Weeding out bad waves: towards selective cannabinoid circuit control in epilepsy.

Endocannabinoids are lipid-derived messengers, and both their synthesis and breakdown are under tight spatiotemporal regulation. As retrograde signalling molecules, endocannabinoids are synthesized postsynaptically but activate presynaptic cannabinoid receptor 1 (CB1) receptors to inhibit neurotransmitter release. In turn, CB1-expressing inhibitory and excitatory synapses act as strategically placed control points for activity-dependent regulation of dynamically changing normal and pathological oscillatory network activity. Here, we highlight emerging principles of cannabinoid circuit control and plasticity, and discuss their relevance for epilepsy and related comorbidities. New insights into cannabinoid signalling may facilitate the translation of the recent interest in cannabis-related substances as antiseizure medications to evidence-based treatment strategies.

The expression of cannabinoid type 1 receptor and 2-arachidonoyl glycerol synthesizing/degrading enzymes is altered in basal ganglia during the active phase of levodopa-induced dyskinesia.

Management of levodopa-induced dyskinesias (LID) is one of the main challenges in the treatment of Parkinson's disease patients. Mechanisms involved in the appearance of these involuntary movements are not well known but modifications in the activity of different neurotransmitter pathways seem to play an important role. The objective of this study was to determine differences in the expression levels of the endocannabinoid system (ECS) elements that would support a role in LID. The basal ganglia nuclei, putamen, external segment of the globus pallidus (GPe), internal segment of the globus pallidus (GPi), subthalamic nucleus (STN) and substantia nigra (SN) were dissected out from cryostat sections obtained from two groups of parkinsonian monkeys treated with levodopa to induce dyskinesias. One group of dyskinetic animals was sacrificed under the effect of levodopa, during the active phase of LID, and the other group 24 h after the last levodopa dose (OFF levodopa). Biochemical analysis by real-time PCR for ECS elements was performed. CB1 receptor expression was upregulated in the putamen, GPe and STN during the active phase of dyskinesia and downregulated in the same nuclei and in the SN when dyskinetic animals were OFF levodopa. Changes in the 2-arachidonoyl glycerol (2-AG) synthesizing/degrading enzymes affecting the pallidal-subthalamic projections in dyskinetic animals OFF levodopa would suggest that 2-AG may play a role in LID. Anandamide (AEA) synthesizing/degrading enzymes were altered specifically in the GPe of untreated parkinsonian monkeys, suggesting that increased AEA levels may be a compensatory mechanism. These results indicate that the expression of the ECS elements is influenced by alterations in dopaminergic neurotransmission. On one hand, changes in CB1 receptor expression and in the 2-AG synthesizing/degrading enzymes suggest that they could be a therapeutic target for the active phase of LID. On the other hand, AEA metabolism could provide a non-dopaminergic target for symptomatic relief. However, further research is needed to unravel the mechanism of action of the ECS and how they could be modulated for a therapeutic purpose.

Increased expression of type 1 cannabinoid (CB1) receptor among patients with rotator cuff lesions and shoulder stiffness.

BACKGROUND: Shoulder stiffness is a disease manifested by pain, limited range of motion, and functional disability. The inflammatory and fibrosis processes play a substantial role in the pathogenesis of shoulder stiffness. The CB1 receptor has been recognized to mediate the processes of pathologic fibrosis. This study investigated the role of the CB1 pathway in pathogenesis of rotator cuff lesions with shoulder stiffness. METHODS: All of the patients undergoing repair surgery for rotator cuff lesions were recruited and subcategorized into subjects with and without shoulder stiffness. Reverse transcription-polymerase chain reaction assay was used to evaluate the expression level of CB1 and interleukin 1ß (IL-1ß) in the subacromial bursae, and enzyme-linked immunosorbent assay was used to measure the concentration of CB1 and IL-1ß in the subacromial fluid. Tenocytes treated with CB1 agonists and antagonists were also studied for the relationship of CB1 and the inflammatory cytokine IL-1ß. RESULTS: The patients with shoulder stiffness had higher messenger RNA (mRNA) expression (P = .040) and immunohistochemistry staining (P < .001) of CB1 in the subacromial bursa and higher CB1 concentration in the subacromial fluid (P = .008). Tenocytes treated with the CB1 agonist WIN 55,212-2 and antagonist AM251 showed increased expression of IL-1ß mRNA (P = .049) and suppressed expression of IL-1ß mRNA (P = .001), respectively. DISCUSSION: The CB1 pathway is involved in the pathogenesis of shoulder stiffness. It may be a promising target for the treatment of rotator cuff lesions with shoulder stiffness.

