Activation Of Cannabinoid Receptors Research Articles

Maladaptive changes in the homeostasis of AEA-TRPV1/CB1R induces pain-related hyperactivity of nociceptors after spinal cord injury

BackgroundNeuropathic pain resulting from spinal cord injury (SCI) is associated with persistent hyperactivity of primary nociceptors. Anandamide (AEA) has been reported to modulate neuronal excitability and synaptic transmission through activation of cannabinoid type-1 receptors (CB1Rs) and transient receptor potential vanilloid 1 (TRPV1). However, the role of AEA and these receptors in the hyperactivity of nociceptors after SCI remains unclear.ResultsIn this study, we investigated the effects of AEA and its receptors on the hyperexcitability of mouse dorsal root ganglion (DRG) neurons after SCI. Using a whole-cell patch-clamp technique, we found that the timing of SCI-induced hyperexcitability in nociceptors paralleled an increase in the endocannabinoid AEA content. The expression of TRPV1 and CB1R was also upregulated at different time points after SCI. High-dose extracellular administration of AEA increased the excitability of naive DRG neurons, leading to the transition from a rapidly accommodating (RA) hypoexcitable state to a highly excitable non-accommodating (NA) state. These AEA-induced transitions were facilitated by increased TRPV1 transcription. Pharmacological and Ca2+ imaging experiments revealed that AEA induced hyperexcitability in nociceptors after SCI via the AEA-TRPV1-Ca2+ pathway, whereas activation of CB1Rs reduced SCI-induced hyperexcitability and maintained cytosolic Ca2+ concentration ([Ca2+]cyto) at low levels in the early stages of SCI. As the AEA and TRPV1 levels increased after SCI, adaptive neuroprotection transitioned to a maladaptive hyperactive state, leading to sustained pain.ConclusionsTaken together, this study provides new insights into how endocannabinoids regulate nociceptor activity after SCI, offering potential targets for the treatment of neuropathic pain.

Design of Small Non-Peptidic Ligands That Alter Heteromerization between Cannabinoid CB1 and Serotonin 5HT2A Receptors.

Activation of cannabinoid CB1 receptors (CB1R) by agonists induces analgesia but also induces cognitive impairment through the heteromer formed between CB1R and the serotonin 5HT2A receptor (5HT2AR). This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Peptides designed from the transmembrane helices of CB1R, which are predicted to bind 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, have been shown to avert CB1R agonist-induced cognitive impairment while preserving analgesia. Using these peptides as templates, we have now designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments. These results provide proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies.

Clinical Efficacy of Endocannabinoid-Mimetic Fatty Acid Amide as a Skin-Soothing Ingredient

Despite the potential benefits of cannabidiol as a skin-soothing ingredient, its regulatory status hampers its general use in cosmetic products in many countries. To develop an alternative to cannabidiol, fatty acid amide molecules mimicking the chemical structure of endocannabinoids were manufactured using a lipase-catalyzed process. A mixture of fatty acid amides from sunflower oil and 1-amino propan-3-ol was synthesized using an immobilized lipase reaction, and the activation of cannabinoid receptor 1 (CB1R) was measured using a cAMP assay. The anti-inflammatory activity of the endocannabimimetic ingredients was evaluated in cultured human monocytes and ex vivo human skin explant models. A clinical study was conducted to address the skin hydration, skin barrier function, and skin redness, and the ratio of the interleukin-1-receptor antagonist (IL1-RA) to IL-1⍺ in corneocytes, as a marker for skin sensitivity, were also measured. As a result, the activation of CB1R by endocannabimimetic ingredients was observed in cAMP assays, and a reduction in inflammatory responses by human monocytes induced by lipopolysaccharide treatment were also observed. External stress-induced inflammatory responses were reduced in ex vivo human skin explants. Improvements in skin hydration and barrier function were observed in a clinical study. A significant decrease in skin redness and the IL-1RA to IL-1⍺ ratio was also observed. Endocannabimimetic ingredients, as alternatives to cannabidiol, can be used in skin-soothing cosmetics to increase skin hydration, improve skin barrier function, and reduce skin sensitivity.

