Toni R. Pak

Binge-Pattern Alcohol Exposure during Puberty Induces Long-Term Changes in HPA Axis Reactivity

Adolescence is a dynamic and important period of brain development however, little is known about the long-term neurobiological consequences of alcohol consumption during puberty. Our previous studies showed that binge-pattern ethanol (EtOH) treatment during pubertal development negatively dysregulated the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis, as manifested by alterations in corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP), and corticosterone (CORT) during this time period. Thus, the primary goal of this study was to determine whether these observed changes in important central regulators of the stress response were permanent or transient. In this study, juvenile male Wistar rats were treated with a binge-pattern EtOH treatment paradigm or saline alone for 8 days. The animals were left undisturbed until adulthood when they received a second round of treatments consisting of saline alone, a single dose of EtOH, or a second binge-pattern treatment paradigm. The results showed that pubertal binge-pattern EtOH exposure induced striking long-lasting alterations of many HPA axis parameters. Overall, our data provide strong evidence that binge-pattern EtOH exposure during pubertal maturation has long-term detrimental effects for the healthy development of the HPA axis.

Binge-pattern alcohol exposure during puberty induces sexually dimorphic changes in genes regulating the HPA axis

Maternal alcohol consumption during critical periods of fetal brain development leads to devastating long-term consequences on adult reproductive physiology, cognitive function, and social behaviors. However, very little is known about the long-term consequences of alcohol consumption during puberty, which is perhaps an equally dynamic and critical period of brain development. Alcohol abuse during adulthood has been linked with an increase in clinically diagnosed anxiety disorders, yet the etiology and neurochemical mechanisms of alcohol-induced anxiety behavior is unknown. In this study, we determined the effects of binge ethanol exposure during puberty on two critical central regulators of stress and anxiety behavior: corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP). Our results showed that ethanol increased plasma corticosterone (CORT) levels in both sexes, yet binge-treated animals had significantly lower CORT levels than animals exposed to a single dose, suggesting that the hypothalamo-pituitary-adrenal (HPA) axis habituated to the repeated stressful stimuli of ethanol. Binge ethanol exposure also significantly increased CRH and AVP gene expression in the paraventricular nucleus of males, but not females. Overall, our results demonstrate that binge ethanol exposure during puberty changes the central expression of stress-related genes in a sex-specific manner, potentially leading to permanent dysregulation of the HPA axis and long-term behavioral consequences.

Alcohol dysregulates corticotropin-releasing-hormone (CRH) promoter activity by interfering with the negative glucocorticoid response element (nGRE).

EtOH exposure in male rats increases corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN), a brain region responsible for coordinating stress and anxiety responses. In this study we identified the molecular mechanisms involved in mediating these effects by examining the direct effects of EtOH on CRH promoter activity in a neuronal cell line derived from the PVN (IVB). In addition, we investigated the potential interactions of EtOH and glucocorticoids on the CRH promoter by concomitantly treating cells with EtOH and the glucocorticoid receptor (GR) antagonist RU486, and by sequentially deleting GR binding sites within glucocorticoid response element (GRE) on the CRH promoter. Cells were transiently transfected with a firefly luciferase reporter construct containing 2.5 kb of the rat wild type (WT) or mutated CRH promoter. Our results showed that EtOH treatment induced a biphasic response in CRH promoter activity. EtOH exposure for 0.5 h significantly decreased promoter activity compared to vehicle treated controls, whereas promoter activity was significantly increased after 2.0 h of EtOH exposure. Treatment with RU486, or deletion of the GR binding sites 1 and 2 within the GRE, abolished the EtOH-induced increase in the promoter activity, however did not affect EtOH-induced decrease in CRH promoter activity at an earlier time point. Overall, our data suggest that alcohol exposure directly regulates CRH promoter activity by interfering with the normal feedback mechanisms of glucocorticoids mediated by GR signaling at the GRE site of the CRH promoter.

Long-Term Effects of Peripubertal Binge EtOH Exposure on Hippocampal microRNA Expression in the Rat

Adolescent binge alcohol abuse induces long-term changes in gene expression, which impacts the physiological stress response and memory formation, two functions mediated in part by the ventral (VH) and dorsal (DH) hippocampus. microRNAs (miRs) are small RNAs that play an important role in gene regulation and are potential mediators of long-term changes in gene expression. Two genes important for regulating hippocampal functions include brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT1), which we identified as putative gene targets of miR-10a-5p, miR-26a, miR-103, miR-495. The purpose of this study was to quantify miR-10a-5p, miR-26a, miR-103, miR-495 expression levels in the dorsal and ventral hippocampus of male Wistar rats during normal pubertal development and then assess the effects of repeated binge-EtOH exposure. In addition, we measured the effects of binge EtOH-exposure on hippocampal Drosha and Dicer mRNA levels, as well as the putative miR target genes, BDNF and SIRT1. Overall, mid/peri-pubertal binge EtOH exposure altered the normal expression patterns of all miRs tested in an age- and brain region-dependent manner and this effect persisted for up to 30 days post-EtOH exposure. Moreover, our data revealed that mid/peri-pubertal binge EtOH exposure significantly affected miR biosynthetic processing enzymes, Drosha and Dicer. Finally, EtOH-induced significant changes in the expression of a subset of miRs, which correlated with changes in the expression of their predicted target genes. Taken together, these data demonstrate that EtOH exposure during pubertal development has long-term effects on miRNA expression in the rat hippocampus.