In vivo and In vitro Identification of Endocannabinoid Signaling in Periodontal Tissues and Their Potential Role in Local Pathophysiology.

The endocannabinoid system (ECS) with its binding receptors CB1 and CB2 impacts multiple pathophysiologies not only limited to neuronal psychoactivity. CB1 is assigned to cerebral neuron action, whereas CB2 is mainly expressed in different non-neuronal tissues and associated with immunosuppressive effects. Based on these tissue-selective CB receptor roles, it was the aim of this study to analyze potential expression in periodontal tissues under physiological conditions and inflammatory states. In vivo, CB receptor expression was investigated on human periodontal biopsies with or without bacterial inflammation and on rat maxillae with or without sterile inflammation. In vitro analyses were performed on human periodontal ligament (PDL) cells at rest or under mechanical strain via qRT-PCR, Western blot, and immunocytochemistry. P < 0.05 was set statistical significant. In vivo, CB1 expression was significantly higher in healthy PDL structures compared to CB2 (13.5% ± 1.3 of PDL tissues positively stained; 7.1% ± 0.9). Bacterial inflammation effected decrease in CB1 (9.7% ± 2.4), but increase in CB2 (14.7% ± 2.5). In contrast, sterile inflammation caused extensive CB1 (40% ± 1.9) and CB2 (41.7% ± 2.2) accumulations evenly distributed in the tooth surrounding PDL. In vitro, CB2 was ubiquitously expressed on gene and protein level. CB1 was constitutively expressed on transcriptional level (0.41% ± 0.09), even higher than CB2 (0.29% ± 0.06), but undetectable on protein level. Analyses further revealed expression changes of both receptors in mechanically loaded PDL cells. CB1 and CB2 are varyingly expressed in periodontal tissues, both adjusted by different entities of periodontal inflammation and by mechanical stress. This indicates potential ECS function as regulatory tool in controlling of periodontal pathophysiology.

Overexpression of cannabinoid receptor 1 in esophageal squamous cell carcinoma is correlated with metastasis to lymph nodes and distant organs, and poor prognosis.

In patients with esophageal squamous cell carcinoma (ESCC), the status of metastasis to lymph nodes is strongly associated with prognosis. Consequently, development of a biomarker to detect the presence of metastasis would be clinically valuable. In this study, we found that overexpression of cannabinoid receptor 1 (CB1R) was applicable as a marker for prediction of metastasis in ESCC. CB1R overexpression was detected immunohistochemically in 54 of 88 cases (61.4%). The intensity of CB1R expression was uniform in both intraepithelial and invasive regions in each case, and was significantly correlated with the status of metastasis to lymph nodes (P = 0.046) and distant organs (P = 0.047). Furthermore, multivariate analysis revealed that CB1R overexpression was independently associated with poor prognosis (P = 0.019). Biological analysis of CB1R overexpression using ESCC cell lines revealed that CB1R activation appeared to promote cell proliferation and invasion. On the basis of these findings, we propose that evaluation of CB1R expression status in biopsy specimens of ESCC using immunohistochemistry might be clinically useful for prediction of metastasis to lymph nodes and distant organs.

Expression of cannabinoid 1 and, 2 receptors and the effects of cannabinoid 1 and, 2 receptor agonists on detrusor overactivity associated with bladder outlet obstruction in rats.

