CB1 Receptor Antagonist SR Research Articles

Cannabinoid CB1 receptors regulate salivation

Saliva serves multiple important functions within the body that we typically take for granted, such as helping prepare food for swallowing and defense against oral pathogens. Dry mouth is a primary symptom of Sjӧgren’s syndrome and is a side effect of many drug treatments. Cannabis users frequently report dry mouth, but the basis for this is still unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of salivation. We examined expression of cannabinoid CB1 receptors in submandibular salivary gland using immunohistochemistry and tested regulation of salivation by THC and cannabinoid-related ligands. We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the submandibular gland. No staining is seen in submandibular gland epithelial cells (acinar and ductal), or myoepithelial cells (MECs). Treatment with THC (4 mg/kg, IP) or the cannabinoid receptor agonist CP55940 (0.5 mg/kg) reduced salivation in both male and female mice 1 h after treatment. CBD had no effect on its own but reversed the effect of THC in a concentration-dependent manner. Neither the CB1 receptor antagonist SR141716 (4 mg/kg) nor the CB2-selective agonist JWH133 (4 mg/kg) had an effect on salivation. We also found that fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide and related lipids, regulates salivation. Salivation was reduced in FAAH knockout mice as well as mice treated with the FAAH blocker URB597 (4 mg/kg). URB597 had no effect in CB1 knockout mice. FAAH protein is detected intracellularly in acinar but not ductal epithelial cells. In lipidomics experiments, we found that FAAH knockout mice chiefly had elevated levels of acylethanolamines, including anandamide, and reduced levels of acyglycines. Our results are consistent with a model wherein endocannabinoids activate CB1 receptors on cholinergic axons innervating the submandibular gland. THC likely acts by plugging into this system, activating CB1 receptors to reduce salivation, thus offering a mechanism underlying the dry mouth reported by cannabis users.

Immunosuppressive activity of non-psychoactive Cannabis sativa L. extract on the function of human T lymphocytes

BackgroundCannabis sativa L. extracts (CSE) are used for treating inflammatory conditions, but little is known about their immunomodulatory effects. We investigated a novel CSE with high (14%) CBD and low (0.2%) THC concentration in comparison with pure CBD on primary human lymphocytes. MethodsProliferation, cell cycle distribution, apoptosis/necrosis and viability were analysed with standard methods. Genotoxicity was evaluated with the comet-assay. The effect on T lymphocyte activation was evaluated via CD25/CD69 marker expression, degranulation assays and the production of cytokines. The influence on the transcription factors was analysed using Jurkat reporter cell lines. Specific CB2 receptor antagonist SR144528 and TRPV1 receptor antagonist A78416B were used to study the involvement of CB2 or TRPV1 receptors. ResultsCSE inhibited the proliferation of activated T lymphocytes in a dose-dependent manner without inducing apoptosis, necrosis, or affecting cell viability and DNA integrity. The inhibitory effect was mediated via the suppression of T lymphocytes activation, particularly by the suppression of CD25 surface marker expression. Furthermore, CSE interferes with the functionality of the T lymphocytes, as indicated by inhibition of degranulation, IL-2, and IFN-γ production. AP-1-and-NFAT-reporter activation was reduced implicating an AP-1-and-NFAT-mediated mode of action. The effects were in part reversed by SR144528 and A78416B, showing that the effects were mainly mediated by CB2 and TRPV1 receptors. ConclusionCSE and CBD have immunomodulatory effects and interfere with the activation and functionality of T lymphocytes. A comparison between CSE and CBD suggests that the immunosuppressive effect of CSE is mostly due to the effect of CBD.

Anticonvulsive and anti-epileptogenesis effects of Echinacea purpurea root extract, an involvement of CB2 receptor.

