Cannabinoid CB1 Receptor Research Articles

Alcohol induced behavioral and immune perturbations are attenuated by activation of CB2 cannabinoid receptors.

The endocannabinoidome (eCBome) is the expanded endocannabinoid system (ECS) and studies show that there is a link between this system and how it modulates alcohol induced neuroinflammation. Using conditional knockout (cKO) mice with selective deletion of cannabinoid type 2 receptors (CB2Rs) in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2), we investigated how CB2Rs modulate behavioral and neuroinflammation induced by alcohol. Behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests were used to evaluate behavioral changes induced by alcohol. Using ELISA assay, we investigated the level of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1α (IL-1α), and interleukin-1β (IL-1β) in the hippocampus of mice. The findings demonstrated that locomotor activity, wheel running, and rotarod performance activities were significantly affected by cell-type specific deletion of CB2Rs in dopamine neurons and microglia. The non-selective CB2R agonist, WIN 55,212-2, reduced alcohol preference in the wild type and cell-type specific CB2R cKO mice. In addition, the result showed that cell-type specific deletion of CB2Rs per se and administration of alcohol to CB2R cKO mice increased the expression of proinflammatory cytokines in the hippocampus. These findings suggest the involvement of CB2Rs in modulating behavioral and immune alterations induced by alcohol.

The Endocannabinoid System and Its Relationship to Human Reproduction.

The endocannabinoid system is among the most important regulators of human reproduction. It already applies at the level of the sperm and the egg, plays an important role in the fertilization of the egg, its implantation, regulates the function of the placenta and participates in childbirth. The aim of this work is to summarize the knowledge accumulated so far and to show that the endocannabinoid system must be perfectly regulated in order to maintain a physiological pregnancy from implantation to delivery. Only an exceptional interplay of enzymes such as NAPE-PDL or FAAH, endogenous cannabinoids and cannabinoid receptors CB1 and CB2 can ensure the proper functioning of the reproductive organs and thus lead to delivery on time. Changes in the endocannabinoid system can lead to a number of pathological conditions, e.g., during blastocyst implantation, retardation of embryo development, impaired placental function or miscarriage. Soon, we can expect not only an understanding of all the regulatory events associated with the endocannabinoid system and other regulatory systems that participate in reproduction, but also several possibilities for pharmacotherapeutic interventions that can modify the formation, degradation and effect of endocannabinoids. It cannot be ruled out that some components of the endocannabinoid system could become a marker for monitoring pregnancy and childbirth.

Activation of the cannabinoid type 2 (CB2) receptor improves cardiac contractile performance in fish, Brycon amazonicus

In addition to their well-known classical effects, cannabinoid CB1 and CB2 receptors have also been involvement in both deleterious and protective actions on the heart under various pathological conditions. While the potential therapeutic applications of the endocannabinoid system in the context of cardiovascular function are indeed a viable prospect, significant debate exists within the literature regarding whether CB1, CB2, or a combination of both receptors exert a favorable influence on cardiac function. Hence, the aim of this study was to investigate the effects of CB1 + CB2 or CB2 agonists on cardiac excitation-contraction (E-C) coupling, utilizing fish (Brycon amazonicus) as an experimental model. The CB2 agonist elicited marked positive inotropic and lusitropic responses in isolated ventricular myocardium, induced cyclic adenosine 3′,5′-monophosphate (cAMP) production, and upregulated critical Ca2+ handling proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Our current study demonstrated, for the first time, that CB2 receptor activation-induced effects improved the efficiency of Ca2+ cycling, excitation-contraction coupling (E-C coupling), and cardiac performance in under physiological conditions. Hence, CB2 receptors could be considered a potential therapeutic target for modulating cardiac contractile dysfunctions.