Activation of cannabinoid receptor 2 by turmeric oleoresin reduces inflammation and oxidative stress in an osteoarthritis in vitro model

IntroductionOsteoarthritis (OA) is a chronic degenerative joint disease characterized by the progressive degradation of articular cartilage, resulting in pain and reduced mobility. Turmeric (Curcuma longa L.) has been widely recognized for its anti-inflammatory and antioxidant properties, but the molecular mechanisms underlying its therapeutic effects remain inadequately explored. This study investigates the potential of turmeric oleoresin (TUR) to activate Cannabinoid Receptor 2 (CBR2) and its role in mediating anti-inflammatory and antioxidant effects in an in vitro OA model.Material and methodsMolecular docking and cAMP quantification assays were used to evaluate TUR’s agonistic activity on CBR2. Human chondrosarcoma cells (SW-1353) were treated with TUR under oxidative stress induced by menadione or inflammatory conditions simulated with IL-1β and TNF-α. The effects of TUR were assessed in the presence and absence of the CBR2 antagonist SR144528. Outcomes included changes in reactive oxygen species (ROS) production, inflammatory marker expression, oxidative defense markers and endocannabinoid system components and receptors.ResultsTUR was confirmed as a CBR2 agonist and significantly reduced ROS production, downregulated pro-inflammatory cytokines (IL-6, COX-2, metalloproteases), and suppressed signaling pathways such as NFKB1, ERK 1/2, and c-Myc. These effects were reversed upon CBR2 inhibition. TUR also enhanced HMOX-1 expression and modulated endocannabinoid-related enzymes, highlighting its impact on oxidative stress and the endocannabinoid system.DiscussionThese findings suggest that CBR2 activation is central to TUR’s anti-inflammatory and antioxidant effects. By modulating key pathways and endocannabinoid system components, TUR demonstrates potential as a novel therapeutic agent for OA management. Future studies could explore its clinical applications and further validate its molecular mechanisms in vivo.

Combinatorial effects of cannabinoid receptor 1 and 2 agonists on characteristics and proteomic alteration in MDA-MB-231 breast cancer cells.

Breast cancer is the most common cancer diagnosed in women worldwide. However, the effective treatment for breast cancer progression is still being sought. The activation of cannabinoid receptor (CB) has been shown to negatively affect breast cancer cell survival. Our previous study also reported that breast cancer cells responded to various combinations of CB1 and CB2 agonists differently. Nonetheless, the mechanism underlying this effect and whether this phenomenon can be seen in other cancer characteristics remain unknown. Therefore, this study aims to further elucidate the effects of highly selective CB agonists and their combination on triple-negative breast cancer proliferation, cell cycle progression, invasion, lamellipodia formation as well as proteomic profile of MDA-MB-231 breast cancer cells. The presence of CB agonists, specifically a 2:1 (ACEA: GW405833) combination, prominently inhibited colony formation and induced the S-phase cell cycle arrest in MDA-MB-231 cells. Furthermore, cell invasion ability and lamellipodia formation of MDA-MB-231 were also attenuated by the exposure of CB agonists and their 2:1 combination ratio. Our proteomic analysis revealed proteomic profile alteration in MDA-MB-231 upon CB exposure that potentially led to breast cancer suppression, such as ZPR1/SHC1/MAPK-mediated cell proliferation and AXL/VAV2/RAC1-mediated cell motility pathways. Our findings showed that selective CB agonists and their combination suppressed breast cancer characteristics in MDA-MB-231 cells. The exposure of CB agonists also altered the proteomic profile of MDA-MB-231, which could lead to cell proliferation and motility suppression.

Extracellular glutamate is not modulated by cannabinoid receptor activity.

Cannabinoid receptor activation has been proposed to trigger glutamate release from astrocytes located in cortical layer 2/3. Here, we measure the basal concentration of extracellular glutamate in layer 2/3 of mouse somatosensory cortex and find it to be 20-30 nM. We further examine the effect of cannabinoid receptor signaling on extracellular glutamate, and find no evidence for increased extracellular glutamate upon cannabinoid receptor agonist application.

