CB1 Expression Research Articles

Hippocampal GR- and CB1-mediated mGluR5 differentially produces susceptibility and resilience to acute and chronic mild stress in rats

The molecular mechanism of individual response of susceptibility and resilience under psychological stress remains controversial and unclear. The present study aimed to explore the relationship of metabotropic glutamate receptor 5 (mGluR5) with glucocorticoid receptor (GR) or cannabinoid receptor (CB1) and further indicate the molecular mechanism of susceptibility and resilience to acute stress (AS) and chronic mild stress (CMS). Sucrose preference test and open field test were used to evaluate the response of susceptibility and resilience under stress in rats. The mRNA levels and protein expressions of mGluR5, GR, and CB1 were detected. AS induced a 35% reduction in the sucrose intake of rats, and these rats were considered as susceptible to stress; 21% of the rats showed resilience to the stress. Thirty-three percent of rats in the CMS group showed reduced sucrose water intake and were considered susceptible, while 20% of rats were considered resilient. Hippocampal mGluR5 mRNA and protein levels were increased in the susceptible rats. Pharmacological testing showed that GR was positively associated with mGluR5 in susceptible rats in the CMS group, while CB1 was negatively related to mGluR5 in susceptible rats in the AS group. The results suggested that GR and CB1 in the hippocampus might regulate mGluR5 protein and mRNA levels, which might be related to individual responses of susceptibility and resilience under AS and CMS.

Changes in gene expression and sensitivity of cocaine reward produced by a continuous fat diet.

Preclinical studies report that free access to a high-fat diet (HFD) alters the response to psychostimulants. The aim of the present study was to examine how HFD exposure during adolescence modifies cocaine effects. Gene expression of CB1 and mu-opioid receptors (MOr) in the nucleus accumbens (N Acc) and prefrontal cortex (PFC) and ghrelin receptor (GHSR) in the ventral tegmental area (VTA) were assessed. Mice were allowed continuous access to fat from PND 29, and the locomotor (10mg/kg) and reinforcing effects of cocaine (1 and 6mg/kg) on conditioned place preference (CPP) were evaluated on PND 69. Another group of mice was exposed to a standard diet until the day of post-conditioning, on which free access to the HFD began. HFD induced an increase of MOr gene expression in the N Acc, but decreased CB1 receptor in the N Acc and PFC. After fat withdrawal, the reduction of CB1 receptor in the N Acc was maintained. Gene expression of GHSR in the VTA decreased during the HFD and increased after withdrawal. Following fat discontinuation, mice exhibited increased anxiety, augmented locomotor response to cocaine, and developed CPP for 1mg/kg cocaine. HFD reduced the number of sessions required to extinguish the preference and decreased sensitivity to drug priming-induced reinstatement. Our results suggest that consumption of a HFD during adolescence induces neurobiochemical changes that increased sensitivity to cocaine when fat is withdrawn, acting as an alternative reward.

Anxious behavior induces elevated hippocampal Cb2 receptor gene expression

Anxiety is differentially expressed across a continuum of stressful/fearful intensity, influenced by endocannabinoid systems and receptors. The hippocampus plays important roles in the regulation of affective behavior, emotion, and anxiety, as well as memory. Location of Cb1/Cb2 receptor action could be important in determining emotional valence, because while the dorsal hippocampus is involved in spatial memory and cognition, the ventral hippocampus has projections to the PFC, BNST, amygdala, and HPA axis, and is important for emotional responses to stress. During repeated social defeat in a Stress-Alternatives Model arena (SAM; an oval open field with escape portals only large enough for smaller mice), smaller C57BL6/N mice are subject to fear conditioning (tone=CS), and attacked by novel larger aggressive CD1 mice (US) over four daily (5min) trials. Each SAM trial presents an opportunity for escape or submission, with stable behavioral responses established by the second day of interaction. Additional groups had access to a running wheel. Social aggression plus fear conditioning stimulates enhanced Cb2 receptor gene expression in the dorsal CA1, dorsal and ventral dentate gyrus subregions in animals displaying a submissive behavioral phenotype. Escape behavior is associated with reduced Cb2 expression in the dorsal CA1 region, with freezing and escape latency correlated with mRNA levels. Escaping and submitting animals with access to running wheels had increased Cb2 mRNA in dorsal DG/CA1. These results suggest that the Cb2 receptor system is rapidly induced during anxiogenic social interactions plus fear conditioning or exercise; with responses potentially adaptive for coping mechanisms.