Effects of intrahippocampal injection of kainate on cytokine expression in cortico-limbic system and the role of cannabinoid system in these effects

According to the International League Against Epilepsy, epilepsy is a chronic condition of the brain that is characterized by a predisposition to epileptic seizures along with related neurobiological, cognitive, psychological, and social consequences. This is a general definition that does not take into account the diversity of epilepsies, including different aetiology, symptoms and mechanisms of epileptogenesis, making the development of a unified disease model challenging. The modeling framework addresses this challenge by selecting a distinct type of epilepsy and its associated manifestations, including electrophysiological, morphological (mainly neurodegeneration), and behavioral aspects [1]. In this study, a status epilepticus model is used with intrahippocampal kainic acid administration to reproduce electrophysiological activity and neurodegeneration. The process of reproducing two aspects simultaneously brings the kainate model closer to the disease called “epilepsy”. Neuroinflammation is a neurobiological process that is associated with a chronic brain condition that is characterized by a persistent susceptibility to epileptic seizures. Specifically, neuroinflammation is the response of the central nervous system (CNS) to various stimuli, including stroke, trauma, infection, autoimmune diseases, stress, and hyperexcitability of the neural network resulting from epileptic seizures. This response involves brain cells, specifically activated microglia and astrocytes, as well as neurons and brain vasculature cells, biosynthesizing and releasing molecules with inflammatory properties [2]. Currently, the study of neuroinflammation in relation to various pathological conditions includes an examination of the influence of the endocannabinoid system (ECS) [3]. However, research on epilepsy primarily focused on the ECS’s effects on network neuronal activity through CB1-mediated changes in synapse function (both excitatory and inhibitory), with only a limited number of studies exploring the interactions between the ECS and neuroinflammation [4]. This study analyzed neuroinflammatory dynamics after kainate administration and the effect of exogenous endocannabinoid receptor modulators on these dynamics. Neuroinflammation was evaluated through the measurement of expression levels of pro- and anti-inflammatory cytokines (IL1b, Il6, Cx3cl1, Ccl2, Tgfb1, Zc3h12a, Tnfa) in various areas including the ipsilateral ventral hippocampus, contralateral dorsal and ventral hippocampuses, neocortex, dura mater, cerebral and hippocampal meninges (undivided arachnoid and pia maters). Expression was quantified using quantitative PCR at 3 and 24 hours following convulsant injection. The study showed that seizures induced by kainate resulted in swift neuroinflammation development in the hippocampus, which resolved nearly entirely after 24 hours. A unique pattern of neuroinflammation was detected in the neocortex, with minor alterations at 3 hours and more pronounced modifications in the expression of inflammatory genes at 24 hours. Using the intrahippocampal kainate administration model, this study was the first to show a significantly delayed neuroinflammatory response in the neocortex compared to the hippocampus across a broad range of genes. Both activation of the cannabinoid CB1 and CB2 receptors and inhibition of the cannabinoid CB1 receptor increased neuroinflammation. However, cannabinoid receptor activation showed a predominantly proinflammatory effect in the neocortex, while CB1 receptor inhibition had a stronger effect in the hippocampus. Our findings indicate that cannabinoid receptor modulators regulate the kainate-induced neuroinflammatory response in the neocortex and hippocampus differently. Moreover, the well-known anti-inflammatory effect of cannabinoids is evident only within a certain range of cannabinoid concentrations and the timing of drug administration. In some cases, however, cannabinoids may have the opposite effect of increasing neuroinflammation.

Discovery of a cannabinoid CB2 receptor fluorescent probe based on a pyridin-2-yl-benzyl-imidazolidine-2,4-dione scaffold

Cannabinoid receptor type 2 (CB2R) agonists have therapeutic potential for the treatment of (neuro)inflammatory diseases. Fluorescent probes enable the detection of CB2R in relevant cell types and serve as a chemical tool in cellular target engagement studies. Here, we report the structure-based design and synthesis of a new CB2R selective fluorescent probe. Based on the cryo-EM structure of LEI-102 in complex with the CB2R, we synthesized 5-fluoropyridin-2-yl-benzyl-imidazolidine-2,4-dione analogues in which we introduced a variety of linkers and fluorophores. Molecular pharmacological characterization showed that compound 22 containing a Cy5-fluorophore with an alkyl-spacer was the most potent probe with a pKi of 6.2 ± 0.6. It was selective over the cannabinoid CB1 receptor and behaved as an inverse agonist (EC50 5.3 ± 0.1, Emax -63% ± 6). Probe 22 may serve as a chemical tool in target and lead validation studies for the CB2R.