BACKGROUND: This study investigated changes in the expression of cannabinoid (CB) receptors and the effects of CB1 and CB2 agonists on detrusor overactivity (DO) associated with bladder outlet obstruction (BOO) in rats. METHODS: Male Sprague Dawley rats were randomly assigned to four groups (n = 10) in each group. The control group comprised sham-operated rats. A animals in the BOO, CB1 agonist and CB2 agonist groups all underwent BOO surgery. Three weeks postoperatively, cystometrography (CMG) was performed on all rats. After confirming the presence of DO in the CB1 and CB2 agonist groups, a CB1 agonist (WIN 55,212-2) and a CB2 agonist (CB65) were instilled intravesically, and CMG was repeated. CMG parameters, including the contraction interval (CI) and contraction pressure (CP) were then analyzed. The bladders of rats in all four groups were excised following CMG. Immunofluorescence staining and Western blotting were performed to localize CB1 and CB2 and measure their expression levels in the urothelium and detrusor muscle. RESULTS: The CI was significantly longer and the CP was significantly lower in the CB1 agonist group than in the BOO group. CI and CP in the CB2 agonist group showed the same results. CB1 receptor immunofluorescence staining signals and immunoreactive bands in Western blotting were increased in the BOO group compared with results in the control group. Similarly, results for the CB2 receptor were also increased in the BOO group, although this difference was not significant. The CMG parameters in the BOO group were significantly improved by the inhibitory effects of CB1 and CB2 agonists on BOO-associated DO. The expression of CB1 was significantly increased in the urothelium and detrusor muscle in BOO-associated DO, but no significant change in CB2 expression was observed. CONCLUSIONS: CB1 and CB2 receptors, especially CB1, play a role in the pathophysiology of BOO-associated DO, and could serve as therapeutic targets.

Chronic Δ9-Tetrahydrocannabinol during Adolescence Differentially Modulates Striatal CB1 Receptor Expression and the Acute and Chronic Effects on Learning in Adult Rats.

The purpose of this study was to determine whether chronic administration of Δ(9)-tetrahydrocannabinol (THC) during adolescence would (1) modify any sex-specific effects of THC on learning and (2) affect the development of tolerance to THC as an adult. Male and female rats received daily injections of saline or 5.6 mg/kg of THC from postnatal day 35-75, yielding four groups (female/saline, female/THC, male/saline, and male/THC). Rats were then trained on a procedure that assayed both learning and performance behavior and administered 0.32-18 mg/kg of THC acutely as adults (experiment 1). THC produced rate-decreasing and error-increasing effects in both sexes; however, female rats were more sensitive than male rats were to the rate-decreasing effects. Rats were then chronically administered 10 mg/kg of THC (experiment 2). Rats that received THC during adolescence developed tolerance to the rate-decreasing effects more slowly and less completely than did rats that received saline; in addition, females developed tolerance to the error-increasing effects of THC slower than males did. Western blot analysis of brain tissue indicated long-term changes in hippocampal and striatal cannabinoid type-1 receptor (CB1R) levels despite levels that were indistinguishable immediately after chronic treatment during adolescence. Striatal CB1R levels were increased in adult rats that received THC during adolescence; hippocampal CB1R levels varied by sex. In summary, female rats were more sensitive than male rats were to the acute and chronic effects of THC, and chronic administration of THC during adolescence produced long-term changes in CB1R levels that correlated with decreased tolerance development to the rate-decreasing effects of THC.

Evaluation of cannabinoid CB1 and CB2 receptors expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients' survival.