Cognitive improvement via cortical cannabinoid receptors and choline-containing lipids.

Recent research linking choline-containing lipids to degeneration of basal forebrain cholinergic neurons in neuropathological states illustrates the challenge of balancing lipid integrity with optimal acetylcholine levels, essential for memory preservation. The endocannabinoid system influences learning and memory processes regulated by cholinergic neurotransmission. Therefore, we hypothesised that activation of the endocannabinoid system may confer neuroprotection against cholinergic degeneration. We examined the neuroprotective potential of sub-chronic treatments with the cannabinoid agonist WIN55,212-2, using ex vivo organotypic tissue cultures including nucleus basalis magnocellularis and cortex and in vivo rat models of specific cholinergic damage induced by 192IgG-saporin. Levels of lipids, choline and acetylcholine were measured with histochemical and immunofluorescence assays, along with [35S]GTPγS autoradiography of cannabinoid and muscarinic GPCRs and MALDI-mass spectrometry imaging analysis. Learning and memory were assessed by the Barnes maze and the novel object recognition test in rats and in the 3xTg-AD mouse model. Degeneration, induced by 192IgG-saporin, of baso-cortical cholinergic pathways resulted in memory deficits and decreased cortical levels of lysophosphatidylcholines (LPC). WIN55,212-2 restored cortical cholinergic transmission and LPC levels via activation of cannabinoid receptors. This activation altered cortical lipid homeostasis mainly by reducing sphingomyelins in lesioned animals. These modifications were crucial for memory recovery. We hypothesise that WIN55,212-2 facilitates an alternative choline source by breaking down sphingomyelins, leading to elevated cortical acetylcholine levels and LPCs. These results imply that altering choline-containing lipids via activation of cannabinoid receptors presents a promising therapeutic approach for dementia linked to cholinergic dysfunction.

Exploring Endocannabinoid System: Unveiling New Roles in Modulating ER Stress.

The endoplasmic reticulum (ER) is the organelle mainly involved in maintaining cellular homeostasis and driving correct protein folding. ER-dependent defects or dysfunctions are associated with the genesis/progression of several pathological conditions, including cancer, inflammation, and neurodegenerative disorders, that are directly or indirectly correlated to a wide set of events collectively named under the term "ER stress". Despite the recent increase in interest concerning ER activity, further research studies are needed to highlight all the mechanisms responsible for ER failure. In this field, recent discoveries paved the way for the comprehension of the strong interaction between ER stress development and the endocannabinoid system. The activity of the endocannabinoid system is mediated by the activation of cannabinoid receptors (CB), G protein-coupled receptors that induce a decrease in cAMP levels, with downstream anti-inflammatory effects. CB activation drives, in most cases, the recovery of ER homeostasis through the regulation of ER stress hallmarks PERK, ATF6, and IRE1. In this review, we focus on the CB role in modulating ER stress, with particular attention to the cellular processes leading to UPR activation and oxidative stress response extinguishment, and to the mechanisms underlying natural cannabinoids' modulation of this complex cellular machine.

Waxy- or Putty-Like Materials as a Novel Drug Preparation for Synthetic Cannabinoid Receptor Agonists: Detection in Prisons and InVitro Cannabinoid Receptor Activity.

After the Scottish Prison Service (SPS) introduced mail photocopying procedures in December 2021, a shift in smuggling methods was observed for synthetic cannabinoid receptor agonists (SCRAs) and other new psychoactive substances (NPS) from drug-infused papers back to traditional sample matrices (e.g., tablets and powders), although new matrices also emerged. This study reports on waxy- or putty-like materials as a novel drug preparation for SCRAs and other drugs seized from UK prisons. In 2023, 22 of these new preparations were seized from Scottish prisons, with eight found in sealed vape pods. The materials were positive for SCRAs, phytocannabinoids, novel benzodiazepines, and/or gabapentinoids. Additionally, 11 preparations were seized from an English prison, all containing the SCRAs MDMB-4en-PINACA and MDMB-INACA. MDMB-INACA was pharmacologically characterized using invitro CB1 and CB2 bioassays, revealing moderate efficacy but low potency at CB1. Furthermore, the invitro CB1 bioassay was also used to evaluate the CB1 activating potential of extracts from eight seized samples. Six of these showed high CB1 activity, whereas the samples lacking SCRAs or containing only MDMB-INACA showed no or only weak CB1 activity, respectively. Lastly, applying the bioassay as an activity-based "untargeted" screening method effectively identified the presence of SCRAs in one waxy preparation, which was initially not detected by gas chromatography-mass spectrometry (GC-MS). This underscores the effectiveness of the bioassay for evaluating these new waxy- or putty-like materials for the presence of SCRAs.