Phytocannabinoids beyond the Δ9-tetrahy-drocannabinol have shown anticonvulsive effects. Also, alkylamides from Echinacea purpurea have been proved as cannabinomimetics. We examined the effect of the hydroalcoholic root extract of E.purpurea on pentylenetetrazol (PTZ)-induced tonic-clonic seizures and kindling model of epileptogenesis and the involvement of CB2 receptors as the mediator of this effect. Male Wistar rats (200±20g) were used. Single intraperitoneal (i.p.) injection of PTZ (80mg/kg) was used to induce tonic-clonic seizures. The kindling model of epileptogenesis was induced by daily injections of PTZ (37mg/kg; i.p. for 15days). Latency and duration of the stages were monitored for analysis. The hydroalcoholic root extract of E.purpurea was injected (i.p.) 20min before seizure induction at the doses of 10, 50, 100 and 200mg/kg. CB2 receptor antagonist SR144528 was injected (0.1mg/kg; i.p.) 20min before the Echinacea injection. In the tonic-clonic model, pretreatment with E.purpurea at the doses of 100 and 200mg/kg significantly increased latencies to S2-S6, while it significantly decreased S6 duration and mortality rate. SR144528 injection before the injection of 100mg/kg of E.purpurea significantly prevented the effects of the extract on S4-S6 latencies. In the kindling model, E.purpurea at the doses of 100 and 200mg/kg significantly delayed epileptogenesis and decreased mortality rate, while SR144528 injection before the injection of 100mg/kg of E. purpurea significantly blocked this effect of the extract. These findings revealed the anticonvulsive and antiepileptogenesis effects of the E.purpurea root extract, which can be mediated by CB2 receptors.

Divergent Effects of Systemic and Intracollicular CB Receptor Activation Against Forebrain and Hindbrain-Evoked Seizures in Rats.

Cannabinoid (CB) receptor agonists are of growing interest as targets for anti-seizure therapies. Here we examined the effect of systemic administration of the CB receptor agonist WIN 55,212-2 (WIN) against audiogenic seizures (AGSs) in the Genetically Epilepsy Prone Rat (GEPR)-3 strain, and against seizures evoked focally from the Area Tempestas (AT). We compared these results to the effect of focal administration of the CB1/2 receptor agonist CP 55940 into the deep layers of the superior colliculus (DLSC), a brain site expressing CB1 receptors. While systemic administration of WIN dose-dependently decreased AGS in GEPR-3s, it was without effect in the AT model. By contrast, intra-DLSC infusion of CP 55940 decreased seizures in both models. To determine if the effects of systemic WIN were dependent upon activation of CB1 receptors in the DSLC, we next microinjected the CB1 receptor antagonist SR141716, before WIN systemic treatment, and tested animals for AGS susceptibility. The pretreatment of the DLSC with SR141716 was without effect on its own and did not alter the anti-convulsant action of WIN systemic administration. Thus, while CB receptors in the DLSC are a potential site of anticonvulsant action, they are not necessary for the effects of systemically administered CB agonists.

Subchronic Central Administration of Cannabinoid Ligands Modulates Nociception in Bulbectomized Rats.

Endocannabinoid system is involved in neuropsychiatric disorders such as major depression. The bilaterally olfactory bulbectomized rat is widely used as an animal model of depression. The removal of the olfactory bulbs produces behavioural, physiological, and neurochemical alterations resembling clinical depression. There is increasing evidence that highlights the important role of cannabinoid signalling in depression and nociception. To investigate the effect of CB1 receptor agonist HU 210 and CB1 receptor antagonist SR 141716A administered icv subchronically (for 7 days) on nociception of rats with model of depression - bilateral olfactory bulbectomy (OBX). Experimental model of depression - bilateral olfactory bulbectomy (OBX). Bilaterally olfactory bulbectomized rats were used as an experimental model of depression. HU 210 (5 µg) or SR 141716A (3 µg) were infused icv for 7 consecutive days, starting 15 days after the olfactory bulbectomy. Nociception was examined by applying paw pressure test (analgesy-meter) evaluating the rat pain threshold. On day 7, five minutes after the last microinjection, the rats were tested in an analgesy-meter and their mechanically evoked pain responses were measured in arbitrary units (AU). Microinjections of HU 210 (5 µg) significantly decreased the pain threshold in olfactory bulbectomized rats, while SR 141716A (3 µg) exerted antinociceptive effect by increasing the pain threshold. Data point to an involvement of CB1 receptors in depression-like behaviour and nociception in olfactory bulbectomized rats and support the data for the association between depressive disorder and pain pathways.