CB2 agonist mitigates cocaine-induced reinstatement of place preference and modulates the inflammatory response in mice.

Chronic exposure to cocaine is known to have profound effects on the brain, leading to the dysregulation of inflammatory signalling pathways, the activation of microglia, and the manifestation of cognitive and motivational behavioural impairments. The endocannabinoid system has emerged as a potential mediator of cocaine's deleterious effects. In this study, we sought to investigate the therapeutic potential of the cannabinoid CB2 receptor agonist, JWH-133, in mitigating cocaine-induced inflammation and associated motivational behavioural alterations in an in vivo model. Our research uncovered compelling evidence that JWH-133, a selective CB2 receptor agonist, exerts a significant dampening effect on the reinstatement of cocaine-induced conditioned place preference. This effect was accompanied by notable changes in the neurobiological landscape. Specifically, JWH-133 administration was found to upregulate Δ-FOSB expression in the nucleus accumbens (Nac), elevate CX3CL1 levels in both the ventral tegmental area and prefrontal cortex (PFC), and concurrently reduce IL-1β expression in the PFC and NAc among cocaine-treated animals. These findings highlight the modulatory role of CB2 cannabinoid receptor activation in altering the reward-seeking behaviour induced by cocaine. Moreover, they shed light on the intricate interplay between the endocannabinoid system and cocaine-induced neurobiological changes, paving the way for potential therapeutic interventions targeting CB2 receptors in the context of cocaine addiction and associated behavioural deficits.

Computational and Experimental Drug Repurposing of FDA-Approved Compounds Targeting the Cannabinoid Receptor CB1.

The cannabinoid receptor 1 (CB1R) plays a pivotal role in regulating various physiopathological processes, thus positioning itself as a promising and sought-after therapeutic target. However, the search for specific and effective CB1R ligands has been challenging, prompting the exploration of drug repurposing (DR) strategies. In this study, we present an innovative DR approach that combines computational screening and experimental validation to identify potential Food and Drug Administration (FDA)-approved compounds that can interact with the CB1R. Initially, a large-scale virtual screening was conducted using molecular docking simulations, where a library of FDA-approved drugs was screened against the CB1R's three-dimensional structures. This in silico analysis allowed us to prioritize compounds based on their binding affinity through two different filters. Subsequently, the shortlisted compounds were subjected to in vitro assays using cellular and biochemical models to validate their interaction with the CB1R and determine their functional impact. Our results reveal FDA-approved compounds that exhibit promising interactions with the CB1R. These findings open up exciting opportunities for DR in various disorders where CB1R signaling is implicated. In conclusion, our integrated computational and experimental approach demonstrates the feasibility of DR for discovering CB1R modulators from existing FDA-approved compounds. By leveraging the wealth of existing pharmacological data, this strategy accelerates the identification of potential therapeutics while reducing development costs and timelines. The findings from this study hold the potential to advance novel treatments for a range of CB1R -associated diseases, presenting a significant step forward in drug discovery research.

Hexahydrocannabinol Pharmacology, Toxicology, and Analysis: The First Evidence for a Recent New Psychoactive Substance.

