Ingrid Nylander

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.

Enzymatic degradation is a major concern in peptide analysis. Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations. It is therefore vital to find reliable, reproducible, and easy-to-use procedures to inhibit enzymatic activity in fresh tissues before subjecting them to qualitative and quantitative analyses. The aim of this study was to test a benchtop thermal stabilization method to optimize measurement of endogenous opioids in brain tissue. Endogenous opioid peptides are generated from precursor proteins through multiple enzymatic steps that include conversion of one bioactive peptide to another, often with a different function. Ex vivo metabolism may, therefore, lead to erroneous functional interpretations. The efficacy of heat stabilization was systematically evaluated in a number of postmortem handling procedures. Dynorphin B (DYNB), Leu-enkephalin-Arg ⁶ (LARG), and Met-enkephalin-Arg ⁶ -Phe ⁷ (MEAP) were measured by radioimmunoassay in rat hypothalamus, striatum (STR), and cingulate cortex (CCX). Also, simplified extraction protocols for stabilized tissue were tested. Stabilization affected all peptide levels to varying degrees compared to those prepared by standard dissection and tissue handling procedures. Stabilization increased DYNB in hypothalamus, but not STR or CCX, whereas LARG generally decreased. MEAP increased in hypothalamus after all stabilization procedures, whereas for STR and CCX, the effect was dependent on the time point for stabilization. The efficacy of stabilization allowed samples to be left for 2 hours in room temperature (20°C) without changes in peptide levels. This study shows that conductive heat transfer is an easy-to-use and efficient procedure for the preservation of the molecular composition in biological samples. Region- and peptide-specific critical steps were identified and stabilization enabled the optimization of tissue handling and opioid peptide analysis. The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

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 of 360 min (MS360) was used to study the effect of ELS, and a short maternal separation 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 maternal separation. Positive correlations, restricted to the hypothalamus and ELS group, were observed between the expression of the HPA 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.

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.

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.

Rationale Early-life events can cause long-term neurobiological and behavioural changes with a resultant effect upon reward and addiction processes that enhance risk to develop alcohol use disorders. Maternal separation (MS) is used to study the mediating mechanisms of early-life influences in rodents. In MS studies, the pups are exposed to maternal absence during the first postnatal weeks. The outcome of MS experiments exhibits considerable variation and questions have been raised about the validity of MS models. Objectives This short review aims to provide information about experimental conditions that are important to consider when assessing the impact of early-life environment on voluntary ethanol consumption. Results The results from currently used MS protocols are not uniform. However, studies consistently show that longer separations of intact litters predispose for higher ethanol consumption and/or preference in adult male rats as compared to shorter periods of MS. Studies using individual pup MS paradigms, other controls, low ethanol concentrations, adult females or examining adolescent consumption reported no differences or inconsistent results. Conclusions There is no “a rodent MS model”, there are several models and they generate different results. The compiled literature shows that MS is a model of choice for analysis of early-life effects on voluntary ethanol consumption but there are examples of MS paradigms that are not suitable. These studies emphasize the importance to carefully designed MS experiments to supply the optimal conditions to definitely test the research hypothesis and to be particulate in the interpretation of the outcome.

The brain is constantly exposed to external and internal input and to function in an ever-changing environment we are dependent on processes that enable the brain to adapt to new stimuli. Exposure to postnatal environmental stimuli can interfere with vital adaption processes and cause long-term changes in physiological function and behavior. Early-life alterations in brain function may result in impaired ability to adapt to new situations, in altered sensitivity to challenges later in life and thereby mediate risk or protection for psychopathology such as alcohol use disorders (AUD). In clinical research the studies of mechanisms, mediators, and causal relation between early environmental factors and vulnerability to AUD are restricted and attempts are made to find valid animal models for studies of the early-life influence on the brain. This review focuses on rodent models and the effects of adverse and naturalistic conditions on peptide networks within the brain and pituitary gland. Importantly, the consequences of alcohol addiction are not discussed but rather neurobiological alterations that can cause risk consumption and vulnerability to addiction. The article reviews earlier results and includes new data and multivariate data analysis with emphasis on endogenous opioid peptides but also oxytocin and vasopressin. These peptides are vital for developmental processes and it is hypothesized that early-life changes in peptide networks may interfere with neuronal processes and thereby contribute the individual vulnerability for AUD. The summarized results indicate a link between early-life rearing conditions, opioids, and ethanol consumption and that the ethanol-induced effects and the treatment with opioid antagonists later in life are dependent on early-life experiences. Endogenous opioids are therefore of interest to further study in the early-life impact on individual differences in vulnerability to AUD and treatment outcome.

Genes involved in alcoholism have consensus sites for the transcription factor activator protein (TFAP) 2β. In the present study, we investigated TFAP-2β protein levels in the ethanol-preferring alko, alcohol (AA) and the ethanol-avoiding alko, non-alcohol (ANA) rat lines. Furthermore, basal and ethanol-induced TFAP-2β levels were examined in Wistar rats exposed to different early postnatal environments that are known to affect later ethanol consumption. Taken together, we found differences in brainstem TFAP-2β protein between the AA and ANA rats.