The impact of periconceptional ethanol exposure on maternal and offspring hypothalamic-pituitary-adrenal axis function and associated behaviours in a rat model

Abstract

The consumption of alcohol during pregnancy is frequent despite clear guidelines that indicate that abstinence is the safest option to prevent adverse offspring outcomes. These outcomes range from overt craniofacial abnormalities through to outcomes such as mental illness, hyperactivity and social difficulties. Human and animal studies have demonstrated that these neurological outcomes may be due to impaired function of the hypothalamic-pituitary-adrenal axis (HPA) in offspring, resulting in altered basal glucocorticoid tone and disrupted responsiveness to stress. However, little is known of the impact of alcohol consumption around the time of conception, known as the periconceptional period, on offspring HPA function. Therefore, this study aimed to use a well-established rat model of ethanol consumption during the periconceptional period (PC:EtOH) to investigate offspring HPA activity, including behaviours, stress responsiveness and underlying molecular pathways. As alcohol consumption directly alters HPA function, this study also aimed to examine if PC:EtOH exposure impairs maternal HPA activity and related physiological pathways, including renal and metabolic function.Female Sprague-Dawley rats were treated with PC:EtOH (12.5% v/v EtOH liquid diet) or a control diet from 4 days before conception, until embryonic day (E) 4. Behavioural tests were performed on offspring at three months of age to assess mental illness-like phenotypes (utilising the Forced Swim Test [FST] and Social Interaction [SI] paradigm), and at five months of age, HPA reactivity tests (combined dexamethasone suppression test [DST] and corticotropin-releasing hormone stimulation test [CST] and restraint stress) were performed. In a separate study, basal corticosterone concentrations were measured at 6 months, and adrenal glands were collected for analysis of steroidogenic gene expression. Aged cohorts (12-14 months) were utilised to measure basal plasma corticosterone, followed by the collection of adrenal glands, pituitary glands, hypothalamus and hippocampal tissue for analysis of various steroidogenesis and glucocorticoid signalling genes and pathology. In a separate cohort of aged rats, telemetry was used to asses blood pressure, heart rate and plasma corticosterone concentrations during 30-minute restraint stress.Maternal hormones (corticosterone, aldosterone), renal function and plasma glucose and lipids were assessed at various stages in gestation. Adrenal glands were collected from dams at E5, E15 and E20 for analysis of steroidogenic gene expression. Placental samples were collected at E20 and genes expression of the glucocorticoid (Nr3c1) and corticotrophin hormone receptor (Crh-r1) measured.This study revealed that PC:EtOH exposure resulted in altered offspring behavioural outcomes, including increased depressive-like behaviour in the forced swim test and altered social interaction with a novel rat. Adult offspring also demonstrated HPA hyperactivity, with elevated responses to the DST/CST challenge. Although there was no difference observed in adult offspring, aged PC:EtOH female offspring demonstrated an altered response to restraint, with reduced stress-induced plasma corticosterone and pressor response. Interestingly, PC:EtOH exposure also resulted in reduced basal plasma corticosterone concentrations in adult and aged female but not male offspring. Furthermore, female offspring showed pituitary gland abnormalities and increased mRNA for Nr3c1 and heat shock protein 90 (Hsp901a) in the hippocampus, suggesting altered HPA signalling and regulatory pathways. Adrenal and hypothalamic mRNA expression of genes regulating glucocorticoid production were not overtly altered by PC:EtOH in aged offspring.PC:EtOH significantly increased plasma corticosterone in the dam prior to mating (E-2). During pregnancy, PC:EtOH resulted in lower concentrations at E5, no differences at E15, and an increase at E20. Only minor changes in the expression of genes which regulate adrenal steroidogenesis were observed in PC:EtOH dams at E5 and E15, with the latter likely to have contributed to the observed increase in plasma corticosterone at E20. PC:EtOH had no impact on metabolic parameters (high and low-density lipoproteins and triglycerides) or renal function (food, water, urinary flow and renal electrolytes) in late gestation. However, placental markers of glucocorticoid exposure were elevated in response to exposure.This study supports the hypothesis that periconceptional ethanol exposure alters the HPA of the mother and programs sex-specific alterations in offspring in a rat model. Maternal HPA and related physiological changes as a consequence of PC:EtOH is likely to contribute to the HPA hyperresponsiveness, and underlie behavioural outcomes observed in this study. Furthermore, these changes to the HPA may be independent of the adrenal gland, with central regulatory pathways involving the hippocampus altered by PC:EtOH.This thesis has provided novel and important evidence that alcohol exposure around the time of conception impairs offspring mental-health like outcomes and induces HPA dysregulation. This work reinforces the concept that the maternal stress axis is highly sensitive to perturbations during early pregnancy. As this system is critical in many major physiological pathways, this can have significant long-term disease implications for both the mother and the child, supporting the critical need for education of appropriate health and wellbeing in preparation for pregnancy.