During the last two years, hexahydrocannabinol (HHC), the hydrogenated derivative of tetrahydrocannabinol has been freely sold by internet websites as a "legal" replacement to THC and cannabis in a range of highly attractive branded and unbranded products, some of which are sold as "legal highs". Potentially, there could be a large demand for HHC products by individuals in Europe and internationally. Studies reporting HHC pharmacology, toxicology and analysis were identified from Pubmed and Scopus databases, and official international organizations' websites were considered. HHC showed the effects of the typical cannabinoid on the central nervous system, with lower potency than Δ9-THC. A few studies highlighted that 9(R)-HHC is more potent than 9(S)-HHC. This molecule showed an affinity for cannabinoid receptor CB1 both in vitro and in vivo, suggesting a possible therapeutic effect in several pathologies. However, the affinity for the CB1 receptor suggests a possible addiction potential, inducing the users to misuse it. Since actual intoxication cases have not yet been reported, the HHC harmful potential was not described, probably due to the lack of effective analytical methods to detect HHC in biological matrices. Conversely, different analytical assays were developed and validated to separate HHC epimers in natural and non-natural sources. Similarly to other NPS, the HHC represents a cheaper alternative to the controlled Δ9-THC. Its monitoring is a crucial challenge for toxicological and forensic purposes. To this concern, it is essential to further investigate HHC to support health providers in the identification of related intoxications.

Evaluating signaling bias for synthetic cannabinoid receptor agonists at the cannabinoid CB2 receptor.

The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern. Despite representing one of the largest classes of new psychoactive substances, pharmacological data on new SCRAs is limited, particularly at the cannabinoid CB2 receptor (CB2 ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB2 , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11. The activity of SCRAs was assessed in a battery of invitro assays in CB2 -expressing HEK 293 cells: G protein activation (Gαi3 and GαoB ), phosphorylation of ERK1/2, and β-arrestin 1/2 translocation. The activity profiles of the ligands were further evaluated using the operational analysis to identify ligand bias. All SCRAs activated the CB2 signaling pathways in a concentration-dependent manner, although with varying potencies and efficacies. Despite the detection of numerous instances of statistically significant bias, compound activities generally appeared only subtly distinct in comparison with the reference ligand, CP55940. In contrast, the phytocannabinoid THC exhibited an activity profile distinct from the SCRAs; most notably in the translocation of β-arrestins. These findings demonstrate that CB2 is able to accommodate a structurally diverse array of SCRAs to generate canonical agonist activity. Further research is required to elucidate whether the activation of CB2 contributes to the toxicity of these compounds.

Role of CB1 Cannabinoid Receptors in Vascular Responses and Vascular Remodeling of the Aorta in Female Mice.

Both the endocannabinoid system (ECS) and estrogens have significant roles in cardiovascular control processes. Cannabinoid type 1 receptors (CB1Rs) mediate acute vasodilator and hypotensive effects, although their role in cardiovascular pathological conditions is still controversial. Estrogens exert cardiovascular protection in females. We aimed to study the impact of ECS on vascular functions. Experiments were performed on CB1R knockout (CB1R KO) and wild-type (WT) female mice. Plasma estrogen metabolite levels were determined. Abdominal aortas were isolated for myography and histology. Vascular effects of phenylephrine (Phe), angiotensin II, acetylcholine (Ach) and estradiol (E2) were obtained and repeated with inhibitors of nitric oxide synthase (NOS, Nω-nitro-L-arginine) and of cyclooxygenase (COX, indomethacin). Histological stainings (hematoxylin-eosin, resorcin-fuchsin) and immunostainings for endothelial NOS (eNOS), COX-2, estrogen receptors (ER-α, ER-β) were performed. Conjugated E2 levels were higher in CB1R KO compared to WT mice. Vasorelaxation responses to Ach and E2 were increased in CB1R KO mice, attenuated by NOS-inhibition. COX-inhibition decreased Phe-contractions, while it increased Ach-relaxation in the WT group but not in the CB1R KO. Effects of indomethacin on E2-relaxation in CB1R KO became opposite to that observed in WT. Histology revealed lower intima/media thickness and COX-2 density, higher eNOS and lower ER-β density in CB1R KO than in WT mice. CB1R KO female mice are characterized by increased vasorelaxation associated with increased utilization of endothelial NO and a decreased impact of constrictor prostanoids. Our results indicate that the absence or inhibition of CB1Rs may have beneficial vascular effects.