Cannabinoid receptors (CB1R and CB2R) constitute essential members of the endocannabinoid system (ECS) which participates in many different functions indispensable to homeostatic regulation in several tissues, exerting also antitumorigenic effects. The present study aimed to assess the clinical significance of CB1R and CB2R protein expression in mobile tongue squamous cell carcinoma (SCC). CB1R and CB2R expression was assessed immunohistochemically on 28 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics and overall and disease-free patients' survival. CB1R, CB2R, and concomitant CB1R/CB2R expression was significantly increased in older compared to younger mobile tongue SCC patients (p = 0.0243, p = 0.0079, and p = 0.0366, respectively). Enhanced CB2R and concomitant CB1R/CB2R expression was significantly more frequently observed in female compared to male mobile tongue SCC patients (p = 0.0025 and p = 0.0016, respectively). Elevated CB2R expression was significantly more frequently observed in mobile tongue SCC patients presenting well-defined tumor shape compared to those with diffuse (p = 0.0430). Mobile tongue SCC patients presenting enhanced CB1R, CB2R, or concomitant CB1R/CB2R expression showed significantly longer overall (log-rank test, p = 0.004, p = 0.011, p = 0.018, respectively) and disease-free (log-rank test, p = 0.003, p = 0.007, p = 0.027, respectively) survival times compared to those with low expression. In multivariate analysis, CB1R was identified as an independent prognostic factor for disease-free patients' survival (Cox-regression analysis, p = 0.032). The present study provides evidence that CB1R and CB2R may play a role in the pathophysiological aspects of the mobile tongue SCC and even each molecule may constitute a potential target for the development of novel anti-cancer drugs for this type of malignancy.

Dronabinol has preferential antileukemic activity in acute lymphoblastic and myeloid leukemia with lymphoid differentiation patterns.

BACKGROUND: It has been previously demonstrated in several cancer models, that Dronabinol (THC) may have anti-tumor activity--however, controversial data exists for acute leukemia. We have anecdotal evidence that THC may have contributed to disease control in a patient with acute undifferentiated leukemia. METHODS: To test this hypothesis, we evaluated the antileukemic efficacy of THC in several leukemia cell lines and native leukemia blasts cultured ex vivo. Expression analysis for the CB1/2 receptors was performed by Western immunoblotting and flow cytometry. CB-receptor antagonists as well as a CRISPR double nickase knockdown approach were used to evaluate for receptor specificity of the observed proapoptotic effects. RESULTS: Meaningful antiproliferative as well as proapoptotic effects were demonstrated in a subset of cases--with a preference of leukemia cells from the lymphatic lineage or acute myeloid leukemia cells expressing lymphatic markers. Induction of apoptosis was mediated via CB1 as well as CB2, and expression of CB receptors was a prerequisite for therapy response in our models. Importantly, we demonstrate that antileukemic concentrations are achievable in vivo. CONCLUSION: Our study provides rigorous data to support clinical evaluation of THC as a low-toxic therapy option in a well defined subset of acute leukemia patients.

Spontaneous involution of pediatric low-grade gliomas: high expression of cannabinoid receptor 1 (CNR1) at the time of diagnosis may indicate involvement of the endocannabinoid system.

BACKGROUND: Pediatric low-grade gliomas (P-LGG) consist of a mixed group of brain tumors that correspond to the majority of CNS tumors in children. Notably, they may exhibit spontaneous involution after subtotal surgical removal (STR). In this study, we investigated molecular indicators of spontaneous involution in P-LGG. METHODS: We performed an integrated molecular analysis including high throughput gene expression (GE), microRNA (miRNA) expression data of primary, untreated tumors from patients with P-LGG who underwent STR at our institution, with at least 10 years follow-up. RESULTS: We identified a set of protein-coding genes and miRNAs significantly differentially expressed in P-LGG that presented spontaneous involution (involution-I) or without progression (stable-S) after STR alone. The cannabinoid receptor 1 (CNR1 or CB1) gene (FC = 2.374; p value = 0.007) was at the top of the list and predicted to be regulated by hsa-miR-29b-3p (FC = -2.353, p value = 0.0001). CNR1 also showed a trend to be higher expressed in S/I by immunohistochemistry. CONCLUSIONS: The P-LGG, which remained stable or that presented spontaneous involution after STR, showed significantly higher CNR1 expression at the time of diagnosis. We hypothesize that high expression levels of CNR1 provide tumor susceptibility to the antitumor effects of circulating endocannabinoids like anandamide, resulting in tumor involution. This corroborates with reports suggesting that CNR1 agonists and activators of the endocannabinoid system may represent therapeutic opportunities for children with LGG. We also suggest that CNR1 may be a prognostic marker for P-LGG. This is the first time spontaneous involution of P-LGG has been suggested to be induced by endocannabinoids.

