Cannabinoid Type 1 Receptor Activation Research Articles

Cannabidiol ameliorates PTSD-like symptoms by inhibiting neuroinflammation through its action on CB2 receptors in the brain of male mice

Post-traumatic stress disorder (PTSD) is a debilitating mental health disease related to traumatic experience, and its treatment outcomes are unsatisfactory. Accumulating research has indicated that cannabidiol (CBD) exhibits anti-PTSD effects, however, the underlying mechanism of CBD remains inadequately investigated. Although many studies pertaining to PTSD have primarily focused on aberrations in neuronal functioning, the present study aimed to elucidate the involvement and functionality of microglia/macrophages in PTSD while also investigated the modulatory effects of CBD on neuroinflammation associated with this condition. We constructed a modified single-prolonged stress (SPS) mice PTSD model and verified the PTSD-related behaviors by various behavioral tests (contextual freezing test, elevated plus maze test, tail suspension test and novel object recognition test). We observed a significant upregulation of Iba-1 and alteration of microglial/macrophage morphology within the prefrontal cortex and hippocampus, but not the amygdala, two weeks after the PTSD-related stress, suggesting a persistent neuroinflammatory phenotype in the PTSD-modeled group. CBD (10 mg/kg, i.p.) inhibited all PTSD-related behaviors and reversed the alterations in both microglial/macrophage quantity and morphology when administered prior to behavioral assessments. We further found increased pro-inflammatory factors, decreased PSD95 expression, and impaired synaptic density in the hippocampus of the modeled group, all of which were also restored by CBD treatment. CBD dramatically increased the level of anandamide, one of the endocannabinoids, and cannabinoid type 2 receptors (CB2Rs) transcripts in the hippocampus compared with PTSD-modeled group. Importantly, we discovered the expression of CB2Rs mRNA in Arg-1-positive cells in vivo and found that the behavioral effects of CBD were diminished by CB2Rs antagonist AM630 (1 mg/kg, i.p.) and both the behavioral and molecular effects of CBD were abolished in CB2Rs knockout mice. These findings suggest that CBD would alleviate PTSD-like behaviors in mice by suppressing PTSD-related neuroinflammation and upregulation and activation of CB2Rs may serve as one of the underlying mechanisms for this therapeutic effect. The present study offers innovative experimental evidence supporting the utilization of CBD in PTSD treatment from the perspective of its regulation of neuroinflammation, and paves the way for leveraging the endocannabinoid system to regulate neuroinflammation as a potential therapeutic approach for psychiatric disorders.

Adenosine receptors are the on-and-off switch of astrocytic cannabinoid type 1 (CB1) receptor effect upon synaptic plasticity in the medial prefrontal cortex.

The medial prefrontal cortex (mPFC) is involved in cognitive functions such as working memory. Astrocytic cannabinoid type 1 receptor (CB1R) induces cytosolic calcium (Ca2+) concentration changes with an impact on neuronal function. mPFC astrocytes also express adenosine A1 and A2A receptors (A1R, A2AR), being unknown the crosstalk between CB1R and adenosine receptors in these cells. We show here that a further level of regulation of astrocyte Ca2+ signaling occurs through CB1R-A2AR or CB1R-A1R heteromers that ultimately impact mPFC synaptic plasticity. CB1R-mediated Ca2+ transients increased and decreased when A1R and A2AR were activated, respectively, unveiling adenosine receptors as modulators of astrocytic CB1R. CB1R activation leads to an enhancement of long-term potentiation (LTP) in the mPFC, under the control of A1R but not of A2AR. Notably, in IP3R2KO mice, that do not show astrocytic Ca2+ level elevations, CB1R activation decreases LTP, which is not modified by A1R or A2AR. The present work suggests that CB1R has a homeostatic role on mPFC LTP, under the control of A1R, probably due to physical crosstalk between these receptors in astrocytes that ultimately alters CB1R Ca2+ signaling.

Activation of Cannabinoid Type 2 Receptor in Microglia Reduces Neuroinflammation through Inhibiting Aerobic Glycolysis to Relieve Hypertension.