Dual allosteric and orthosteric pharmacology of synthetic analog cannabidiol-dimethylheptyl, but not cannabidiol, on the cannabinoid CB2 receptor

Cannabinoid CB2 receptor (CB2R) is a class A G protein-coupled receptor (GPCR) involved in a broad spectrum of physiological processes and pathological conditions. For that reason, targeting CB2R might provide therapeutic opportunities in neurodegenerative disorders, neuropathic pain, inflammatory diseases, and cancer. The main components from Cannabis sativa, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), have been therapeutically exploited and synthetically-derived analogs have been generated. One example is cannabidiol-dimethylheptyl (CBD-DMH), which exhibits anti-inflammatory effects. Nevertheless, its pharmacological mechanism of action is not yet fully understood and is hypothesized for multiple targets, including CB2R. The aim of this study was to further investigate the molecular pharmacology of CBD-DMH on CB2R while CBD was taken along as control. These compounds were screened in equilibrium and kinetic radioligand binding studies and various functional assays, including G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. In dissociation studies, CBD-DMH allosterically modulated the radioligand binding. Furthermore, CBD-DMH negatively modulated the G protein activation of reference agonists CP55,940, AEA and 2-AG, but not the agonist-induced ß-arrestin-2 recruitment. Nevertheless, CBD-DMH also displayed competitive binding to CB2R and partial agonism on G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. CBD did not exhibit such allosteric behavior and only very weakly bound CB2R without activation. This study shows a dual binding mode of CBD-DMH, but not CBD, to CB2R with the suggestion of two different binding sites. Altogether, it encourages further research into this dual mechanism which might provide a new class of molecules targeting CB2R.

Myeloid cannabinoid receptor CB1 deficiency confers atheroprotection in a sex-specific manner

Abstract Aim Cardiovascular diseases (CVDs) are the leading cause of death globally, claiming an estimated 17.9 million lives each year. A better understanding of the underlying chronic inflammatory disease mechanisms in atherosclerosis might help finding novel treatments and reduce CVD deaths. In particular, the cannabinoid receptor CB1 has been implicated in atherosclerosis, while its cell-specific effects in this disease are not well understood. Methods Myeloid Cnr1 (CB1 encoding gene) knockout mice were generated on apolipoprotein E deficiency (Apoe-/-) background by crossing CB1flox mice with transgenic mice carrying Cre under control of the lysozyme M (LysM) promoter for selective expression in myeloid cells. Age - and sex-matched groups were analyzed at baseline, 4 and 16 weeks Western diet (0,15% cholesterol). For in vitro experiments, murine bone marrow-derived macrophages (BMDM) were used. Results Male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores compared to controls, while only minor effects in females were observed. Male Cnr1 deficient mice had reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in vitro in BMDMs and blunted by female hormone estradiol treatment. Array-based kinase activity profiling of BMDMs stimulated with a synthetic CB1 agonist revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling via bulk RNA-sequencing of CB1 agonist-stimulated BMDMs further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, again only in male mice. Conclusion Impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The biological effects of macrophage CB1 signaling seem to be more pronounced in male mice. This highlights the need to consider the biological sex as an important variable in preclinical studies.

Canine immune cells express high levels of CB1 and CB2 cannabinoid receptors and cannabinoid-mediated alteration of canine cytokine production is vehicle-dependent

