Pharmacological Evaluation of Cannabinoid Receptor Modulators Using GRABeCB2.0 Sensor.

Cannabinoid receptors CB1R and CB2R are G-protein coupled receptors acted upon by endocannabinoids (eCBs), namely 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (AEA), with unique pharmacology and modulate disparate physiological processes. A genetically encoded GPCR activation-based sensor that was developed recently-GRABeCB2.0-has been shown to be capable of monitoring real-time changes in eCB levels in cultured cells and preclinical models. However, its responsiveness to exogenous synthetic cannabinoid agents, particularly antagonists and allosteric modulators, has not been extensively characterized. This current study expands upon the pharmacological characteristics of GRABeCB2.0 to enhance the understanding of fluorescent signal alterations in response to various functionally indiscriminate cannabinoid ligands. The results from this study could enhance the utility of the GRABeCB2.0 sensor for in vitro as well as in vivo studies of cannabinoid action and may aid in the development of novel ligands.

CB1 receptor expression and signaling are required for dexamethasone-induced aversive memory consolidation.

The molecular processes that underlie long-term memory formation involve signaling pathway activation by neurotransmitter release, which induces the expression of immediate early genes, such as Zif268, having a key role in memory formation. In this work, we show that the cannabinoid CB1 receptor signaling is necessary for the effects of dexamethasone on the behavioral response in an inhibitory avoidance task, on dexamethasone-induced ERK phosphorylation, and on dexamethasone-dependent Zif268 expression. Furthermore, we provide primary evidence for the mechanism responsible for this crosstalk between cannabinoid and glucocorticoid-mediated signaling pathways, showing that dexamethasone regulates endocannabinoid metabolism by inhibiting the activity of the Fatty acid amide hydrolase (FAAH), an integral membrane enzyme that hydrolyzes endocannabinoids and related amidated signaling lipids. Our results provide novel evidence regarding the role of the endocannabinoid system, and in particular of the CB1 receptor, as a mediator of the effects of glucocorticoids on the consolidation of aversive memories.

The conserved endocannabinoid anandamide modulates olfactory sensitivity to induce hedonic feeding in C. elegans.

The ability of cannabis to increase food consumption has been known for centuries. In addition to producing hyperphagia, cannabinoids can amplify existing preferences for calorically dense, palatable food sources, a phenomenon called hedonic amplification of feeding. These effects result from the action of plant-derived cannabinoids that mimic endogenous ligands called endocannabinoids. The high degree of conservation of cannabinoid signaling at the molecular level across the animal kingdom suggests hedonic feeding may also be widely conserved. Here, we show that exposure of Caenorhabditis elegans to anandamide, an endocannabinoid common to nematodes and mammals, shifts both appetitive and consummatory responses toward nutritionally superior food, an effect analogous to hedonic feeding. We find that anandamide's effect on feeding requires the C. elegans cannabinoid receptor NPR-19 but can also be mediated by the human CB1 cannabinoid receptor, indicating functional conservation between the nematode and mammalian endocannabinoid systems for the regulation of food preferences. Furthermore, anandamide has reciprocal effects on appetitive and consummatory responses to food, increasing and decreasing responses to inferior and superior foods, respectively. Anandamide's behavioral effects require the AWC chemosensory neurons, and anandamide renders these neurons more sensitive to superior foods and less sensitive to inferior foods, mirroring the reciprocal effects seen at the behavioral level. Our findings reveal a surprising degree of functional conservation in the effects of endocannabinoids on hedonic feeding across species and establish a new system to investigate the cellular and molecular basis of endocannabinoid system function in the regulation of food choice.

Electrophysiology of Endocannabinoid Signaling.

Electrophysiological technique is an efficient tool for investigating the synaptic regulatory effects mediated by the endocannabinoid system. Stimulation of presynaptic type 1 cannabinoid receptor (CB1) is the principal mode by which endocannabinoids suppress transmitter release in the central nervous system, but a non-retrograde manner of functioning and other receptors have also been described. Endocannabinoids are key modulators of both short- and long-term plasticity. Here, we discuss ex vivo electrophysiological approaches to examine synaptic signaling induced by cannabinoid and endocannabinoid molecules in the mammalian brain.