Studies have shown that the chronic use of cannabis is associated with a decrease in blood pressure. Our previous studies prove that activating the cannabinoid type 2 (CB2) receptor in the brain can effectively reduce blood pressure in spontaneously hypertensive rats; however, the exact mechanism has not been clarified. The objective of this study is to demonstrate that activation of microglial CB2 receptors can effectively reduce the levels of TNF-α, IL-1β, and IL-6 in the paraventricular nucleus (PVN) through inhibiting aerobic glycolysis, thereby relieving hypertension. AngiotensinII (AngII) was administered to BV2 cells and C57 mice to induce hypertension and the release of proinflammatory cytokines. The mRNA and protein expression of the CB2 receptor, TNF-α, IL-1β, IL-6, and the PFK and LDHa enzymes were detected using RT-qPCR and Western blotting. The Seahorse XF Energy Metabolism Analyzer was used to measure the oxidative phosphorylation and aerobic glycolysis metabolic pathways in BV2 cells. The long-term effects of injecting JWH133, a selective CB2 receptor agonist, intraperitoneally on blood pressure were ascertained. ELISA was used to measure norepinephrine and lactic acid levels while immunofluorescence labeling was used to locate the CB2 receptor and c-Fos. By injecting pAAV-F4/80-GFP-mir30shRNA (AAV2-r-CB2shRNA) into the lateral cerebral ventricle, the CB2 receptor in microglia was specifically knocked down. Activation of CB2 receptors by the agonist JWH133 suppressed TNF-α, IL-1β, and IL-6 by inhibiting PFK and LDHa enzymes involved in glycolysis, as well as lactic acid accumulation, along with a reduction in glycoPER levels (marks of aerobic glycolysis) in AngII-treated BV2 cells. In AngII-treated mice, the administration of JWH133 specifically activated CB2 receptors on microglia, resulting in decreased expression levels of PFK, LDHa, TNF-α, IL-1β, and IL-6, subsequently leading to a decrease in c-Fos protein expression within PVN neurons as well as reduced norepinephrine levels in plasma, ultimately contributing to blood pressure reduction. The results suggest that activation of the microglia CB2 receptor decreases the neuroinflammation to relieve hypertension; the underlying mechanism is related to inhibiting aerobic glycolysis of microglia.

Electroacupuncture Exerts Analgesic Effects by Restoring Hyperactivity via Cannabinoid Type 1 Receptors in the Anterior Cingulate Cortex in Chronic Inflammatory Pain.

As one of the commonly used therapies for pain-related diseases in clinical practice, electroacupuncture (EA) has been proven to be effective. In chronic pain, neurons in the anterior cingulate cortex (ACC) have been reported to be hyperactive, while the mechanism by which cannabinoid type 1 receptors (CB1Rs) in the ACC are involved in EA-mediated analgesic mechanisms remains to be elucidated. In this study, we investigated the potential central mechanism of EA analgesia. A combination of techniques was used to detect the expression and function of CB1R, including quantitative real-time PCR (q-PCR), western blot (WB), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and in vivo multichannel optical fibre recording, and neuronal activity was examined by in vivo two-photon imaging and in vivo electrophysiological recording. We found that the hyperactivity of pyramidal neurons in the ACC during chronic inflammatory pain is associated with impairment of the endocannabinoid system. EA at the Zusanli acupoint (ST36) can reduce the hyperactivity of pyramidal neurons and exert analgesic effects by increasing the endocannabinoid ligands anandamide (AEA), 2-arachidonoylglycerol (2-AG) and CB1R. More importantly, CB1R in the ACC is one of the necessary conditions for the EA-mediated analgesia effect, which may be related to the negative regulation of the N-methyl-D-aspartate receptor (NMDAR) by the activation of CB1R downregulating NR1 subunits of NMDAR (NR1) via histidine triad nucleotide-binding protein 1 (HINT1). Our study suggested that the endocannabinoid system in the ACC plays an important role in acupuncture analgesia and provides evidence for a central mechanism of EA-mediated analgesia.

Activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in a chronic migraine rat model

BackgroundCentral sensitization has been widely accepted as an underlying pathophysiological mechanism of chronic migraine (CM), activation of cannabinoid type-1 receptor (CB1R) exerts antinociceptive effects by relieving central sensitization in many pain models. However, the role of CB1R in the central sensitization of CM is still unclear.MethodsA CM model was established by infusing inflammatory soup (IS) into the dura of male Wistar rats for 7 days, and hyperalgesia was assessed by the mechanical and thermal thresholds. In the periaqueductal gray (PAG), the mRNA and protein levels of CB1R and hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) were measured by qRT–PCR and western blotting. After intraventricular injection of Noladin ether (NE) (a CB1R agonist), ZD 7288 (an HCN2 blocker), and AM 251 (a CB1R antagonist), the expression of tyrosine phosphorylation of N-methyl-D-aspartate receptor subtype 2B (pNR2B), calcium-calmodulin-dependent kinase II (CaMKII), and phosphorylated cAMP-responsive element binding protein (pCREB) was detected, and central sensitization was evaluated by the expression of calcitonin gene-related peptide (CGRP), c-Fos, and substance P (SP). Synaptic-associated protein (postsynaptic density protein 95 (PSD95) and synaptophysin (Syp)) and synaptic ultrastructure were detected to explore synaptic plasticity in central sensitization.ResultsWe observed that the mRNA and protein levels of CB1R and HCN2 were both significantly increased in the PAG of CM rats. The application of NE or ZD 7288 ameliorated IS-induced hyperalgesia; repressed the pNR2B/CaMKII/pCREB pathway; reduced CGRP, c-Fos, SP, PSD95, and Syp expression; and inhibited synaptic transmission. Strikingly, the application of ZD 7288 relieved AM 251-evoked elevation of pNR2B, CGRP, and c-Fos expression.ConclusionsThese data reveal that activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in CM rats. The activation of CB1R might have a positive influence on the prevention of CM by mitigating central sensitization.