With the increased popularity and societal acceptance of marijuana and cannabidiol (CBD) use in humans, there is an interest in using cannabinoids in veterinary medicine. There have been a few placebo-controlled clinical trials in dogs suggesting that cannabis-containing extracts are beneficial for dogs with inflammatory diseases such as osteoarthritis, and there is growing interest in their immunosuppressive potential for the treatment of immune-mediated diseases. Since cannabinoids exhibit anti-inflammatory and immunosuppressive effects in many species, the purpose of these studies was to examine whether the plant-derived cannabinoids, CBD and Δ9-tetrahydrocannabinol (THC), would also suppress immune function in canine peripheral blood mononuclear cells (PBMCs). Another goal was to characterize expression of the cannabinoid receptors, CB1 and CB2, in canine immune cells. We hypothesized that CBD and THC would suppress stimulated cytokine expression and that both cannabinoid receptors would be expressed in canine immune cells. Surprisingly, cannabinoid suppressive effects in canine PMBCs were quite modest, with the most robust effect occurring at early stimulation times and predominantly by THC. We further showed that cannabinoid-mediated suppression was dog- and vehicle-dependent with CBD and THC delivered in dimethyl sulfoxide (DMSO) producing more immune suppressive effects as compared to ethanol (ETOH). PCR, flow cytometry, and immunohistochemical staining demonstrated that both CB1 and CB2 are expressed in canine immune cells. Together these data show that canine immune cells are sensitive to suppression by cannabinoids, but more detailed studies are needed to further understand the mechanisms and broad effects of these compounds in the dog.

Complex Regional Pain Syndrome Type I: Evidence for the CB1 and CB2 Receptors Immunocontent and Beneficial Effect of Local Administration of Cannabidiol in Mice.

Introduction: Complex regional pain syndrome type I (CRPS-I) is a debilitating neuropathic painful condition associated with allodynia, hyperalgesia, sudomotor and/or vasomotor dysfunctions, turning investigation of its pathophysiology and new therapeutic strategies into an essential topic. We aim to investigate the impact of ischemia/reperfusion injury on the immunocontent of CB1 and CB2 cannabinoid receptor isoforms in the paws of mice submitted to a chronic postischemia pain (CPIP) model and the effects of local administration of cannabidiol (CBD) on mechanical hyperalgesia. Methods: Female Swiss mice, 30-35 g, were submitted to the CPIP model on the right hind paw. Skin and muscle samples were removed at different periods for western blot analysis. Results: No changes in the immunocontent of CB1 and CB2 receptors in paw muscle tissues after ischemia-reperfusion were observed. CBD promoted an antihyperalgesic effect in both phases. AM281 reversed the effect of CBD, whereas ruthenium red abolished the late phase. Conclusion: Our results point to the possible beneficial effects of local administration of CBD in modulating CRPS-I in humans. As possible targets for CBD antihyperalgesia in this model, the contribution of cannabinoid receptor CB1, in addition to TRPM8 is suggested.

Participation of the cannabinoid system and the NO/cGMP/KATP pathway in serotonin-induced peripheral antinociception

It has already been shown that serotonin can release endocannabinoids at the spinal cord level, culminating in inhibition of the dorsal horn. At the peripheral level, cannabinoid receptors modulate primary afferent neurons by inhibiting calcium conductance and increasing potassium conductance. Studies have shown that after the activation of opioid receptors and cannabinoids, there is also the activation of the NO/cGMP/KATP pathway, inducing cellular hyperpolarization. In this study, we evaluated the participation of the cannabinoid system with subsequent activation of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect of serotonin. The paw pressure test of mice was used in animals that had their sensitivity to pain increased due to an intraplantar injection of PGE2 (2 μg). Serotonin (250 ng/paw), administered locally in the right hind paw, induced antinociceptive effect. CB1 and CB2 cannabinoid receptors antagonists, AM251 (20, 40 and 80 μg) and AM630 (25, 50 and 100 μg), respectively, reversed the serotonin-induced antinociceptive effect. MAFP (0.5 μg), an inhibitor of the FAAH enzyme that degrades anandamide, and JZL184 (3.75 μg), an inhibitor of the enzyme MAGL that degrades 2-AG, as well as the VDM11 (2.5 μg) inhibitor of anandamide reuptake, potentiated the antinociceptive effect induced by a low dose (62. 5 ng) of serotonin. In the evaluation of the participation of the NO/cGMP/KATP pathway, the antinociceptive effect of serotonin was reversed by the administration of the non-selective inhibitor of NOS isoforms L-NOarg (12.5, 25 and 50 μg) and by the selective inhibitor for the neuronal isoform LNPA (24 μg), as well as by the soluble guanylate cyclase inhibitor ODQ (25, 50 and 100 μg). Among potassium channel blockers, only Glibenclamide (20, 40 and 80 μg), an ATP-sensitive potassium channel blocker, reversed the effect of serotonin. In addition, intraplantar administration of serotonin (250 ng) was shown to induce a significant increase in nitrite levels in the homogenate of the plantar surface of the paw of mice. Taken together, these data suggest that the antinociceptive effect of serotonin occurs by activation of the cannabinoid system with subsequent activation of the NO/cGMP/KATP pathway.