[Research progress on anti-inflammatory effects of plant-derived cannabinoid type 2 receptor modulators].

Excessive and persistent inflammatory responses are a potential pathological condition that can lead to diseases of various systems, including nervous, respiratory, digestive, circulatory, and endocrine systems. Cannabinoid type 2 receptor(CB2R) belongs to the G protein-coupled receptor family and is widely distributed in immune cells, peripheral tissues, and the central nervous system. It plays a role in inflammatory responses under various pathological conditions. The down-regulation of CB2R activity is an important marker of inflammation and and CB2R modulators have been shown to have anti-inflammatory effects. This study explored the relationship between CB2R and inflammatory responses, delved into its regulatory mechanisms in inflammatory diseases, and summarized the research progress on CB2R modulators from plants other than cannabis, including plant extracts and monomeric compounds, in exerting anti-inflammatory effects. The aim is to provide new insights into the prevention and treatment of inflammatory diseases.

Coordinated Regulation of CB1 Cannabinoid Receptors and Anandamide Metabolism Stabilizes Network Activity during Homeostatic Downscaling.

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the postsynaptic compartments to regulate synaptic transmission, plasticity, and neuronal excitability primarily through retrograde activation of presynaptic cannabinoid receptor type 1 (CB1). The eCB system is well situated to regulate neuronal network properties and coordinate presynaptic and postsynaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. To address this issue, we used Western blotting and targeted lipidomics to measure adaptations in eCB system to bicuculline (BCC)-induced chronic hyperexcitation in mature cultured rat cortical neurons, and used multielectrode array (MEA) recording and live-cell imaging of glutamate dynamics to test the effects of pharmacological manipulations of eCB on network activities. We show that BCC-induced chronic hyperexcitation triggers homeostatic downscaling and a coordinated adaptation to enhance tonic eCB signaling. Hyperexcitation triggers first the downregulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide, then an accumulation of anandamide and related metabolites, and finally a delayed upregulation of surface and total CB1. Additionally, we show that BCC-induced downregulation of surface AMPA-type glutamate receptors (AMPARs) and upregulation of CB1 occur through independent mechanisms. Finally, we show that endocannabinoids support baseline network activities before and after downscaling and is engaged to suppress network activity during adaptation to hyperexcitation. We discuss the implications of our findings in the context of downscaling and homeostatic regulation of in vitro oscillatory network activities.

Special issue editorial: Cannabinoid signalling in the brain: New vistas.

The endocannabinoid system is widely expressed both in the brain and in the periphery. This system regulates a plethora of physiological functions and is composed of cannabinoid receptors, their endogenous ligands, and the enzymes involved in their metabolic processes. In the last few years, the development of new imaging and molecular tools has demonstrated that these receptors are distributed in many cell types (e.g., neuronal or glial cells) and intracellular compartments (e.g., mitochondria). Interestingly, cellular or molecular effects are differentially mediated by cannabinoid receptors according to their specific localization in different cell-types or in different subcellular locations. Moreover, the endocannabinoid system is also expressed throughout the body where it can serve to modulate the connection between the brain and the periphery. Finally, better understanding of the cannabinoid receptors structure and pharmacology has led researchers to propose interesting and new allosteric modulators of synaptic communication. The latest advances and innovative research in the cannabinoid field will provide new insights and better approaches to improve its interesting potential therapeutic profile. This special issue intends to bring together a series of empirical papers, targeted reviews and opinions from leaders in the field that will highlight the new advances in cannabinoid research.

Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects.

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects-including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism-without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors-ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators.

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-ß-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

Cannabidiol and substance use disorder: Dream or reality.