Metabolic Syndrome Induced Bladder Cannabinoid Receptor Changes in the Fructose-Fed Rats.

To investigate the effect of metabolic syndrome on the bladder cannabinoid receptors 1 and 2 (CB1/CB2) expression and function in the fructose-fed rats (FR). Adult male Sprague-Dawley rats were divided into two groups: (i) Control rats fed with normal chow; and (ii) Rats fed with high-fructose diet (FR) for 9 weeks. The body weight, blood pressure, plasma sugar, insulin, triglyceride and cholesterol were measured. Bladder muscle strips were prepared in organ bath and pre-contracted with 1 µM/L acetylcholine (ACh) or 50 mM/L KCl. The relaxation responses to CB1/CB2 agonist Bay59-3047 (0.01-1 µM/L) were recorded. The effects of CB1 antagonist AM251, CB2 antagonist AM630, protein kinase A (PKA) inhibitor H-89 and ATP-sensitive potassium channel (KATP) inhibitor glibenclamide on the Bay59-3047-induced response were tested. Western blotting and real-time polymerase chain reaction (RT-PCR) analyses were performed for bladder CB1/CB2 receptors. Significant increases of body weight, blood pressure, plasma glucose, insulin, cholesterol and triglyceride levels were found in the FR. Bay59-3047 reduced ACh and KCl pre-contracted bladder strip tension in a dose-dependent fashion. The relaxation responses were significantly decreased in the FR. The Bay59-3047-induced relaxation was attenuated by AM251, glibenclamide and H-89. Western blotting and RT-PCR showed decreased expressions of FR bladder CB1 and CB2 receptor protein and mRNA. CB1/CB2 receptors mediate rat bladder relaxation through the PKA and KATP pathway. The CB1 receptor may play a more prominent role. The response is decreased in the FR bladder due to reduced expressions of the cannabinoid receptors.

Regulation of Cell Surface CB2 Receptor during Human B Cell Activation and Differentiation.

Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20+ B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD+/CD38Dim). While naïve mature and quiescent memory B cells (IgD-/CD38-) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD-/CD38+) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

In vivo TSPO and cannabinoid receptor type 2 availability early in post-stroke neuroinflammation in rats: a positron emission tomography study

BackgroundUpregulated levels of 18-kDa translocator proteins (TSPO) and type 2 endocannabinoid receptors (CB2) are considered to reflect different aspects of microglia-related neuroinflammatory responses in the brain. Relative to the increase in the TSPO expression that occurs slightly later during neuroinflammation in a proinflammatory fashion, CB2 activation is considered to relate to the neuroprotective responses that occurs predominantly at an early stage of brain disorders. These findings, however, were deduced from studies with different animal samples under different experimental settings. Here, we aimed to examined the differences in TSPO binding and CB2 availability at an early stage of stroke in the same animal using positron emission tomography (PET).MethodsWe used a total of eight Sprague-Dawley rats that underwent photothrombotic stroke surgery. The binding levels of a TSPO tracer [11C](R)PK11195 and a CB2 tracer [11C]NE40 were measured at 24 h after the surgery in the same animal using PET in combination with immunohistochemistry for CB2 and several other markers. A morphological inspection was also performed with X-ray computed tomography for small animals.ResultsThe levels of [11C]NE40 binding potential (BPND) were significantly higher in the cerebral cortical region on the lesion side than those on the non-lesion side, whereas no difference was found in the levels of [11C](R)PK11195 BPND between hemispheres. The tracer influx index (R1) data were all reduced on the lesion side irrespective of tracers. This increase in [11C]NE40 BPND was concomitant with an elevation in CB2 expression mainly within the microglia in the peri-infarct area, as shown by immunohistochemical examinations with Iba-1, CD11b/c+, and NG2+ staining.ConclusionsThe present results provide in vivo evidence of different responses of microglia occurring in the acute state of stroke. The use of the CB2 tracer [11C]NE40 allows us to evaluate the roles played by the neuroprotective aspect of microglia in acute neuroinflammatory processes.

