Robert J. Handa - US grants

Young adult female rats exposed to alcohol in utero exhibit several hormonal abnormalities. One of these is a reduced ability to secrete luteinizing hormone (LH) in response to estrogen (positive feedback), This deficit in positive feedback is similar to that previously described in the middle aged female rat immediately prior to the onset of sterility and suggest that the female rat expose to ethanol in utero may undergo premature reproductive senescence. This grant application will describe some mechanisms which may underlie the reduced positive feedback response of young adult female rats exposed to ethanol in utero. In addition, a strategy is proposed and examined in hopes of ameliorating the deficits in LH secretion in this population of fetal alcohol exposed (FAE) females. The reproductive history of the FAE female will be characterized in terms of 1) onset of puberty, 2) regularity of cyclicity and onset of persistent vaginal estrous, 3) proestrus gonadotropin secretion and gonadotropin response to estrogen and progesterone, 4) reproductive and non-reproductive behaviors. The mechanisms to be examined which might underlie reduced LH secretion in FAE females are: 1) Alterations in estrogen sensitivity as revealed by changes in estrogen receptor (ER). ER concentration and synthesis (determined by in vitro binding assay, solution hybridization and in situ hybridization. 2) Reductions in gonadotropin releasing hormone (GnRH) concentration as determined by RIA, synthesis as determined by in situ hybridization or GnRH secretion as determined using an in vitro perfusion system. Based on our existing knowledge, as therapeutic strategy involving the prepuberal administration of low doses of estrogen will be evaluated to determine its potential in ameliorating the observed reduced LH secretion of FAE females. These studies are important in determining the factors underlying the reduced reproductive potential of FAE females and to determine the efficacy of a therapeutic strategy which can be used clinically to ameliorate reproductive deficits in FAE females.

DESCRIPTION: (Adapted from the Investigator's Abstract) The reproductive neuroendocrine axis of the female undergoes well described age-related changes which result in the loss of cyclicity during middle age. We have recently shown that adult females exposed to ethanol while in utero exhibit regular estrous cyclicity following puberty but become acyclic at a much earlier age than control females. Studies in this rat model have demonstrated that fetal exposure to ethanol will disrupt the hypothalamo-pituitary-gonadal axis of the adult female. Adult fetal alcohol exposed (FAE) females exhibit a delay in the onset of puberty, reductions in the preovulatory-like surge of anterior pituitary gonadotropic hormones, reductions in anterior pituitary gonadotropin subunit mRNAs and reductions in gonadotropin-releasing hormone mRNA in a specific population of neurons in the basal forebrain. These data demonstrate that the young adult FAE female exhibits neuroendocrine impairments which can limit reproductive potential. Many of the neuroendocrine changes in young adult FAE females are similar to those described for the normal middle-aged female rat immediately prior to the onset of age-related anovulatory sterility. Thus, it appears that the FAE female is at increased risk to enter reproductive senescence at an early age. The elucidation of the consequences of fetal alcohol exposure on reproductive hormone secretion supports the possibility of a heightened risk of an early age-related loss of cyclicity in adult human female populations, not limited to those diagnosed with fetal alcohol syndrome, but including populations exposed to much more modest levels of ethanol in utero. In this proposal, we outline studies to investigate the neuroendocrine mechanisms which underlie the deficits in hypothalamo-pituitary-gonadal function and the early onset of anovulatory sterility in the adult female rat exposed to alcohol in utero. We hypothesize that hormone secretion in adult FAE females is impaired due to the decrease in hypothalamic release of gonadotropin-releasing hormone. This is a consequence of the loss of neural systems driving GnRH synthesis or secretion. The long-term goals of these studies are to define the neuroendocrine mechanisms which regulate age-related reproductive deficits in adult females especially in terms of those which are susceptible to damage by prenatal exposure to teratogenic agents such as alcohol.