BACKGROUND: Cannabidiol (CBD) is one of the major constituents of Cannabis sativa L. that lacks psychotomimetic and rewarding properties and inhibits the rewarding and reinforcing effects of addictive drugs such as cocaine, methamphetamine (METH), and morphine. Additionally, CBD's safety profile and therapeutic potential are currently evaluated in several medical conditions, including pain, depression, movement disorders, epilepsy, multiple sclerosis, Alzheimer's disease, ischemia, and substance use disorder. There is no effective treatment for substance use disorders such as addiction, and this review aims to describe preclinical and clinical investigations into the effects of CBD in various models of opioid, psychostimulant, cannabis, alcohol, and nicotine abuse. Furthermore, the possible mechanisms underlying the therapeutic potential of CBD on drug abuse disorders are reviewed. METHODS: The current review considers and summarizes the preclinical and clinical investigations into CBD's effects in various models of drug abuse include opioids, psychostimulants, cannabis, alcohol, and nicotine. RESULTS: Several preclinical and clinical studies have proposed that CBD may be a reliable agent to inhibit the reinforcing and rewarding impact of drugs. CONCLUSIONS: While the currently available evidence converges to suggest that CBD could effectively reduce the rewarding and reinforcing effects of addictive drugs, more preclinical and clinical studies are needed before CBD can be added to the therapeutic arsenal for treating addiction.

The neuropharmacology of cannabinoid receptor ligands in central signaling pathways.

The endocannabinoid system is a complex neuronal system involved in a number of biological functions, like attention, anxiety, mood, memory, appetite, reward, and immune responses. It is at the centre of scientific interest, which is driven by therapeutic promise of certain cannabinoid ligands and the changing legalization of herbal cannabis in many countries. The endocannabinoid system is a modulatory system, with endocannabinoids as retrograde neurotransmitters rather than direct neurotransmitters. Neuropharmacology of cannabinoid ligands in the brain can therefore be understood in terms of their modulatory actions through other neurotransmitter systems. The CB1 receptor is chiefly responsible for effects of endocannabinoids and analogous ligands in the brain. An overview of the neuropharmacology of several cannabinoid receptor ligands, including endocannabinoids, herbal cannabis and synthetic cannabinoid receptor ligands is given in this review. Their mechanism of action at the endocannabinoid system is described, mainly in the brain. In addition, effects of cannabinoid ligands on other neurotransmitter systems will also be described, such as dopamine, serotonin, glutamate, noradrenaline, opioid, and GABA. In light of this, therapeutic potential and adverse effects of cannabinoid receptor ligands will also be discussed.

Preclinical investigation of ß-caryophyllene as a therapeutic agent in an experimental murine model of Dravet syndrome.

Dravet Syndrome (DS) is caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel Nav1.1, which results in febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved for the management of seizures in DS patients, but it appears to be also active against associated comorbidities. In this new study, we have investigated ß-caryophyllene (BCP), a cannabinoid with terpene structure that appears to also have a broad-spectrum profile, as a useful therapy against both seizuring activity and progression of associated comorbidities. This has been studied in heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the Central Nervous System (Syn-Cre/Scn1aWT/A1783V), using two experimental approaches. In the first approach, an acute treatment with BCP was effective against seizuring activity induced by pentylenetetrazole (PTZ) in wildtype (Scn1aWT/WT) and also in Syn-Cre/Scn1aWT/A1783V mice, with these last animals having a greater susceptibility to PTZ. Such benefits were paralleled by a BCP-induced reduction in PTZ-induced reactive astrogliosis (labelled with GFAP) and microgliosis (labelled with Iba-1) in the prefrontal cortex and the hippocampal dentate gyrus, which were visible in both wildtype (Scn1aWT/WT) and Syn-Cre/Scn1aWT/A1783V mice. In the second approach, both genotypes were treated repeatedly with BCP to investigate its effects on several DS comorbidities. Thus, BCP corrected important behavioural abnormalities of Syn-Cre/Scn1aWT/A1783V mice (e.g. delayed appearance of hindlimb grasp reflex, induction of clasping response, motor hyperactivity, altered social interaction and memory impairment), attenuated weight loss, and slightly delayed premature mortality. Again, these benefits were paralleled by a BCP-induced reduction in reactive astrogliosis and microgliosis in the prefrontal cortex and the hippocampal dentate gyrus typical of Syn-Cre/Scn1aWT/A1783V mice. In conclusion, BCP was active in Syn-Cre/Scn1aWT/A1783V mice against seizuring activity (acute treatment) and against several comorbidities (repeated treatment), in both cases in association with its capability to reduce glial reactivity in areas related to these behavioural abnormalities. This situates BCP in a promising position for further preclinical evaluation towards a close translation to DS patients.