The endocannabinoid anandamide affects the synthesis of human syncytiotrophoblast-related proteins.

The human syncytiotrophoblast (hST) has a major role in the production of important placental hormones. Several molecules regulate hST endocrine function but the role of endocannabinoids in this process is still unknown. Here, we report that the endocannabinoid anandamide (AEA) decreased cAMP levels, impaired human chorionic gonadotropin secretion, placental alkaline phosphatase activity and decreased aromatase mRNA levels and protein expression, through cannabinoid (CB) receptor activation. AEA also downregulated leptin and placental protein 13 transcription, though via a CB receptor-independent mechanism. All this evidence suggests AEA is a novel modulator of hormone synthesis by the syncytiotrophoblast, supporting the importance of the endocannabinoid signalling in placental function.

Prenatal exposure to the CB1 and CB2 cannabinoid receptor agonist WIN 55,212-2 alters migration of early-born glutamatergic neurons and GABAergic interneurons in the rat cerebral cortex.

The endocannabinoid system, composed of cannabinoid receptors, endocannabinoids, and synthesis and degradation enzymes, is present since early stages of brain development. During this period, the endocannabinoid system is involved in the regulation of neural progenitor proliferation and specification as well as the migration and differentiation of pyramidal neurons and interneurons. Marijuana consumption during pregnancy represents a serious risk in relation to the fetal brain development since Δ(9) -tetrahidrocannabinol, the main active compound of cannabis, can reach the fetus through placenta and hemato-encephalic barrier. Cohort studies performed on children and adolescents of mothers who consumed marijuana during pregnancy reported cognitive and comportamental abnormalities. In the present study, we examined the expression of the cannabinoid receptor CB1 R during corticogenesis in radially and tangentially migrating post-mitotic neurons. We found that prenatal exposure to WIN impaired tangential and radial migration of post-mitotic neurons in the dorsal pallium. In addition, we described alterations of two transcription factors associated with proliferating and newly post-mitotic glutamatergic cells in the dorsal pallium, Tbr1 and Tbr2, and disruption in the number of Cajal-Retzius cells. The present results contribute to the knowledge of neurobiological substrates that determine neuro-comportamental changes that will persist through post-natal life.

Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/ß-arrestin activation of ERK1/2 and Src kinase.

We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two Gßγ-dependent pathways (PLC-ß3 and phosphatidylinositol 3-kinase/integrin-linked kinase). Here we show that the anticipated Gßγ-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via Gαi, signaling resulted from Gßγ-independent phosphorylation of CB1 receptors by GRK5, recruitment of ß-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by pertussis toxin (PTx) or Gi minigene nor expression of a Gßγ-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing ß-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated phosphoinositide hydrolysis or enhance Gαq:RGS4 association and inactivation of Gαq. Activation of Src kinase by anandamide enhanced both myosin phosphatase RhoA-interacting protein (M-RIP):RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity, leading to increase of myosin light chain (MLC) phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage Gßγ but signaled via GRK5/ß-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of Gαq by RGS4, and Src kinase enhanced MLC phosphatase activity, leading to inhibition of ACh-stimulated contraction.

Molecular basis for the improvement in muscle metaboreflex and mechanoreflex control in exercise-trained humans with chronic heart failure.

