Repeated binge-like eating episodes in female rats alter adenosine A2A and dopamine D2 receptor genes regulation in the brain reward system.

OBJECTIVE: Binge-eating disorder is an eating disorder characterized by recurrent binge-eating episodes, during which individuals consume excessive amounts of highly palatable food (HPF) in a short time. This study investigates the intricate relationship between repeated binge-eating episode and the transcriptional regulation of two key genes, adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R), in selected brain regions of rats. METHOD: Binge-like eating behavior on HPF was induced through the combination of food restrictions and frustration stress (15 min exposure to HPF without access to it) in female rats, compared to control rats subjected to only restriction or only stress or none of these two conditions. After chronic binge-eating episodes, nucleic acids were extracted from different brain regions, and gene expression levels were assessed through real-time quantitative PCR. The methylation pattern on genes' promoters was investigated using pyrosequencing. RESULTS: The analysis revealed A2AAR upregulation in the amygdala and in the ventral tegmental area (VTA), and D2R downregulation in the nucleus accumbens in binge-eating rats. Concurrently, site-specific DNA methylation alterations at gene promoters were identified in the VTA for A2AAR and in the amygdala and caudate putamen for D2R. DISCUSSION: The alterations on A2AAR and D2R genes regulation highlight the significance of epigenetic mechanisms in the etiology of binge-eating behavior, and underscore the potential for targeted therapeutic interventions, to prevent the development of this maladaptive feeding behavior. These findings provide valuable insights for future research in the field of eating disorders. PUBLIC SIGNIFICANCE: Using an animal model with face, construct, and predictive validity, in which cycles of food restriction and frustration stress evoke binge-eating behavior, we highlight the significance of epigenetic mechanisms on adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R) genes regulation. They could represent new potential targets for the pharmacological management of eating disorders characterized by this maladaptive feeding behavior.

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

A possible role for G-quadruplexes formation and DNA methylation at IMOOD gene promoter in Obsessive Compulsive Disorder.

Obsessive Compulsive Disorder (OCD) is a mental health condition still classified and diagnosed with subjective interview-based assessments and which molecular clues have not completely been elucidated. We have recently identified a new regulator of anxiety and OCD-like behavior called Immuno-moodulin (IMOOD) and, here, we report that IMOOD gene promoter is differentially methylated in OCD subjects when compared to genomic material collected from healthy controls and this alteration is significantly correlated with the increased expression of the gene in OCD. We also demonstrated that IMOOD promoter can form G-quadruplexes and we suggest that, in homeostatic conditions, these structures could evoke DNA-methylation silencing the gene, whereas in pathological conditions, like OCD, could induce gene expression making the promoter more accessible to transcriptional factors. We here thus further suggest IMOOD as a new biomarker for OCD and also hypothesize new mechanisms of gene regulation.

Methylation Dynamics on 5'-UTR of DAT1 Gene as a Bio-Marker to Recognize Therapy Success in ADHD Children.

Attention-deficit/hyperactivity disorder (ADHD), a neuropsychiatric condition characterized by inattention, hyperactivity, and impulsivity, afflicts 5% of children worldwide. Each ADHD patient presents with individual cognitive and motivational peculiarities. Furthermore, choice of appropriate therapy is still up to clinicians, who express somewhat qualitative advice on whether a child is being successfully cured or not: it would be more appropriate to use an objective biomarker to indicate whether a treatment led to benefits or not. The aim of our work is to search for such clinical biomarkers. We recruited 60 ADHD kids; psychopathological scales were administered at recruitment and after six weeks of therapy. Out of such a cohort of ADHD children, we rigorously extracted two specific subgroups; regardless of the initial severity of their disease, we compared those who obtained the largest improvement (ΔCGAS > 5) vs. those who were still characterized by a severe condition (CGAS < 40). After such a therapy, methylation levels of DNA extracted from buccal swabs were measured in the 5'-UTR of the DAT1 gene. CpGs 3 and 5 displayed, in relation to the other CpGs, a particular symmetrical pattern; for "improving" ADHD children, they were methylated together with CpG 2 and CpG 6; instead, for "severe" ADHD children, they accompanied a methylated CpG 1. These specific patterns of methylation could be used as objective molecular biomarkers of successful cures, establishing if a certain therapy is akin to a given patient (personalized medicine). Present data support the use of post-therapy molecular data obtained with non-invasive techniques.