Antinociceptive and antineuropathic effects of Trifolium resupinatum L. on formalin-induced nociception and cervical spinal cord hemi-contusion: Underlying Mechanisms

Ethnopharmacological relevanceTrifolium resupinatum L. (Fabaceae), known as Persian clover, ethnomedicinally used in Persian folk medicine to treat peritoneal inflammation, rheumatism, and back pain. Aim of the studyTo investigate the antineuropathic and antinociceptive activities of Trifolium resupinatum leaves essential oil (TREO) in male Wistar rats, as well as to explore the potential mechanisms of action. Materials and methodsThe antinociceptive activity of TREO and its main constituents, quercetin (Qc) was assessed using the formalin-induced paw licking test. Moreover, the potential mechanisms of antinociception were evaluated through various competitive and non-competitive antagonisms. Additionally, the antineuropathic potential was investigated using the cervical spinal cord hemi-contusion (CCS) model, and the role of phosphorylated Stat-3 was analyzed using Western blotting. ResultsTREO exerted significant antinociceptive activity (P < 0.01) in both phases of the formalin-induced test; however, its effects were more pronounced in the second phase. Modulators of the NO-cGMP-K+ channel pathway significantly reversed the antinociceptive activity of TREO (P < 0.05). Additionally, antagonists of TRPV1 and TRPV2, as well as CB1 and GABAA receptors, significantly reversed the antinociceptive effects of TREO (P < 0.05). In another study, both TREO and Qc significantly attenuated hyperalgesia and mechanical allodynia (P < 0.01) when evaluated using the CCS-induced nociception model. Notably, TREO also reduced the expression levels of interleukin-1 beta, interleukin-2, and tumor necrosis factor alpha in CCS-induced rats (P < 0.05). ConclusionTREO and Qc exhibit both antinociceptive and anti-neuropathic activities. The antinociceptive effects are partially mediated through the NO-cGMP-K+ channel pathways, along with the activation of TRPV, GABA, and cannabinoid receptors. Furthermore, the anti-neuropathic activity of TREO may be partially regulated through the inhibition of cytokines.

Z-ligustilide alleviates atherosclerosis by reconstructing gut microbiota and sustaining gut barrier integrity through activation of cannabinoid receptor 2

BackgroundZ-Ligustilide (ZL) is an essential phthalide found in Ligusticum chuanxiong Hort, a commonly used traditional Chinese medicine for treating atherosclerosis (AS) clinically. ZL has been shown to be effective in treating AS. However, the underlying mechanism of ZL against AS and its potential targets remain elusive. PurposeThe purpose of this research was to assess the influence of ZL on AS and explore the role of the gut microbiome in mediating this effect. MethodsA well-established AS mouse model, apolipoprotein E deficient (ApoE-/-) mice was used to examine the effects of ZL on AS, inflammation, and the intestinal barrier. To analyze the changes in gut microbial community, we employed the 16S rRNA gene sequencing. Antibiotic cocktail and fecal microbiota transplantation (FMT) were employed to clarify the contribution of the gut microbiota to the anti-AS effects of ZL. The mechanism through which ZL provided protective effects on AS and the intestinal barrier was explored by untargeted metabolomics, as well as by validating the involvement of cannabinoid receptor 2 (CB2R) in mice and Caco-2 cells. ResultsOral administration of ZL inhibited the development of atherosclerotic lesions, improved plaque stability, inhibited the increase in serum and atherosclerotic inflammation, and improved intestinal barrier function. Fecal bacteria from ZL-treated mice induced similar beneficial effects on AS and the intestinal barrier. We used 16S RNA gene sequencing to reveal a significant increase in Rikenella abundance in both ZL-treated mice and ZL-FMT mice, which was associated with the beneficial effects of ZL. Further function prediction analysis of the gut microbiota and CB2R antagonist intervention experiment in mice and Caco-2 cells showed that the activation of CB2R resulted in the enhancement of the intestinal barrier by ZL. Furthermore, the analysis of metabolomic profiling revealed the enrichment of capsaicin upon ZL treatment, which induced the activation of CB2R in human colon epithelial cells. ConclusionOur study is the first to demonstrate that oral treatment with ZL has the potential to alleviate AS by reducing inflammation levels and enhancing the intestinal barrier function. This mechanism relies on the gut microbiota in a CB2R-dependent manner, suggesting promising strategies and ideas for managing AS. This study provides insights into a novel mechanism for treating AS with ZL.

