Etomidate Depresses Spontaneous Complex Spikes Activity of Cerebellar Purkinje Cells via Cannabinoid 1 Receptor in vivo in Mice.

INTRODUCTION: Complex spikes (CSs) activity of cerebellar Purkinje cells plays critical roles in motor coordination and motor learning by transferring information to cerebellar cortex, which is an accessible and useful model for neurophysiological investigation. Etomidate is an ultrashort-acting nonbarbiturate intravenous anesthetic, which inhibits the spontaneous activity of cerebellar Purkinje cells through activation of GABAA and glycine receptors in vivo in mice. However, the effect of etomidate on the spontaneous CSs activity of cerebellar Purkinje cells in living mouse is not clear. METHODS: We here investigated the effects of etomidate on spontaneous CSs activity of cerebellar Purkinje cell in urethane-anesthetized mice by electrophysiology recording technique and pharmacological methods. RESULTS: Our results showed that cerebellar surface perfusion of etomidate significantly depressed the activity of spontaneous CSs, which exhibited decreases in the number of spikelets and the area under curve (AUC) of the CSs. The etomidate-produced inhibition of CSs activity was persisted in the presence of GABAA and glycine receptors antagonists. However, application of cannabinoid 1 (CB1) receptor antagonist, AM-251, completely blocked the etomidate-induced inhibition of CSs. Furthermore, application of the CB1 receptor agonist, WIN55212-2, induced a decrease of CSs. Moreover, in the presence of a specific protein kinase A (PKA) inhibitor, KT5720, etomidate failed to produce decreases in the spikelets number and the AUC of the spontaneous CSs. CONCLUSION: These results indicate that cerebellar surface application of etomidate facilitates CB1 receptor activity resulting in a depression of spontaneous CSs activity of Purkinje cells via PKA signaling pathway in mouse cerebellar cortex. Our present results suggest that the etomidate administration may impair the function of cerebellar cortical neuronal circuitry by inhibition of the climbing fiber - Purkinje cells synaptic transmission through activation of CB1 receptors in vivo in mice.

Age, gender and body-mass-index relationships with in vivo CB1 receptor availability in healthy humans measured with [11C]OMAR PET.

Brain cannabinoid 1 receptors (CB1Rs) contribute importantly to the regulation of autonomic tone, appetite, mood and cognition. Inconsistent results have been reported from positron emission tomography (PET) studies using different radioligands to examine relationships between age, gender and body mass index (BMI) and CB1R availability in healthy individuals. In this study, we examined these variables in 58 healthy individuals (age range: 18-55 years; 44 male; BMI=27.01±5.56), the largest cohort of subjects studied to date using the CB1R PET ligand [11C]OMAR. There was a significant decline in CB1R availability (VT) with age in the pallidum, cerebellum and posterior cingulate. Adjusting for BMI, age-related decline in VT remained significant in the posterior cingulate among males, and in the cerebellum among women. CB1R availability was higher in women compared to men in the thalamus, pallidum and posterior cingulate. Adjusting for age, CB1R availability negatively correlated with BMI in women but not men. These findings differ from those reported using [11C]OMAR and other radioligands such as [18F]FMPEP-d2 and [18F]MK-9470. Although reasons for these seemingly divergent findings are unclear, the choice of PET radioligand and range of BMI in the current dataset may contribute to the observed differences. This study highlights the need for cross-validation studies using both [11C]OMAR and [18F]FMPEP-d2 within the same cohort of subjects.

Synaptic changes induced by cannabinoid drugs and cannabis use disorder.

The legalization of cannabis in many countries, as well as the decrease in perceived risks of cannabis, have contributed to the increase in cannabis use medicinally and recreationally. Like many drugs of abuse, cannabis and cannabis-derived drugs are prone to misuse, and long-term usage can lead to drug tolerance and the development of Cannabis Use Disorder (CUD). These drugs signal through cannabinoid receptors, which are expressed in brain regions involved in the neural processing of reward, habit formation, and cognition. Despite the widespread use of cannabis and cannabinoids as therapeutic agents, little is known about the neurobiological mechanisms associated with CUD and cannabinoid drug use. In this article, we discuss the advances in research spanning animal models to humans on cannabis and synthetic cannabinoid actions on synaptic transmission, highlighting the neurobiological mechanisms following acute and chronic drug exposure. This article also highlights the need for more research elucidating the neurobiological mechanisms associated with CUD and cannabinoid drug use.