Cannabis use impacts pre-stimulus neural activity in the visual cortices of people with HIV.

People with HIV (PWH) use cannabis at a higher rate than the general population, but the influence on neural activity is not well characterized. Cannabis use among PWH may have a beneficial effect, as neuroinflammation is known to be a critical problem in PWH and cannabis use has been associated with a reduction in proinflammatory markers. Thus, it is important to understand the net impact of cannabis use on brain and cognitive function in PWH. In this study, we collected magnetoencephalographic (MEG) brain imaging data on 81 participants split across four demographically matched groups (i.e., PWH using cannabis, controls using cannabis, non-using PWH, and non-using controls). Participants completed a visuospatial processing task during MEG. Time-frequency resolved voxel time series were extracted to identify the dynamics of oscillatory and pre-stimulus baseline neural activity. Our results indicated strong theta (4-8 Hz), alpha (10-16 Hz), and gamma (62-72 Hz) visual oscillations in parietal-occipital brain regions across all participants. PWH exhibited significant behavioral deficits in visuospatial processing, as well as reduced theta oscillations and elevated pre-stimulus gamma activity in visual cortices, all of which replicate prior work. Strikingly, chronic cannabis use was associated with a significant reduction in pre-stimulus gamma activity in the visual cortices, such that PWH no longer statistically differed from controls. These results provide initial evidence that cannabis use may normalize some neural aberrations in PWH. This study fills an important gap in understanding the impact of cannabis use on brain and cognitive function in PWH.

Endocannabinoids, cannabinoids and the regulation of anxiety.

Cannabis has been used for hundreds of years, with its ability to dampen feelings of anxiety often reported as a primary reason for use. Only recently has the specific role cannabinoids play in anxiety been thoroughly investigated. Here we discuss the body of evidence describing how endocannabinoids and exogenous cannabinoids are capable of regulating the generation and termination of anxiety states. Disruption of the endogenous cannabinoid (eCB) system following genetic manipulation, pharmacological intervention or stress exposure reliably leads to the generation of an anxiety state. On the other hand, upregulation of eCB signaling is capable of alleviating anxiety-like behaviors in multiple paradigms. When considering exogenous cannabinoid administration, cannabinoid receptor 1 (CB1) agonists have a biphasic, dose-dependent effect on anxiety such that low doses are anxiolytic while high doses are anxiogenic, a phenomenon that is evident in both rodent models and humans. Translational studies investigating a loss of function mutation in the gene for fatty acid amide hydrolase, the enzyme responsible for metabolizing AEA, have also shown that AEA signaling regulates anxiety in humans. Taken together, evidence reviewed here has outlined a convincing argument for cannabinoids being powerful regulators of both the manifestation and amelioration of anxiety symptoms, and highlights the therapeutic potential of targeting the eCB system for the development of novel classes of anxiolytics. This article is part of the special issue on 'Cannabinoids'.

Cannabinoid Receptor Modulation of Neurogenesis: ST14A Striatal Neural Progenitor Cells as a Simplified In Vitro Model.

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.

Could Cannabidiol Be a Treatment for Coronavirus Disease-19-Related Anxiety Disorders?