Cannabinoid type 2 receptor activation inhibits MPP+-induced M1 differentiation of microglia through activating PI3K/Akt/Nrf2 signal pathway.

Growing evidence indicates that cannabinoid type 2 (CB2) receptor activation inhibits neuroinflammation in the pathogenesis of Parkinson's disease (PD). Nonetheless, the precise mechanisms of CB2 receptor-mediated neuroprotection have not been fully elucidated. The differentiation of microglia from the M1 to M2 phenotype plays a vital role in neuroinflammation. In the present study, we investigated the effect of CB2 receptor activation on the M1/M2 phenotypic transformation of microglia treated with 1-methyl-4-phenylpyridinium (MPP+). The M1 phenotype microglia markers, including inducible nitric oxide (iNOS), interleukin 6 (IL-6), and CD86, and the M2 phenotype microglia markers, including arginase-1 (Arg-1), IL-10, and CD206, were detected by western blots and flow cytometry. The levels of phosphoinositide-3-kinase (PI3K)/Akt and nuclear factor erythroid 2-related factor 2 (Nrf2) were determined by Western blots. Subsequent addition of Nrf2 inhibitors initially revealed the specific mechanism by which CB2 receptors affect phenotypic changes in microglia. Our results showed that pretreatment with JWH133 significantly inhibited the MPP+-induced up-regulation of M1 phenotype microglia markers. Meanwhile, JWH133 increased the levels of M2 phenotype microglia markers. JWH133-mediated effects were blocked by co-treatment with AM630. Mechanism studies found that MPP+ treatment downregulated PI3K, Akt phosphorylated proteins, and nuclear Nrf2 protein. JWH133 pretreatment promoted PI3K/Akt activation and facilitated nuclear translocation of Nrf2, which was reversed by the PI3K inhibitor. Further studies showed that Nrf2 inhibitors inverted the effect of JWH133 on microglia polarization. The results indicate that CB2 receptor activation promotes MPP+-induced microglia transformation from M1 to M2 phenotype through PI3K/Akt/Nrf2 signaling pathway.

CB2R activation ameliorates late adolescent chronic alcohol exposure-induced anxiety-like behaviors during withdrawal by preventing morphological changes and suppressing NLRP3 inflammasome activation in prefrontal cortex microglia in mice

BackgroundChronic alcohol exposure (CAE) during late adolescence increases the risk of anxiety development. Alcohol-induced prefrontal cortex (PFC) microglial activation, characterized by morphological changes and increased associations with neurons, plays a critical role in the pathogenesis of anxiety. Alcohol exposure increases NLRP3 inflammasome expression, increasing cytokine secretion by activated microglia. Cannabinoid type 2 receptor (CB2R), an essential receptor of the endocannabinoid system, regulates microglial activation and neuroinflammatory reactions. We aimed to investigate the role of CB2R activation in ameliorating late adolescent CAE-induced anxiety-like behaviors and microglial activation in C57BL/6J mice. MethodsSix-week-old C57BL/6J mice were acclimated for 7 days and then were administered alcohol by gavage (4 g/kg, 25 % w/v) for 28 days. The mice were intraperitoneally injected with the specific CB2R agonist AM1241 1 h before alcohol treatment. Anxiety-like behaviors during withdrawal were assessed by open field test and elevated plus maze test 24 h after the last alcohol administration. Microglial activation, microglia-neuron interactions, and CB2R and NLRP3 inflammasome-related molecule expression in the PFC were measured using immunofluorescence, immunohistochemical, qPCR, and Western blotting assays. Microglial morphology was evaluated by Sholl analysis and the cell body-to-total cell size index. Additionally, N9 microglia were activated by LPS in vitro, and the effects of AM1241 on NLRP3 and N9 microglial activation were investigated. ResultsAfter CAE, mice exhibited severe anxiety-like behaviors during withdrawal. CAE induced obvious microglia-neuron associations, and increased expression of microglial activation markers, CB2R, and NLRP3 inflammasome-related molecules in the PFC. Microglia also showed marked filament retraction and reduction and cell body enlargement after CAE. AM1241 treatment ameliorated anxiety-like behaviors in CAE model mice, and it prevented microglial morphological changes, reduced microglial activation marker expression, and suppressed the microglial NLRP3 inflammasome activation and proinflammatory cytokine secretion induced by CAE. AM1241 suppressed the LPS-induced increase in NLRP3 inflammasome-related molecules, IL-1β release, and M1 phenotype markers (iNOS and CD86) in N9 cell, which was reversed by CB2R antagonist treatment. ConclusionsCAE caused anxiety-like behaviors in late adolescent mice at least partly by inducing microglial activation and increasing microglia-neuron associations in the PFC. CB2R activation ameliorated these effects by preventing morphological changes and suppressing NLRP3 inflammasome activation in PFC microglia.

