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

Abstract

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.

Maternal separation affects expression of stress response genes and increases vulnerability to ethanol consumption

“…We did not observe any differences in the expression of Avp after T1, similar to what has been observed in other early life stress studies (Oreland, Gustafsson‐Ericson, & Nylander, ; Todkar et al., ). Nevertheless, higher expression levels of Avp and Nr3c1 after maternal separation have been previously reported (Murgatroyd et al., ).…”

“…Nevertheless, Todkar et al. () reported more pronounced effects of maternal separation on stress‐related genes and ethanol consumption when they used a control litter that was not subjected to maternal separation, instead of the MS15 control. This confirms the previously reported effects of handling (Jaworski, Francis, Brommer, Morgan, & Kuhar, ; Pryce & Feldon, ) and highlights the importance of carefully choosing the control group.…”

“…Previous studies in rodents have shown the effects of postnatal stress and ethanol consumption on the HPA axis and on gene expression in the dopaminergic system (Bendre et al., ; García‐Gutiérrez et al., ; Todkar et al., ). Nevertheless, these studies were limited as they analyzed only a small set of genes and did not correlate the results with serotonergic transmission genes or possible behavioral changes.…”

DNA methylation at stress‐related genes is associated with exposure to early life institutionalization

“…Particularly with limited subject numbers, targeted gene analyses supported by molecular studies and neuroscience, minimize the potential for false positive findings, an issue particularly relevant to genome wide studies that fail to leverage the substantial existing neurobiologically‐informed research related to the impact of early adversity. Future studies that examine methylation in additional candidate genes involved in stress‐response systems, such as the gene that encodes the glucocorticoid receptor ( NR3C1 ), or the corticotrophin hormone receptors ( CRHR1, CRHR2 ) would be of interest (Palma‐Gudiel et al, ; Todkar et al, ). In studies with larger sample sizes, potential interactions between these candidate genes could also be tested, to determine if methylation states across these genes contribute to any long term psychological or biological outcomes.…”

Studies using macaque monkeys to address excessive alcohol drinking and stress interactions

“…Specifically, the risk for future alcohol consumption may be measurable in DNA methylation profiles that have been found to link parental alcohol exposure and the HPA axis activity of alcohol-naïve offspring (Asimes et al, 2016; Todkar et al, 2016). These studies have opened a door to understanding environmental impacts, such as early life stress and paternal/maternal stressors including alcohol intake, on the epigenetic changes that may confer a risk for the development of AUDs and may provide new avenues for the development of pharmacological interventions.…”