Central Neuropathic Pain.

This article provides an approach to the assessment, diagnosis, and treatment of central neuropathic pain. Recent studies of the pathophysiology of central neuropathic pain, including evidence of changes in the expression of voltage-gated sodium channels and N-methyl-d-aspartate (NMDA) receptors, may provide the basis for new therapies. Other areas of current research include the role of cannabinoid-receptor activity and microglial cell activation in various animal models of central neuropathic pain. New observations regarding changes in primary afferent neuronal activity in central neuropathic pain and the preliminary observation that peripheral nerve blocks may relieve pain due to central neuropathic etiologies provide new insights into both the mechanism and treatment of central neuropathic pain. In the patient populations treated by neurologists, central neuropathic pain develops most frequently following spinal cord injury, multiple sclerosis, or stroke. A multimodal, individualized approach to the management of central neuropathic pain is necessary to optimize pain relief and may require multiple treatment trials to achieve the best outcome.

Mechanistic insights into the impact of WIN 55, 212-2, a synthetic cannabinoid, on adhesion molecules PECAM-1 and VE-cadherin in HeLa cells: implications on cancer processes

The endocannabinoid (eCB) system comprises endogenous ligands, cannabinoid receptors (CBRs), and their regulatory proteins; its alteration leads to many diseases including cancer. Thus, becomes a therapeutic target for synthetic cannabinoids aimed to control cancer cell proliferation, migration, adhesion, and invasion. However, little is known about adhesion molecules regulation through CBRs activation. The aim of this study was to evaluate the effects of a CB1/CB2 agonist, WIN-55, 212-2 (WIN), on the regulation of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial cadherin (VE-cadherin) in HeLa cells. CBRs expression was evaluated by immunofluorescence staining in HeLa cells and cell viability (thiazolyl blue tetrazolium bromide), cell adhesion (crystal violet), adhesion molecules expression and location (Western blot and immunofluorescence staining assays) were all assessed on cells treated with different WIN concentrations. Receptors CB1, CB2, and G-protein-coupled receptor 55 were expressed in HeLa cells. Additionally, biphasic effects were observed in their metabolic activity and adhesive properties: low WIN concentrations resulted in significant increases whereas, high ones decreased them compared to controls (p < 0.0001), demonstrating that WIN elicits opposite effects depending on the concentration and exposure time. PECAM-1 was detected in HeLa cell’s cytoplasm, membrane, and perinuclear region, whereas VE-cadherin had a nuclear distribution. There were no significant differences in PECAM-1 and VE-cadherin expression and location, suggesting that WIN does not modulate these proteins. These findings support the potential use of WIN due to its anticancer properties without dysregulating adhesion molecules. WIN possible contribution to inhibit cancer progression should be further investigated.

Differential Behavior of Conformational Dynamics in Active and Inactive States of Cannabinoid Receptor 1.

Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor that regulates critical physiological processes including pain, appetite, and cognition. Understanding the conformational dynamics of CB1 associated with transitions between inactive and active signaling states is imperative for developing targeted modulators. Using microsecond-level all-atom molecular dynamics simulations, we identified marked differences in the conformational ensembles of inactive and active CB1 in apo. The inactive state exhibited substantially increased structural heterogeneity and plasticity compared to the more rigidified active state in the absence of stabilizing ligands. Transmembrane helices TM3 and TM7 were identified as distinguishing factors modulating the state-dependent dynamics. TM7 displayed amplified fluctuations selectively in the inactive state simulations attributed to disruption of conserved electrostatic contacts anchoring it to surrounding helices in the active state. Additionally, we identified significant reorganizations in key salt bridge and hydrogen bond networks contributing to the CB1 activation/inactivation. For instance, D213-Y224 hydrogen bond and D184-K192 salt bridge showed marked rearrangements between the states. Collectively, these findings reveal the specialized role of TM7 in directing state-dependent CB1 dynamics through electrostatic switch mechanisms. By elucidating the intrinsic enhanced flexibility of inactive CB1, this study provides valuable insights into the conformational landscape enabling functional transitions. Our perspective advances understanding of CB1 activation mechanisms and offers opportunities for structure-based drug discovery targeting the state-specific conformational dynamics of this receptor.

Depression exacerbates myocardial ischemia-reperfusion injury in mice via CNR2 gene and MIF-AMPK signaling pathway

BackgroundMyocardial ischemia-reperfusion(I/R)injury constitute the fundamental pathophysiology of acute myocardial infarction (AMI). Ischemic heart releases macrophage migration inhibitory factor (MIF), which activates MIF- AMPK signaling pathway. Depression is a significant risk factor for AMI. In a state of depression, peripheral expression of cannabinoid receptor 2 (CNR2) genes was downregulated. AimsWe investigated the mechanism by which depression exacerbates myocardial I/R injury through the CNR2 and MIF-AMPK signaling pathways. MethodsWe established mouse models of depression and myocardial I/R. Left ventricular function was assessed using cardiac ultrasound and TTC staining. The protein levels of myocardial CNR2, MIF, AMPK, and ACC were determined by Western blot, while the expression level of CNR2 was measured using RT-qPCR. Additionally, MIF content in peripheral blood was quantified using ELISA. ResultsAfter I/R, the expression level of CNR2 was found to be lower in the depression group, leading to a deterioration in left heart function. Depressed mice exhibited lower secretion of MIF, accompanied by a decrease in the activation of the MIF-AMPK signaling pathway. However, injection of CNR2 agonist JWH133 prior to ischemia increased the activation of the MIF-AMPK signaling pathway, while CNR2 inhibitor AM630 decreased the activation. LimitationsFurther research is needed to investigate the specific neuroendocrine mechanism affecting myocardial CNR2 expression in depression. And these experimental conclusions require further verification at the cellular level. ConclusionsThe activation of CNR2 in myocardium following I/R is impeded by depression, thereby exacerbating myocardial I/R injury through attenuation of the MIF-AMPK signaling pathway activation.

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes in male mice

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of d-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter d-serine, by its precursor l-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards l-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes.

Dorsomedial Striatum CB1R signaling is required for Pavlovian outcome devaluation in male Long Evans rats and reduces inhibitory synaptic transmission in both sexes.

Cannabinoid-1 receptor (CB1R) signaling in the dorsal striatum regulates the shift from flexible to habitual behavior in instrumental outcome devaluation. Based on prior work establishing individual, sex, and experience-dependent differences in Pavlovian behaviors, we predicted a role for dorsomedial striatum CB1R signaling in driving rigid responding in Pavlovian autoshaping and outcome devaluation. We trained male and female Long Evans rats in Pavlovian Lever Autoshaping (PLA). We gave intra-dorsomedial striatum (DMS) infusions of the CB1R inverse agonist, rimonabant, before satiety-induced outcome devaluation test sessions, where we sated rats on training pellets or home cage chow and tested them in brief nonreinforced Pavlovian Lever Autoshaping sessions. Overall, inhibition of DMS CB1R signaling prevented Pavlovian outcome devaluation but did not affect behavior in reinforced PLA sessions. Males were sensitive to devaluation while females were not and DMS CB1R inhibition impaired devaluation sensitivity in males. We then investigated how DMS CB1R signaling impacts local inhibitory synaptic transmission in male and female Long Evans rats. We recorded spontaneous inhibitory postsynaptic currents (sIPSC) from DMS neurons at baseline and before and after application of a CB1R agonist, WIN 55,212-2. We found that male rats showed decreased sIPSC frequency compared to females, and that CB1R activation reduced DMS inhibitory transmission independent of sex. Altogether our results demonstrate that DMS CB1Rs regulate Pavlovian devaluation sensitivity and inhibitory synaptic transmission and suggest that basal sex differences in inhibitory synaptic transmission may underly sex differences in DMS function and behavioral flexibility.