Development of N-(1-Adamantyl)benzamides as Novel Anti-Inflammatory Multitarget Agents Acting as Dual Modulators of the Cannabinoid CB2 Receptor and Fatty Acid Amide Hydrolase.

Cannabinoid type 2 receptor (CB2R), belonging to the endocannabinoid system, is overexpressed in pathologies characterized by inflammation, and its activation counteracts inflammatory states. Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the degradation of the main endocannabinoid anandamide; thus, the simultaneous CB2R activation and FAAH inhibition may be a synergistic anti-inflammatory strategy. Encouraged by principal component analysis (PCA) data identifying a wide chemical space shared by CB2R and FAAH ligands, we designed a small library of adamantyl-benzamides, as potential dual agents, CB2R agonists, and FAAH inhibitors. The new compounds were tested for their CB2R affinity/selectivity and CB2R and FAAH activity. Derivatives 13, 26, and 27, displaying the best pharmacodynamic profile as CB2R full agonists and FAAH inhibitors, decreased pro-inflammatory and increased anti-inflammatory cytokines production. Molecular docking simulations complemented the experimental findings by providing a molecular rationale behind the observed activities. These multitarget ligands constitute promising anti-inflammatory agents.

Synthetic cannabinoids reduce the inflammatory activity of microglia and subsequently improve neuronal survival in vitro

Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB1) or type 2 (CB2) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdhQ7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB1 receptors (ACEA) or CB2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB1 or CB2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons.

ACPA Alleviates Bleomycin-Induced Pulmonary Fibrosis by Inhibiting TGF-β-Smad2/3 Signaling-Mediated Lung Fibroblast Activation.

Pulmonary fibrosis is a group of life-threatening diseases with limited therapeutic options. The involvement of cannabinoid type 1 receptors (CB1R) has been indicated in fibrotic diseases, but whether or not the activation of CB1R can be a benefit for fibrosis treatment is controversial. In this study, we investigated the effects of arachidonoylcyclopropylamide (ACPA), as a selective CB1R agonist, on bleomycin (BLM)-induced pulmonary fibrosis. We showed that ACPA treatment significantly improved the survival rate of BLM-treated mice, alleviated BLM-induced pulmonary fibrosis, and inhibited the expressions of extracellular matrix (ECM) markers, such as collagen, fibronectin, and α-SMA. The enhanced expressions of ECM markers in transforming growth factor-beta (TGF-β)-challenged primary lung fibroblasts isolated from mouse lung tissues were inhibited by ACPA treatment in a dose-dependent manner, and the fibroblast migration triggered by TGF-β was dose-dependently diminished after ACPA administration. Moreover, the increased mRNA levels of CB1R were observed in both lung fibroblasts of BLM-induced fibrotic mice in vivo and TGF-β-challenged primary lung fibroblasts in vitro. CB1R-specific agonist ACPA significantly diminished the activation of TGF-β–Smad2/3 signaling, i.e., the levels of p-Smad2 and p-Smad3, and decreased the expressions of downstream effector proteins including slug and snail, which regulate ECM production, in TGF-β-challenged primary lung fibroblasts. Collectively, these findings demonstrated that CB1R-specific agonist ACPA exhibited antifibrotic efficacy in both in vitro and in vivo models of pulmonary fibrosis, revealing a novel anti-fibrosis approach to fibroblast-selective inhibition of TGF-β-Smad2/3 signaling by targeting CB1R.