The Systemic Administration of the Chemokine CCL1 Evokes Thermal Analgesia in Mice Through the Activation of the Endocannabinoid System.

Apart from its involvement in immune functions, the chemokine CCL1 can participate in the modulation of nociceptive processing. Previous studies have demonstrated the hypernociceptive effect produced by CCL1 in the spinal cord, but its possible action on peripheral nociception has not yet been characterized. We describe here that the subcutaneous administration of CCL1 (1-10µg/kg) produces dose-dependent and long-lasting increases in thermal withdrawal latencies measured by the unilateral hot plate test in mice. The antinociceptive nature of this effect is further supported by the reduction of spinal neurons expressing Fos protein in response to a noxious thermal stimulus observed after the administration of 10µg/kg of CCL1. CCL1-induced antinociception was inhibited after systemic, but not spinal administration of the selective antagonist R243 (0.1-1mg/kg), demonstrating the participation of peripheral CCR8 receptors. The absence of this analgesic effect in mice treated with a dose of cyclophosphamide that produces a drastic depletion of leukocytes suggests its dependency on white blood cells. Furthermore, whereas the antinociceptive effect of CCL1 was unaffected after the treatment with either the antagonist of opioid receptors naloxone or the cannabinoid type 1 receptor blocker AM251, it was dose-dependently inhibited after the administration of the CB2 receptor antagonist SR144528 (0.1-1mg/kg). The detection by ELISA of an increased presence of the endocannabinoid 2-arachidonoylglycerol after the administration of an analgesic dose of CCL1 supports the notion that CCL1 can evoke thermal analgesia through the release of this endocannabinoid from circulating leukocytes.

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.

A real time screening assay for cannabinoid CB1 receptor-mediated signaling

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K+ (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus. In whole-cell patch clamp experiments application of the cannabinoid agonist WIN 55,212-2 to AtT20 cells expressing the CB1 receptor (AtT20/CB1) activated GIRK currents that were blocked by BaCl2. WIN 55,212-2 activation of the GIRK channels was associated with a time- and concentration-dependent (EC50 = 309 nM) hyperpolarization of the membrane potential in the AtT20/CB1 cells when monitored using a fluorescent membrane potential-sensitive dye. The WIN 55,212-2-induced fluorescent signal was inhibited by pretreatment of the cells with either the GIRK channel blocker tertiapin-Q or the CB1 receptor antagonist SR141716. The cannabinoids displayed a response of WIN 55,212-2 ≈ anandamide (AEA) > CP 55,940 > Δ9-tetrahydrocannabinol (THC) when maximal concentrations of the four ligands were tested in the assay. Thus, the AtT20/CB1 cell fluorescent assay will provide a straightforward and efficient methodology for examining cannabinoid-stimulated Gi signaling.

Single and combined effects of plant-derived and synthetic cannabinoids on cognition and cannabinoid-associated withdrawal signs in mice.

