expression of microglial maker Iba1 and astrocytic marker GFAP (glial fibrillary acidic protein) in the spinal dorsal horn in an AM22-52-sensitive manner. Furthermore, the application of AM1-50 (10 nM, 3 h) to dorsal root ganglion (DRG) explant cultures induced an increase in the expression of transient receptor potential vanilloid 1 (TRPV1). The treatment with AM1-50 did not change TRPV1 expression in DRG in the presence of AM22-52 (2 μM). These results suggest that the increased AM bioactivity induced mechanical allodynia and may contribute to the mechanical pain hypersensitivity under pathological conditions. The mechanisms may involve the activation of ERK signaling pathway and spinal glia as well as the recruitment of nNOS and TRPV1 in the spinal dorsal horn or DRG. The present study indicates that inhibition of the activation AM receptor might provide a fruitful strategy to relieving chronic pain.

Intermedin in Paraventricular Nucleus Attenuates Sympathoexcitation and Decreases TLR4-Mediated Sympathetic Activation via Adrenomedullin Receptors in Rats with Obesity-Related Hypertension.

Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.

Prenatal immune activation potentiates endocannabinoid-related plasticity of inhibitory synapses in the hippocampus of adolescent rat offspring

There is strong evidence that immune activation from prenatal infection increases the risk for offspring to develop schizophrenia. The endocannabinoid (eCB) system has been implicated in the pathophysiology of schizophrenia while models of cortical dysfunction postulate an imbalance between neuronal excitation and inhibition in the disorder. The current study examined the impact of prenatal immune activation on eCB-mediated inhibitory mechanisms. We compared two forms of eCB-related plasticity of evoked inhibitory postsynaptic currents, namely depolarization-induced suppression of inhibition (DSI) and metabotropic glutamate receptor-induced long term depression (mGluR-iLTD), in both the dorsal and ventral hippocampus between adolescent offspring from rat dams that received either saline or bacterial lipopolysaccharide (LPS) during pregnancy. Compared to prenatal saline offspring, prenatal LPS offspring displayed prolonged DSI and stronger mGluR-iLTD in the dorsal and ventral hippocampus, respectively. The sensitivity of mGluR-iLTD to the CB1 receptor antagonist AM251 was also lower in the dorsal hippocampus of prenatal LPS compared to prenatal saline offspring. Testing whether changes in eCB receptor signaling or levels could contribute to these changes in inhibitory transmission, we found region specific increases in 2-arachidonoylglycerol-stimulated signaling and in basal and mGluR-induced levels of anandamide in prenatal LPS offspring when compared to prenatal saline offspring. Our findings indicate that prenatal immune activation can lead to long-term changes in eCB-related plasticity of hippocampal inhibitory synaptic transmission in adolescent rat offspring. Perturbation of the eCB system resulting from prenatal immune activation could represent a mechanism linking early life immune events to the development of psychopathology in adolescence.