9909858
Handa

This award supports the participation of American scientists in a U.S.-Japan seminar on Neuroplasticity, Development and Steroid Hormone Action, to be held in Hawaii from September 29 to October 3, 2000. The co-organizers are Professors Robert Handa at Colorado State University, Boulder and Professor Shinji Hayashi at the Tokyo Metropolitan Institute of Neuroscience in Japan. The focus of this seminar will be in elucidating the effects of steroid hormones on neuroplastic changes in the brain throughout the lifespan of the animal. The five major topics to be discussed include: 1) development and differentiation of the hypothalamus and LHRH neuronal systems; 2) steroid dependent brain differentiation; 3) central regulation of hormone secretion; 4) steroid hormones and neuroplasticity in the mature brain; and 5) steroid mediated mechanisms of cell growth and survival.

Steroid hormones are key modulators of the intercellular communications network used by the central nervous system. These simple hormones have the capacity to influence virtually all neural functions from the maintenance and organization of neurons and their connections during development to the activity and function of neurons in adulthood, to the death of neurons during aging and neuropathology. Steroid sensitive neural circuits are implicated in reproductive function, stress responses, emotion, aggression, cognition, activity, feeding and others. The Seminar organizers have made a special effort to involve younger researchers as both participants and observers. The exchange of ideas and data with Japanese experts in this field will enable U.S. participants to advance their own work, and will set the stage for future collaborative projects. It is anticipated that dissemination of proceedings of the meeting will be published in the journal "Frontiers in Neuroendocrinology. The information will also be available on the website http://lamar.colostate.edu/~bhanda/us-japan.htm.

This proposal requests funding for a joint symposium to include neuroendocrinologists from the United States and from Japan. The Symposium is entitled: "US/Japan Symposium on Neuroplasticity, Development and Steroid Hormone Action". The symposium will be held September 26-29, 2000 at the East-West Center in Honolulu, HI. The primary goal of this symposium s to bring together neurobiologists from the United States and Japan to interact and discuss their current research related to the study of steroid hormones and their action in the brain. This is often difficult given the financial and geographical constraints of travel between the United States and Japan, and thus, a site situated between both countries has been chosen. A second goal of this symposium is to foster in- depth exchange of new ideas and to encourage collaborative and cross- disciplinary interactions. This will be achieved by intentionally limiting the size of the symposium and not associating it with a meeting of a national organization. The Organizing committee has selected session topics and speakers within each session that address the most important areas in steroid hormone action in the brain and that span the lifetime of the animal, from fetal development to age-related changes. The makeup of each session has been deliberately designed to be interdisciplinary in order to foster discussion. Within each session, scientists from early, middle and late stages of their careers have been chosen to present their research. Investigators using a range of model systems and technical approaches have been included. The final goal of this symposium is to establish a conduit for future scientific exchange between the United States and Japan that will ultimately benefit the neuroendocrine communities of both countries.

This award enables young scientists to attend the Workshop on Steroid Hormones and Brain Function and give a talk in a section designed for promising new scientists, the Young Investigator Symposium. The Workshop on Steroid Hormones and Brain Function is a forum in which state-of-the-art concepts and technologies are discussed among neuroendocrinologists. The workshop has been in existence for 7 years and the National Science Foundation has funded the Young Investigator Symposium during the last 5 years. Since the number of participants is limited to approximately 100, it provides a venue for investigators to present their data in an informal atmosphere and fosters interactions among participants, especially between young and established investigators. The experience and exposure gained by the new investigators attending the meeting can facilitate the establishment of future collaborations and can broaden their scientific perspective of the field.