Molecular signaling of synthetic cannabinoids: Comparison of CB1 receptor and TRPV1 channel activation

Recreational use of synthetic cannabinoids (SCs) is associated with desirable euphoric and relaxation effects as well as adverse effects including anxiety, agitation and psychosis. These SC-mediated actions represent a combination of potentiated cannabinoid receptor signaling and “off-target” receptor activity. The goal of this study was to compare the efficacy of various classes of SCs in stimulating CB1 receptors and activating “off-target” transient receptor potential (TRP) channels. Cannabinoid-type 1 (CB1) receptor activity was determined by measuring SC activation of G protein-gated inward rectifier K+ (GIRK) channels using a membrane potential-sensitive fluorescent dye assay. SC opening of vanilloid type-1 (TRPV1) channels was measured by recording intracellular Ca2+ transients. All of the SCs tested activated the GIRK channel with an efficacy of 4-fluoro MDMB-BUTINACA > 5-fluoro MDMB-PICA > MDMB-4en-PINACA ≈ WIN 55,212-2 > AB-FUBINACA > AM1220 ≈ JWH-122 N-(5-chloropentyl) > AM1248 > JWH-018 ≈ XLR-11 ≈ UR-144. The potency of the SCs at the CB1 receptor was 5-fluoro MDMB-PICA ≈ 4-fluoro MDMB-BUTINACA > AB-FUBINACA ≈ MDMB-4en-PINACA > JWH-018 > AM1220 > XLR-11 > JWH-122 N-(5-chloropentyl) > WIN 55,212-2 ≈ UR-144 > AM1248. In contrast, when tested at a SC concentration that produced a maximal effect on the Gi/GIRK channel, only XLR-11, UR-144 and AM1220 caused a significant activation of the TRPV1 channels. The TRPV1 channel/Ca2+ signal measured during application of 10 μM XLR-11 was similar to the signal induced by the endocannabinoid N-arachidonoylethanolamine (AEA). Thus, while various SCs share the ability to stimulate CB1 receptor/Gi signaling, they display limited efficacy in opening TRPV1 channels.

CB1R activation in nucleus accumbens core promotes stress-induced reinstatement of cocaine seeking by elevating extracellular glutamate in a drug-paired context

Preclinical models of stress-induced relapse to drug use have shown that the dysregulation of glutamatergic transmission within the nucleus accumbens (NA) contributes notably to the reinstatement of cocaine-seeking behavior in rodents. In this sense, there has been increasing interest in the cannabinoid type-1 receptor (CB1R), due to its crucial role in modulating glutamatergic neurotransmission within brain areas involved in drug-related behaviors. This study explored the involvement of CB1R within the NA subregions in the restraint stress-induced reinstatement of cocaine-conditioned place preference (CPP), as well as in the regulation of glutamatergic transmission, by using a pharmacological approach and the in vivo microdialysis sampling technique in freely moving rats. CB1R blockade by the antagonist/inverse agonist AM251 (5 nmol/0.5 μl/side) or CB1R activation by the agonist ACEA (0.01 fmol/0.5 μl/side), prevented or potentiated restraint stress-induced reinstatement of cocaine-CPP, respectively, after local administration into NAcore, but not NAshell. In addition, microdialysis experiments demonstrated that restraint stress elicited a significant increase in extracellular glutamate in NAcore under reinstatement conditions, with the local administration of AM251 or ACEA inhibiting or potentiating this, respectively. Interestingly, this rise specifically corresponded to the cocaine-associated CPP compartment. We also showed that this context-dependent change in glutamate paralleled the expression of cocaine-CPP, and disappeared after the extinction of this response. Taken together, these findings demonstrated the key role played by CB1R in mediating reinstatement of cocaine-CPP after restraint stress, through modulation of the context-specific glutamate release within NAcore. Additionally, CB1R regulation of basal extracellular glutamate was demonstrated and proposed as the underlying mechanism.