Investigating the Effects of Exogenous and Endogenous 2-Arachidonoylglycerol on Retinal CB1 Cannabinoid Receptors and Reactive Microglia in Naive and Diseased Retina.

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina.

Cannabinoid type-2 receptors modulate terpene induced anxiety-reduction in zebrafish

Terpenes are the most extensive and varied group of naturally occurring compounds mostly found in plants, including cannabis, and have an array of potential therapeutic benefits for pathological conditions. The endocannabinoid system can potently modulate anxiety in humans, rodents, and zebrafish. The ‘entourage effect’ suggests terpenes may target cannabinoid CB1 and CB2 receptors, among others, but this requires further investigation. In this study we first tested for anxiety-altering effects of the predominant ‘Super-Class’ terpenes, bisabolol (0.001%, 0.0015%, and 0.002%) and terpinolene (TPL; 0.01%, 0.05%, and 0.1%), in zebrafish with the open field test. Bisabolol did not have an effect on zebrafish behaviour or locomotion. However, TPL caused a significant increase in time spent in the inner zone and decrease in time spent in the outer zone of the arena indicating an anxiolytic (anxiety decreasing) effect. Next, we assessed whether CB1 and CB2 receptor antagonists, rimonabant and AM630 (6-Iodopravadoline) respectively, could eliminate or reduce the anxiolytic effects of TPL (0.1%) and β-caryophyllene (BCP; 4%), another super-class terpene previously shown to be anxiolytic in zebrafish. Rimonabant and AM630 were administered prior to terpene exposure and compared to controls and fish exposed to only the terpenes. AM630, but not rimonabant, eliminated the anxiolytic effects of both BCP and TPL. AM630 modulated locomotion on its own, which was potentiated by terpenes. These findings suggest the behavioural effects of TPL and BCP on zebrafish anxiety-like behaviour are mediated by a selective preference for CB2 receptor sites. Furthermore, the CB2 pathways mediating the anxiolytic response are likely different from those altering locomotion.

Cannabinoid CB2 receptor agonist reduces local and systemic inflammation associated with pneumonia-induced sepsis in mice

Sepsis is a severe condition secondary to dysregulated host response to infection leading to tissue damage and organ dysfunction. Cannabinoid CB2 receptor has modulatory effects on the immune response. Therefore, this study investigated the effects of a cannabinoid CB2 receptor agonist on the local and systemic inflammatory process associated with pneumonia-induced sepsis. Pneumonia-induced sepsis was induced in mice by intratracheal inoculation of Klebsiella pneumoniae. Tissue and bronchoalveolar lavage (BAL) were collected 6, 24, or 48 h after surgery. Mice were treated with CB2 agonist (AM1241, 0.3 and 3 mg/kg, i.p.) and several parameters of inflammation were evaluated 24 h after sepsis induction. Polymorphonuclear cell migration to the infectious focus peaked 24 h after pneumonia-induced sepsis induction in male and female animals. Septic male mice presented a significant reduction of cannabinoid CB2 receptor density in the lung tissue after 24 h, which was not observed in females. CB2 expression in BAL macrophages was also reduced in septic animals. Treatment of septic mice with AM1241 reduced cell migration, local infection, myeloperoxidase activity, protein extravasation, and NOS-2 expression in the lungs. In addition, the treatment reduced plasma IL-1β, increased IL-10 and reduced the severity and mortality of septic animals. These results suggest that AM1241 promotes an interesting balance in the inflammatory response, maintaining lung function and preventing organ injury. Therefore, cannabinoid CB2 receptors are potential targets to control the excessive inflammatory process that occurs in severe conditions, and agonists of these receptors can be considered promising adjuvants in pneumonia-induced sepsis treatment.