Assessing Gene Expression of the Endocannabinoid System Components by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction.

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a major development in PCR technology, is a powerful and sensitive gene analysis technique that has revolutionized the field of gene expression assays. In this chapter, we describe in detail RNA extraction, reverse transcription (RT), and relative quantification of genes forming the endocannabinoid system in different experimental models. In particular, we here provide specific and sensitive assays to be used to assess gene expression of the endocannabinoid system components in mouse, rat, or human samples.

DNA Methylation Analysis of Cnr1 Gene Promoter.

DNA methylation pattern could be considered a biomarker to be exploited for the study and management of several human diseases. In this chapter, detailed protocols are provided for two experimental approaches used for quantitative methylation analysis of bisulfite converted DNA: methylation-specific PCR (MSP) and pyrosequencing.

Endocannabinoid System Regulation in Female Rats with Recurrent Episodes of Binge Eating.

Recurrent Binge Eating (BE) episodes characterize several eating disorders. Here, we attempted to reassemble a condition closer to BE disorder, and we analyzed whether recurrent episodes might evoke molecular alterations in the hypothalamus of rats. The hypothalamus is a brain region which is sensitive to stress and relevant in motivated behaviors, such as food intake. A well-characterized animal model of BE, in which a history of intermittent food restriction and stress induce binge-like palatable food consumption, was used to analyze the transcriptional regulation of the endocannabinoid system (ECS). We detected, in rats showing the BE behavior, an up-regulated gene expression of cannabinoid type-1 receptor (CB1), sn-1-specific diacylglycerol lipase, as well as fatty acid amide hydrolase (Faah) and monoacylglycerol lipase. A selective reduction in DNA methylation was also observed at the promoter of Faah, which is consistent with the changes in the gene expression. Moreover, BE behavior in rats was associated with an increase in anandamide (AEA) levels. Our findings support the relevant role of the ECS in the regulation of food intake in rats subjected to repeated BE episodes, and, in particular, on AEA signaling, acting via CB1 and FAAH modulation. Notably, the epigenetic regulation of the Faah gene might suggest this enzyme as a possible target for developing new therapeutical approaches.

Methylation patterns within 5'-UTR of DAT1 gene as a function of allelic 3'-UTR variants and their maternal or paternal origin: May these affect the psychopathological phenotypes in children? An explorative study.

Psychopathological symptoms such as depression/anxiety vs attention or aggression problems, in children, have been associated to altered expression of the DAT1/SLC6A3 gene. Inheriting specific 9- or 10-repeat VNTR alleles could modify the pattern of methylation in the CpGs islands at the 5'-UTR of the DAT1 gene. Through accurate recruitment at primary schools, we ended up with four subgroups of children: 9/9 and 10/10 homozygous; 9/10 heterozygous born from 9/10 mothers and 10/10 fathers (called heM); 9/10 heterozygous born from 10/10 mothers and 9/10 fathers (called heF). (Epi)genetical changes were found to be in relation to internalizing and externalizing symptoms: compared to other genotypes, our 9/9 children exhibited mainly internalizing symptoms, while 10/10 genotype was previously associated with ADHD severity. We found that 10/10 children bear 5'-UTR motifs showing a CpGs 1-2-3-5 unity with anticorrelated CpG 6, while 9/9 children showed rather a demethylated CpG 1 linked to demethylated CpG 6. We found two different patterns between heMs and heFs: a feature of heM children is in CpGs 1-3 methylated pattern with CpGs 2, 5 and 6 demethylated together, supporting a "split" unitary destiny. Within the heF children, the status for CpGs 3 + 6 remained opposite, yet pattern of (de)methylation was not well defined. The prevailing one between inherited parental alleles may somewhat influence the motif destiny of heterozigous children. Present work aimed to identify novel epigenetic biomarkers, to be exploited as fairly indicators of children's psychopathological vulnerability.