Coronavirus disease-19 (COVID-19)-related anxiety and post-traumatic stress symptoms (PTSS) or post-traumatic stress disorder (PTSD) are likely to be a significant long-term issue emerging from the current pandemic. We hypothesize that cannabidiol (CBD), a chemical isolated from Cannabis sativa with reported anxiolytic properties, could be a therapeutic option for the treatment of COVID-19-related anxiety disorders. In the global over-the-counter CBD market, anxiety, stress, depression, and sleep disorders are consistently the top reasons people use CBD. In small randomized controlled clinical trials, CBD (300-800 mg) reduces anxiety in healthy volunteers, patients with social anxiety disorder, those at clinical high risk of psychosis, in patients with Parkinson's disease, and in individuals with heroin use disorder. Observational studies and case reports support these findings, extending to patients with anxiety and sleep disorders, Crohn's disease, depression, and in PTSD. Larger ongoing trials in this area continue to add to this evidence base with relevant patient cohorts, sample sizes, and clinical end-points. Pre-clinical studies reveal the molecular targets of CBD in these indications as the cannabinoid receptor type 1 and cannabinoid receptor type 2 (mainly in fear memory processing), serotonin 1A receptor (mainly in anxiolysis) and peroxisome proliferator-activated receptor gamma (mainly in the underpinning anti-inflammatory/antioxidant effects). Observational and pre-clinical data also support CBD's therapeutic value in improving sleep (increased sleep duration/quality and reduction in nightmares) and depression, which are often comorbid with anxiety. Together these features of CBD make it an attractive novel therapeutic option in COVID-related PTSS that merits investigation and testing through appropriately designed randomized controlled trials.

Cannabis based medicines and cannabis dependence: A critical review of issues and evidence.

Cannabis has been legalised for medical use in an ever-increasing number of countries. A growing body of scientific evidence supports the use of medical cannabis for a range of therapeutic indications. In parallel with these developments, concerns have been expressed by many prescribers that increased use will lead to patients developing cannabis use disorder. Cannabis use disorder has been widely studied in recreational users, and these findings have often been projected onto patients using medical cannabis. However, studies exploring medical cannabis dependence are scarce and the appropriate methodology to measure this construct is uncertain. This article provides a narrative review of the current research to discern if, how and to what extent, concerns about problems of dependence in recreational cannabis users apply to prescribed medical users. We focus on the main issues related to medical cannabis and dependence, including the importance of dose, potency, cannabinoid content, pharmacokinetics and route of administration, frequency of use, as well as set and setting. Medical and recreational cannabis use differs in significant ways, highlighting the challenges of extrapolating findings from the recreational cannabis literature. There are many questions about the potential for medical cannabis use to lead to dependence. It is therefore imperative to address these questions in order to be able to minimise harms of medical cannabis use. We draw out seven recommendations for increasing the safety of medical cannabis prescribing. We hope that the present review contributes to answering some of the key questions surrounding medical cannabis dependence.

Relevance of Peroxisome Proliferator Activated Receptors in Multitarget Paradigm Associated with the Endocannabinoid System.

Cannabinoids have shown to exert their therapeutic actions through a variety of targets. These include not only the canonical cannabinoid receptors CB1R and CB2R but also related orphan G protein-coupled receptors (GPCRs), ligand-gated ion channels, transient receptor potential (TRP) channels, metabolic enzymes, and nuclear receptors. In this review, we aim to summarize reported compounds exhibiting their therapeutic effects upon the modulation of CB1R and/or CB2R and the nuclear peroxisome proliferator-activated receptors (PPARs). Concomitant actions at CBRs and PPARα or PPARγ subtypes have shown to mediate antiobesity, analgesic, antitumoral, or neuroprotective properties of a variety of phytogenic, endogenous, and synthetic cannabinoids. The relevance of this multitargeting mechanism of action has been analyzed in the context of diverse pathologies. Synergistic effects triggered by combinatorial treatment with ligands that modulate the aforementioned targets have also been considered. This literature overview provides structural and pharmacological insights for the further development of dual cannabinoids for specific disorders.