It has been suggested that the non-euphorogenic phytocannabinoid cannabidiol (CBD) can ameliorate adverse effects of Δ9 -tetrahydrocannabinol (THC). We determined whether CBD ameliorates cognitive deficits and withdrawal signs induced by cannabinoid CB1 /CB2 receptor agonists or produces these pharmacological effects on its own. The effects of THC or the CB1 /CB2 receptor full agonist WIN55212 alone, CBD alone or their combination were tested across a range of doses. Cognitive effects were assessed in C57BL/6 mice in a conditional discrimination task and in the Barnes maze. Cannabinoid withdrawal signs were assessed following precipitated withdrawal by acute administration of the CB1 receptor antagonist SR141716, the 5-HT1A receptor antagonist WAY100635, the TRPV1 receptor antagonist capsazepine or the adenosine A2A receptor antagonist SCH58261. THC produced significant motor and cognitive impairment in the Barnes maze task, none of which were attenuated by the addition of CBD. CBD alone did not affect cognitive performance. Precipitation of withdrawal signs by SR141716 occurred in mice chronically treated with THC or WIN55,212. These withdrawal signs were not attenuated by addition of chronic CBD. Chronic treatment with CBD alone did not induce withdrawal signs precipitated by SR141716 or WAY100635. Chronic CBD treatment also produced anxiolysis, which was not altered by attempting to precipitate withdrawal-induced anxiety with a range of antagonists. CBD as a monotherapy may prove to be a safer pharmacological agent, than CB1 receptor agonists alone or in combination with CBD, for the treatment of several disorders. This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Antagonism of cannabinoid receptor 1 attenuates the anti-inflammatory effects of electroacupuncture in a rodent model of migraine.

The anti-nociceptive effects of electroacupuncture (EA) in migraine have been documented in multiple randomised controlled trials. Neurogenic inflammation plays a key role in migraine attacks, and the anti-inflammatory effects of acupuncture have been associated with the type 1 cannabinoid (CB1) receptor. To investigate whether CB1 receptors mediate the anti-inflammatory effects of EA on migraine attacks. A migraine model was produced in Sprague-Dawley rats by unilateral electrical stimulation of the trigeminal ganglion (TGES). Rats received EA daily on the 5 days preceding TGES with (TGES+EA+SR141716 group) or without (TGES+EA group) intraperitoneal injections of the CB1 receptor antagonist SR141716. Another group of TGES rats (TGES+MA group) and a non-TGES sham-operated group of rats (Sham+MA group) received minimal acupuncture (MA). Calcitonin gene-related peptide (CGRP) and prostaglandin E2 (PGE2) concentrations were determined in serum obtained from the ipsilateral jugular vein at initiation of TGES and 5 min after. Postmortem interleukin (IL)-1β and cyclooxygenase (COX)2 protein levels in the trigeminal ganglion (TG) and plasma protein extravasation (PPE) in the dura mater were assessed. TGES induced increases in serum CGRP and PGE2 levels (TGES+MA vs baseline and vs Sham: all p<0.001), as well as IL-1β and COX2 protein expression in the TG, and neurogenic PPE levels (TGES+MA vs Sham+MA: all p<0.001). EA attenuated TGES-induced increases in the levels of these proteins (TGES+EA vs TGES+MA: all p<0.001). CB1 receptor antagonism reversed the effects of EA (TGES+EA+SR141716 vs TGES+EA: all p<0.05). CB1 receptors appear to mediate anti-inflammatory effects of EA in a rat model of migraine.

CB1 ligands modulate learning and memory of OBX rats

Purpose. The brain endocannabinoid system has been shown to play a role in learning and memory processes, although the exact mechanisms by which it interferes in these processes are not well established. We aimed at studying the effects of CB1 receptor ligands on learning and memory of rats with an experimental model of depression - olfactory bulbectomy (OBX). Material and methods. Cannabinoid CB1 receptor agonist HU-210 (5 µg) and cannabinoid CB1 receptor antagonist SR 141716A (3 µg) were microinjected i.c.v. in male Wistar rats with olfactory bulbectomy (OBX) model of depression. A passive avoidance task (step through) was used as a test for learning and memory. Results. In the non-OBX group, HU-210 impaired learning and memory processes expressed by the shortened latency time on the retention tests (3 hours and 24 hours after training) and by the decreased percentage of rats that have reached the learning criterion, as compared to the saline-treated controls. SR 141716A did not affect significantly the performance of rats in the step-through task. In the OBX rats HU-210 prolonged the latency time as compared to the saline-treated OBX controls, while SR 141716A did not affect significantly the OBX-related learning and memory deficits. Conclusion. These findings suggest complex modulatory effect of CB1 receptor agonist on learning and memory processes in non-OBX and OBX rats.

