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.

Fatty Acid Amide Hydrolase and Cannabinoid Receptor Type 1 Genes Regulation is Modulated by Social Isolation in Rats.

Social isolation is a state of lack of social connections, involving the modulation of different molecular signalling cascades and associated with high risk of mental health issues. To investigate if and how gene expression is modulated by social experience at the central level, we analyzed the effects of 5 weeks of social isolation in rats focusing on endocannabinoid system genes transcription in key brain regions involved in emotional control. We observed selective reduction in mRNA levels for fatty acid amide hydrolase (Faah) and cannabinoid receptor type 1 (Cnr1) genes in the amygdala complex and of Cnr1 in the prefrontal cortex of socially isolated rats when compared to controls, and these changes appear to be partially driven by trimethylation of Lysine 27 and acetylation of Lysine 9 at Histone 3. The alterations of Cnr1 transcriptional regulation result also directly correlated with those of oxytocin receptor gene. We here suggest that to counteract the effects of SI, it is of relevance to restore the endocannabinoid system homeostasis via the use of environmental triggers able to revert those epigenetic mechanisms accounting for the alterations observed.

Selective alterations of endocannabinoid system genes expression in obsessive compulsive disorder.

Obsessive Compulsive Disorder (OCD) is listed as one of the top 10 most disabling neuropsychiatric conditions in the world. The neurobiology of OCD has not been completely understood and efforts are needed in order to develop new treatments. Beside the classical neurotransmitter systems and signalling pathways implicated in OCD, the possible involvement of the endocannabinoid system (ECS) has emerged in pathophysiology of OCD. We report here selective downregulation of the genes coding for enzymes allowing the synthesis of the endocannabinoids. We found reduced DAGLα and NAPE-PLD in blood samples of individuals with OCD (when compared to healthy controls) as well as in the amygdala complex and prefrontal cortex of dopamine transporter (DAT) heterozygous rats, manifesting compulsive behaviours. Also mRNA levels of the genes coding for cannabinoid receptors type 1 and type 2 resulted downregulated, respectively in the rat amygdala and in human blood. Moreover, NAPE-PLD changes in gene expression resulted to be associated with an increase in DNA methylation at gene promoter, and the modulation of this gene in OCD appears to be correlated to the progression of the disease. Finally, the alterations observed in ECS genes expression appears to be correlated with the modulation in oxytocin receptor gene expression, consistently with what recently reported. Overall, we confirm here a role for ECS in OCD at both preclinical and clinical level. Many potential biomarkers are suggested among its components, in particular NAPE-PLD, that might be of help for a prompt and clear diagnosis.

Mild internet use is associated with epigenetic alterations of key neurotransmission genes in salivary DNA of young university students.

The potentially problematic use of the Internet is a growing concern worldwide, which causes and consequences are not completely understood yet. The neurobiology of Internet addiction (IA) has attracted much attention in scientific research, which is now focusing on identifying measurable biological markers. Aim of this study was to investigate epigenetic and genetic regulation of oxytocin receptor (OXTR), dopamine transporter (DAT1) and serotonin transporter (SERT) genes using DNA obtained from saliva samples of young university students: the Internet Addiction Test (IAT) was administered to evaluate the potential existence and intensity of IA. Significant changes in DNA methylation levels at OXTR, DAT1 and SERT genes were observed in the 30 < IAT < 49 group (mild-risk internet users) compared to the IAT < 29 subjects (complete control of internet use) and IAT > 50 subjects (considered as moderately addicted). Moreover, epigenetic markers were significantly correlated, either directly (for OXTR and DAT1) or inversely (OXTR and DAT1 versus SERT), to the psychometric properties. Our data confirmed the association of OXTR, DAT1 and SERT genes in processes related to behavioural addictions and might be of relevance to suggest possible biological predictors of altered behaviours and the eventual vulnerability to develop an IA. Different other genetic pathways have been suggested to play a role in IA and research is ongoing to better define them, in order to help in the early diagnosis as well as in the development of new potential treatments.

Neurobehavioral alterations induced by third-trimester gestation-equivalent ethanol exposure are inhibited by folate administration.

Prenatal ethanol exposure (PEE) causes several neurobehavioral impairments in the fetus. Postnatal days (PDs) 4-9 in rodents are considered equivalent to the third trimester of gestation in humans. This period is characterized by high rates of synaptogenesis and myelination and the maturation of key structures and transmitter systems. Nutritional supplements, such as folate, have gained attention as putative treatments to mitigate detrimental effects of PEE. Folate is crucial for DNA synthesis and amino acid metabolism and heightens antioxidant defenses. The present study examined neurobehavioral effects of the concurrent administration of folate (20 mg/kg/day) and ethanol (5 g/kg/day) during PDs 4-9 in male and female Wistar rats. During PDs 16-18, the rat pups were tested for anxiety-like and exploratory activity in the light-dark box (LDB), open field (OF), and concentric square field (CSF) tests. After weaning, they were tested for sucrose preference and ethanol intake. Neonatal ethanol exposure reduced body weight in infancy but did not enhance ethanol self-administration or significantly affect performance in the OF or LDB. Neonatal ethanol exposure also reduced sucrose intake in the preference test and increased shelter-seeking in the CSF, and folate significantly inhibited these effects. The present findings suggest that folate, a treatment that is devoid of serious side effects, can ameliorate some neurobehavioral effects of PEE.

Bored with boredom? Trait boredom predicts internet addiction through the mediating role of attentional bias toward social networks.

Internet addiction is an emerging issue, impacting people's psychosocial functioning and well-being. However, the prevalence and the mechanisms underlying internet misuse are largely unknown. As with other behavioral addiction disorders, the increase and persistence of internet addiction may be favored by negative affect such as boredom. In this study, we examined the role of boredom susceptibility, as a personality trait, in predicting the risk of internet addiction. Furthermore, we analyzed the attentional mechanisms that may exacerbate dysfunctional internet behaviors. Specifically, we assessed the mediating role of attentional bias toward social media cues on the relation between boredom susceptibility and internet addiction. Sixty-nine young adults were administered a dot-probe task assessing internet-related attentional bias (AB) and questionnaires measuring internet addiction (IAT) and boredom susceptibility (BS-BSSS). Correlation and t-test analyses confirmed that the tendency to experience boredom and selective attention toward social network information was related to internet addiction. Furthermore, the mediation model indicated that AB fully explains the link between BS-BSSS and IAT. The study highlighted the crucial role of selective attentional processing behind internet addiction. The current results are useful for both researchers and clinicians as they suggest that intervention programs for internet addiction should include strategies to cope with dysfunctional cognitive processes.

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.

Peripheral control of psychiatric disorders: Focus on OCD. Are we there yet?

"We are all in this together" - we often hear this phrase when we want to flag up a problem that is not for a single individual but concerns us all. A similar reflection has been recently made in the field of mental disorders where brain-centric scientists have started to zoom out their brain-focused graphical representations of the mechanisms regulating psychiatric diseases to include other organs or mediators that did not belong historically to the world of neuroscience. The brain itself - that has long been seen as a master in command secluded in its fortress (the blood brain barrier), has now become a collection of Airbnb(s) where all sorts of cells come in and out and sometimes even rearrange the furniture! Under this new framework of reference, mental disorders have become multisystem pathologies where different biological systems - not just the CNS -contribute 'all together' to the development and severity of the disease. In this narrative review article, we will focus on one of the most popular biological systems that has been shown to influence the functioning of the CNS: the immune system. We will specifically highlight the two main features of the immune system and the CNS that we think are important in the context of mental disorders: plasticity and memory.

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.

Folate administration ameliorates neurobehavioral effects of prenatal ethanol exposure.

Background: Prenatal ethanol exposure (PEE) induces heightened ethanol intake at adolescence in preclinical studies. Ethanol intake alters the absorption of folate, a methyl-group donor critical for numerous cellular functions. The prenatal administration of folate is, therefore, a promising approach to reduce the effects of PEE.Objectives: Experiment 1 determined if prenatal folate modulated the effects of PEE on ethanol intake, anxiety-like response, and exploratory behaviors (Experiment 1) in Wistar rats. Experiment 2 assessed, in rats not given PEE, if postnatal folate reversed effects of ethanol exposure at postnatal days 28-42. Experiment 3 assessed if folate altered blood ethanol levels (BELs).Methods: Experiment 1 involved 242 (125 male) adolescent Wistar rats derived from dams given folate (20 mg/kg, gestational days - GD- 13-20) + ethanol (2.0 g/kg, GD 17-20), ethanol, or vehicle only at pregnancy. Experiment 2 involved 29 male adolescents administered vehicle or ethanol doses co-administered or not with folate. In Experiment 3 twelve adult females were tested for BELs after folate administration. These tests were applied: intake tests, light dark box (LDB), elevated plus maze, open field and concentric square field.Results: PEE heightened ethanol intake (η2 ps = 0.06-07) and induced hyperactivity and a reduced latency to exit the white area of the LDB (η2 ps = 0.12-17). These effects were partially inhibited by folate (p > .05). Rats exposed to ethanol exposure at adolescence exhibited reduced motor activity (η2 p = .17), regardless of folate treatment. Folate did not affect BELs.Conclusion: Folate administration should be considered as a preventive or acute treatment to attenuate the neurobehavioral effects of PEE.

The Effects of Peripubertal THC Exposure in Neurodevelopmental Rat Models of Psychopathology.

Adolescent exposure to cannabinoids as a postnatal environmental insult may increase the risk of psychosis in subjects exposed to perinatal insult, as suggested by the two-hit hypothesis of schizophrenia. Here, we hypothesized that peripubertal Δ9-tetrahydrocannabinol (aTHC) may affect the impact of prenatal methylazoxymethanol acetate (MAM) or perinatal THC (pTHC) exposure in adult rats. We found that MAM and pTHC-exposed rats, when compared to the control group (CNT), were characterized by adult phenotype relevant to schizophrenia, including social withdrawal and cognitive impairment, as revealed by social interaction test and novel object recognition test, respectively. At the molecular level, we observed an increase in cannabinoid CB1 receptor (Cnr1) and/or dopamine D2/D3 receptor (Drd2, Drd3) gene expression in the prefrontal cortex of adult MAM or pTHC-exposed rats, which we attributed to changes in DNA methylation at key regulatory gene regions. Interestingly, aTHC treatment significantly impaired social behavior, but not cognitive performance in CNT groups. In pTHC rats, aTHC did not exacerbate the altered phenotype nor dopaminergic signaling, while it reversed cognitive deficit in MAM rats by modulating Drd2 and Drd3 gene expression. In conclusion, our results suggest that the effects of peripubertal THC exposure may depend on individual differences related to dopaminergic neurotransmission.

Are the epigenetic changes predictive of therapeutic efficacy for psychiatric disorders? A translational approach towards novel drug targets.

The etiopathogenesis of mental disorders is not fully understood and accumulating evidence support that clinical symptomatology cannot be assigned to a single gene mutation, but it involves several genetic factors. More specifically, a tight association between genes and environmental risk factors, which could be mediated by epigenetic mechanisms, may play a role in the development of mental disorders. Several data suggest that epigenetic modifications such as DNA methylation, post-translational histone modification and interference of microRNA (miRNA) or long non-coding RNA (lncRNA) may modify the severity of the disease and the outcome of the therapy. Indeed, the study of these mechanisms may help to identify patients particularly vulnerable to mental disorders and may have potential utility as biomarkers to facilitate diagnosis and treatment of psychiatric disorders. This article summarizes the most relevant preclinical and human data showing how epigenetic modifications can be central to the therapeutic efficacy of antidepressant and/or antipsychotic agents, as possible predictor of drugs response.

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.

Bioinformatics of the Endocannabinoid System: Study of DNA Methylation at Rat Cnr1 Gene Promoter.

In this chapter, we will describe the bioinformatic tools that allow verifying the presence of CpG islands in a gene promoter region. We will also describe the tools needed to identify consensus motifs for specific transcription factors, focusing on the study of rat type-1 cannabinoid receptor gene (R_Cnr1) as a case study.

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.

Cannabis use and related clinical variables in patients with obsessive-compulsive disorder.

