CB1 Gene Research Articles

Appetite regulation: The central role of melatonin in Danio rerio

Melatonin is the hormonal mediator of photoperiodic information to the central nervous system in vertebrates and allows the regulation of energy homeostasis through the establishment of a proper balance between energy intake and energy expenditure. The aim of this study was to evaluate the role of melatonin in appetite central control analyzing the involvement of this hormone in the regulation of feeding behavior in the zebrafish Danio rerio. For this purpose, the effect of two different melatonin doses (100 nM and 1 μM) administered for 10 days, via water, to zebrafish adults was evaluated at both physiological and molecular level and the effect of melatonin was considered in relation to the most prominent systems involved in appetite regulation. For the first time, in fact, melatonin control of food intake by the modulation of leptin, MC4R, ghrelin, NPY and CB1 gene expression was evaluated. The results obtained indicate that melatonin significantly reduces food intake and the reduction is in agreement with the changes observed at molecular level. A significant increase in genes codifying for molecules involved in feeding inhibition, such as leptin and MC4R, and a significant reduction in the major orexigenic signals including ghrelin, NPY and CB1 are showed here. Taken together these results support the idea that melatonin falls fully into the complex network of signals that regulate food intake thus playing a key role in central appetite regulation.

Functional Interactions between Endogenous Cannabinoid and Opioid Systems: Focus on Alcohol, Genetics and Drug-Addicted Behaviors

Although the first studies regarding the endogenous opioid system and addiction were published during the 1940s, addiction and cannabinoids were not addressed until the 1970s. Currently, the number of opioid addiction studies indexed in PubMed-Medline is 16 times greater than the number of cannabinoid addiction reports. More recently, functional interactions have been demonstrated between the endogenous cannabinoid and opioid systems. For example, the cannabinoid brain receptor type 1 (CB1) and mu opioid receptor type 1 (MOR1) co-localize in the same presynaptic nerve terminals and signal through a common receptor-mediated G-protein pathway. Here, we review a great variety of behavioral models of drug addiction and alcohol-related behaviors. We also include data providing clear evidence that activation of the cannabinoid and opioid endogenous systems via WIN 55,512-2 (0.4-10 mg/kg) and morphine (1.0-10 mg/kg), respectively, produces similar levels of relapse to alcohol in operant alcohol self-administration tasks. Finally, we discuss genetic studies that reveal significant associations between polymorphisms in MOR1 and CB1 receptors and drug addiction. For example, the SNP A118G, which changes the amino acid aspartate to asparagine in the MOR1 gene, is highly associated with altered opioid system function. The presence of a microsatellite polymorphism of an (AAT)n triplet near the CB1 gene is associated with drug addiction phenotypes. But, studies exploring haplotypes with regard to both systems, however, are lacking.

G1359A polymorphism of the cannabinoid receptor gene (CNR1) and insulin resistance in patients with diabetes mellitus type 2.

A silent intragenic biallelic polymorphism (1359 G/A) (rs1049353) of the CB1 gene resulting in the substitution of the G to A at nucleotide position 1359 in codon 435 (Thr), was reported as a common polymorphism in Caucasian populations. The aim of our study was to investigate the influence of the missense polymorphism (G1359A) of CB1 receptor gene on obesity anthropometric parameters, cardiovascular risk factors and adipocytokines in patients with obesity and diabetes mellitus type 2. A population of 60 naïve diabetic patients was analyzed. An indirect calorimetry, tetrapolar electrical bioimpedance, blood pressure, a serial assessment of nutritional intake with 3 days written food records and biochemical analysis (lipid profile, adipocytokines, insulin, CRP and lipoprotein-a) were performed. The statistical analysis was performed for the combined G1359A and A1359A as a group and wild type G1359G as second group, with a dominant model. The mean age was 57.44 +/- 11.7 years and the mean BMI 37.84 +/- 6.4, with 14 males and 46 females. Thirty-five patients (58.3%) had genotype G1359G (wild type group) and 25 (42.7%) patients G1359A (mutant type group). Age was similar in both groups (wild type: 56.3 +/- 11.8 years vs mutant group: 58.7 +/- 10 years:ns). Sex distribution was similar in both groups (wild vs mutant type groups), males (22.9% vs 24%) and females (77.1% vs 76%). No differences were detected between groups in anthropometric parameters, cardiovascular risk factors, dietary intake and adipocytokines levels. The finding of this study is the lack of association of G1359A polymorphism of CB receptor 1 gene with obesity, cardiovascular risk factors and adipocytokines.