Regulation of oxytocin receptor gene expression in obsessive-compulsive disorder: a possible role for the microbiota-host epigenetic axis.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a prevalent and severe clinical condition. Robust evidence suggests a gene-environment interplay in its etiopathogenesis, yet the underlying molecular clues remain only partially understood. In order to further deepen our understanding of OCD, it is essential to ascertain how genes interact with environmental risk factors, a cross-talk that is thought to be mediated by epigenetic mechanisms. The human microbiota may be a key player, because bacterial metabolites can act as epigenetic modulators. We analyzed, in the blood and saliva of OCD subjects and healthy controls, the transcriptional regulation of the oxytocin receptor gene and, in saliva, also the different levels of major phyla. We also investigated the same molecular mechanisms in specific brain regions of socially isolated rats showing stereotyped behaviors reminiscent of OCD as well as short chain fatty acid levels in the feces of rats. RESULTS: Higher levels of oxytocin receptor gene DNA methylation, inversely correlated with gene expression, were observed in the blood as well as saliva of OCD subjects when compared to controls. Moreover, Actinobacteria also resulted higher in OCD and directly correlated with oxytocin receptor gene epigenetic alterations. The same pattern of changes was present in the prefrontal cortex of socially-isolated rats, where also altered levels of fecal butyrate were observed at the beginning of the isolation procedure. CONCLUSIONS: This is the first demonstration of an interplay between microbiota modulation and epigenetic regulation of gene expression in OCD, opening new avenues for the understanding of disease trajectories and for the development of new therapeutic strategies.

Polyphenols and Cannabidiol Modulate Transcriptional Regulation of Th1/Th2 Inflammatory Genes Related to Canine Atopic Dermatitis.

Canine atopic dermatitis (AD) is a multifactorial allergic disease associated with immune and abnormal skin barrier dysfunction and it is one of the primary causes of pruritus. Using a novel in vitro model of AD, here we tried to revert the alteration of transcriptional regulation of AD canine key genes testing a nutraceutical mixture containing flavonoids, stilbene, and cannabinoids, which are already well-known for their applications within dermatology diseases. The nutraceutical mixture induced in inflamed cells a significant downregulation (p < 0.05) of the gene expression of ccl2, ccl17, and tslp in keratinocytes and of ccl2, ccl17, and il31ra in monocytes. Consistent with the observed alterations of tslp, ccl2, ccl17, and il31ra messenger RNA (mRNA) levels, a significant increase (p < 0.05) of DNA methylation at specific CpG sites on the gene regulatory regions was found. These results lay the foundation for the use of these natural bioactives in veterinary medicine and provide a model for deeper understanding of their mechanisms of action, with potential translation to human research.

Epigenetic regulation of DAT gene promoter modulates the risk of externalizing and internalizing behaviors on a normative population: An explorative study.

Accumulating research addressed epigenetic modifications and their role on behavioral phenotypes. We recently proposed to study methylation dynamics of two CpG motifs within the 5'-UTR of dopamine transporter (DAT) gene. Starting from a normative population sample of young adults, we selected three sub-groups based on their prevalent symptoms: subjects were assigned to Internalizing, Externalizing and Low-risk sub-groups according to elevated scores in specific phenotypic scales. Using a new approach, we calculated three independent matrixes of cross-correlation between CpG methylation levels, one within each phenotypic sub-group, to determine in which dynamics did the sub-groups differ. We found specific cross-correlation patterns in Externalizing (CpG1, 2 and 3, opposite to the methylation at CpG6) and Internalizing individuals (CpG1 methylation opposite to CpG2, 3 and 6), while Low-risk individuals could follow both trends. The aim of our study was to look for a specific DAT methylation pattern, providing a biomarker that allows early identification of the risk for psycho-pathological deviance.

On the Role of Central Type-1 Cannabinoid Receptor Gene Regulation in Food Intake and Eating Behaviors.