DESCRIPTION (applicant's abstract): The focus of this proposal is to determine the mechanisms by which estrogen and testosterone act to influence peptide containing neurons in the paraventricular nucleus of the hypothalamus (PVN) that are involved in regulating endocrine responses to stress. Females exhibit a more robust adrenocorticotropic hormone (ACTH) and corticosterone (CORT) response to stress when compared to males. This sex difference arises as a result of circulating adult hormone levels. Estrogen augments and testosterone suppresses stress related ACTH and CORT secretion. A novel form of estrogen receptor, termed beta (ER-beta), has been found in magnocellular and parvocellular neurons of the PVN, whereas the classically described alpha form is not present. ER-beta-containing neurons are uniquely distributed and may allow transduction of the estrogen signal to neuropeptide genes. In contrast androgen receptors (AR) are not found in neuroendocrine neurons of the PVN, but are found in neurons in the medial preoptic area (MPOA) and bed nucleus of the stria terminalis (BnST) that project to the PVN. This proposal describes studies to test the hypothesis that estrogen acts directly upon corticotropin releasing hormone (CRH), vasopressin (AVP) or oxytocin (OXY) neurons in the PVN, whereas androgen acts indirectly by modulating GABAergic neurons in the MPOA and BnST that project to the PVN. 5 specific aims are proposed: 1) to determine if the local application of estrogen or testosterone to the PVN alters stress responsive hormone secretion, neuropeptide mRNA, protein content and heteronuclear RNA levels in the PVN. 2) To determine if androgen receptor or estrogen receptor act upon nucleotide sequences in the CRH, AVP or OXY gene promoters. 3) To determine if AR positive, GABA positive neurons in the BnST project to the PVN. 4) To determine if estrogen or androgen modulate glucocorticoid receptor mediated negative feedback regulation of neuropeptide genes in the PVN. 5) To determine if ER-beta or AR interact with glucocorticoid receptor to regulate transcription of reporter genes downstream of the CRH, AVP or OXY gene promoters. Since a dysregulation of stress-responsive hormone secretion is a characteristic of affective disorders, and these are more prevalent in females than males, the long-term goals of this project are to define the role played by estrogen and androgen in the pathology of affective disorders.

The hippocampus, a part of the mammalian brain involved in cognitive functions, is also sensitive to the steroid hormone estrogen. Estrogen is known to influence the sexual differentiation of the hippocampus, but the molecular mechanisms for this development are not clear. Numbers of estrogen receptor (ER) molecules in the hippocampus increase during development, triggered by transient expression of both the alpha and beta forms of ER, which suggests a possible novel critical period for estrogen action. The transient expression of ER-beta is predominantly of an isoform splice variant termed -delta 3 (-d3), which acts through a unique molecular mechanism to regulate target genes, unlike the classically described estrogen-response element (ERE). This project uses molecular biology to examine the function in vitro of these -d3 splice variants of ER-beta. Transient transfection, reporter gene analysis, and other transcription and trafficking assays will determine how -d3 variants drive transcription and interact with other regulatory proteins and other ER receptor types. The project also bridges the effects seen in vitro with those normal physiological events seen in the living animal.
Results will clarify a novel mechanism of how estrogens affect hippocampal development, and will be important beyond neuroendocrinology, on developmental biology and potentially on cognitive neuroscience. The project also involves training undergraduates and graduate students in an excellent laboratory research environment.

Prenatal exposure to alcohol has profound effects on the developing nervous system. This can result in neurobehavioral pathology without growth deficits, mental retardation or the dysmorphological characteristics associated with Fetal Alcohol Syndrome (FAS). The effects of fetal alcohol exposure (FAE) are of concern to national health because the FAE population is so much larger that that of FAS. In contrast to FAS, deficits in FAE children can also include changes associated with autonomic and neuroendocrine function. Developmental damage to the suprachiasmatic nucleus (SCN) of the hypothalamus may be a common thread linking many FAE associated neuropathologies. The SCN acts to entrain multiple biological rhythms which normally fluctuate across the 24 hour day. When entrainment is weak or slow to respond to normal entrainment cues such as light, behavioral and physiological disturbances can occur. Preliminary data indicate that fetal alcohol exposure alters adult circadian rhythms in core body temperature and plasma corticosterone. This is accompanied by changes in vasopressin and Per2 expression within the SCN. Vasopressin serves a key effector function of the SCN to modulate amplitude and sensitivity of systems related to the control of rhythms. Per2 is an intrinsic clock gene that is expressed in a circadian fashion within the SCN. Therefore, the experiments described in this application will test the hypothesis that prenatal alcohol exposure disrupts hypothalamic development resulting in a dysregulation of the neural mechanisms involved in the entrainment of autonomic and neuroendocrine rhythms. This application will determine: 1) If FAE causes circadian rhythm dysfunction in adult male and female rats due to an insensitivity of the SCN to photic entrainment. 2) If FAE disrupts the function of local circuits within the SCN that regulate diurnal rhythms of neuropeptides and intrinsic clock genes. 3) If FAE disrupts the functional outputs of the SCN to the paraventricular nucleus of the hypothalamus, a brain region that regulates autonomic and neuroendocrine function. The long term goals of these studies are to establish the extent of rhythm dysregulation caused by FAE and the mechanisms underlying such a dysregulation. This will lay the foundation for future neurodevelopmental studies which will help determine intervention strategies useful in counteracting the effects of FAE on SCN function