The central role of melatonin in Danio rerio appetite regulation

Event Abstract Back to Event The central role of melatonin in Danio rerio appetite regulation Beatrice Migliarini1*, Chiara C. Piccinetti1, Ike Olivotto1 and Oliana Carnevali1 1 Polytechnic University of Marche, Department of Marine Sciences, Italy Melatonin is the hormonal mediator of photoperiodic information to the central nervous system in vertebrates and allows the regulation of energy homeostasis through the establishment of a proper balance between energy intake and energy expenditure. The aim of this study was to evaluate the role of melatonin in the regulation of feeding behavior in the zebrafish Danio rerio. For this purpose, the effect of two different melatonin doses (100 nM and 1µM) administered for ten days via water to zebrafish adults was evaluated at both physiological and molecular level and the effect of melatonin was considered in relation to the most prominent systems involved in appetite regulation. The melatonin control of food intake by the modulation of leptin, MC4R, ghrelin, NPY and CB1 gene expression was evaluated. The results obtained indicate that melatonin significantly reduces food intake and the reduction is in agreement with the changes observed at the molecular level. A significant increase in genes codifying for molecules involved in feeding inhibition, such as leptin and MC4R, and a significant reduction in the major orexigenic signals including ghrelin, NPY and CB1 was demonstrated. These results support the idea that melatonin falls fully into the complex network of signals that regulate food intake and therefore this hormone plays a key role in central appetite regulation. Keywords: comparative endocrinology Conference: 25th Conference of the European Comparative Endocrinologists, Pécs, Hungary, 31 Aug - 4 Sep, 2010. Presentation Type: Conference Presentation Topic: Comparative endocrinology Citation: Migliarini B, Piccinetti CC, Olivotto I and Carnevali O (2010). The central role of melatonin in Danio rerio appetite regulation. Front. Endocrinol. Conference Abstract: 25th Conference of the European Comparative Endocrinologists. doi: 10.3389/conf.fendo.2010.01.00014 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Aug 2010; Published Online: 29 Aug 2010. * Correspondence: Dr. Beatrice Migliarini, Polytechnic University of Marche, Department of Marine Sciences, Ancona, Italy, b.migliarini@univpm.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Beatrice Migliarini Chiara C Piccinetti Ike Olivotto Oliana Carnevali Google Beatrice Migliarini Chiara C Piccinetti Ike Olivotto Oliana Carnevali Google Scholar Beatrice Migliarini Chiara C Piccinetti Ike Olivotto Oliana Carnevali PubMed Beatrice Migliarini Chiara C Piccinetti Ike Olivotto Oliana Carnevali Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cannabinoid CB1 receptor expression in relation to visceral adipose depots, endocannabinoid levels, microvascular damage, and the presence of the Cnr1 A3813G variant in humans

Dysregulation of the endocannabinoid system in the visceral adipose tissue (VAT) is associated with metabolic and cardiovascular complications of obesity. We studied perirenal VAT CB1 receptor expression in relation to anthropometry, VAT area and endocannabinoid levels, kidney microvascular damage (MVDa), and the presence of the CB1 gene A3813G variant, the frequency of which was also evaluated in a large population of obese-hypertensive (OH) patients with or without the metabolic syndrome (MetS). Perirenal VAT and kidney samples were obtained from 30 patients undergoing renal surgery. Total and perirenal VAT areas were determined by computed tomography. CB1 messenger RNA expression and endocannabinoid levels in perirenal VAT were determined by quantitative reverse transcriptase polymerase chain reaction and liquid chromatography–mass spectrometry, respectively. The MVDa was evaluated in healthy portions of kidney cortex. The A3813G alleles were identified by genotyping in these patients and in 280 nondiabetic OH patients (age ≤65 years). Metabolic syndrome was defined according to the Adult Treatment Panel III criteria. Perirenal VAT CB1 expression was 40% lower in patients with the A3813G polymorphism, and correlated positively with perirenal and total VAT area and with perirenal VAT levels of the endocannabinoid anandamide. A 2-fold higher CB1 expression was associated with MVDa. The OH patients with the A3813G allele had lower prevalence of MetS in both unadjusted and adjusted models. Genetics influence perirenal VAT CB1 expression and the prevalence of MetS in OH. Increased VAT is associated with increased perirenal VAT endocannabinoid tone, which in turn correlates with increased MVDa. Endocannabinoid overactivity might be involved in human visceral obesity and its renal complications.

Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5' exons of both CB2 isoforms are untranslated 5'UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.

Interactions between the anxiogenic effects of CB1 gene disruption and 5-HT3 neurotransmission

Neuroanatomical findings revealed that CB1 cannabinoid and 5-HT3 receptors are coexpressed by a subtype of gamma-aminobutyric acid (GABA)ergic interneurons in the prefrontal cortex, hippocampus, and basolateral amygdala, three brain regions that are crucial for the control of anxiety. In these regions, serotonergic inputs increase GABA release through 5-HT3 receptors, the phenomenon being retrogradely controlled by cannabinoid neurotransmission. This suggests a functional interaction between 5-HT3 neurotransmission and CB1 signaling. In a first attempt to investigate the behavioral relevance of these interactions, we studied the effects of the selective 5-HT3 agonist 1-(m-chlorophenyl)-biguanide (mCPBG), on plus-maze behavior in NMRI, CD1 wild type, and CB1-KO mice. The genetic disruption of CB1 receptors consistently increased anxiety. This effect was significantly decreased by the 5-HT3 agonist, mCPBG. The dose-response curve was bell-shaped. Surprisingly, mCPBG did not affect the behavior of CD1 wild type and NMRI mice. We hypothesize that in the aforementioned regions, 5-HT3 activation decreases anxiety by promoting GABA release, but this effect is dampened by CB1 signaling. The disruption of CB1 receptors in CB1-KOs released GABA neurons from retrograde inhibition and made the effects of 5-HT3 stimulation conspicuous. Altogether, our findings reveal a functional interaction between 5-HT3 neurotransmission and CB1 signaling. Besides this interaction being an interesting aspect of anxiety control, it may also explain the notoriously inconsistent effects of 5-HT3 ligands on anxiety. If 5-HT3 neurotransmission and CB1 signaling interact, the anxiety-related effects of 5-HT3 ligands may depend on species, strain, and situation-related differences in both 5-HT3 and CB1 receptor expression and function.

Species differences in cannabinoid receptor 2 (CNR2) gene: identification of novel human and rodent CB2 isoforms, differential tissue expression, and regulation by cannabinoid receptor ligands

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5′ exons of both CB2 isoforms are untranslated 5′UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.

Functional expression of brain neuronal CB2 cannabinoid receptors are involved in the effects of drugs of abuse and in depression.

Major depression and addiction are mental health problems associated with stressful events in life with high relapse and recurrence even after treatment. Many laboratories were not able to detect the presence of CB2 cannabinoid receptors (CB2-Rs) in healthy brains, but CB2-R expression has been demonstrated in rat microglial cells and other brain-associated cells during inflammation. Thus, neuronal expression of CB2-Rs has been ambiguous and controversial, and its role in depression and substance abuse is unknown. In this study we tested the hypothesis that genetic variants of the CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances, including opiates, cocaine, and ethanol, in rodents. Here we demonstrate that a high incidence of Q63R but not H316Y polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated after exposure to stressors and administration of abused drugs. Mice that developed an alcohol preference had reduced CB2 gene expression, and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 antisense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. Using electron microscopy we report the subcellular localization of CB2-Rs that are mainly on postsynaptic elements in rodent brain. Our data demonstrate the functional expression of CB2-Rs in the brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuroimmunocannabinoid activity.