Previous studies have demonstrated that muscle mechanoreflex and metaboreflex controls are altered in heart failure (HF), which seems to be due to changes in cyclooxygenase (COX) pathway and changes in receptors on afferent neurons, including transient receptor potential vanilloid type-1 (TRPV1) and cannabinoid receptor type-1 (CB1). The purpose of the present study was to test the hypotheses: 1) exercise training (ET) alters the muscle metaboreflex and mechanoreflex control of muscle sympathetic nerve activity (MSNA) in HF patients. 2) The alteration in metaboreflex control is accompanied by increased expression of TRPV1 and CB1 receptors in skeletal muscle. 3) The alteration in mechanoreflex control is accompanied by COX-2 pathway in skeletal muscle. Thirty-four consecutive HF patients with ejection fractions <40% were randomized to untrained (n = 17; 54 ± 2 yr) or exercise-trained (n = 17; 56 ± 2 yr) groups. MSNA was recorded by microneurography. Mechanoreceptors were activated by passive exercise and metaboreceptors by postexercise circulatory arrest (PECA). COX-2 pathway, TRPV1, and CB1 receptors were measured in muscle biopsies. Following ET, resting MSNA was decreased compared with untrained group. During PECA (metaboreflex), MSNA responses were increased, which was accompanied by the expression of TRPV1 and CB1 receptors. During passive exercise (mechanoreflex), MSNA responses were decreased, which was accompanied by decreased expression of COX-2, prostaglandin-E2 receptor-4, and thromboxane-A2 receptor and by decreased in muscle inflammation, as indicated by increased miRNA-146 levels and the stable NF-κB/IκB-α ratio. In conclusion, ET alters muscle metaboreflex and mechanoreflex control of MSNA in HF patients. This alteration with ET is accompanied by alteration in TRPV1 and CB1 expression and COX-2 pathway and inflammation in skeletal muscle.

MicroRNA-665 is involved in the regulation of the expression of the cardioprotective cannabinoid receptor CB2 in patients with severe heart failure.

The myocardial endocannabinoid system has been linked to stress response and cardioprotection. In chronic heart failure (CHF), protective CB2 receptors are markedly up-regulated while CB1 receptors are slightly down-regulated. We here provide evidence that myocardial CB receptors are subject to microRNA regulation. By a combined computational and experimental approach we show that CB1 receptors are regulated by miR-494, and CB2 receptors are targeted by miR-665. Moreover, we demonstrate that in CHF, miR-665 expression is significantly decreased while miR-494 is slightly increased, which is concordant with the previously reported alterations of CB receptors. These results suggest that in CHF, altered expression of specific miRNAs may contribute to a compensatory response of the diseased myocardium.

Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB1 receptors and apoptotic cell death.

The abuse of herbal products containing synthetic cannabinoids has become an issue of public concern. The purpose of this paper was to evaluate the acute cytotoxicity of synthetic cannabinoids on mouse brain neuronal cells. Cytotoxicity induced by synthetic cannabinoid (CP-55,940, CP-47,497, CP-47,497-C8, HU-210, JWH-018, JWH-210, AM-2201, and MAM-2201) was examined using forebrain neuronal cultures. These synthetic cannabinoids induced cytotoxicity in the forebrain cultures in a concentration-dependent manner. The cytotoxicity was suppressed by preincubation with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630. Furthermore, annexin-V-positive cells were found among the treated forebrain cells. Synthetic cannabinoid treatment induced the activation of caspase-3, and preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity. These synthetic cannabinoids induced apoptosis through a caspase-3-dependent mechanism in the forebrain cultures. Our results indicate that the cytotoxicity of synthetic cannabinoids towards primary neuronal cells is mediated by the CB1 receptor, but not by the CB2 receptor, and further suggest that caspase cascades may play an important role in the apoptosis induced by these synthetic cannabinoids. In conclusion, excessive synthetic cannabinoid abuse may present a serious acute health concern due to neuronal damage or deficits in the brain.

Acute resistance exercise induces antinociception by activation of the endocannabinoid system in rats.

BACKGROUND: Resistance exercise (RE) is also known as strength training, and it is performed to increase the strength and mass of muscles, bone strength, and metabolism. RE has been increasingly prescribed for pain relief. However, the endogenous mechanisms underlying this antinociceptive effect are still largely unexplored. Thus, we investigated the involvement of the endocannabinoid system in RE-induced antinociception. METHODS: Male Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by a mechanical nociceptive test (paw pressure) before and after exercise. To investigate the involvement of cannabinoid receptors and endocannabinoids in RE-induced antinociception, cannabinoid receptor inverse agonists, endocannabinoid metabolizing enzyme inhibitors, and an anandamide reuptake inhibitor were injected before RE. After RE, CB1 cannabinoid receptors were quantified in rat brain tissue by Western blot and immunofluorescence. In addition, endocannabinoid plasma levels were measured by isotope dilution-liquid chromatography mass spectrometry. RESULTS: RE-induced antinociception was prevented by preinjection with CB1 and CB2 cannabinoid receptor inverse agonists. By contrast, preadministration of metabolizing enzyme inhibitors and the anandamide reuptake inhibitor prolonged and enhanced this effect. RE also produced an increase in the expression and activation of CB1 cannabinoid receptors in rat brain tissue and in the dorsolateral and ventrolateral periaqueductal regions and an increase in endocannabinoid plasma levels. CONCLUSIONS: The present study suggests that a single session of RE activates the endocannabinoid system to induce antinociception.