Peripheral CB1R as a modulator of metabolic inflammation.

Obesity is associated with chronic inflammation in insulin-sensitive tissues, including liver and adipose tissue, and causes hormonal/metabolic complications, such as insulin resistance. There is growing evidence that peripheral cannabinoid-type 1 receptor (CB1R) is a crucial participant in obesity-induced pro-inflammatory responses in insulin-target tissues, and its selective targeting could be a novel therapeutic strategy to break the link between insulin resistance and metabolic inflammation. In this review, we introduce the role of peripheral CB1R in metabolic inflammation and as a mediator of hormonal/metabolic complications that underlie metabolic syndrome, including fatty liver, insulin resistance, and dyslipidemia. We also discuss the therapeutic potential of second- and third-generation peripherally restricted CB1R antagonists for treating obesity-induced metabolic inflammation without eliciting central CB1R-mediated neurobehavioral effects, predictive of neuropsychiatric side effects, in humans.

(+)-trans-Cannabidiol-2-hydroxy pentyl is a dual CB1R antagonist/CB2R agonist that prevents diabetic nephropathy in mice.

Natural cannabidiol ((-)-CBD) and its derivatives have increased interest for medicinal applications due to their broad biological activity spectrum, including targeting of the cannabinoid receptors type 1 (CB1R) and type 2 (CB2R). Herein, we synthesized the (+)-enantiomer of CBD and its derivative (+)-CBD hydroxypentylester ((+)-CBD-HPE) that showed enhanced CB1R and CB2R binding and functional activities compared to their respective (-) enantiomers. (+)-CBD-HPE Ki values for CB1R and CB2R were 3.1 ± 1.1 and 0.8 ± 0.1 nM respectively acting as CB1R antagonist and CB2R agonist. We further tested the capacity of (+)-CBD-HPE to prevent hyperglycemia and its complications in a mouse model. (+)-CBD-HPE significantly reduced streptozotocin (STZ)-induced hyperglycemia and glucose intolerance by preserving pancreatic beta cell mass. (+)-CBD-HPE significantly reduced activation of NF-κB by phosphorylation by 15% compared to STZ-vehicle mice, and CD3+ T cell infiltration into the islets was avoided. Consequently, (+)-CBD-HPE prevented STZ-induced apoptosis in islets. STZ induced inflammation and kidney damage, visualized by a significant increase in plasma proinflammatory cytokines, creatinine, and BUN. Treatment with (+)-CBD-HPE significantly reduced 2.5-fold plasma IFN-γ and increased 3-fold IL-5 levels compared to STZ-treated mice, without altering IL-18. (+)-CBD-HPE also significantly reduced creatinine and BUN levels to those comparable to healthy controls. At the macroscopy level, (+)-CBD-HPE prevented STZ-induced lesions in the kidney and voided renal fibrosis and CD3+ T cell infiltration. Thus, (+)-enantiomers of CBD, particularly (+)-CBD-HPE, have a promising potential due to their pharmacological profile and synthesis, potentially to be used for metabolic and immune-related disorders.

Design, synthesis, and pharmacological profiling of cannabinoid 1 receptor allosteric modulators: Preclinical efficacy of C2-group GAT211 congeners for reducing intraocular pressure.