DESCRIPTION (provided by applicant): This project will define the mechanism by which equol, an isoflavone metabolite, acts to inhibit normal and pathological growth of the prostate gland. In the United States, prostate cancer is the most frequently diagnosed cancer in men, and ranks second among cancer-related male deaths. The development of prostate cancer involves a complex interplay of numerous factors, however lifetime exposure to androgens, and advancing age, are two necessary etiological factors. Similarly, the development of benign prostatic hypertrophy (BPH) is also dependent upon androgen exposure and it is estimated that over half of the male population in the United States over the age of 60 have symptoms of BPH. The importance of androgen in prostate gland pathology is evidenced by current therapies for these diseases, which include reduction in circulating testosterone;inhibition of 5-alpha reductase, an enzyme that synthesizes the potent androgen, dihydrotestosterone;or blocking the actions of the androgen receptor. Diet is an important consideration when examining risk factors for hormone-dependent diseases such as prostate cancer. Asian men, on an eastern diet, have the lowest incidence of prostate cancer in the world. The basis for this correlation may be the presence of estrogen-like isoflavones (genistein and daidzein), in their diet. We have recently shown that equol, a product of isoflavone metabolism, has anti- androgenic properties as well. Equol selectively binds dihydrotestostserone and prevents androgen receptor activation to reduce androgen-dependent prostate growth. Equol is the principal metabolite of daidzein. It circulates at high levels, and is concentrated in the prostate. Moreover, equol can selectively bind estrogen receptor beta to activate an anti-proliferative cascade in prostate. Thus, equol possesses a unique dual action to inhibit prostate growth by 1) preventing proliferative actions of androgens, and 2) activating anti-proliferative actions of ERbeta. However, only about 30% of all humans have the correct intestinal flora to produce equol. In these studies, we will test the hypothesis that pathological growth of the prostate gland can be prevented or delayed by dietary equol. We will also determine the cellular mechanisms whereby equol provides such protection. These studies utilize the TRAMP mouse line where spontaneous prostate tumors develop in adult males making this particularly useful for studying the chemopreventive actions of equol. In addition, we will utilize an orthotopic injection model whereby human prostate carcinoma lines that express a luciferase reporter gene are injected into host mice and monitored by in vivo imaging techniques to determine the effects of equol, on growth of prostate tumors. Lastly, we will determine if equol can act by altering the polyamine biosynthetic pathway, thereby inhibiting cell cycle and tumor growth. PUBLIC HEALTH RELEVANCE: Prostate cancer is the most frequently diagnosed cancer in men, and ranks second among cancer-related male deaths in the United States. In this application, we will test the hypothesis that pathological growth of the prostate gland can be prevented or delayed by dietary administration of equol, a product of isoflavone metabolism that has anti-androgenic and estrogen receptor beta activating properties. The long-term goal of this project is to define the mechanism by which equol acts to inhibit the pathological growth of the prostate gland

OISE-0737946 (Handa, University of Arizona)
U.S.-Japan Symposium: Steroid Hormone Receptors and Neural Sex Differences?

Abstract

This award supports an international workshop entitled: ?Steroid Hormone Receptors and Neural Sex Differences? to be held in Gifu, Japan in September, 2008. The workshop will bring together speakers from the United States and Japan. Drs. Nobuhara Harada (Fujita Health University) and Mitsuhiro Kawata (Kyoto Prefectural Univ.) are the primary organizers from the Japan side. Given the overwhelming recent advances in the field of neuroscience and steroid hormone biology, the goal of this workshop is to reinvigorate collaborations and provide new linkages between neuroscientists in the United States and Japan. The workshop will be held over a 3 ½ day period with subsequent visits for US scientists to laboratory facilities in the Nagoya, Japan region. The workshop will cover the following topics: (1) Sex steroid hormone action in the brain: transcriptional regulation and cellular functions; (2) Alternative mechanisms of steroid hormone action in brain: rapid membrane and ligand-independent responses; (3) Role of G-protein coupled receptors and their signaling partners in the control of reproduction; (4) Regulation of GnRH neurons and central control of steroid hormone secretion; (5) Developmental effects of steroid hormones: sexual differentiation of the brain; and (6) Steroid hormones and adult sexually differentiated function.

