The contribution of the early postnatal environment to the pervasive effects of prenatal alcohol exposure (PAE) is poorly understood. Moreover, PAE often carries increased risk of exposure to adversity/stress during early life. Dysregulation of immune function may play a role in how pre- and/or postnatal adversity/stress alters brain development. Here, we combine two animal models to examine whether PAE differentially increases vulnerability to immune dysregulation in response to early-life adversity. PAE and control litters were exposed to either limited bedding (postnatal day [PNI 8-12) to model early-life adversity or normal bedding, and maternal behavior and pup vocalizations were recorded. Peripheral (serum) and central (amygdala) immune (cytokines and C-reactive protein - CRP) responses of PAE animals to early-life adversity were evaluated at PN12. Insufficient bedding increased negative maternal behavior in both groups. Early-life adversity increased vocalization in all animals; however, PAE pups vocalized less than controls. Early-life adversity reduced serum TNF-alpha, KC/GRO, and IL-10 levels in control but not PAE animals. PAE increased serum CRP, and levels were even higher in pups exposed to adversity. Finally, PAE reduced KC/GRO and increased IL-10 levels in the amygdala. Our results indicate that PAE alters immune system development and both behavioral and immune responses to early-life adversity, which could have subsequent consequences for brain development and later life health. (C) 2017 Elsevier Inc. All rights reserved.

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the previous 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37–44) and late (PND 67–74) adolescent development. Animals were mated 24 h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring.

BACKGROUND Prenatal alcohol exposure (PAE) results in dysregulation of the offspring hypothalamic-pituitary-adrenal (HPA) axis, increasing sensitivity to stressors and vulnerability to stress-related disorders. We have previously shown that exposure to chronic mild stress (CMS) in adulthood significantly increases anxiety-like behaviors (elevated plus maze) in PAE males and females compared to controls. To explore neurobiological mechanisms linking HPA dysregulation and altered anxiety-like behavior, we investigated neuropeptide (corticotropin-releasing hormone [CRH] and arginine vasopressin [AVP]) expression in brain areas involved in the stress neurocircuitry of animals from this previous behavioral study. METHODS Adult PAE, pair-fed (PF), and ad libitum fed control (C) male and female offspring exposed to CMS or remaining undisturbed (non-CMS) were terminated 30 minutes following behavioral testing. RESULTS In the paraventricular nucleus, CMS increased CRH mRNA levels in PAE compared to PF and C males and increased AVP mRNA levels in PAE compared to C males, with no differential effects for CRH or AVP in females. In the central nucleus of the amygdala, there was an increase in CRH mRNA expression overall, regardless of CMS condition or sex, in PAE compared to C animals. Moreover, in PF males, CMS increased AVP mRNA levels in the paraventricular nucleus, resulting in a decreased CRH/AVP ratio compared to PAE males, and decreased amygdala CRH mRNA compared to that in the non-CMS condition. CONCLUSIONS CMS differentially altered central HPA peptide expression in PAE and PF animals compared to their control counterparts, with a possible shift toward greater CRH mediation of HPA regulation in PAE males, and greater AVP mediation of HPA regulation in PF males. However, changes in CRH and AVP expression do not align fully with the anxiogenic profile observed in our previous behavior study, suggesting that other neuronal substrates and limbic forebrain regions also contribute to increased anxiety-like behavior following CMS.

BACKGROUND:During late prenatal and early postnatal life, the reproductive system in males undergoes an extensive series of physiological and morphological changes. Prenatal ethanol (EtOH) exposure has marked effects on the development of the reproductive system, with long-term effects on function in adulthood. The present study tested the hypothesis that prenatal EtOH exposure will delay the onset of spermatogenesis.METHODS:Development of the seminiferous tubules and the onset of spermatogenesis were examined utilizing a rat model of fetal alcohol spectrum disorder (FASD). Male offspring from ad libitum-fed control (C), pair-fed (PF), and EtOH-fed (prenatal alcohol exposure [PAE]) dams were terminated on postnatal (PN) days 5, 15, 18, 20, 25, 35, 45, and 55, to investigate morphological changes through morphometric analysis of the testes from early neonatal life through young adulthood.RESULTS:PAE males had lower relative (adjusted for body weight) testis weights compared with PF and/or C males from PN15 through puberty (PN45). In addition, fewer gonocytes (primordial germ cells) were located on the basal lamina on PN5, while more of those touching the basal lamina were dividing in PAE compared with PF and C males, suggesting delayed cell division and migration processes. As well, the percentage of tubules with open lumena was lower in PAE compared with PF and C males on PN18 and 20, and PAE males had fewer primary spermatocytes per tubule on PN18 and round spermatids per tubule on PN25 compared with C males. Finally, the percentage of tubules at stages VII and VIII, when mature spermatids move to the apex of the epithelium and are released, was lower in PAE compared with PF and/or C males in young adulthood (PN55).CONCLUSIONS:Maternal EtOH consumption appears to delay both reproductive development and the onset of spermatogenesis in male offspring, with effects persisting at least until young adulthood.

