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DNA methylation and gene expression can be altered by early life stress (ELS) and/or ethanol consumption. The present study aimed to investigate whether DNA methylation of the Vesicular Glutamate Transporters (Vglut)1-3 is related to previously observed Vglut1-3 transcriptional differences in the ventral tegmental area (VTA), nucleus accumbens (Acb), dorsal striatum (dStr) and medial prefrontal cortex (mPFC) of adult rats exposed to ELS, modelled by maternal separation, and voluntary ethanol consumption. Targeted next-generation bisulfite sequencing was performed to identify the methylation levels on 61 5′-cytosine-phosphate-guanosine-3′ sites (CpGs) in potential regulatory regions of Vglut1, 53 for Vglut2, and 51 for Vglut3. In the VTA, ELS in ethanol-drinking rats was associated with Vglut1-2 CpG-specific hypomethylation, whereas bidirectional Vglut2 methylation differences at single CpGs were associated with ELS alone. Exposure to both ELS and ethanol, in the Acb, was associated with lower promoter and higher intronic Vglut3 methylation; and in the dStr, with higher and lower methylation in 26% and 43% of the analyzed Vglut1 CpGs, respectively. In the mPFC, lower Vglut2 methylation was observed upon exposure to ELS or ethanol. The present findings suggest Vglut1-3 CpG-specific methylation signatures of ELS and ethanol drinking, underlying previously reported Vglut1-3 transcriptional differences in the mesocorticolimbic brain.

Discordant associations between monoamine oxidase A (MAOA) genotype and high alcohol drinking have been reported in human and non-human primates. Environmental influences likely moderate genetic susceptibility. The biological basis for this interplay remains elusive, and inconsistencies call for translational studies in which conditions can be controlled and brain tissue is accessible. The present study investigated whether early life stress and subsequent adult episodic alcohol consumption affect Maoa expression in stress-and reward-related brain regions in the rat. Outbred Wistar rats were exposed to rearing conditions associated with stress (prolonged maternal separation) or no stress during early life, and given free choice between alcohol and/or water in adulthood. Transcript levels of Maoa were assessed in the ventral tegmental area, nucleus accumbens (NAc), medial prefrontal cortex, cingulate cortex, amygdala and dorsal striatum (DS). Blood was collected to assess corticosterone levels. After alcohol consumption, lower blood corticosterone and Maoa expression in the NAc and DS were found in rats exposed to early life stress compared with control rats. An interaction between early life stress and voluntary alcohol intake was found in the NAc. Alcohol intake before death correlated negatively with Maoa expression in DS in high alcohol-drinking rats exposed to early life stress. Maoa expression is sensitive to adulthood voluntary alcohol consumption in the presence of early life stress in outbred rats. These findings add knowledge of the molecular basis of the previously reported associations between early life stress, MAOA and susceptibility to alcohol misuse.

The vulnerability to develop alcoholism is dependent on both genetic and environmental factors. The neurobiological mechanisms underlying these factors are not fully understood but individual divergence in the endogenous opioid peptide system may contribute. We have previously reported that early-life experiences can affect endogenous opioids and also adult voluntary ethanol intake. In the present study, this line of research was continued and the effects of long-term voluntary ethanol drinking on the opioid system are described in animals reared in different environmental settings. Rat pups were subjected to 15 min (MS15) or 360 min (MS360) of daily maternal separation during postnatal days 1-21. At 10 weeks of age, male rats were exposed to voluntary ethanol drinking in a four-bottle paradigm with 5%, 10% and 20% ethanol solution in addition to water for 2 months. Age-matched controls received water during the same period. Immunoreactive (ir) Met-enkephalin-Arg6Phe7 (MEAP) and dynorphin B (DYNB) peptide levels were thereafter measured in the pituitary gland and several brain areas. In water-drinking animals, lower ir MEAP levels were observed in the MS360 rats in the hypothalamus, medial prefrontal cortex, striatum and the periaqueductal gray, whereas no differences were seen in ir DYNB levels. Long-term ethanol drinking induced lower ir MEAP levels in MS15 rats in the medial prefrontal cortex and the periaqueductal gray, whereas higher levels were detected in MS360 rats in the hypothalamus, striatum and the substantia nigra. Chronic voluntary drinking affected ir DYNB levels in the pituitary gland, hypothalamus and the substantia nigra, with minor differences between MS15 and MS360. In conclusion, manipulation of the early environment caused changes in the opioid system and a subsequent altered response to ethanol. The altered sensitivity of the opioid peptides to ethanol may contribute to the previously reported differences in ethanol intake between MS15 and MS3

In order to investigate the involvement of the opioid and nociceptin/orphanin FQ (N/OFQ) system in alcohol drinking behaviour, N/OFQ and the opioid peptides dynorphin B (DYNB) and Met-enkephalin-Arg(6) Phe(7) (MEAP) were examined in the alcohol-preferring C57BL/6J mice. Basal peptide levels were compared in the brain and the pituitary gland with basal levels in the alcohol-avoiding DBA/2J mice. Furthermore, the effects of chronic alcohol self-administration on peptides were studied in the C57BL/6J mice. Compared to the DBA/2J mice, C57BL/6J mice had low immunoreactive (ir) levels of DYNB and MEAP in the nucleus accumbens, the hippocampus, and the substantia nigra, low ir-DYNB levels in the striatum and low ir-MEAP levels in the frontal cortex. Higher ir-DYNB levels in the pituitary gland and in the periaqueductal gray (PAG) and higher ir-N/OFQ levels in the frontal cortex and in the hippocampus were detected in C57BL/6J mice compared to the DBA/2J mice. After 4 weeks of voluntary alcohol consumption, only minor changes in steady-state peptide levels were identified. However, 5 days after the alcohol-drinking period, lower levels of all peptides were detected in the ventral tegmental area and ir-DYNB levels were also lower in the amygdala and in the substantia nigra. Twenty-one days after cessation of alcohol self-administration, the opioid peptides in alcohol-consuming C57BL/6J mice were lower in the PAG, the N/OFQ was lower in the frontal cortex and DYNB was higher in the amygdala and substantia nigra as compared to control C57BL/6J mice. This study demonstrates strain differences between C57BL/6J mice and DBA/2J mice that could contribute to divergent drug-taking behaviour, and it also demonstrates time- and structure-specific changes in neuropeptide levels after alcohol self-administration in the C57BL/6J mice.