Arginine vasopressin regulation in pre- and postpubertal male rats by the androgen metabolite 3β-diol

Arginine vasopressin (AVP) is a nonapeptide expressed in several brain regions. In addition to its well-characterized role in osmoregulation, AVP regulates paternal behavior, aggression,circadian rhythms, and the stress response. In the bed nucleus of the stria terminalis (BST), AVP gene expression is tightly regulated by gonadal steroid hormones. However, the degree by which AVP is regulated by gonadal steroid hormones in the suprachiasmatic nucleus (SCN) and medial amygdala (MeA) is unclear. Previous studies have shown that AVP expression in the brain of gonadectomized rats is restored with testosterone, 17beta-estradiol, and 5alpha-dihydrotestosterone(DHT) replacement. In addition, we have demonstrated that 3beta-diol, a metabolite of DHT,increased AVP promoter activity in a neuronal cell line and that the effects of 3beta-diol on AVP promoter activity were mediated by estrogen receptor-beta. To test whether 3beta-diol has a physiological role in the regulation of central AVP expression in vivo, we gonadectomized pre- and postpubertal male rats and followed with once daily injections of estradiol benzoate (EB),DHT-propionate, 3beta-diol-dipropionate, or vehicle. The SCN, BST, and MeA were analyzed for AVP mRNA expression using in situ hybridization. In the BST, intact juveniles had significantly fewer AVP-expressing cells than adults. GDX abolished all AVP mRNA expression in the BST in both age groups, whereas treatment with EB restored >80% and DHTP <10% of the AVP expression. Interestingly, 3beta-diol-proprionate was more effective at inducing AVP expression in juveniles than in adults, suggesting that the regulation of AVP by 3beta-diol might be age dependent [corrected].

Characterisation of human oestrogen receptor beta (ERβ) splice variants in neuronal cells.

Oestrogen receptor (ER)α and ERβ are members of the ligand‐activated superfamily of nuclear receptors and mediate most facets of oestrogen signalling. Several naturally occurring splice variants of each ER have been identified in the human brain, yet the biological significance of these splice variants in the brain remains unknown. In the present study, we exploit the unique structural differences of the human ERβ splice variants to determine the functional significance of individual ER domains in the brain. We previously established that full‐length rodent ERβ (i.e. rERβ1) has constitutive transcriptional activity in neuronal cells in the absence of ligand. By contrast to the rodent splice variants, the human ERβ splice variants used in the present study contain varying length truncations of exon 8, which encodes for the E/F domains. Our results reveal that, in neuronal cells, each human‐specific ERβ splice variant constitutively activated promoters mediated by a canonical oestrogen response element and repressed promoters mediated by activator protein‐1 sites via p38 activity. From these data, we conclude that the C‐terminus, encoding the AF‐2 region and F domain, is not essential for the constitutive properties of human ERβ. Taken together, these studies show that human‐specific ERβ variants are constitutively active and also provide novel insight into the contributions of the functional domains of ERβ towards mediating constitutive transcription at various promoters in neuronal cells.

Effects of kisspeptin on parameters of the HPA axis

The hypothalamo–pituitary–adrenal (HPA) and hypothalamo–pituitary–gonadal (HPG) axes have an intricate cross talk that results in the inhibition of reproductive functions during periods of chronic physiological or psychological stress. Recent studies have shown that kisspeptin neurons have projections to many non-reproductive areas of the brain including the paraventricular nucleus (PVN) of the hypothalamus, thereby providing evidence of an anatomical framework for kisspeptin to regulate the HPA axis. In this study, we tested as to whether kisspeptin modulates the HPA axis at three potential levels of regulation: (1) transcription of stress-related genes CRH, AVP, and oxytocin (OXY); (2) release of neuropeptides from PVN-derived neuronal cells via mobilization of intracellular calcium stores; and (3) in vivo regulation of the HPA axis under basal and stress-induced conditions in adult male rats. Overall, our data showed that kisspeptin did not alter basal, or stress-induced HPA axis activity (plasma corticosterone (CORT) and adrenocorticotropin hormone (ACTH)) in adult male rats and had modest, yet significant effects on CRH, AVP, and OXY gene expressions.