The endocannabinoid receptor, CB1, is required for normal axonal growth and fasciculation

Endocannabinoids are retrograde neurotransmitters, which act upon the presynaptically located, G-protein coupled receptor CB1, to modulate synaptic transmission in the adult brain. Recently, however, a number of lines of evidence have suggested that endocannabinoid signalling may play an important role in early neuronal development. In this study, we show that the CB1 receptor has a wide expression pattern in the developing nervous system and that its expression follows neuronal differentiation in the embryo from the earliest stages. We also show that the enzymes involved in 2-AG synthesis are expressed in an overlapping manner at these stages. We further show that interfering with CB1 function using a pharmacological inhibitor causes problems in axon pathfinding and fasciculation. Similarly, CB1 gene knock down in the zebrafish by morpholino injection results in defects in axonal growth and fasciculation in these embryos. Thus CB1 function is required in the early embryo for axonal growth and fasciculation.

Brain Neuronal CB2 Cannabinoid Receptors in Drug Abuse and Depression: From Mice to Human Subjects

BackgroundAddiction and major depression are mental health problems associated with stressful events in life with high relapse and reoccurrence even after treatment. Many laboratories were not able to detect the presence of cannabinoid CB2 receptors (CB2-Rs) in healthy brains, but there has been demonstration of CB2-R expression in rat microglial cells and other brain associated cells during inflammation. Therefore, neuronal expression of CB2-Rs had been ambiguous and controversial and its role in depression and substance abuse is unknown.Methodology/Principal FindingsIn this study we tested the hypothesis that genetic variants of CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances including opiates, cocaine and ethanol in rodents. Here we demonstrate that a high incidence of (Q63R) but not (H316Y) polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated following exposure to stressors and administration of abused drugs. Mice that developed alcohol preference had reduced CB2 gene expression and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 anti-sense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. We report for the using electron microscopy the sub cellular localization of CB2-Rs that are mainly on post-synaptic elements in rodent brain.Conclusions/SignificanceOur data demonstrate the functional expression of CB2-Rs in brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuro-immunocannabinoid activity.

Estrogenic induction of cannabinoid CB1 receptor in human colon cancer cell lines

Objective. Cannabinoids are a class of compounds that have the ability to activate two specific receptor subtypes, the cannabinoid CB1 and CB2 receptors. CB1 receptor is a G-protein-coupled receptor that is linked to the signal transduction pathways. The cumulative effects of this receptor have important implications in the control of cell survival and cell death having the potential to regulate tumor cell growth. In this connection, interest has been focused on factors such as sex steroid hormones, which regulate CB1 receptor expression. The aim of this study was to investigate the effects of 17β-estradiol exposure on the CB1 receptor gene and its protein expression in human primary tumor colon cancer cell lines, such as DLD-1, HT-29 and one lymph node metastatic cell line, SW620. Material and methods. CB1 gene expression was determined using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in DLD-1, HT-29 and SW620 cells treated at different times and doses of 17β-estradiol exposure. CB1 protein expression was detected by Western immunoblot. Results. 17β-estradiol induced CB1 gene expression in all the human colon cancer cells studied. The early induction of CB1 receptor mRNA in DLD-1 and SW620 cells was mediated by the estrogen receptor because the pure estrogen antagonist, ICI 182,780, was able to counteract this effect. Estrogenic induction of the CB1 receptor was also detectable at protein level in all cell types tested. Conclusions. The CB1 receptor can be considered an estrogen-responsive gene in DLD-1, HT-29 and SW620 cells. Up-regulation of CB1 expression by 17β-estradiol is a further mechanism of estrogens to control colon cancer proliferation.

Analysis of Promoter Regions Regulating Basal and Interleukin-4-Inducible Expression of the Human CB1 Receptor Gene in T Lymphocytes

The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) -3086 to -2490 and nts -1950 to -1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts -648 and -559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt -2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R(+)-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells.