Using cellphone technology to monitor and increase dosing adherence

Aims: The observation that dual blockade of endogenous cannabinoid degradative enzymes monoacylglycerol (MAGL) and fatty acid amide hydrolase (FAAH) elicits THC-like psychotropic effects suggest FAAH & MAGL function as dual brakes in curtailing the pharmacological effects of endogenous cannabinoids. To test this hypothesis, we examined whether blockade of FAAH and MAGL would produce an interoceptive stimulus in mice, using the drugdiscriminationparadigm.Drugdiscrimination is an assayused to infer the subjective effects of drugs. Herewe testedwhether elevating levels of the naturally occurring endogenous cannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) via inhibition of both degradative enzymes would serve as an interoceptive stimulus in mice, N=7–8. Towards this end, we employed the dual FAAH & MAGL inhibitor SA-57 as the training drug. Methods:Micewere trainedusingadouble alternationbetween SA-57 and vehicle (DDVVDD) and acquired the task over 40 days. Results: The discriminative stimulus effects of 10mg/kg SA57 were dose-related (ED50 (95% CI) = 4.49 (3.77–5.35)mg/kg) and were antagonized by the CB1 receptor antagonist rimonabant (3mg/kg), but not the CB2 receptor antagonist SR144528 (3mg/kg). CP55, 940 as well as the dual FAAH/MAGL inhibitor JZL195 substituted for SA-57, and rimonabant blocked the substitution of both. Lowdoses of SA-57 (<3mg/kg),which completely inhibit FAAH, but not MAGL, did not substitute for SA-57. Conclusions: These findings suggest that inhibition of FAAH alone is not sufficient to elicit SA-57-like subjective effects. Taken together, this study represents the first example that elevating levels of naturally occurring endogenous cannabinoids serves as a discriminative stimulus. Thus, MAGL and possibly FAAH serve as brakes in curtailing the psychotropic effects of endogenous cannabinoids. Financial support:Dr. Aron Lichtman, Virginia Commonwealth University.

The effects of endocannabinoid receptor agonist anandamide and antagonist rimonabant on opioid analgesia and tolerance in rats.

The role of the cannabinoid (CB) system in the tolerance to analgesic effect of opioid remains obscure. The aim of the present study was to evaluate the effects of the endocannabinoid nonselective receptor agonist anandamide (AEA) and CB1 receptor antagonist rimonabant (SR141716) on morphine analgesia and tolerance in rats. Male Wistar albino rats weighing 215-230 g were used in these experiments. To constitute morphine analgesic tolerance, a 3-day cumulative dosing regimen was used. The analgesic effects of AEA (10 mg/kg), SR141716 (10 mg/kg), and morphine (5 mg/kg) were considered at 30-min intervals by tail flick (TF) and hot plate (HP) analgesia tests. The analgesic effects of the drugs were measured as TF and HP latencies in all groups for each rat and converted to %MPE. The data were analysed by analysis of variance followed by Tukey test. The findings suggested that AEA in combination with morphine produced a significant increase in expression of analgesic tolerance to morphine. Conversely, cannabinoid receptor antagonist SR141716 attenuated morphine analgesic tolerance. In addition, administration of AEA with morphine increased morphine analgesia. In conclusion, we observed that the cannabinoid receptor agonist anandamide and CB1 receptor antagonist SR141716 plays a significant role in the opioid analgesia and tolerance.

Cannabidiol fails to reverse hypothermia or locomotor suppression induced by Δ(9) -tetrahydrocannabinol in Sprague-Dawley rats.