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.

Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

BDNF-induced endocannabinoid release modulates neocortical glutamatergic neurotransmission.

Endocannabinoids (eCBs) and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), are potent neuromodulators found throughout the mammalian neocortex. Both eCBs and BDNF play critical roles in many behavioral and neurophysiological processes and are targets for the development of novel therapeutics. The effects of eCBs and BDNF are primarily mediated by the type 1 cannabinoid (CB1) receptor and the trkB tyrosine kinase receptor, respectively. Our laboratory and others have previously established that BDNF potentiates excitatory transmission by enhancing presynaptic glutamate release and modulating NMDA receptors. In contrast, we have shown that BDNF attenuates inhibitory transmission by inducing postsynaptic release of eCBs that act retrogradely to suppress GABA release in layer 2/3 of somatosensory cortex. Here, we hypothesized that BDNF also induces release of eCBs at excitatory synapses, which could have a mitigating or opposing effect on the direct presynaptic effects of BDNF. We found the highest levels of expression of CB1 and trkB and receptors in layers 2/3 and 5. Surprisingly, BDNF did not increase the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) onto layer 5 pyramidal neurons in somatosensory cortex, in contrast to its effects in the hippocampus and visual cortex. However, the effect of BDNF on mEPSC frequency in somatosensory cortex was unmasked by blocking CB1 receptors or disrupting eCB release. Thus, BDNF-trKB signaling regulates glutamate release in the somatosensory cortex via opposing effects, a direct presynaptic enhancement of release probability, and simultaneous postsynaptically-induced eCB release that decreases release probability via presynaptic CB1 receptors.

Lipid signaling and fat storage in the dark-eyed junco

Seasonal hyperphagia and fattening promote survivorship in migratory and wintering birds, but reduced adiposity may be more advantageous during the breeding season. Factors such as photoperiod, temperature, and food predictability are known environmental determinants of fat storage, but the underlying neuroendocrine mechanisms are less clear. Endocannabinoids and other lipid signaling molecules regulate multiple aspects of energy balance including appetite and lipid metabolism. However, these functions have been established primarily in mammals; thus the role of lipid signals in avian fat storage remains largely undefined. Here we examined relationships between endocannabinoid signaling and individual variation in fat storage in captive white-winged juncos (Junco hyemalis aikeni) following a transition to long-day photoperiods. We report that levels of the endocannabinoid 2-arachidonoylglycerol (2-AG), but not anandamide (AEA), in furcular and abdominal fat depots correlate negatively with fat mass. Hindbrain mRNA expression of CB1 endocannabinoid receptors also correlates negatively with levels of fat, demonstrating that fatter animals experience less central and peripheral endocannabinoid signaling when in breeding condition. Concentrations of the anorexigenic lipid, oleoylethanolamide (OEA), also inversely relate to adiposity. These findings demonstrate unique and significant relationships between adiposity and lipid signaling molecules in the brain and periphery, thereby suggesting a potential role for lipid signals in mediating adaptive levels of fat storage.