Cocaine-induced increases in motivation require 2-arachidonoylglycerol mobilization and CB1 receptor activation in the ventral tegmental area

A wide body of evidence supports an integral role for mesolimbic dopamine (DA) in motivated behavior. In brief, drugs that increase DA in mesolimbic terminal regions, like cocaine, enhance motivation, while drugs that decrease DA concentration reduce motivation. Data from our laboratory and others shows that phasic activation of mesolimbic DA requires signaling at cannabinoid type-1 (CB1) receptors in the ventral tegmental area (VTA), and systemic delivery of CB1 receptor antagonists reduces DA cell activity and attenuates motivated behaviors. Recent findings demonstrate that cocaine mobilizes the endocannabinoid 2-arachidonoylglycerol (2-AG) in the VTA to cause phasic activation of DA neurons and terminal DA release. It remains unclear, however, if cocaine-induced midbrain 2-AG signaling contributes to the motivation-enhancing effects of cocaine. To examine this, we trained male and female rats on a progressive ratio (PR) task for a food reinforcer. Each rat underwent a series of tests in which they were pretreated with cocaine alone or in combination with systemic or intra-VTA administration of the CB1 receptor antagonist rimonabant or the 2-AG synthesis inhibitor tetrahydrolipstatin (THL). Cocaine increased motivation, measured by augmented PR breakpoints, while rimonabant dose-dependently decreased motivation. Importantly, intra-VTA administration of rimonabant or THL, at doses that did not decrease breakpoints on their own, blocked systemic cocaine administration from increasing breakpoints in male and female rats. These data suggest that cocaine-induced increases in motivation require 2-AG signaling at CB1 receptors in the VTA and may provide critical insight into cannabinoid-based pharmacotherapeutic targets for the successful treatment of substance abuse.

Contrasting Roles of Ang II and ACEA in the Regulation of IL10 and IL1β Gene Expression in Primary SHR Astroglial Cultures.

Angiotensin (Ang) II is well-known to have potent pro-oxidant and pro-inflammatory effects in the brain. Extensive crosstalk between the primary Ang II receptor, Ang type 1 receptor (AT1R), and the cannabinoid type 1 receptor (CB1R) has been demonstrated by various groups in the last decade. Since activation of glial CB1R has been demonstrated to play a key role in the resolution of inflammatory states, we investigated the role of Ang II (100 nM) and/or ACEA (10 nM), a potent CB1R-specific agonist in the regulation of inflammatory markers in astrocytes from spontaneously hypertensive rats (SHR) and Wistar rats. Astrocytes were cultured from brainstems and cerebellums of SHR and Wistar rats and assayed for IL1β and IL10 gene expression and secreted fraction, in treated and non-treated cells, by employing qPCR and ELISA, respectively. mRNA expression of both IL10 and IL1β were significantly elevated in untreated brainstem and cerebellar astrocytes isolated from SHR when compared to Wistar astrocytes. No changes were observed in the secreted fraction. While ACEA-treatment resulted in a significant increase in IL10 gene expression in Wistar brainstem astrocytes (Log2FC ≥ 1, p < 0.05), its effect in SHR brainstem astrocytes was diminished. Ang II treatment resulted in a strong inhibitory effect on IL10 gene expression in astrocytes from both brain regions of SHR and Wistar rats (Log2FC ≤ −1, p < 0.05), and an increase in IL1β gene expression in brainstem astrocytes from both strains (Log2FC ≥ 1, p < 0.05). Co-treatment of Ang II and ACEA resulted in neutralization of Ang II-mediated effect in Wistar brainstem and cerebellar astrocytes, but not SHR astrocytes. Neither Ang II nor ACEA resulted in any significant changes in IL10 or IL1β secreted proteins. These data suggest that Ang II and ACEA have opposing roles in the regulation of inflammatory gene signature in astrocytes isolated from SHR and Wistar rats. This however does not translate into changes in their secreted fractions.

Activation of astroglial CB1R mediates cerebral ischemic tolerance induced by electroacupuncture

There are no effective treatments for stroke. The activation of endogenous protective mechanisms is a promising therapeutic approach, which evokes the intrinsic ability of the brain to protect itself. Accumulated evidence strongly suggests that electroacupuncture (EA) pretreatment induces rapid tolerance to cerebral ischemia. With regard to mechanisms underlying ischemic tolerance induced by EA, many molecules and signaling pathways are involved, such as the endocannabinoid system, although the exact mechanisms have not been fully elucidated. In the current study, we employed mutant mice, neuropharmacology, microdialysis, and virus transfection techniques in a middle cerebral artery occlusion (MCAO) model to explore the cell-specific and brain region-specific mechanisms of EA-induced neuroprotection. EA pretreatment resulted in increased ambient endocannabinoid (eCB) levels and subsequent activation of ischemic penumbral astroglial cannabinoid type 1 receptors (CB1R) which led to moderate upregulation of extracellular glutamate that protected neurons from cerebral ischemic injury. These findings provide a novel cellular mechanism of EA and a potential therapeutic target for ischemic stroke.