Growing evidence shows cannabidiol (CBD) modulates some of the effects of Δ(9) -tetrahydrocannabinol (THC). CBD is a constituent of some strains of recreational cannabis but its content is highly variable. High CBD strains may have less memory-impairing effects than low-CBD strains and CBD can reverse behavioural effects of THC in monkeys. CBD/THC interactions in rodents are more complicated as CBD can attenuate or exacerbate the effects of THC. This study was undertaken to determine if CBD could reverse hypothermia or hypolocomotor effects caused by THC in rats. Male Sprague-Dawley rats were prepared with radiotelemetry devices and then given doses of THC (10-30 mg·kg(-1) , i.p.) with or without CBD. Experiments determined the effect of simultaneous or 30 min pretreatment with CBD in a 1:1 ratio with THC, as well as the effect of CBD in a 3:1 ratio. Additional experiments determined the effects of pretreatment with the cannabinoid CB1 receptor antagonist SR141716 (rimonabant). CBD did not attentuate THC-induced hypothermia or hypolocomotion but instead exaggerated these effects in some conditions. The antagonist SR141716 blocked hypolocomotor effects of THC for the first hour after injection and the hypothermia for 6 h; thus validating the pharmacological model. There is no evidence from this study that elevated CBD content in cannabis could provide protection from the physiological effects of THC, in rats.

Abstract 542: Blockade of Angiotensin-(1-7)-mediated Vascular Remodeling by a CB2 Cannabinoid Receptor Antagonist

Angiotensin-(1-7) [Ang-(1-7)] inhibits vascular smooth muscle cell (VSMC) growth and reduces cardiac hypertrophy and vascular remodeling in Ang II-infused hypertensive rats. We previously showed that Ang-(1-7) increased the endocannabinoid 2-AG in VSMCs and that inhibition of VSMC growth by Ang-(1-7) was prevented by blockade of CB2 cannabinoid receptors. VSMCs isolated from mice with ablated CB2 receptors were treated with platelet-derived growth factor to stimulate growth; neither Ang-(1-7) nor the CB2 receptor agonist HU308 reduced VSMC growth (98.7±9.9% and 105.4±13.0% of stimulated growth, respectively, n = 3-6), suggesting that growth inhibition by Ang-(1-7) is mediated through activation of the CB2 receptor. Ang II-infused Sprague-Dawley rats were treated for 28 days with Ang-(1-7), in the presence or absence of the CB2 receptor antagonist SR144528, to determine whether CB2 receptor blockade prevents the Ang-(1-7)-mediated reductions in Ang II-stimulated cardiac hypertrophy and vascular remodeling. Systolic blood pressure was similar in both groups, before or after treatment (115±4.6 mmHg with SR144528 and 115.7±10.2 mmHg with Ang-(1-7)/SR144528 before treatment and 186.7±6.2 mmHg and 182.0±2.1 mmHg after Ang II treatment, respectively, n=4). Ang II increased the mean cross-sectional area of cardiac myocytes which was reduced 55% by Ang-(1-7); CB2 receptor blockade prevented the Ang-(1-7)-mediated reduction in myocyte size. Fibrosis (interstitial and perivascular) and vessel size (M/L) were unchanged by Ang-(1-7) in the presence of SR144528 compared to a 79%, 85% and 90% reduction of the Ang II-mediated increase in interstitial fibrosis, perivascular fibrosis and M/L, respectively, by Ang-(1-7) alone. Brain natriuretic peptide and atrial natriuretic peptide also were unchanged by Ang-(1-7) in the hearts of rats treated with Ang II and SR144528 compared to a 91% and 99% reduction by Ang-(1-7) in Ang II-treated rats. Thus, treatment with a CB2R antagonist blunts the anti-hypertrophic and anti-fibrotic responses to Ang-(1-7) in hypertensive Ang II-treated rats, independent of blood pressure, suggesting that the growth inhibitory properties of Ang-(1-7) involve a novel pathway which includes activation of the CB2 receptor.