Decreased expression of fatty acid amide hydrolase in women with polycystic ovary syndrome

This study aimed to investigate the correlation between endocannabinoids and polycystic ovary syndrome (PCOS), and the expression levels of endocannabinoids in different phases of menstruation. The expression of cannabinoid receptors (CB1) and fatty acid amide hydrolase (FAAH) in the endometrium were immunohistochemically stained and compared between women with PCOS and the control group. Integrated optical density (IOD) was assessed to analyze their expression levels. The CB1 and FAAH were expressed in endometrial epithelial cytoplasm. No significant difference in CB1 level was observed between PCOS and non-PCOS women. Additionally, the expression of CB1 did not fluctuate with menstrual cycle. However, the FAAH levels were lower in the PCOS group than the non-PCOS group (p < 0.05). FAAH levels in secretory phases were significantly elevated compared to menstrual and proliferative phases (p < 0.05). Our results demonstrate that the endocannabinoid system may play an important role in menstruation, and dysregulation of the system may result in PCOS.

Cannabinoid receptor type 2 (CB2) as one of the candidate genes in human carotid plaque imaging: Evaluation of the novel radiotracer [11C]RS-016 targeting CB2 in atherosclerosis

IntroductionEndarterectomized human atherosclerotic plaques are a valuable basis for gene expression studies to disclose novel imaging biomarkers and therapeutic targets, such as the cannabinoid receptor type 2 (CB2). In this work, CB2 is expressed on activated immune cells, which are abundant in inflamed plaques. We evaluated the CB2-specific radiotracer [11C]RS-016 for imaging vascular inflammation in human and mouse atherosclerotic lesions. MethodsThe differential gene expression of microscopically classified human carotid plaques was evaluated using quantitative polymerase chain reaction. In addition, CB2 expression levels in human plaques were investigated by in vitro autoradiography. As an appropriate animal model we used apolipoprotein E knockout mice (ApoE KO) with shear stress-induced atherosclerosis to evaluate CB2 levels in vivo. Positron emission tomography (PET) was performed with both the CB2 radioligand [11C]RS-016 and the metabolic radiotracer [18F]fluorodeoxyglucose ([18F]FDG) at various time points. Retrospectively, carotids were dissected for histopathology and gene expression analysis. ResultsWe identified 28 human genes differentially expressed in atherosclerotic plaques compared to normal arteries of which 12 were upregulated preferentially in vulnerable plaques. The latter group included members of matrix metalloproteinase family and the T-lymphocyte activation antigens CD80 and CD86. CB2 was upregulated by 2-fold in human atherosclerotic plaques correlating with CD68 expression levels. Specific in vitro binding of [11C]RS-016 was predominantly observed to plaques. In vivo PET imaging of ApoE KO mice revealed accumulation of [11C]RS-016 and [18F]FDG in atherosclerotic plaques. Development of advanced plaques with elevated CB2 and CD68 levels were found in vitro in ApoE KO mice resembling human vulnerable plaques. ConclusionWe identified human genes associated with plaque vulnerability, which potentially could serve as novel imaging or therapeutic targets. The CB2-specific radiotracer [11C]RS-016 detected human plaques by in vitro autoradiography and accumulated in vivo in plaques of ApoE KO mice, however not exclusively in vulnerable plaques.

Fatty acid amide hydrolase (FAAH) inhibitor PF-3845 reduces viability, migration and invasiveness of human colon adenocarcinoma Colo-205 cell line: an in vitro study.