β-Caryophyllene, a natural bicyclic sesquiterpene attenuates β-adrenergic agonist-induced myocardial injury in a cannabinoid receptor-2 dependent and independent manner

The downregulation of cannabinoid type-2 receptors (CB2R) have been reported in numerous diseases including cardiovascular diseases (CVDs). The activation of CB2R has recently emerged as an important therapeutic target to mitigate myocardial injury. We examined whether CB2R activation can protect against isoproterenol (ISO)-induced myocardial injury (MI) in rats. In the present study, we investigated the cardioprotective effect of β-caryophyllene (BCP), a naturally occurring dietary cannabinoid in rat model of MI. Rats were pre- and co-treated with BCP (50 mg/kg, orally) twice daily for 10 days along with subcutaneous injection of ISO (85 mg/kg) at an interval of 24 h for two days (9th and 10th days). AM630 (1 mg/kg), a CB2 receptor antagonist, was injected intraperitoneal as a pharmacological challenge prior to BCP treatment to reveal CB2R-mediated cardioprotective mechanisms of BCP. Desensitization of beta-adrenergic receptor (β-AR) signaling, receptor phosphorylation and recruitment of adapter β-arrestins were observed in ISO-induced MI in rats. ISO injections caused impaired cardiac function, elevated the levels of serum cardiac marker enzymes, and enhanced oxidative stress markers along with altered PI3K/Akt and NrF2/Keap1/HO-1 signaling pathways. ISO also promoted lysosomal dysfunction along with activation of NLRP3 inflammasomes and TLR4-NFκB/MAPK signaling and triggered rise in proinflammatory cytokines. There was a concomitant mitochondrial dysfunction followed by the activation of endoplasmic reticulum (ER) stress-mediated Hippo signaling and intrinsic pathway of apoptosis as well as altered autophagic flux/mTOR signaling in ISO-induced MI. Furthermore, ISO also triggered dyslipidemia evidenced by altered lipids, lipoproteins and lipid marker enzymes along with ionic homeostasis malfunction. However, treatment with BCP resulted in significant protective effects on all biochemical and molecular parameters analyzed. The cardioprotective effects were further strengthened by preservation of cardiomyocytes and cell organelles as observed in histopathological and ultrastructural studies. Interestingly, treatment with AM630, a CB2R antagonist was observed to abrogate the protective effects of BCP on the biochemical and molecular parameters except hyperlipidemia and ionic homeostasis in ISO-induced MI in rats. The present study findings demonstrate that BCP possess the potential to protect myocardium against ISO-induced MI in a CB2-dependent and independent manner.

Short-Term Plasticity in Cortical GABAergic Synapses on Olfactory Bulb Granule Cells Is Modulated by Endocannabinoids

Olfactory bulb and higher processing areas are synaptically interconnected, providing rapid regulation of olfactory bulb circuit dynamics and sensory processing. Short-term plasticity changes at any of these synapses could modulate sensory processing and potentially short-term sensory memory. A key olfactory bulb circuit for mediating cortical feedback modulation is granule cells, which are targeted by multiple cortical regions including both glutamatergic excitatory inputs and GABAergic inhibitory inputs. There is robust endocannabinoid modulation of excitatory inputs to granule cells and here we explored whether there was also endocannabinoid modulation of the inhibitory cortical inputs to granule cells. We expressed light-gated cation channel channelrhodopsin-2 (ChR2) in GABAergic neurons in the horizontal limb of the diagonal band of Broca (HDB) and their projections to granule cells in olfactory bulb. Selective optical activation of ChR2 positive axons/terminals generated strong, frequency-dependent short-term depression of GABAA-mediated-IPSC in granule cells. As cannabinoid type 1 (CB1) receptor is heavily expressed in olfactory bulb granule cell layer (GCL) and there is endogenous endocannabinoid release in GCL, we investigated whether activation of CB1 receptor modulated the HDB IPSC and short-term depression at the HDB→granule cell synapse. Activation of the CB1 receptor by the exogenous agonist Win 55,212-2 significantly decreased the peak amplitude of individual IPSC and decreased short-term depression, while blockade of the CB1 receptor by AM 251 slightly increased individual IPSCs and increased short-term depression. Thus, we conclude that there is tonic endocannabinoid activation of the GABAergic projections of the HDB to granule cells, similar to the modulation observed with glutamatergic projections to granule cells. Modulation of inhibitory synaptic currents and frequency-dependent short-term depression could regulate the precise balance of cortical feedback excitation and inhibition of granule cells leading to changes in granule cell mediated inhibition of olfactory bulb output to higher processing areas.

The impact of cannabinoid type 2 receptors (CB2Rs) in neuroprotection against neurological disorders.