Different neuromodulatory systems are involved in long-term energy balance and body weight and, among these, evidence shows that the endocannabinoid system, in particular the activation of type-1 cannabinoid receptor, plays a key role. We here review current literature focusing on the role of the gene encoding type-1 cannabinoid receptors in the CNS and on the modulation of its expression by food intake and specific eating behaviors. We point out the importance to further investigate how environmental cues might have a role in the development of obesity as well as eating disorders through the transcriptional regulation of this gene in order to prevent or to treat these pathologies.

Involvement of DAT1 Gene on Internet Addiction: Cross-Correlations of Methylation Levels in 5'-UTR and 3'-UTR Genotypes, Interact with Impulsivity and Attachment-Driven Quality of Relationships.

Internet influences our communication, social and work interactions, entertainment, and many other aspects of life. Even if the original purpose was to simplify our lives, an excessive and/or maladaptive use of it may have negative consequences. The dopamine transporter (DAT1) gene was studied in relation to addictions, including excessive use of the Internet. The crucial role of DAT1 was previously underlined in modulating emotional aspects, such as affiliative behaviors. The present research follows a new approach based on cross-correlation between (de)methylation levels in couples of CpG loci, as previously shown. We investigated the possible relationships between Internet addiction, impulsivity, quality of attachment, DAT1 genotypes (from the 3'-untranslated region (UTR) variable number of tandem repeats (VNTR) poly-morphism), and the dynamics of methylation within the 5'-UTR of the DAT1 gene. From a normative sample of 79 youths, we extrapolated three subgroups a posteriori, i.e., one "vulnerable" with high Internet Addiction Test (IAT) scores (and high Barrat Impulsivity Scale (BIS) scores; n = 9) and two "controls'' with low BIS scores and 10/10 vs. 9/x genotype (n = 12 each). Controls also had a "secure" attachment pattern, while genotypes and attachment styles were undistinguished in the vulnerable subgroup (none showed overt Internet addiction). We found a strongly positive correlation in all groups between CpG2 and CpG3. An unsuspected relationship between the 3'-UTR genotype and a 5'-UTR intra-motif link was revealed by CpG5-CpG6 comparison. The negative correlation between the CpG3-CpG5 positions was quite significant in the control groups (both with genotype 10/10 and with genotype 9/x), whereas a tendency toward positive correlation emerged within the high IAT group. In conclusion, future attention shall be focused on the intra- and inter-motif interactions of methylation on the CpG island at the 5'-UTR of DAT1.

Cross-correlations between motifs in the 5'-UTR of DAT1 gene: Findings from Parkinson's disease.

Parkinson's disease (PD) is a neuro-degenerative disorder affecting the striatal motor system, caused by the loss of neuronal cells in the mid-brain, where reduced amounts of dopamine do cause involuntary movements and others symptoms. Alterations of methylome have been reported in PD epigenomic studies, and also human dopamine transporter gene (DAT1, SLC6A3) is considered as a candidate risk factor for PD. Since the DNA methylation on DAT promoter may well have a role in the development of this disease, we aimed to further assess the epigenetic control, by focusing on specific CpG sites located in the 5'-untranslated region (5'-UTR) of the DAT1 gene. Significant changes in DAT 5'-UTR methylation were already found in peripheral blood mononuclear cells (PBMCs) of PD subjects (Rubino et al., 2020). Of note, methylation values at the CpG 5 were increased. We run on same data a novel statistical approach: cross-correlation between pairs of loci. CpG 5 was the only always-differing variable but, alternatively, CpGs 2 and 6 or CpGs 1 and 3 were also significantly correlated with CpG 5. Interestingly, this picture emerged for those patients whose M2xM6 index was above-median; loci were rather independent for below-median patients. Present data may shed light into dynamics occurring at 5'-UTR of DAT1, a gene involved in PD but also in many psycho-physiological pathologies.

Search for an epigenetic biomarker in ADHD diagnosis, based on the DAT1 gene 5'-UTR methylation: a new possible approach.