Insulin differentially modulates the peripheral endocannabinoid system in human subcutaneous abdominal adipose tissue from lean and obese individuals

Human obesity has been associated with a dysregulation of the peripheral and adipose tissue (AT) endocannabinoid system (ES). The aim of this study was to elucidate the acute in vivo effects of insulin on gene expression of the cannabinoid type 1 (CB-1) and type 2 (CB-2) receptors, as well as of the fatty acid amide hydrolase (FAAH) in the sc abdominal adipose tissue (SCAAT). Nine lean (L) and 9 obese (OB), but otherwise healthy males were studied in the fasting state and during a euglycemic hyperinsulinemic clamp (40 mU/m2 * min(-1)). SCAAT biopsies were obtained at baseline and after 270 min of i.v. maintained hyperinsulinemia. The basal SCAAT gene expression pattern revealed an upregulation of the FAAH in the OB (p=0.03 vs L), whereas similar CB-1 and CB-2 mRNA levels were seen. Following hyperinsulinemia, the FAAH mRNA levels significantly increased approximately 2-fold in the L (p=0.01 vs baseline) but not in the OB. In contrast, insulin failed to significantly change both the adipose CB-1 and CB-2 gene expression. Finally, the FAAH gene expression positively correlated with the fasting serum insulin concentration (r 0.66; p=0.01), whereas an inverse association with the whole-body glucose disposal (r -0.58; p<0.05) was seen. Taken together, these first time observations demonstrate that the ES-related genes in the SCAAT differentially respond to hyperinsulinemia in lean/insulin-sensitive and in obese/insulin-resistant individuals. We suggest that insulin may play a key role in the obesity-linked dysregulation of the adipose ES at the gene level.

Involvement of cannabinoid CB2 receptor in alcohol preference in mice and alcoholism in humans

We tested if cannabinoid type 2 receptor (CB2) in the central nervous system plays a role in alcohol abuse/dependence in animal model and then examined an association between the CB2 gene polymorphism and alcoholism in human. Mice experiencing more alcohol preference by drinking showed reduced Cb2 gene expression, whereas mice with little preference showed no changes of it in ventral midbrain. Alcohol preference in conjunction with chronic mild stress were enhanced in mice treated with CB2 agonist JWH015 when subjected to chronic stress, whereas antagonist AM630 prevented development of alcohol preference. There is an association between the Q63R polymorphism of the CB2 gene and alcoholism in a Japanese population (P=0.007; odds ratio 1.25, 95% CI, (1.06-1.47)). CB2 under such environment is associated with the physiologic effects of alcohol and CB2 antagonists may have potential as therapies for alcoholism.

Transcriptional regulation of the cannabinoid receptor type 1 gene in T cells by cannabinoids

Effects of cannabinoids (CBs) are mediated by two types of receptors, CB1 and CB2. In this report, we investigated whether CBs regulate gene expression of their cognate receptors in T cells and studied underlying mechanisms in CD4+ Jurkat T cells. Transcription of the CB1 gene was strongly induced in response to Delta9-tetrahydrocannabinol (THC), whereas the CB2 gene was not regulated. The induction of CB1 gene expression is mediated by CB2 receptors only, as demonstrated by using the CB1 and CB2 agonists R(+)-methanandamide and JWH 015, respectively, and combinations of THC plus CB1- and CB2-specific antagonists. After activation of CB2 receptors, the transcription factor STAT5 is phosphorylated. STAT5 then transactivates IL-4. Induction of IL-4 mRNA as well as IL-4 protein release from the cells are necessary for the following induction of the CB1 gene. This was demonstrated by using decoy oligonucleotides against STAT5, which blocked IL-4 and CB1 mRNA induction, and by using the IL-4 receptor antagonist IL-4 [R121D,Y124D], which blocked the up-regulation of CB1 gene transcription. Transactivation of the CB1 gene in response to IL-4 is then mediated by the transcription factor STAT6, as shown by using decoy oligonucleotides against STAT6. An increase in CB1-mediated phosphorylation of MAPK in cells prestimulated with CB2-specific agonists suggests up-regulation of functional CB1 receptor proteins. In summary, up-regulation of CB1 in T lymphocytes in response to CBs themselves may facilitate or enhance the various immunomodulatory effects related to CBs.