Allosteric modulators of cannabinoid 1 receptor (CB1R) show translational promise over orthosteric ligands due to their potential to elicit therapeutic benefit without cannabimimetic side effects. The prototypic 2-phenylindole CB1R allosteric modulator, GAT211 (1), demonstrates preclinical efficacy in various disease models. The limited systematic structure-activity relationship (SAR) data at the C2 position of the indole ring within GAT211 invites the opportunity for further modifications to improve GAT211's pharmacological profile while serving to amplify and variegate this library of therapeutically attractive agents. These considerations prompted this focused SAR study in which we substituted the GAT211 C2-phenyl ring with heteroaromatic substituents. The synthesized GAT211 analogs were then evaluated in vitro as CB1R allosteric modulators in cAMP and ß-arrestin2 assays with CP55,940 as the orthosteric ligand. Furan and thiophene rings (15c-f and 15m) were the best-tolerated substituents at the C2 position of GAT211 for engagement with human CB1R (hCB1R). The SAR around the novel ligands reported allowed direct experimental characterization of the interaction profile of that pharmacophore with its binding domain in functional, human CB1R, thus offering guidance for accessing subsequent-generation hCB1R allosteric modulators as potential therapeutics. The most potent analog, 15d, markedly promoted orthosteric ligand binding to hCB1R. Pharmacological profiling in the GTPγS and mouse vas deferens assays demonstrated that 15d behaves as a CB1R agonist-positive allosteric modulator (ago-PAM), as confirmed electrophysiologically in autoptic neurons. In vivo, 15d was efficacious as a topical agent that significantly reduced intraocular pressure (IOP) in the ocular normotensive murine model of glaucoma. Since elevated IOP is a decisive risk factor for glaucoma and attendant vision loss, our data support the proposition that the 2-phenylindole class of CB1R ago-PAMs has therapeutic potential for glaucoma and other diseases where potentiation of CB1R signaling may be therapeutic.

Association between cannabinoid 1 receptor availability and glutamate levels in healthy controls and drug-free patients with first episode psychosis: a multi-modal PET and 1H-MRS study.

Cannabinoid 1 receptor and glutamatergic dysfunction have both been implicated in the pathophysiology of schizophrenia. However, it remains unclear if cannabinoid 1 receptor alterations shown in drug-naïve/free patients with first episode psychosis may be linked to glutamatergic alterations in the illness. We aimed to investigate glutamate levels and cannabinoid 1 receptor levels in the same region in patients with first episode psychosis. Forty volunteers (20 healthy volunteers, 20 drug-naïve/free patients with first episode psychosis diagnosed with schizophrenia/schizoaffective disorder) were included in the study. Glutamate levels were measured using proton magnetic resonance spectroscopy. CB1R availability was indexed using the distribution volume (VT (ml/cm3)) of [11C]MePPEP using arterial blood sampling. There were no significant associations between ACC CB1R levels and ACC glutamate levels in controls (R = - 0.24, p = 0.32) or patients (R = - 0.10, p = 0.25). However, ACC glutamate levels were negatively associated with CB1R availability in the striatum (R = - 0.50, p = 0.02) and hippocampus (R = - 0.50, p = 0.042) in controls, but these associations were not observed in patients (p > 0.05). Our findings extend our previous work in an overlapping sample to show, for the first time as far as we're aware, that cannabinoid 1 receptor alterations in the anterior cingulate cortex are shown in the absence of glutamatergic dysfunction in the same region, and indicate potential interactions between glutamatergic signalling in the anterior cingulate cortex and the endocannabinoid system in the striatum and hippocampus.

Disease-associated polymorphisms within the conserved ECR1 enhancer differentially regulate the tissue-specific activity of the cannabinoid-1 receptor gene promoter; implications for cannabinoid pharmacogenetics.

Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Toward addressing the hypothesis that genetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we characterized the effects of disease-associated allelic variation within a conserved regulatory sequence (ECR1) in CNR1 intron 2 that had previously been shown to modulate cannabinoid response, alcohol intake, and anxiety-like behavior. We used primary cell analysis of reporters carrying different allelic variants of the human ECR1 and found that human-specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in primary hippocampal cells than did the ancestral T-allele and demonstrated a differential response to CB1 agonism. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity and evidence that the ancestral t-allele variant of ECR1 interacted with higher affinity with the insulator binding factor CTCF. The cell-specific approaches used in our study represent an important step in gaining a mechanistic understanding of the roles of noncoding polymorphic variation in disease and in the increasingly important field of cannabinoid pharmacogenetics.

Distinctive Evidence Involved in the Role of Endocannabinoid Signalling in Parkinson's Disease: A Perspective on Associated Therapeutic Interventions.