Earlier reports suggest that the endocannabinoids may play a role of endogenous tumor growth modulators. In this study, we investigated whether inhibition of the enzymes involved in the synthesis and degradation of endocannabinoids may reduce colorectal cancer cell invasion and migration. The human colon adenocarcinoma Colo-205 cells were incubated with PF-3845, JZL-184 and RHC-80267 (fatty acid amide hydrolase (FAAH), mono- (MAGL) and diacylglycerol lipase (DAGL) inhibitors, respectively) for 48 h. The MTT colorimetric assay was performed to quantify cell viability. Next, Colo-205 cells were incubated with PF-3845 alone or with PF-3845 together with selected antagonists: AM 251, AM 630, SB 366791, RN 1734 and G-15 (CB1, CB2, TRPV1, TRPV4 and GPR30 antagonists, respectively). Western blot assay was applied to identify the changes in CB1 and CB2 receptor expression. Migration and invasion assays were employed to characterize the effect of PF-3845 on colorectal cancer cell invasion. We found that of all the inhibitors used, the FAAH inhibitor PF-3845 reduced the Colo-205 cell line viability the most effectively (IC50=52.55 μM). We also showed that the effect of decreased cell viability was enhanced when Colo-205 cells were incubated with PF-3845 and RN-1734, a TRPV4 antagonist (IC50=30.54 μM). Western blot assay revealed significantly decreased CB1 receptor expression levels, while CB2 expression was increased in response to PF-3845 when compared to control. Furthermore, PF-3845 inhibited migration and invasion of Colo-205 cell line. These results suggest that pharmacological inhibition of FAAH and consequent enhancement of the endocannabinoid levels may reduce the colorectal cancer growth and progression.

Polyunsaturated Fatty Acids Differentially Modulate Cell Proliferation and Endocannabinoid System in Two Human Cancer Lines

Evidence suggests that quantity and quality of dietary polyunsaturated fatty acids (PUFAs) play a role in the development of cancer. However, the mechanisms involved in this interaction(s) are not clear. Endocannabinoids are lipid metabolites known to have growth modulatory actions. We studied the effect of supplementation with PUFAs ω-6 and ω-3 (essential fatty acids, EFAs), saturated and monounsaturated fatty acids (non-EFAs) on the growth of tumor cells and modifications in their endocannabinoid content. Cell cultures of human glioblastoma (T98G) and breast cancer (MCF7) were supplemented with 50 or 100mmol EFAs and non-EFAs for 72h. Cell proliferation was then determined by MTT, anandamide (AEA) levels by HPLC, total fatty acids profiles by GLC, CB1 receptor expression by WB and FAAH activity by spectrophotometric method. Fatty acids profile reflected the incorporation of the lipids supplemented in each assay. Arachidonic acid (EFA ω-6) supplementation increased AEA levels and inhibited the growth of T98G, whereas palmitic acid (non-EFA) enhanced their proliferation. In breast cancer (MCF7) cells, eicosapentaenoic acid (EFA ω-3) reduced and oleic acid (non-EFA) enhanced their proliferation. CB1 expression was higher in T98G and no differences were observed in FAAH activity. The growth of tumor cells can be differentially modulated by fatty acids and, at least in part, can be attributed to their ability to act on the components of the endocannabinoid system.

Effects of JWH015 in cytokine secretion in primary human keratinocytes and fibroblasts and its suitability for topical/transdermal delivery.

BackgroundJWH015 is a cannabinoid (CB) receptor type 2 agonist that produces immunomodulatory effects. Since skin cells play a key role in inflammatory conditions and tissue repair, we investigated the ability of JWH015 to promote an anti-inflammatory and pro-wound healing phenotype in human primary skin cells.MethodsHuman primary keratinocytes and fibroblasts were stimulated with lipopolysaccharide. The mRNA expression of cannabinoid receptors was determined using RT-PCR. The effects of JWH015 (0.05, 0.1, 0.5, and 1 µM) in pro- and anti-inflammatory factors were tested in lipopolysaccharide-stimulated cells. A scratch assay, using a co-culture of keratinocytes and fibroblasts, was used to test the effects of JWH015 in wound healing. In addition, the topical and transdermal penetration of JWH015 was studied in Franz diffusion cells using porcine skin and LC-MS.ResultsThe expression of CB1 and CB2 receptors (mRNA) and the production of pro- and anti-inflammatory factors enhanced in keratinocytes and fibroblasts following lipopolysaccharide stimulation. JWH015 reduced the concentration of major pro-inflammatory factors (IL-6 and MCP-1) and increased the concentration of a major anti-inflammatory factor (TGF-β) in lipopolysaccharide-stimulated cells. JWH015 induced a faster scratch gap closure. These JWH015’seffects were mainly modulated through both CB1 and CB2 receptors. Topically administered JWH015 was mostly retained in the skin and displayed a sustained and low level of transdermal permeation.ConclusionsOur findings suggest that targeting keratinocytes and fibroblasts with cannabinoid drugs could represent a therapeutic strategy to resolve peripheral inflammation and promote tissue repair.