Attention Deficit/Hyperactivity Disorder (ADHD) is the most common neuro-developmental alteration in childhood. To date, its diagnosis is exclusively clinical, however recent studies focused on searching for objective biomarkers. We recently reported a selective alteration of DNA methylation in the 5'-UTR of dopamine transporter (DAT1) gene, in a 1CGG2CGG3CGG and a 5CG6CG motif, for ADHD patients (compared to controls). Presently, we looked for DNA methylation of the corresponding CpG sites but complementary on the opposite strand ("COS"). Exploiting a novel cross-correlation approach, we found a core M5 - M5 COS and M2 - M2 COS relationship with relatively free M1 and M6 COS extremes. Our data might be relevant, to find a new biomarker to diagnose ADHD in affected subjects.

Selective alterations in endogenous opioid system genes expression in rats selected for high ethanol intake during adolescence.

Historically, the roots of alcoholism have been linked to either environment or heredity. However, the interaction between these factors is still largely unexplored. The evidence supports a link between alcohol consumption and the endogenous opioid system. We here studied the opioid genes expression in male and female Wistar rats derived from a short-term breeding program which selected -- at adolescence -- for high (ADHI line) or low (ADLO line) ethanol drinking. Specifically, in this work we analyzed central opioid gene expression in the rats of the second filial generation (S2-ADLO and S2-ADHI). Selective downregulation of pronociceptin (Pnoc) and its receptor (Oprl1) mRNA levels were observed in the prefrontal cortex of male S2-ADHI rats when compared to S2-ADLO, and for Oprl1 also in the nucleus accumbens. An increase in gene expression was instead observed for pro-opiomelanocortin (Pomc) in the nucleus accumbens of S2-ADHI males when compared to S2-ADLO, as well as for mu opioid receptor (Oprm1) but in females. The differences in mRNA levels may be due to the different alcohol consumption between the two groups of rats or may represent pre-existing differences between them. Moreover, we show a sex-specific modulation of the expression of these genes, thus pointing out the importance of sex on ethanol responses. The results might lead to more specific and effective pharmacological treatments for alcoholism.

Epigenetic regulation of the cannabinoid receptor CB1 in an activity-based rat model of anorexia nervosa.

OBJECTIVE: Both environmental and genetic factors are known to contribute to the development of anorexia nervosa (AN), but the exact etiology remains poorly understood. Herein, we studied the transcriptional regulation of the endocannabinoid system, an interesting target for body weight maintenance and the control of food intake and energy balance. METHOD: We used two well-characterized animal models of AN: (a) the activity-based anorexia (ABA) model in which rats, housed with running wheels and subjected to daily food restriction, show reductions in body weight and increase in physical activity; (b) the genetic anx/anx mouse displaying the core features of AN: low food intake and emaciation. RESULTS: Among the evaluated endocannabinoid system components, we observed a selective and significant down-regulation of the gene encoding for the type 1 cannabinoid receptor (Cnr1) in ABA rats' hypothalamus and nucleus accumbens and, in the latter area, a consistent, significant and correlated increase in DNA methylation at the gene promoter. No changes were evident in the anx/anx mice except for a down-regulation of Cnr1, in the prefrontal cortex. DISCUSSION: Our findings support a possible role for Cnr1 in the ABA animal model of AN. In particular, its regulation in the nucleus accumbens appears to be triggered by environmental cues due to the consistent epigenetic modulation of the promoter. These data warrant further studies on Cnr1 regulation as a possible target for treatment of AN.

Methylation of Brain Derived Neurotrophic Factor (BDNF) Val66Met CpG site is associated with early onset bipolar disorder.

BACKGROUND: The brain-derived neurotrophic factor (BDNF) rs6265 (Val66Met) Met allele is associated with early onset (≤ 19 years old) bipolar disorder (BD). Val66Met (G196A) creates a CpG site when the Val/G allele is present. We sought to study the methylation of the BDNF promoter and its interaction with Val66Met genotype in BD. METHODS: Sex/age-matched previously genotyped DNA samples from BD-Type 1 cases [N = 166: early onset (≤ 19 years old) n = 79, late onset (> 20 years old) n = 87] and controls (N = 162) were studied. Pyrosequencing of four CpGs in Promoter-I, four CpGs in promoter-IV, and two CpGs in Promoter-IX (CpG2 includes G= Val allele) was performed. Logistic regression adjusting for batch effect was used to compare cases vs. controls. Analyses also included stratification by disease onset and adjustment for Val66Met genotype. Secondary exploratory analyses for the association of life stressors, comorbid substance abuse, and psychotropic use with methylation patterns were performed. RESULTS: Comparing all BD cases vs. controls and adjusting for Val66Met genotype, BD cases had significantly higher methylation in promoter -IX/CPG-2 (p = 0.0074). This was driven by early onset cases vs. controls (p = 0.00039) and not late onset cases vs. controls (p = 0.2). LIMITATION: Relatively small sample size. CONCLUSION: Early onset BD is associated with increased methylation of CpG site created by Val=G allele of the Val66Met variance. Further studies could include larger sample size and postmortem brain samples in an attempt to replicate these findings.