Prenatal alcohol exposure (PAE) has adverse effects on the development of numerous physiological systems, including the hypothalamic-pituitary-adrenal (HPA) axis and the immune system. HPA hyper-responsiveness and impairments in immune competence have been demonstrated. The present study investigated immune function in PAE females utilizing an adjuvant-induced arthritis (AA) model, widely used as a model of human rheumatoid arthritis. Given the effects of PAE on HPA and immune function, and the known interaction between HPA and immune systems in arthritis, we hypothesized that PAE females would have heightened autoimmune responses, resulting in increased severity of arthritis, compared to controls, and that altered HPA activity might play a role in the immune system changes observed. The data demonstrate, for the first time, an adverse effect of PAE on the course and severity of AA in adulthood, indicating an important long-term alteration in functional immune status. Although overall, across prenatal treatments, adjuvant-injected animals gained less weight, and exhibited decreased thymus and increased adrenal weights, and increased basal levels of corticosterone and adrenocorticotropin, PAE females had a more prolonged course of disease and greater severity of inflammation compared to controls. In addition, PAE females exhibited blunted lymphocyte proliferative responses to concanavalin A and a greater increase in basal ACTH levels compared to controls during the induction phase, before any clinical signs of disease were apparent. These data suggest that prenatal alcohol exposure has both direct and indirect effects on inflammatory processes, altering both immune and HPA function, and likely, the normal interactions between these systems.

BACKGROUND:Rats prenatally exposed to ethanol (E) typically show increased hypothalamic-pituitary-adrenal (HPA) responses to stressors in adulthood. Importantly, prenatal ethanol may differentially alter stress responsiveness in male and female offspring, suggesting a role for the gonadal hormones in mediating the effects of ethanol on HPA activity. We investigated the role of ethanol-induced changes in hypothalamic-pituitary-gonadal (HPG) activity in the differential HPA regulation observed in E compared to control females across the estrous cycle.METHODS:Peripheral hormones and changes in central neuropeptide mRNA levels were measured across the estrous cycle in adult female offspring from E, pair-fed (PF) and ad libitum-fed control (C) dams.RESULTS:Ethanol females showed normal estrous cyclicity (vaginal smears) but delayed sexual maturation (vaginal opening). Both HPG and HPA activity were differentially altered in E (and in some cases, PF) compared to control females as a function of estrous cycle stage. In relation to HPG activity, E and PF females had higher basal and stress estradiol (E(2)) levels in proestrus compared to other phases of the cycle, and decreased GnRH mRNA levels compared to C females in diestrus. Further, E females had greater variation in LH than PF and C females across the cycle, and in proestrus, only E females showed a significant LH increase following stress. In relation to HPA activity, both basal and stress CORT levels and overall ACTH levels were greater in E than in C females in proestrus. Furthermore, AVP mRNA levels were increased overall in E compared to PF and C females.CONCLUSIONS:These data demonstrate ethanol-induced changes in both HPG and HPA activity that are estrous phase-specific, and support the possibility that changes in HPA activity in E females may reflect differential sensitivity to ovarian steroids. E females appear to have an increased HPA sensitivity to E(2), and a possible shift toward AVP regulation of HPA activity. That PF were similar to E females on some measures suggests that nutritional effects of diet or food restriction played a role in mediating at least some of the changes observed.

Prenatal ethanol (E) exposure programs the fetal hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes such that E rats show HPA hyperresponsiveness to stressors and altered HPG and reproductive function in adulthood. Importantly, prenatal ethanol may differentially alter stress responsiveness in adult male and female offspring compared to their control counterparts. To test the hypothesis that alterations in HPA activity in E males are mediated, at least in part, by ethanol-induced changes in the capacity of testosterone to regulate HPA activity, we explored dose-related effects of testosterone on HPA and HPG function in adult male offspring from prenatal E, pair-fed (PF) and ad libitum-fed control (C) dams. Our data suggest that E males show changes in both HPA and HPG regulation, as well as altered sensitivity to the inhibitory effects of testosterone. While gonadectomy (GDX) reduced weight gain in all animals, low testosterone replacement restored body weights in PF and C but not E males. Further, sensitivity of the thymus and adrenal to circulating testosterone was reduced in E rats. In addition, stress-induced corticosterone (CORT) levels were increased in PF and C but not E males following GDX, and while low dose testosterone replacement restored CORT levels for PF and C, high testosterone levels were needed to normalize CORT levels for E males. A negative correlation between pre-stress testosterone and post-stress CORT levels in C but not in E and PF males further supports the finding of reduced sensitivity to testosterone. Importantly, testosterone appeared to have reduced effects on central corticotrophin releasing hormone (CRH) pathways in E, but greater effects on central arginine vasopressin (AVP) pathways in E and/or PF compared to C males. Testosterone also had less of an inhibitory effect on stress-induced luteinizing hormone increases in E than in PF and C males following GDX. In addition, androgen receptor mRNA levels in the medial preoptic nucleus and the principal nucleus of posterior bed nucleus of the stria terminalis were lower in E and PF compared to C males under intact conditions. Together, these data support our previous work suggesting altered sensitivity to testosterone in E males. Furthermore, differential effects of testosterone on the complex balance between central CRH and central AVP pathways may play a role in the HPA alterations observed. That some findings were similar in E and PF males suggest that nutritional effects of diet may have played a role in mediating at least some of the changes seen in E animals.