Targeting the Endocannabinoid/CB1 Receptor System For Treating Major Depression Through Antidepressant Activities of Curcumin and Dexanabinol-Loaded Solid Lipid Nanoparticles

Background/Aims: This study investigated the underlying mechanisms of the antidepressant effects of curcumin and dexanabinol-loaded solid lipid nanoparticles in corticosterone-induced cell and mice depression models. Methods: Curcumin and dexanabinol-loaded solid lipid nanoparticles (Cur/SLNs-HU-211) were synthesized via an emulsifcation and low-temperature solidification method. Antidepressant activities of nanoparticles in a corticosterone-induced major depression model were investigated by MTT assay, cellular uptake by flow cytometry, behaviour by Forced Swimming Test and rotarod test, neurotransmitters by High Performance Liquid Chromatography, Western blotting, qPCR and immunofluorescence. Results: Treatment with Cur/SLNs-HU-211 induced greater dopamine (DA)/5-hydroxytryptamine (5-HT) release with reduced corticosterone-induced apoptotic cell death in PC12 cells. Additionally, in vivo Cur/SLNs-HU-211 significantly induced recovery from depressive behaviour with increased DA/5-HT levels, CB1 mRNA levels and CB1, p-MEK1 and p-ERK1/2 protein expression levels in the hippocampus and striatum. Cur/SLNs-HU-211 improved CB1 expression and inspired the proliferation of astrocytes in the hippocampus and striatum, exerted neuroprotective effects by preventing corticosterone -induced BDNF/NeuN expression reduction. Conclusion: Our study implies that Cur/SLNs-HU-211 may be a useful approach for treatment of major depression.

Development of Calbindin- and Calretinin-Immunopositive Neurons in the Enteric Ganglia of Rats.

Calbindin D28K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.

Cannabinoid modulation of zebrafish fear learning and its functional analysis investigated by c-Fos expression

It has been shown that zebrafish fear learning proceeds in the same way as reported for rodents. However, in zebrafish fear learning it is possible to substitute the use of electric shocks as unconditioned stimulus and utilize the inborn fear responses to the alarm substance Schreckstoff, instead. The skin extract Schreckstoff elicits typical fear reactions such as preferred bottom dwelling, swimming in a tighter shoal, erratic movements and freezing. This natural fear behavior can be transferred from Schreckstoff to any other sensory stimulus by associative conditioning (fear learning). We presented Schreckstoff simultaneously with a red light stimulus and tested the effectiveness of fear learning during memory retrieval. The two brain regions known to be relevant for learning in zebrafish are the medial and the lateral pallium of the dorsal telencephalon, both containing rich expressions of the endocannabinoid receptor CB1. To test the influence of the zebrafish endocannabinoid system on fear acquisition learning, an experimental group of ten fish was pretreated with the CB1 receptor agonist THC (Δ9-tetrahydrocannabinol; 100nM for 1h). We found that CB1 activation significantly inhibited acquisition of fear learning, possibly by impairing stimulus encoding processes in pallial areas. This was supported by analyzes of c-Fos expression in the brains of experimental animals. Schreckstoff exposure during fear acquisition learning and memory retrieval during red light presentation increased the number of labelled cells in pallial structures, but in no other brain region investigated (e.g. striatum, thalamus, and habenula). THC administration before fear conditioning significantly decreased c-Fos expression in these structures to a level similar to the control group without Schreckstoff experience, suggesting that Schreckstoff induced fear learning requires brain circuits restricted mainly to pallial regions of the dorsal telencephalon.