Bimodal control of fear-coping strategies by CB1 cannabinoid receptors.

To maximize their chances of survival, animals need to rapidly and efficiently respond to aversive situations. These responses can be classified as active or passive and depend on the specific nature of threats, but also on individual fear coping styles. In this study, we show that the control of excitatory and inhibitory brain neurons by type-1 cannabinoid (CB1) receptors is a key determinant of fear coping strategies in mice. In classical fear conditioning, a switch between initially predominant passive fear responses (freezing) and active behaviors (escape attempts and risk assessment) develops over time. Constitutive genetic deletion of CB1 receptors in CB1⁻/⁻ mice disrupted this pattern by favoring passive responses. This phenotype can be ascribed to endocannabinoid control of excitatory neurons, because it was reproduced in conditional mutant mice lacking CB1 receptors from cortical glutamatergic neurons. CB1 receptor deletion from GABAergic brain neurons led to the opposite phenotype, characterized by the predominance of active coping. The CB1 receptor agonist Δ9-tetrahydrocannabinol exerted a biphasic control of fear coping strategies, with lower and higher doses favoring active and passive responses, respectively. Finally, viral re-expression of CB1 receptors in the amygdala of CB1⁻/⁻ mice restored the normal switch between the two coping strategies. These data strongly suggest that CB1 receptor signaling bimodally controls the spontaneous adoption of active or passive coping strategies in individuals. This primary function of the endocannabinoid system in shaping individual behavioral traits should be considered when studying the mechanisms of physiological and pathological fear.

Crosstalk between dopamine D2 receptors and cannabinoid CB1 receptors regulates CNR1 promoter activity via ERK1/2 signaling.

Previously, we found that chronic methamphetamine treatment altered cannabinoid type 1 receptor (CB1R)-dependent cAMP/PKA/dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32)/T34/PP2B signaling and decreased levels of CB1R protein and mRNA in the nucleus accumbens. These findings suggested the existence of signaling interplay between mesolimbic dopamine and CB1R. In this study, we further investigate interactions between CB1R and dopamine D2 receptor (D2R) signaling. Activation of either CB1R or D2R increased extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, while co-stimulation of CB1R and D2 R evoked an additive effect on the phospho-ERK1/2 signal. This effect was mediated through a pertussis toxin-sensitive Gαi/o pathway in primary striatal cells. Furthermore, the mRNA level of CB1R was increased via dopamine D2 receptor short form (D(2S)R) by treatment with D2R agonist quinpirole in D(2S)R/C6 glioma cells. This effect could be suppressed by co-treatment with the ERK1/2 inhibitor U0126. To test if D(2S)R could transcriptionally regulate CB1R, the 5'-untranslated region (5'-UTR) of the cannabinoid receptor 1 (CNR1) gene was sequenced from rat brain. Results showed that the CNR1 gene includes two exons, which contain 375 bp of 5'-UTR and are separated by a 17-kb intron. A luciferase reporter assay showed that the maximal D(2S)R-responsive promoter activity is located in the -1 to -222 region of CNR1 promoter. Overall, we demonstrate previously unidentified crosstalk between D2R and CB1R via ERK1/2 signaling that enhances the expression of CB1R by modulating its promoter activity.

Repeated low-dose administration of the monoacylglycerol lipase inhibitor JZL184 retains cannabinoid receptor type 1-mediated antinociceptive and gastroprotective effects.

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug-induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [(3)H]SR141716A binding and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ(9)-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.