The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.

Endocannabinoid system in Xenopus laevis development: CB1 receptor dynamics

This study investigates for the first time the dynamics of endocannabinoid system appearance during low vertebrate Xenopus laevis development. We observed that the CB1 gene started to be expressed during the organogenesis period (±1 dpf, st. 28) and expression persisted throughout the three further stages analyzed. Attention was focused on the localization of the CB1 messenger that was found both at the central level (in romboencephalon and in olfactory placods) and at the peripheral level (in the gastrointestinal tract) at ±3 dpf (st. 41), ±4 dpf (st. 46) and ±12 dpf (st. 49). We also considered the synthesis of CB1 protein that occurred from st. 41 onwards and, from this stage, we tested the receptor functionality in response to anandamide using cytosensor microphysiometry. CB1 functionality increased with development at both central and peripheral level. These data provide sufficient evidence to encourage further analysis on endocannabinoid physiological roles during embryonic and larval X. laevis growth.

Genetic and Pharmacogenetic Aspects of Alcohol-Dependence

Alcohol dependence is a frequent and complex disorder involving both genetic (h2≉ 0.5) and environmental factors. The definition of what is inherited with alcohol dependence is still unknown. Shared liability has been detected with other addictive disorders (mainly nicotine dependence), and certain psychiatric disorders (such as bipolar disorder) and/or personality disorders (specifically antisocial personality). Such a broad spectrum may also explain the development of antidepressants, mood regulators and/or anti-impulsive drugs in the treatment of alcohol dependence. Genome wide scans, analyses of alcohol metabolism and reward circuits have identified many candidate genes or locations. Some genes linked to the reward pathway of alcohol, may have pharmacogenetic relevancy, such as the GABRA6 gene (regarding the role of benzodiazepine in alcohol withdrawal), SLC6A4 gene (as serotonin reuptake inhibitors may reduce alcohol intake in subgroups of patients), CB1 gene (because the CB1 agonists modify alcohol consumption, at least in rodents), and the OPRM1 gene (the 118G allele being associated with increased chances of Naltrexone efficacy). High-throughput approach for genotyping of polymorphisms, transcriptomics, and proteomics are useful tools that will help to identify susceptibility and protection genes for alcohol diseases. Together, these tools could be used to develop a rational pharmacogenomics strategy to test specific individual treatment for alcohol dependence. Keywords: Opioids, Flushing Syndrome, DRD2 receptors, neurotransmitter, CB1 gene, MAP-kinase

Depolarization and Ca 2+ downregulate CB1 receptors and CB1-mediated signaling in cerebellar granule neurons

Presynaptic terminals of cerebellar granule neurons are primary targets of cannabinoids, which act through type 1 Gα i/o-coupled cannabinoid receptors (CB1) to modulate glutamate release. To study CB1 signaling investigators use primary cultures of granule neurons, typically grown in medium supplemented with elevated KCl to improve long-term survival. Herein, we demonstrate that CB1 expression and signaling are perturbed under these conditions. Specifically, immunochemical and RT–PCR assays indicate that depolarizing growth conditions decrease CB1 protein, mRNA and CB1-mediated inhibition of adenylyl cyclase compared to cultures grown in physiologic medium containing 5 mM KCl. Depolarization-dependent downregulation of CB1 mRNA, like survival, is attenuated by L-type VDCC antagonists but not the Na +-channel antagonist, tetrodotoxin. Comparison of oligonucleotide microarrays from cultures grown in 5 mM versus 25 mM KCl confirms that depolarization reduces CB1 mRNA, but not mRNAs encoding several G-protein subunits or adenylyl cyclases. However, significant alterations in synaptic signaling proteins that likely lie downstream of CB1 are observed, including K + channels, α-neurexins, cAMP-GEFII, Munc13-3, secretogranin and synaptotagmin. These findings make a compelling argument to adopt cultures grown in 5 mM KCl for future study of CB1 signaling in granule neurons. Further, they suggest that a depolarization and Ca 2+-dependent signaling pathway represses CB1 gene transcription.