The award also supports the travel of young investigators (postdoctoral and graduate student level) to present their recent research findings in workshop panels and poster sessions, thereby allowing them to interact with new and established investigators in this growing field.

DESCRIPTION (provided by applicant): The long-term goals of this project are to determine the neurobiological mechanisms that underlie the sex differences in the function of the adult hypothalamo-pituitary-adrenal (HPA) axis. In humans and animals, sex differences in HPA reactivity are well established;females exhibit a more robust activation of the HPA axis following stress than do males. Our hypothesis is that sex differences in adult hormonal stress responses are primarily the result of the opposing actions of testosterone (T) and estrogen (E) on HPA function. Activation of the HPA axis is a basic response of animals to environmental perturbations that threaten homeostasis. These responses are regulated by neurons residing in the paraventricular nucleus of the hypothalamus (PVN) that synthesize and secrete corticotropin-releasing hormone (CRH). Other PVN neuropeptides, such as vasopressin (AVP) and oxytocin (OT), modulate activity of CRH neurons at the level of the PVN as well as enhancing CRH secretogogue activity at the anterior pituitary gland. The reproductive steroids, E and T can also modulate stress responses. Circulating E enhances stress activated ACTH and CORT secretion. In contrast, T decreases the gain of the HPA axis. Published and preliminary data show that androgens can act directly on PVN neurons in the male rat through a novel pathway that involves estrogen receptor beta (ERbeta), whereas E acts predominantly through ERalpha. Thus, we hypothesize that in males, T suppresses HPA function by androgen metabolites that bind ERbeta. Clues to the neurobiological mechanisms underlying such a novel action can be gleaned from studies showing extensive colocalization of ERbeta in OT-containing cells of the PVN. Hence, in this application we address the possibility that testosterone inhibits HPA reactivity by metabolizing to a compound that binds ERbeta and regulates OT containing neurons of the PVN. Furthermore, we hypothesize that this action is distinct from that found in females where estradiol is the ligand acting through ERalpha. Five specific aims are proposed. Aim 1 will identify the location and regulation of steroid metabolizing enzymes in neurons of the PVN that allow synthesis of 32-Diol. Aim 2 will use mouse models to test the hypothesis that locally synthesized 32-Diol acts upon ERbeta neurons in the PVN to inhibit HPA reactivity. Aim 3 will determine if ERbeta containing neurons regulate HPA axis function through local release of OT or extra-PVN oxytocinergic connections with limbic nuclei. Aim 4 will test the hypothesis that 32 Diol and ERbeta functionally interact with the oxytocin promoter in a ligand dependent fashion. Aim 5 will test the hypothesis that the nature of the ligand bound to ERbeta determines the assembly of co-regulatory factors recruited to target sites on the OT promoter. PUBLIC HEALTH RELEVANCE: Sex differences exist in the hormonal response of animals to physical and psychological stressors. These sex differences arise as a result of the actions of estrogen and androgen acting upon neural circuits within the hypothalamus. In this application we will test the hypothesis that the actions of testosterone act to inhibit neuroendocrine responses to stress through metabolism to 32-Diol and subsequent binding to estrogen receptor beta found in oxytocin neurons of the hypothalamus.

The steroid estrogen regulates many functions of the brain including behavior and cognition, while the prefrontal cortex (PFC) is a brain region that controls aspects of cognitive function and emotional behavior. Not surprisingly the PFC is estrogen sensitive, but there is currently little known about how it responds to estrogen. The goal of this research is therefore to determine how estrogen regulates the PFC. The research tests the prediction that of the two main types of estrogen receptors, alpha and beta, that the PFC is regulated by beta (ERb). Successful completion of these studies will provide novel information about an important but unexplored neuronal phenotype in the PFC. Innovative approaches are used to examine ERb neurons and the results of these studies can impact multiple research arenas. This research project will be used for the training of high school, undergraduate and graduate students, particularly underrepresented minorities, in neuroscience. Results will be disseminated through peer-reviewed publication and by student presentations at local and national meetings.