Current pharmacotherapy of Parkinson's disease (PD) is symptomatic and palliative, with levodopa/carbidopa therapy remaining the prime treatment, and nevertheless, being unable to modulate the progression of the neurodegeneration. No available treatment for PD can enhance the patient's life-quality by regressing this diseased state. Various studies have encouraged the enrichment of treatment possibilities by discovering the association of the effects of the endocannabinoid system (ECS) in PD. These reviews delineate the reported evidence from the literature on the neuromodulatory role of the endocannabinoid system and expression of cannabinoid receptors in symptomatology, cause, and treatment of PD progression, wherein cannabinoid (CB) signalling experiences alterations of biphasic pattern during PD progression. Published papers to date were searched via MEDLINE, PubMed, etc., using specific key words in the topic of our manuscript. Endocannabinoids regulate the basal ganglia neuronal circuit pathways, synaptic plasticity, and motor functions via communication with dopaminergic, glutamatergic, and GABAergic signalling systems bidirectionally in PD. Further, gripping preclinical and clinical studies demonstrate the context regarding the cannabinoid compounds, which is supported by various evidence (neuroprotection, suppression of excitotoxicity, oxidative stress, glial activation, and additional benefits) provided by cannabinoid-like compounds (much research addresses the direct regulation of cannabinoids with dopamine transmission and other signalling pathways in PD). More data related to endocannabinoids efficacy, safety, and pharmacokinetic profiles need to be explored, providing better insights into their potential to ameliorate or even regress PD.

Additive effect of 5-HT2C and CB1 receptor blockade on the regulation of sleep-wake cycle.

BACKGROUND: Previous data show that serotonin 2C (5-HT2C) and cannabinoid 1 (CB1) receptors have a role in the modulation of sleep-wake cycle. Namely, antagonists on these receptors promoted wakefulness and inhibited rapid eye movement sleep (REMS) in rodents. The interaction of these receptors are also present in other physiological functions, such as the regulation of appetite. Blockade of 5-HT2C receptors modulat the effect of CB1 receptor antagonist, presumably in consecutive or interdependent steps. Here we investigate, whether previous blockade of 5-HT2C receptors can affect CB1 receptor functions in the sleep-wake regulation. RESULTS: Wistar rats were equipped with electroencephalography (EEG) and electromyography (EMG) electrodes. Following the recovery and habituation after surgery, animals were injected intraperitoneally (ip.) with SB-242084, a 5-HT2C receptor antagonist (1.0 mg/kg) at light onset (beginning of passive phase) followed by an injection with AM-251, a CB1 receptor antagonist (5.0 or 10.0 mg/kg, ip.) 10 min later. EEG, EMG and motor activity were analyzed for the subsequent 2 h. Both SB-242084 and AM-251 increased the time spent in active wakefulness, while decreased the time spent in non-REMS and REMS stages in the first 2 h of passive phase. In combination, the effect of the agents were additive, furthermore, statistical analysis did not show any interaction between the effects of these drugs in the modulation of vigilance stages. CONCLUSIONS: Our results suggest that 5-HT2C receptor blockade followed by blockade of CB1 receptors evoked additive effect on the regulation of sleep-wake pattern.

Divalent cannabinoid-1 receptor ligands: A linker attachment point survey of SR141716A for development of high-affinity CB1R molecular probes.

The cannabinoid-1 receptor (CB1R) inverse agonist SR141716A has proven useful for study of the endocannabinoid system, including development of divalent CB1R ligands possessing a second functional motif attached via a linker unit. These have predominantly employed the C3 position of the central pyrazole ring for linker attachment. Despite this precedent, a novel series of C3-linked CB1R-D2R divalent ligands exhibited extremely high affinity at the D2R, but only poor affinity for the CB1R. A systematic linker attachment point survey of the SR141716A pharmacophore was therefore undertaken, establishing the C5 position as the optimal site for linker conjugation. This linker attachment survey enabled the identification of a novel divalent ligand as a lead compound to inform ongoing development of high-affinity CB1R molecular probes.

Integrating endocannabinoid signaling in the regulation of anxiety and depression.

Brain endogenous cannabinoid (eCB) signaling seems to harmonize appropriate behavioral responses, which are essential for the organism's long-term viability and homeostasis. Dysregulation of eCB signaling contributes to negative emotional states and increased stress responses. An understanding of the underlying neural cell populations and neural circuit regulation will enable the development of therapeutic strategies to mitigate behavioral maladaptation and provide insight into the influence of eCB on the neural circuits involved in anxiety and depression. This review focuses on recent evidence that has added a new layer of complexity to the idea of targeting the eCB system for therapeutic benefits in neuropsychiatric disease and on the future research direction of neural circuit modulation.