Preclinical and Clinical Evidence for a Distinct Regulation of Mu Opioid and Type 1 Cannabinoid Receptor Genes Expression in Obesity.

Among endogenous signaling networks involved in both rewarding and homeostatic mechanisms of obesity, a relevant role is played by the endocannabinoid (ECS) and the opioid (EOS) systems. We here studied the transcriptional regulation of ECS and EOS genes in the hypothalamus of Diet-induced obesity rats, a preclinical model of obesity, as well as in humans with obesity and healthy controls. A significant and selective increase in type 1 cannabinoid receptor gene (Cnr1) expression was observed at the beginning of obesity development (5 weeks on high fat diet) as well as after 21 weeks of high diet exposure. After 5 weeks on high fat diet, selective up-regulation of mu opioid receptor gene (Oprm1) expression was also observed. Consistently, epigenetic studies showed a selective and significant decrease in DNA methylation at specific CpG sites at both gene promoters in overweight rats, but only after 5 weeks on high fat diet. Moreover, significantly lower levels of DNA methylation were observed at selected CpG sites of both receptor gene promoters, analyzed in peripheral blood mononuclear cells from younger (<30 years old) humans with obesity, as well as in those with shorter time length from disease onset. Taken together, we here provide evidence of selective, synergistic and time-dependent transcriptional regulation of CNR1 and OPRM1 genes in overweight rats, as well as in human subjects. These alterations in genes regulation could contribute to the development of the obese phenotype, and we thus suggest CNR1 and OPRM1 epigenetic modulation as possible biomarkers of obesity development. Due to the reversible nature of the epigenetic hallmark, our data might also open new avenue to early environmental strategies of intervention.

Regulation of adenosine A2A receptor gene expression in a model of binge eating in the amygdaloid complex of female rats.

BACKGROUND: Pharmacological treatment approaches for eating disorders, such as binge eating disorder and bulimia nervosa, are currently limited. METHODS AND AIMS: Using a well-characterized animal model of binge eating, we investigated the epigenetic regulation of the A2A Adenosine Receptor (A2AAR) and dopaminergic D2 receptor (D2R) genes. RESULTS: Gene expression analysis revealed a selective increase of both receptor mRNAs in the amygdaloid complex of stressed and restricted rats, which exhibited binge-like eating, when compared to non-stressed and non-restricted rats. Consistently, pyrosequencing analysis revealed a significant reduction of the percentage of DNA methylation but only at the A2AAR promoter region in rats showing binge-like behaviour compared to the control animals. Focusing thus on A2AAR agonist (VT 7) administration (which inhibited the episode of binge systemically at 0.1 mg/kg or intra-central amygdala (CeA) injection at 900 ng/side) induced a significant increase of A2AAR mRNA levels in restricted and stressed rats when compared to the control group. In addition, we observed a significant decrease in A2AAR mRNA levels in rats treated with the A2AAR antagonist (ANR 94) at 1 mg/kg. Consistent changes in the DNA methylation status of the A2AAR promoter were found in restricted and stressed rats after administration of VT 7 or ANR 94. CONCLUSION: We confirm the role of A2AAR in binge eating behaviours, and we underline the importance of epigenetic regulation of the A2AAR gene, possibly due to a compensatory mechanism to counteract the effect of binge eating. We suggest that A2AAR activation, inducing receptor gene up-regulation, could be relevant to reduction of food consumption.