CB1 receptor antagonism in the granular insular cortex or somatosensory area facilitates consolidation of object recognition memory

Cannabinoid agonists typically impair memory, whereas CB1 receptor antagonists enhance memory performance under specific conditions. The insular cortex has been implicated in object memory consolidation. Here we show that infusions of the CB1 receptor antagonist SR141716 enhances long-term object recognition memory in rats in a dose-dependent manner (facilitation with 1.5, but not 0.75 or 3μg/μL) when administered into the granular insular cortex; the SR141716 facilitation was seen with a memory delay of 72h, but not when the delay was shorter (1h), consistent with enhancement of memory consolidation. Moreover, a sub-group of rats with cannulas placed in the somatosensory area were also facilitated. These results highlight the robust potential of cannabinoid antagonists to facilitate object memory consolidation, as well as the capacity for insular and somatosensory cortices to contribute to object processing, perhaps through enhancement of tactile representation.

Involvement of the Cannabinoid CB1 Receptor in Modulation of Dopamine Output in the Prefrontal Cortex Associated with Food Restriction in Rats

Increase in dopamine output on corticolimbic structures, such as medial prefrontal cortex (mPFC) and nucleus accumbens, has been related to reward effects associated with palatable food or food presentation after a fasting period. The endocannabinoid system regulates feeding behavior through a modulatory action on different neurotransmitter systems, including the dopaminergic system. To elucidate the involvement of type 1 cannabinoid receptors in the regulation of dopamine output in the mPFC associated with feeding in hungry rats, we restricted the food availability to a 2-h period daily for 3 weeks. In food-restricted rats the extracellular dopamine concentration in the mPFC increased starting 80 min before food presentation and returned to baseline after food removal. These changes were attenuated in animals treated with the CB1 receptor antagonist SR141716. To better understand how food restriction can change the response of mesocortical dopaminergic neurons, we studied several components of the neuronal circuit that regulates dopamine output in the mPFC. Patch-clamp experiments revealed that the inhibitory effect of the CB1 receptor agonist WIN 55,212-2 on GABAergic sIPSC frequency was diminished in mPFC neurons of FR compared to fed ad libitum rats. The basal sIPSC frequency resulted reduced in mPFC neurons of food-restricted rats, suggestive of an altered regulation of presynaptic GABA release; these changes were accompanied by an enhanced excitability of mPFC and ventral tegmental area neurons. Finally, type 1 cannabinoid receptor expression in the mPFC was reduced in food-restricted rats. Together, our data support an involvement of the endocannabinoid system in regulation of dopamine release in the mPFC through changes in GABA inhibitory synapses and suggest that the emphasized feeding-associated increase in dopamine output in the mPFC of food-restricted rats might be correlated with an altered expression and function of type 1 cannabinoid receptor in this brain region.

Blockade of CB1 receptors prevents retention of extinction but does not increase low preincubated conditioned fear in the fear incubation procedure

We recently developed a procedure to study fear incubation, in which rats given 100 tone-shock pairings over 10 days show low fear 2 days after conditioned fear training and high fear after 30 days. Notably, fear 2 days after 10 sessions of fear conditioning is lower than fear seen 2 days after a single session of fear conditioning, suggesting that fear is suppressed. Here, we investigate the potential role of CB1 receptor activation by endocannabinoids in this fear suppression. We subjected rats to 10 days of fear conditioning and then administered systemic injections of the CB1 receptor antagonist SR141716 before a conditioned fear test was conducted 2 days later under extinction conditions. A second test was conducted without any injections on the following day (3 days after training) to examine retention of fear extinction. SR141716 injections did not increase fear expression 2 days after extended fear conditioning or affect within-session extinction; however, it impaired retention of between-session fear extinction in the day 3 test. These data suggest that CB1 receptor activation does not suppress fear soon after extended fear conditioning in the fear incubation task. The data also add to the existing literature on the role of CB1 receptors in extinction of conditioned fear.