Etomidate Modulates the Tactile Stimulation-Evoked Field Potential Responses in Cerebellar Granule Cell Layer in vivo in Mice.

Etomidate (ET) produces sedation by binding on the γ-aminobutyric acid type A (GABAA) receptors. We previously found that ET inhibited cerebellar Purkinje cells activity via both GABAA and glycine receptors in vivo in mice, suggesting that ET modulated sensory information synaptic transmission in cerebellar cortex. In this study, we investigated the effect of ET on the sensory stimulation-evoked responses in the cerebellar granule layer (GL) in urethane-anesthetized mice, using electrophysiological and pharmacological methods. Our results showed that cerebellar surface perfusion of ET (100 µmol/L) significantly decreased amplitude and area under the curve (AUC) of the sensory stimulation-evoked excitatory component (N1) in the cerebellar GL. Application of GABAA receptor antagonist, SR95531 (20 µmol/L) significantly attenuated, but not abolished the ET-induced decrease in amplitude and AUC of facial stimulation-evoked responses. However, application of a mixture of SR95531 (20 µmol/L) and cannabinoid 1 receptor (CB1) antagonist, AM-251 (5 µmol/L), completely blocked the ET-induced decrease in amplitude and AUC of facial stimulation-evoked responses. Furthermore, application of the CB1 receptor agonist, WIN55212-2, induced a decrease in amplitude and AUC of N1 in the absence of GABAA receptors activity, as well occluded the ET-induced depression of N1. Moreover, the ET-induced changes in amplitude and AUC of N1 in absence of GABAA receptors activity were abolished by a specific protein kinase A (PKA) inhibitor, KT5720. These results indicate that ET facilitates CB1 receptors in the absence of GABAA receptors activity, resulting in a depression of the sensory stimulation-evoked synaptic transmission via PKA signaling pathway in mouse cerebellar GL.

Absence of cannabinoid 1 receptor in beta cells protects against high-fat/high-sugar diet-induced beta cell dysfunction and inflammation in murine islets.

AIMS/HYPOTHESIS: The cannabinoid 1 receptor (CB1R) regulates insulin sensitivity and glucose metabolism in peripheral tissues. CB1R is expressed on pancreatic beta cells and is coupled to the G protein Gαi, suggesting a negative regulation of endogenous signalling in the beta cell. Deciphering the exact function of CB1R in beta cells has been confounded by the expression of this receptor on multiple tissues involved in regulating metabolism. Thus, in models of global genetic or pharmacological CB1R blockade, it is difficult to distinguish the indirect effects of improved insulin sensitivity in peripheral tissues from the direct effects of inhibiting CB1R in beta cells per se. To assess the direct contribution of beta cell CB1R to metabolism, we designed a mouse model that allows us to determine the role of CB1R specifically in beta cells in the context of whole-body metabolism. METHODS: We generated a beta cell specific Cnr1 (CB1R) knockout mouse (ß-CB1R-/-) to study the long-term consequences of CB1R ablation on beta cell function in adult mice. We measured beta cell function, proliferation and viability in these mice in response to a high-fat/high-sugar diet and induction of acute insulin resistance with the insulin receptor antagonist S961. RESULTS: ß-CB1R-/- mice had increased fasting (153 ± 23% increase at 10 weeks of age) and stimulated insulin secretion and increased intra-islet cAMP levels (217 ± 33% increase at 10 weeks of age), resulting in primary hyperinsulinaemia, as well as increased beta cell viability, proliferation and islet area (1.9-fold increase at 10 weeks of age). Hyperinsulinaemia led to insulin resistance, which was aggravated by a high-fat/high-sugar diet and weight gain, although beta cells maintained their insulin secretory capacity in response to glucose. Strikingly, islets from ß-CB1R-/- mice were protected from diet-induced inflammation. Mechanistically, we show that this is a consequence of curtailment of oxidative stress and reduced activation of the NLRP3 inflammasome in beta cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate CB1R to be a negative regulator of beta cell function and a mediator of islet inflammation under conditions of metabolic stress. Our findings point to beta cell CB1R as a therapeutic target, and broaden its potential to include anti-inflammatory effects in both major forms of diabetes. DATA AVAILABILITY: Microarray data have been deposited at GEO (GSE102027).