Cannabinoid Receptor Signaling As a Target for Personalized Therapy in Aggressive B Cell Lymphomas

Non-Hodgkin Lymphoma (NHL) is the most common hematological malignancy, with B-cell lymphoma (NHL-B) accounting for 85% of all lymphomas. In the United States, there are ~500,000 lymphoma patients currently living with this disease and ~20,000 lymphoma-related deaths occur annually. The current overall cure rate for B-cell lymphoma is estimated at ~30%, indicating that new innovative therapeutic approaches are needed to significantly reduce the high mortality rate, particularly of relapsed/refractory (r/r) NHL-B.The poor quality of life in patients suffering from chronic diseases like cancer has forced many patients to pursue alternative treatment options, including medicinal cannabinoids (CB), in order to improve their clinical prospect/outcomes. Medicinal cannabinoids have been legalized in 23 states and DC for several medical conditions such as cachexia, chronic pain, epilepsy and other similar disorders characterized by seizures, glaucoma, HIV- AIDS, Multiple Sclerosis, muscle spasticity and GI enteritis. Lately however, cannabis has been shown to have a broader biologic activity spectrum with various cannabis compounds functioning as ligands binding the two principle cannabinoid-specific G protein-coupled receptors (GPCR) CB1 (in neural cells), and CB2, in immune lymphoid, particularly B cells, but have also been identified, showing aberrant expression in a wide variety of important human cancers. This suggests not only a wider spectrum of cellular usage of cannabinoids and their cognate receptors, but also their potential utility as novel therapeutic targets. Gene expression profiling data has demonstrated, however, that B-cell lymphoma is one of the top three cancers (glioma and gastric are the other two) showing high expression of CB1 and CB2 receptors. Our studies showed that CB1 receptor is highly expressed in aggressive NHL-B, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cells in comparison to normal unstimulated (G0) B cells, and that targeting CB1 using an siRNA approach leads to cell growth inhibition. Furthermore, pharmacological approaches targeting CB1 with small molecule antagonists (Rimonabant and Otenabant) inhibited lymphoma cell viability, leading to the induction of apoptosis and G2M cell cycle arrest. Using proteomic approach via reverse-phase protein array (RPPA), we have demonstrated that lymphoma cells treated with the CB1 antagonist Rimonabant showed a robust effect on apoptosis (increases in caspase 3 and 7, Bad, and bak), cell cycle (increases in p27 and cyclin D1), DNA damage (increases in gH2AX), and autophagy (increases in LC3A) associated proteins. In addition, Rimonabant treatment also inhibited several growth and survival pathways, including STAT3, SRC, and b-catenin, while enhancing the PI3K/ATK pathway. Of note, Rimonabant treatment also activated the DNA damage response (DDR) pathway through stimulating two checkpoint kinases (Chk1 and Chk2). Blocking Rimonabant-induced Chk1 and Chk2 with a selective ATP-competitive inhibitor of Chk1 and Chk2 leads to a robust synergistic effect on cell growth inhibition and apoptotic induction, suggesting that blocking the DDR pathway with Chk kinase inhibitors prevents cells recovering from rimonabant-induced DNA damage. These findings suggest that targeting the cannabinoid receptors and the DDR pathway represents a new therapeutic strategy against resistant r/r NHL-B cells. DisclosuresPham:Vyripharm Biopharmaceuticals: Research Funding. Bryant:Vyripharm Biopharmaceuticals: Equity Ownership. Yang:Vyripharm Biopharmaceuticals: Employment.

Modulation of Type-1 and Type-2 Cannabinoid Receptors by Saffron in a Rat Model of Retinal Neurodegeneration.

Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection.