Early life stress and voluntary alcohol consumption in relation to Maoa methylation in male rats.

Early life stress (ELS) or alcohol consumption can influence DNA methylation and affect gene expression. Monoamine oxidase A (Maoa) encodes the enzyme that metabolizes monoaminergic neurotransmitters crucial for the stress response, alcohol reward, and reinforcement. Previously, we reported lower Maoa expression in the nucleus accumbens and dorsal striatum of male rats exposed to ELS during the first three postnatal weeks, and to voluntary alcohol consumption in adulthood, compared with controls. The present study continued to investigate the effect of ELS and alcohol consumption on Maoa methylation, and its relation to Maoa expression in these animals. We selected candidate CpGs after performing next-generation bisulfite sequencing of the Maoa promoter, intron 1-5, and exons 5 and 6, together composed of 107 CpGs (5'-cytosine-phosphate-guanosine-3'), in a subgroup of rats. Pyrosequencing was used to analyze the methylation of 10 candidate CpGs in the promoter and intron 1 in the entire sample. ELS and alcohol displayed an interactive effect on CpG-specific methylation in the dorsal striatum. CpG-specific methylation correlated with Maoa expression, corticosterone levels, and alcohol consumption in a brain region-specific manner. CpG-specific methylation in the Maoa promoter was a potential moderator of the interaction of ELS with alcohol consumption on Maoa expression in the NAc. However, the findings were sparse, did not survive correction for multiple testing, and the magnitude of differences in methylation levels was small. In conclusion, CpG-specific Maoa methylation in the promoter and intron 1 may associate with ELS, alcohol consumption, and Maoa expression in reward-related brain regions.

Episodic Ethanol Exposure in Adolescent Rats Causes Residual Alterations in Endogenous Opioid Peptides.

Adolescent binge drinking is associated with an increased risk of substance use disorder, but how ethanol affects the central levels of endogenous opioid peptides is still not thoroughly investigated. The aim of this study was to examine the effect of repeated episodic ethanol exposure during adolescence on the tissue levels of three different endogenous opioid peptides in rats. Outbred Wistar rats received orogastric (i.e., gavage) ethanol for three consecutive days per week between 4 and 9 weeks of age. At 2 h and 3 weeks, respectively, after the last exposure, beta-endorphin, dynorphin B and Met-enkephalin-Arg6Phe7 (MEAP) were analyzed with radioimmunoassay. Beta-endorphin levels were low in the nucleus accumbens during ethanol intoxication. Remaining effects of adolescent ethanol exposure were found especially for MEAP, with low levels in the amygdala, and high in the substantia nigra and ventral tegmental area three weeks after the last exposure. In the hypothalamus and pituitary, the effects of ethanol on beta-endorphin were dependent on time from the last exposure. An interaction effect was also found in the accumbal levels of MEAP and nigral dynorphin B. These results demonstrate that repeated episodic exposure to ethanol during adolescence affected opioid peptide levels in regions involved in reward and reinforcement as well as stress response. These alterations in opioid networks after adolescent ethanol exposure could explain, in part, the increased risk for high ethanol consumption later in life.

The expression of opioid genes in non-classical reward areas depends on early life conditions and ethanol intake.

The young brain is highly sensitive to environmental influences that can cause long-term changes in neuronal function, possibly through altered gene expression. The endogenous opioid system continues to mature after birth and because of its involvement in reward, an inadequate maturation of this system could lead to enhanced susceptibility for alcohol use disorder. Recent studies show that the classical reward areas nucleus accumbens and ventral tegmental area are less affected by early life stress whereas endogenous opioids in non-classical areas, e.g. dorsal striatum and amygdala, are highly responsive. The aim was to investigate the interaction between early life conditions and adult voluntary ethanol intake on opioid gene expression. Male Wistar rats were exposed to conventional rearing, 15, or 360min of daily maternal separation (MS) postnatal day 1-21, and randomly assigned to ethanol or water drinking postnatal week 10-16. Rats exposed to early life stress (MS360) had increased opioid receptor gene (Oprm1, Oprd1 and Oprk1) expression in the dorsal striatum. Ethanol drinking was associated with lower striatal Oprd1 and Oprk1 expression solely in rats exposed to early life stress. Furthermore, rats exposed to early life stress had high inherent Pomc expression in the amygdala but low expression after ethanol intake. Thus, adverse events early in life induced changes in opioid gene expression and also influenced the central molecular response to ethanol intake. These long-term consequences of early life stress can contribute to the enhanced risk for excessive ethanol intake and alcohol use disorder seen after exposure to childhood adversity.

Ethanol affects limbic and striatal presynaptic glutamatergic and DNA methylation gene expression in outbred rats exposed to early-life stress.

Alcohol use disorder is the outcome of both genetic and environmental influences and their interaction via epigenetic mechanisms. The neurotransmitter glutamate is an important regulator of reward circuits and implicated in adaptive changes induced by ethanol intake. The present study aimed at investigating corticolimbic and corticostriatal genetic signatures focusing on the glutamatergic phenotype in relation to early-life stress (ELS) and consequent adult ethanol consumption. A rodent maternal separation model was employed to mimic ELS, and a free-choice paradigm was used to assess ethanol intake in adulthood. Gene expression levels of the Vesicular Glutamate Transporters (Vglut) 1, 2 and 3, as well as two key regulators of DNA methylation, DNA (cytosine-5)-methyltransferase 1 (Dnmt1) and methyl-CpG-binding protein 2 (Mecp2), were analyzed. Brain regions of interest were the ventral tegmental area (VTA), nucleus accumbens (Acb), medial prefrontal cortex (mPFC) and dorsal striatum (dStr), all involved in mediating aspects of ethanol reward. Region-specific Vglut, Dnmt1 and Mecp2 expression patterns were observed. ELS was associated with down-regulated expression of Vglut2 in the VTA and mPFC. Rats exposed to ELS were more sensitive to ethanol-induced changes in Vglut expression in the VTA, Acb, and dStr and in Dnmt1 and Mecp2 expression in the striatal regions. These findings suggest long-term glutamatergic and DNA methylation neuroadaptations as a consequence of ELS, and show an association between voluntary drinking in non-preferring, non-dependent, rodents and different Vglut, Dnmt1 and Mecp2 expression depending on early-life history.

Evidence for a Link Between Fkbp5/FKBP5, Early Life Social Relations and Alcohol Drinking in Young Adult Rats and Humans.

Alcohol misuse has been linked to dysregulation of stress, emotion, and reward brain circuitries. A candidate key mediator of this association is the FK506-binding protein (FKBP5), a negative regulator of the glucocorticoid receptor. The aim of the present study was to further understand the Fkbp5/FKBP5-related genetic underpinnings underlying the relationship between early life social relations and alcohol drinking. The effect of maternal separation and voluntary alcohol drinking on Fkbp5 expression was investigated in the brain of young adult rats, whereas the interaction effect of the functional FKBP5 single nucleotide polymorphism rs1360780 genotype and parent-child relationship on problematic drinking was examined in young adult humans. In rats, Fkbp5 expression in the nucleus accumbens and ventral tegmental area, core regions of the reward system, was affected in a region-dependent manner and in opposite direction by maternal separation and alcohol drinking. Fkbp5 expression in the cingulate cortex was affected by the combined effect of maternal separation and alcohol drinking. In humans, the TT genotype, in the presence of a poor relationship between the child and parents, was associated with problematic drinking behavior. The present findings suggest that Fkbp5 expression in mesocorticolimbic dopaminergic regions associates with early life stress-mediated sensitivity to alcohol drinking and that FKBP5 genotype interacts with parent-child relationship to influence alcohol drinking. These findings are the first to point to a role of FKBP5 in propensity to alcohol misuse and call for studies of the underlying molecular mechanisms to identify potential drug targets.

Αlpha 2a-Adrenoceptor Gene Expression and Early Life Stress-Mediated Propensity to Alcohol Drinking in Outbred Rats.

Stressful events early in life, later high alcohol consumption and vulnerability to alcohol use disorder (AUD) are tightly linked. Norepinephrine is highly involved in the stress response and the α2A-adrenoceptor, which is an important regulator of norepinephrine signalling, is a putative target in pharmacotherapy of AUD. The aim of the present study was to investigate the effects of early-life stress and adult voluntary alcohol drinking on the α2A-adrenoceptor. The relative expression and promoter DNA methylation of the Adra2a gene were measured in the hypothalamus, a key brain region in stress regulation. A well-characterized animal model of early-life stress was used in combination with an episodic voluntary drinking in adulthood. Alcohol drinking rats with a history of early-life stress had lower Adra2a expression than drinking rats not exposed to stress. Alcohol intake and Adra2a gene expression were negatively correlated in high-drinking animals, which were predominantly rats subjected to early-life stress. The results provide support for a link between early-life stress, susceptibility for high alcohol consumption, and low Adra2a expression in the hypothalamus. These findings can increase our understanding of the neurobiological basis for vulnerability to initiate risk alcohol consumption and individual differences in the response to α2A-adrenoceptor agonists.

HPA Axis Gene Expression and DNA Methylation Profiles in Rats Exposed to Early Life Stress, Adult Voluntary Ethanol Drinking and Single Housing.

The neurobiological basis of early life stress (ELS) impact on vulnerability to alcohol use disorder is not fully understood. The effect of ELS, adult ethanol consumption and single housing, on expression of stress and DNA methylation regulatory genes as well as blood corticosterone levels was investigated in the hypothalamus and pituitary of adult out-bred Wistar rats subjected to different rearing conditions. A prolonged maternal separation (MS) of 360 min (MS360) was used to study the effect of ELS, and a short MS of 15 min (MS15) was used as a control. Voluntary ethanol drinking was assessed using a two-bottle free choice paradigm to simulate human episodic drinking. The effects of single housing and ethanol were assessed in conventional animal facility rearing (AFR) conditions. Single housing in adulthood was associated with lower Crhr1 and higher Pomc expression in the pituitary, whereas ethanol drinking was associated with higher expression of Crh in the hypothalamus and Crhr1 in the pituitary, accompanied by lower corticosterone levels. As compared to controls with similar early life handling, rats exposed to ELS displayed lower expression of Pomc in the hypothalamus, and higher Dnmt1 expression in the pituitary. Voluntary ethanol drinking resulted in lower Fkbp5 expression in the pituitary and higher Crh expression in the hypothalamus, independently of rearing conditions. In rats exposed to ELS, water and ethanol drinking was associated with higher and lower corticosterone levels, respectively. The use of conventionally reared rats as control group yielded more significant results than the use of rats exposed to short MS. Positive correlations, restricted to the hypothalamus and ELS group, were observed between the expression of the hypothalamus-pituitary-adrenal receptor and the methylation-related genes. Promoter DNA methylation and expression of respective genes did not correlate suggesting that other loci are involved in transcriptional regulation. Concluding, single housing is a confounding factor to be considered in voluntary ethanol drinking paradigms. ELS and ethanol drinking in adulthood exert independent effects on hypothalamic and pituitary related genes, however, in a manner dependent on the control group used.