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells.

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse ß-cell lines, human islets and CB1R-null (CB1R-/- ) mice, we have now investigated the role of CB1Rs in modulating ß-cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP-1-mediated cAMP accumulation and insulin secretion as well as glucose-stimulated insulin secretion in mouse ß-cell lines and human islets. In addition, silencing CB1R in mouse ß cells resulted in an increased expression of pro-insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in ß cells lacking insulin receptor. Furthermore, CB1R-/- mice had increased pro-insulin, GCK and GLUT2 expression in ß cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve ß-cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to ß-cell function in type 2 diabetes.

Analgesic effects of FAAH inhibitor in the insular cortex of nerve-injured rats.

The insular cortex is an important region of brain involved in the processing of pain and emotion. Recent studies indicate that lesions in the insular cortex induce pain asymbolia and reverse neuropathic pain. Endogenous cannabinoids (endocannabinoids), which have been shown to attenuate pain, are simultaneously degraded by fatty acid amide hydrolase (FAAH) that halts the mechanisms of action. Selective inhibitor URB597 suppresses FAAH activity by conserving endocannabinoids, which reduces pain. The present study examined the analgesic effects of URB597 treatment in the insular cortex of an animal model of neuropathic pain. Under pentobarbital anesthesia, male Sprague-Dawley rats were subjected to nerve injury and cannula implantation. On postoperative day 14, rodents received microinjection of URB597 into the insular cortex. In order to verify the analgesic mechanisms of URB597, cannabinoid 1 receptor (CB1R) antagonist AM251, peroxisome proliferator-activated receptor alpha (PPAR alpha) antagonist GW6471, and transient receptor potential vanilloid 1 (TRPV1) antagonist Iodoresiniferatoxin (I-RTX) were microinjected 15 min prior to URB597 injection. Changes in mechanical allodynia were measured using the von-Frey test. Expressions of CB1R, N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), and TRPV1 significantly increased in the neuropathic pain group compared to the sham-operated control group. Mechanical threshold and expression of NAPE-PLD significantly increased in groups treated with 2 nM and 4 nM URB597 compared with the vehicle-injected group. Blockages of CB1R and PPAR alpha diminished the analgesic effects of URB597. Inhibition of TRPV1 did not effectively reduce the effects of URB597 but attenuated expression of NAPE-PLD compared with the URB597-injected group. In addition, optical imaging demonstrated that neuronal activity of the insular cortex was reduced following URB597 treatment. Our results suggest that microinjection of FAAH inhibitor into the insular cortex causes analgesic effects by decreasing neural excitability and increasing signals related to the endogenous cannabinoid pathway in the insular cortex.

Peripheral cannabinoid 1 receptor blockade mitigates adipose tissue inflammation via NLRP3 inflammasome in mouse models of obesity.

AIM: To analyze the metabolic parameters and adipose tissue inflammation via NLRP3 inflammasome following chronic treatment of mouse models of obesity with AJ5018 as the peripherally restricted cannabinoid 1 receptor (CB1R) antagonist. MATERIALS AND METHODS: The selectivity for CB1R over CB2R, brain/plasma concentration ratio, and centrally mediated neurobehavioural effects of AJ5018, were assessed. The long-term effects of AJ5018 and rimonabant on the metabolic parameters and adipose tissue inflammation were analyzed in diet-induced obese (DIO) mice and diabetic db/db mice. RESULTS: AJ5018 had a higher degree of selectivity for CB1R over CB2R and markedly reduced brain penetrance, as reflected by the lower brain/plasma concentration ratio and the attenuated centrally mediated neurobehavioural effects, compared with its brain-penetrant parent compound rimonabant. In DIO and db/db mice, AJ5018 exhibited comparable effects to rimonabant in improving metabolic abnormalities and suppressing macrophage infiltration into white adipose tissue, activation of the NLRP3 inflammasome, and production of proinflammatory cytokines. CONCLUSIONS: These results suggest that peripheral CB1R blockade improves obesity-induced insulin resistance by suppressing adipose tissue inflammation via the NLRP3 inflammasome.

Cannabinoid 1 receptor knockout mice display cold allodynia, but enhanced recovery from spared-nerve injury-induced mechanical hypersensitivity.