Memory impairment was ameliorated by corticolimbic microinjections of arachidonylcyclopropylamide (ACPA) and miRNA-regulated lentiviral particles in a streptozotocin-induced Alzheimer's rat model

The present study aimed to investigate the effect of corticolimbic cannabinoid CB1 receptors activity on memory impairment in the intracerebroventricular (ICV)-streptozotocin (STZ) animal model of Alzheimer's like-disease. This study also assessed whether the corticolimbic overexpression of miRNA-137 or -let-7a could increase the endocannabinoids by inhibiting the monoglyceride lipase (MAGL) to ameliorate STZ response. The results showed that ICV microinjection of STZ (3 mg/kg/10 μl) impaired passive avoidance memory retrieval. The chronic microinjection of arachidonylcyclopropylamide (ACPA; 10 ng/0.5 μl), a selective cannabinoid CB1 receptor agonist, into the hippocampal CA1 region, the central amygdala (CeA) or the medial prefrontal cortex (mPFC) ameliorated the amnesic effect of ICV-STZ. Intra-CA1 or -CeA microinjection of ACPA alone did not affect memory retrieval, while its microinjection into the mPFC impaired memory formation. Based on bioinformatics analysis and verification of the MAGL gene, miRNA-137 and -let-7a were chosen to target the expression levels of MAGL in the corticolimbic regions. The chronic corticolimbic microinjection of lentiviral particles containing miRNA-137 or -let-7a ameliorated ICV-STZ-induced memory impairment. The high transfection efficiency was determined for each virus using comparing fluorescent and conventional vision. Corticolimbic overexpression of miRNA-137 or -let-7a decreased the MAGL gene expression that encodes the MAGL enzyme to increase the endocannabinoids. Thus, among the molecular mechanisms and signaling pathways involved in the pathophysiology of Alzheimer's disease (AD), it is worth mentioning the role of endocannabinoids in the corticolimbic regions. CB1 receptor agonists, miRNA-137 or -let-7a, may be potential therapeutic targets against cognitive decline in AD.

Neuroinflammation in the Central Nervous System: Exploring the Evolving Influence of Endocannabinoid System.

Neuroinflammation is a complex biological process that typically originates as a protective response in the brain. This inflammatory process is triggered by the release of pro-inflammatory substances like cytokines, prostaglandins, and reactive oxygen and nitrogen species from stimulated endothelial and glial cells, including those with pro-inflammatory functions, in the outer regions. While neuronal inflammation is common in various central nervous system disorders, the specific inflammatory pathways linked with different immune-mediated cell types and the various factors influencing the blood-brain barrier significantly contribute to disease-specific characteristics. The endocannabinoid system consists of cannabinoid receptors, endogenous cannabinoids, and enzymes responsible for synthesizing and metabolizing endocannabinoids. The primary cannabinoid receptor is CB1, predominantly found in specific brain regions such as the brainstem, cerebellum, hippocampus, and cortex. The presence of CB2 receptors in certain brain components, like cultured cerebellar granular cells, Purkinje fibers, and microglia, as well as in the areas like the cerebral cortex, hippocampus, and cerebellum is also evidenced by immunoblotting assays, radioligand binding, and autoradiography studies. Both CB1 and CB2 cannabinoid receptors exhibit noteworthy physiological responses and possess diverse neuromodulatory capabilities. This review primarily aims to outline the distribution of CB1 and CB2 receptors across different brain regions and explore their potential roles in regulating neuroinflammatory processes.