Differential fibroblast growth factor 8 (FGF8)-mediated autoregulation of its cognate receptors, Fgfr1 and Fgfr3, in neuronal cell lines.

Fibroblast growth factors (FGFs) mediate a vast range of CNS developmental processes including neural induction, proliferation, migration, and cell survival. Despite the critical role of FGF signaling for normal CNS development, few reports describe the mechanisms that regulate FGF receptor gene expression in the brain. We tested whether FGF8 could autoregulate two of its cognate receptors, Fgfr1 and Fgfr3, in three murine cell lines with different lineages: fibroblast-derived cells (3T3 cells), neuronal cells derived from hippocampus (HT-22 cells), and neuroendocrine cells derived from hypothalamic gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). GnRH is produced by neurons in the hypothalamus and is absolutely required for reproductive competence in vertebrate animals. Several lines of evidence strongly suggest that Fgf8 is critical for normal development of the GnRH system, therefore, the GT1-7 cells provided us with an additional endpoint, Gnrh gene expression and promoter activity, to assess potential downstream consequences of FGF8-induced modulation of FGF receptor levels. Results from this study suggest that the autoregulation of its cognate receptor represents a common downstream effect of FGF8. Further, we show that Fgfr1 and Fgfr3 are differentially regulated within the same cell type, implicating these two receptors in different biological roles. Moreover, Fgfr1 and Fgfr3 are differentially regulated among different cell types, suggesting such autoregulation occurs in a cell type-specific fashion. Lastly, we demonstrate that FGF8b decreases Gnrh promoter activity and gene expression, possibly reflecting a downstream consequence of altered FGF receptor populations. Together, our data bring forth the possibility that, in addition to the FGF synexpression group, autoregulation of FGFR expression by FGF8 represents a mechanism by which FGF8 could fine-tune its regulatory actions.

Estrogen Signaling and the Aging Brain: Context-Dependent Considerations for Postmenopausal Hormone Therapy

Recent clinical studies have spurred rigorous debate about the benefits of hormone therapy (HT) for postmenopausal women. Controversy first emerged based on a sharp increase in the risk of cardiovascular disease in participants of the Womens Health Initiative (WHI) studies, suggesting that decades of empirical research in animal models was not necessarily applicable to humans. However, a reexamination of the data from the WHI studies suggests that the timing of HT might be a critical factor and that advanced age and/or length of estrogen deprivation might alter the bodys ability to respond to estrogens. Dichotomous estrogenic effects are mediated primarily by the actions of two high-affinity estrogen receptors alpha and beta (ERα & ERβ). The expression of the ERs can be overlapping or distinct, dependent upon brain region, sex, age, and exposure to hormone, and, during the time of menopause, there may be changes in receptor expression profiles, post-translational modifications, and protein:protein interactions that could lead to a completely different environment for E2 to exert its effects. In this review, factors affecting estrogen-signaling processes will be discussed with particular attention paid to the expression and transcriptional actions of ERβ in brain regions that regulate cognition and affect.

17β-Estradiol Is Required for the Sexually Dimorphic Effects of Repeated Binge-Pattern Alcohol Exposure on the HPA Axis during Adolescence

Alcohol consumption during adolescence has long-term sexually dimorphic effects on anxiety behavior and mood disorders. We have previously shown that repeated binge-pattern alcohol exposure increased the expression of two critical central regulators of stress and anxiety, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), in adolescent male rats. By contrast, there was no effect of alcohol on these same genes in adolescent females. Therefore, we tested the hypothesis that 17β-estradiol (E2), the predominant sex steroid hormone in females, prevents alcohol-induced changes in CRH and AVP gene expression in the paraventricular nucleus (PVN) of the hypothalamus. To test this hypothesis, postnatal day (PND) 26 females were ovariectomized and given E2 replacement or cholesterol as a control. Next, they were given an alcohol exposure paradigm of 1) saline alone, 2) acute (single dose) or 3) a repeated binge-pattern. Our results showed that acute and repeated binge-pattern alcohol treatment increased plasma ACTH and CORT levels in both E2- and Ch-treated groups, however habituation to repeated binge-pattern alcohol exposure was evident only in E2-treated animals. Further, repeated binge-pattern alcohol exposure significantly decreased CRH and AVP mRNA in Ch-, but not E2-treated animals, which was consistent with our previous observations in gonad intact females. We further tested the effects of E2 and alcohol treatment on the activity of the wild type CRH promoter in a PVN-derived neuronal cell line. Alcohol increased CRH promoter activity in these cells and concomitant treatment with E2 completely abolished the effect. Together our data suggest that E2 regulates the reactivity of the HPA axis to a repeated stressor through modulation of the habituation response and further serves to maintain normal steady state mRNA levels of CRH and AVP in the PVN in response to a repeated alcohol stressor.