Cannabinoids have long been used for their psychotropic and possible medical properties of symptom relief. In the past few years, a vast literature shows that cannabinoids are neuroprotective under different pathological situations. Most of the effects of cannabinoids are mediated by the well-characterized cannabinoid receptors, the cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R). Even though CB1Rs are highly expressed in the central nervous system (CNS), the adverse central side effects and the development of tolerance resulting from CB1R activation may ultimately limit the clinical utility of CB1R agonists. In contrast to the ubiquitous presence of CB1Rs, CB2Rs are less commonly expressed in the healthy CNS but highly upregulated in glial cells under neuropathological conditions. Experimental studies have provided robust evidence that CB2Rs seem to be involved in the modulation of different neurological disorders. In this paper, we summarize the current knowledge regarding the protective effects of CB2R activation against the development of neurological diseases and provide a perspective on the future of this field. A better understanding of the fundamental pharmacology of CB2R activation is essential for the development of clinical applications and the design of novel therapeutic strategies.

A cannabinoid type 2 (CB2) receptor agonist augments NOS-dependent responses of cerebral arterioles during type 1 diabetes

While activation of cannabinoid (CB2) receptors has been shown to be neuroprotective, no studies have examined whether this neuroprotection is directed at cerebral arterioles and no studies have examined whether activation of CB2 receptors can rescue cerebrovascular dysfunction during a chronic disease state such as type 1 diabetes (T1D). Our goal was to test the hypothesis that administration of a CB2 agonist (JWH-133) would improve impaired endothelial (eNOS)- and neuronal (nNOS)-dependent dilation of cerebral arterioles during T1D. In vivo diameter of cerebral arterioles in nondiabetic and T1D rats was measured in response to an eNOS-dependent agonist (adenosine 5′-diphosphate; ADP), an nNOS-dependent agonist (N-methyl-d-aspartate; NMDA), and an NOS-independent agonist (nitroglycerin) before and 1 h following JWH-133 (1 mg/kg IP). Dilation of cerebral arterioles to ADP and NMDA was greater in nondiabetic than in T1D rats. Treatment with JWH-133 increased responses of cerebral arterioles to ADP and NMDA in both nondiabetic and T1D rats. Responses of cerebral arterioles to nitroglycerin were similar between nondiabetic and T1D rats, and JWH-133 did not influence responses to nitroglycerin in either group. The restoration in responses to the agonists by JWH-133 could be inhibited by treatment with a specific inhibitor of CB2 receptors (AM-630; 3 mg/kg IP). Thus, activation of CB2 receptors can potentiate reactivity of cerebral arterioles during physiologic and pathophysiologic states. We speculate that treatment with CB2 receptor agonists may have potential therapeutic benefits for the treatment of cerebral vascular diseases via a mechanism that can increase cerebral blood flow.

Effects of \u22069-tetrahydrocannabinol on aversive memories and anxiety: a review from human studies

BackgroundPosttraumatic stress disorder (PTSD) may stem from the formation of aberrant and enduring aversive memories. Some PTSD patients have recreationally used Cannabis, probably aiming at relieving their symptomatology. However, it is still largely unknown whether and how Cannabis or its psychotomimetic compound Δ9-tetrahydrocannabinol (THC) attenuates the aversive/traumatic memory outcomes. Here, we seek to review and discuss the effects of THC on aversive memory extinction and anxiety in healthy humans and PTSD patients.MethodsMedline, PubMed, Cochrane Library, and Central Register for Controlled Trials databases were searched to identify peer-reviewed published studies and randomized controlled trials in humans published in English between 1974 and July 2020, including those using only THC and THC combined with cannabidiol (CBD). The effect size of the experimental intervention under investigation was calculated.ResultsAt low doses, THC can enhance the extinction rate and reduce anxiety responses. Both effects involve the activation of cannabinoid type-1 receptors in discrete components of the corticolimbic circuitry, which could couterbalance the low “endocannabinoid tonus” reported in PTSD patients. The advantage of associating CBD with THC to attenuate anxiety while minimizing the potential psychotic or anxiogenic effect produced by high doses of THC has been reported. The effects of THC either alone or combined with CBD on aversive memory reconsolidation, however, are still unknown.ConclusionsCurrent evidence from healthy humans and PTSD patients supports the THC value to suppress anxiety and aversive memory expression without producing significant adverse effects if used in low doses or when associated with CBD. Future studies are guaranteed to address open questions related to their dose ratios, administration routes, pharmacokinetic interactions, sex-dependent differences, and prolonged efficacy.