Abstract 182: Medullary Endocannabinoid Content Contributes to the Differential Resting Baroreflex Sensitivity in Rats with Altered Brain Renin-Angiotensin System Expression

Activation of CB1 receptors in the solitary tract nucleus (NTS) over a 100-fold dose range elicits biphasic effects, with high doses inhibiting baroreflex sensitivity (BRS) for control of heart rate and lower doses facilitating BRS. There is growing evidence for signaling interactions between the endocannabinoid system (ECS) and pathogenic actions of angiotensin (Ang) II at AT1 receptors in hypertension and the metabolic syndrome. Hypotensive ASrAOGEN (AS) rats, overexpressing an angiotensinogen antisense in glia, have significantly elevated medullary tissue glutamate levels and enhanced BRS compared to normotensive Sprague-Dawley (SD) rats (1.42 ± 0.11 vs. 1.04 ± 0.05 ms/mmHg; n = 5 - 6; P = 0.02). Transgenic (mRen2)27 rats, a monogenetic Ang II-dependent model of hypertension, have markedly impaired BRS compared to SD rats (0.43 ± 0.06 vs. 1.04 ± 0.05 mmHg; n = 5; P &lt; 0.0001). There is no effect of the CB1 receptor antagonist SR141716 on BRS when microinjected into the NTS of SD rats; however, SR141716 injected into the NTS of hypertensive (mRen2)27 rats dose-dependently improved BRS. In contrast, SR141716 injected into the NTS of hypotensive AS rats dose-dependently reduced BRS. Microinjection of 0.36 pmol SR141716 (n = 3) reduced BRS from a baseline of 1.23 ± 0.05 ms/mmHg to 0.81 ± 0.14 (P = 0.01) and 36 pmol SR141716 (n = 3) decreased BRS from 1.60 ± 0.17 to 0.48 ± 0.06 ms/mmHg (P = 0.001), a level comparable with baseline BRS of hypertensive (mRen2)27 rats. The effects of CB1 receptor blockade on BRS parallel the endogenous levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) as measured by mass spectrometry with lower 2-AG in AS compared to (mRen2)27 rats (1.17 ± 0.09 vs. 2.70 ± 0.28 ng/mg tissue; n = 4 - 5; P = 0.002). Normotensive SD rats had intermediate 2-AG levels (1.85 ± 0.27 ng/mg tissue; n = 5; n.s. vs. other strains). These observations reveal that an upregulated ECS contributes to RAS-dependent suppression of BRS. That there is a reduction in BRS in response to NTS CB1 receptor blockade in hypotensive AS rats exhibiting elevated glutamate levels suggests differential regulatory functions of the brain ECS that may involve alterations in transmitters known to influence BRS and that parallel the Ang II:Ang-(1-7) ratio in these animals.

Effects of Acutely Applied Cannabinoid CB1 Ligands on Nociception in Rats with a Model of Depression

The effects of CB1 receptor agonist HU-210 and cannabinoid CB1 receptor antagonist SR 141716A on nociception of male Wistar rats with a model of depression (bilateral olfactory bulbectomy, OBX) were studied. HU-210 (5 g) and SR 141716A (3 g) were acutely microinjected i.c.v. on the developed depression background. Nociception was examined as applied mechanical pressure on the left hind paw of the rat (analgesy-meter test, Randall & Sellitto). In the OBX rats, it was found that the pain threshold was increased. HU-210 significantly increased the pain threshold in OBX rats, i.e. decreased the pain sensitivity as compared to saline-treated OBX controls and to sham-operated controls, suggesting an implication of CB1 receptors in nociception of OBX rats. SR 141716A did not affect the nociception in OBX rats. These results suggest that stimulation of CB1 receptors in rats with a model of depression exerted antinociceptive effect.