Regulation of Cannabinoid and Opioid Receptor Levels by Endogenous and Pharmacological Chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), δ opioid receptor (DOR), and CB1R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. SIGNIFICANCE STATEMENT: This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of cannabinoid CB1 receptor, delta opioid receptor, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

Therapeutic Application of Modulators of Endogenous Cannabinoid System in Parkinson's Disease.

The endogenous cannabinoid system (ECS) of the brain plays an important role in the molecular pathogenesis of Parkinson's disease (PD). It is involved in the formation of numerous clinical manifestations of the disease by regulating the level of endogenous cannabinoids and changing the activation of cannabinoid receptors (CBRs). Therefore, ECS modulation with new drugs specifically designed for this purpose may be a promising strategy in the treatment of PD. However, fine regulation of the ECS is quite a complex task due to the functional diversity of CBRs in the basal ganglia and other parts of the central nervous system. In this review, the effects of ECS modulators in various experimental models of PD in vivo and in vitro, as well as in patients with PD, are analyzed. Prospects for the development of new cannabinoid drugs for the treatment of motor and non-motor symptoms in PD are presented.

Inhibition of 2-AG hydrolysis alleviates posttraumatic headache attributed to mild traumatic brain injury

BackgroundPosttraumatic headache (PTH) is a common and debilitating symptom following repetitive mild traumatic brain injury (rmTBI), and it mainly resembles a migraine-like phenotype. While modulation of the endocannabinoid system (ECS) is effective in treating TBI and various types of pain including migraine, the role of augmentation of endocannabinoids in treating PTH has not been investigated.MethodsRepetitive mild TBI was induced in male C57BL/6J mice using the non-invasive close-head impact model of engineered rotational acceleration (CHIMERA). Periorbital allodynia was assessed using von Frey filaments and determined by the “Up-Down” method. Immunofluorescence staining was employed to investigate glial cell activation and calcitonin gene-related peptide (CGRP) expression in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) of the rmTBI mice. Levels of 2-arachidonoyl glycerol (2-AG), anandamide (AEA), and arachidonic acid (AA) in the TG, medulla (including TNC), and periaqueductal gray (PAG) were measured by mass spectrometry. The therapeutic effect of endocannabinoid modulation on PTH was also assessed.ResultsThe rmTBI mice exhibited significantly increased cephalic pain hypersensitivity compared to the sham controls. MJN110, a potent and selective inhibitor of the 2-AG hydrolytic enzyme monoacylglycerol lipase (MAGL), dose-dependently attenuated periorbital allodynia in the rmTBI animals. Administration of CGRP at 0.01 mg/kg reinstated periorbital allodynia in the rmTBI animals on days 33 and 45 post-injury but had no effect in the sham and MJN110 treatment groups. Activation of glial cells along with increased production of CGRP in the TG and TNC at 7 and 14 days post-rmTBI were attenuated by MJN110 treatment. The anti-inflammatory and anti-nociceptive effects of MJN110 were partially mediated by cannabinoid receptor activation, and the pain-suppressive effect of MJN110 was completely blocked by co-administration of DO34, an inhibitor of 2-AG synthase. The levels of 2-AG in TG, TNC and PAG were decreased in TBI animals, significantly elevated and further reduced by the selective inhibitors of 2-AG hydrolytic and synthetic enzymes, respectively.ConclusionEnhancing endogenous levels of 2-AG appears to be an effective strategy for the treatment of PTH by attenuating pain initiation and transmission in the trigeminal pathway and facilitating descending pain inhibitory modulation.