BACKGROUND: The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R-/-) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. RESULTS: At baseline, CB1R-/- mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R-/- mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R-/- mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/-) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R-/- mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different among genotypes. CONCLUSION: Cold allodynia and significant recovery from spared-nerve injury-induced mechanical hypersensitivity are two novel phenotypes which characterize the global CB1R-/- mice. An increase in transient receptor potential channel of melastatin 8 channel function in DRG neurons may underlie the cold phenotype. Recovery of mechanical thresholds in the CB1R knockouts was independent of motor function. These results indicate that CB1R expression contributes to the development of persistent mechanical hypersensitivity, protects against the development of robust cold allodynia but is not involved in motor impairment following spared-nerve injury in mice.

The hepatic cannabinoid 1 receptor as a modulator of hepatic energy state and food intake.

The cannabinoid 1 receptor (CB1R) has a well-established role in appetite regulation. Central CB1R antagonists, notably rimonabant, induced weight loss and improved the metabolic profile in obese individuals, but were discontinued due to psychiatric side-effects. The CB1R is also expressed peripherally, where its effects include promotion of liver fat accumulation, which consumes ATP. Type 2 diabetes in obese subjects is linked to excess liver fat, whilst there is a negative correlation between hepatic ATP content and insulin resistance. A decreased hepatic ATP/AMP ratio increases food intake by signals via the vagus nerve to the brain. The hepatic cannabinoid system is highly upregulated in obesity, and the effects of hepatic CB1R activation include increased activity of lipogenic and gluconeogenic transcription factors. Thus, blockade of hepatic CB1Rs could contribute significantly to the weight-reducing and insulin-sensitizing effects of CB1R antagonists. Additionally, upregulation of the hepatic CB1R may contribute to chronic liver inflammation, fibrosis and cirrhosis from causes including obesity, alcoholism and viral hepatitis. Peripheral CB1R antagonists induce weight loss and metabolic improvements in obese rodents; however, as there is evidence that hepatic CB1Rs are predominately intracellular, due to high intrinsic clearance, many drugs may not effectively block these receptors and therefore have limited efficacy. Hepatoselective CB1R antagonists may be effective at reducing hepatic steatosis, insulin resistance and bodyweight in obese, diabetic patients, with far fewer side-effects than first-generation CB1R antagonists. Additionally, such compounds may be effective in treating inflammatory liver disease, such as non-alcoholic steatohepatitis, reducing the likelihood of disease progression to cirrhosis or cancer.

Gastrodin Ameliorates Post-Stroke Depressive-Like Behaviors Through Cannabinoid-1 Receptor-Dependent PKA/RhoA Signaling Pathway.

Post-stroke depression (PSD) is a significant complication in stroke patients, increases long-term mortality, and exaggerates ischemia-induced brain injury. However, the underlying molecular mechanisms and effective therapeutic targets related to PSD have remained elusive. Here, we employed an animal behavioral model of PSD by combining the use of middle cerebral artery occlusion (MCAO) followed by spatial restraint stress to study the molecular underpinnings and potential therapies of PSD. Interestingly, we found that sub-chronic application of gastrodin (Gas), a traditional Chinese medicinal herb Gastrodia elata extraction, relieved depression-related behavioral deficits, increased the impaired expression of synaptic transmission-associated proteins, and restored the altered spine density in hippocampal CA1 of PSD animals. Furthermore, our results indicated that the anti-PSD effect of Gas was dependent on membrane cannabinoid-1 receptor (CB1R) expression. The contents of phosphorated protein kinase A (p-PKA) and phosphorated Ras homolog gene family member A (p(ser188)-RhoA) were decreased in the hippocampus of PSD-mice, which was reversed by Gas treatment, and CB1R depletion caused a diminished efficacy of Gas on p-PKA and p-RhoA expression. In addition, the anti-PSD effect of Gas was partially blocked by PKA inhibition or RhoA activation, indicating that the anti-PSD effect of Gas is associated with the CB1R-mediated PKA/RhoA signaling pathway. Together, our findings revealed that Gas treatment possesses protective effects against the post-stroke depressive-like state; the CB1R-involved PKA/RhoA signaling pathway is critical in mediating Gas's anti-PSD potency, suggesting that Gas application may be beneficial in the prevention and adjunctive treatment of PSD.