OBJECTIVE: Limited studies have investigated cannabis use in patients with obsessive-compulsive disorder (OCD), despite its widespread use by patients with psychiatric illnesses. The aim of this study was to assess the frequency, correlates, and clinical impact of cannabis use in an Italian sample of patients with OCD. METHODS: Seventy consecutive outpatients with OCD were recruited from a tertiary specialized clinic. To assess cannabis-related variables, patients completed a questionnaire developed for the purpose of this study, investigating cannabis use-related habits and the influence of cannabis use on OCD symptoms and treatments. A set of clinician and self-reported questionnaires was administered to measure disease severity. The sample was then divided into three subgroups according to the pattern of cannabis use: "current users" (CUs), "past-users" (PUs), and "non-users" (NUs). RESULTS: Approximately 42.8% of patients reported lifetime cannabis use and 14.3% reported current use. Approximately 10% of cannabis users reported an improvement in OCD symptoms secondary to cannabis use, while 23.3% reported an exacerbation of anxiety symptoms. CUs showed specific unfavorable clinical variables compared to PUs and NUs: a significant higher rate of lifetime use of tobacco, alcohol, and other substances, and a higher rate of pre-OCD onset comorbidities. Conversely, the three subgroups showed a similar severity of illness. CONCLUSION: A considerable subgroup of patients with OCD showed a predisposition towards cannabis use and was associated with some specific clinical characteristics, suggesting the need for targeted consideration and interventions in this population.

Dopamine-transporter heterozygous rats carrying maternal wild-type allele are more vulnerable to the development of compulsive behavior.

Compulsivity is defined as an unstoppable tendency toward repetitive and habitual actions, which are reiterated despite negative consequences. Polydipsia is induced preclinically by intermittent reward, leading rodents to ingest large amounts of fluids. We focused on the role of dopamine transporter (DAT) and inheritance factors in compulsive behavior. Our sample consisted of DAT heterozygous (HET) rats with different genetic inheritance (MAT-HET, born from WT-dams × KO-fathers; MIX-HET, born from HET-dams × KO-fathers). As controls, we used both wild-type (WT) rats and their socially-isolated (WTi) siblings. We ran the schedule-induced polydipsia (SIP) protocol, to induce compulsive behavior; then the Y-maze and marble-burying tests, to verify its actual development. Only MAT-HET (who inherited the functional DAT allele from the WT mother) is vulnerable to developing compulsive behavior. MAT-HET rats drank increasingly more water during SIP; they showed significant perseverance in the Y-maze test and exhibited compulsive actions in the marble-burying test. Interestingly, compulsive behaviors of MAT-HET rats correlated with expression ex vivo of different genes in different areas. Regarding the prefrontal cortex (PFC), D2R correlated with Y-maze "perseverance" in addition to BDNF; considering the amygdala (AMY), both D3R and OXTR correlated with SIP "licks." Indeed, compulsivity may be linked to D2R and BDNF in PFC, while extreme anxiety in MAT-HET rats may be associated with D3R and OXTR in the AMY. These results confirm some similarities between MAT-HET and DAT-KO subjects, and link the epigenetic context of the DAT gene to the development of compulsive behavior.

The Role of Stress and Cognitive Absorption in Predicting Social Network Addiction.

Nowadays, the use of social networks (SNs) is pervasive and ubiquitous. Among other things, SNs have become a key resource for establishing and maintaining personal relationships, as further demonstrated by the emergence of the pandemic. However, easy access to SNs may be a source of addictive behaviour, especially among the younger population. The literature highlights various psychological and physiological factors as possible predictors of vulnerability to SN addiction. This paper explores the joint effects of stress level and cognitive absorption, in the form of temporal dissociation while on SNs, on the addiction of university students to SNs. Here, 312 participants were involved in an online survey. About 14% of the sample presented a risk for SN addiction. Moreover, it was found that stress level predicted SN addiction both directly and indirectly through the effect of individual temporal dissociation, as experienced during SN usage. These results suggest a significant role of perceived stress level on addiction risk, while also pointing out additional vulnerability to SN addiction for cognitive profiles that are relatively more prone to temporal dissociation while online.