These studies explore the function of a select group of cells in the mouse PFC that express ERb. The PFC controls many behaviors including cognition, attention, and neuroendocrine stress responses. Estrogen can alter the development of this brain region, yet, little is known of the neurons in the PFC that express ERb, partly due to the absence validated reagents allowing the detection of ERb in brain. The proposed studies will examine this group of neurons in the PFC using a novel transgenic mouse that express a fluorescent protein in ERb neurons. Preliminary results show that the location of ERb expressing neurons changes across development of the PFC. Studies in this application will address the hypothesis that these changes reflect developmental alterations in estrogen sensitivity in different cortical layers across time. Using anatomical approaches, these studies will determine the identity of the cortical neurons that express ERb. Next, molecular approaches will be used to address the reason why estrogen receptor levels change in amount and location during cortical development and lastly, the function of identified ERb neurons in the PFC will be examined using electrophysiology

? DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the neurobiological mechanisms that underlie effects of estrogen on the adult hypothalamo-pituitary-adrenal (HPA) axis. HPA axis activation in mammals is a basic response to environmental perturbations that threaten homeostasis and such responses, although beneficial in the short-term, have deleterious consequences under chronic conditions. Prolonged elevations of adrenal glucocorticoids (GCs) are neuroendangering and alter behaviors. Moreover, a dysregulation of the HPA activity accompanies neuropsychiatric disorders. In rodents, females show a more robust HPA axis response to stress than do males, partly because of sex-differences in circulating estradiol (E2) levels. Thus, the overarching postulate of this application is that individual differences in adult stress-responses arise from differential E actions on the stress-circuitry. Our studies focus predominantly on estrogen receptor beta (ER?). Rodent studies show that the alpha form of ER (ER?) increases adrenal corticosterone (CORT) and pituitary adrenocorticotropic hormone (ACTH) response to stressors whereas activation of ER? inhibits HPA activity. Importantly, ER? is highly expressed in neurons of the PVN of both male and female mice, allows integration of gonadal hormone levels with stress-related inputs. Using novel transgenic mouse models, we will identify stress responsive ER?-ergic neural circuitry of the mouse hypothalamus, and determine if activation of PVN ER? reduces HPA drive through OTergic pathways. Specific aim 1 will assess populations of ER? and ER? neurons that are incorporated into the stress circuitry of the mouse brain. Aim 2 will test the hypothesis that activation of ER? in male and female PVN neurons inhibits HPA axis function through 1) synaptic connections between ER? and CRH neurons or 2) through reciprocal projections to the Bed n. of the Stria Terminalis (BST) or medial Amygdala (mAmg). Aim 3 will elucidate molecular changes and sex differences that occur in PVN ER? neurons in response to stress or ER (? / ?) agonist treatment. Aim 4 will directly test whether OT is requird for ER? regulation of PVN function. The results of these studies will provide novel insight into the role played by PVN ER? neurons in controlling hypophysiotrophic function with hopes of identifying novel targets for therapeutic approaches to treating stress and associated neurological deficits.

ABSTRACT: The long-term goal of this project is to determine the neurobiological mechanisms that underlie the effects of estrogens on the adult hypothalamo-pituitary-adrenal (HPA) axis. HPA axis activation in mammals is a basic response to environmental perturbations that threaten homeostasis and such responses, although beneficial in the short-term, have deleterious consequences under chronic conditions. Prolonged elevations of adrenal glucocorticoids (GCs) are neuroendangering and alter feeding and autonomic functions. Moreover, a dysregulation of the HPA activity accompanies these disorders. In rodents, females show a more robust HPA axis response to stress than do males, partly because of sex-differences in circulating estradiol (E2) levels. Thus, the overarching postulate of this application is that individual differences in adult stress-responses arise from differential E2 actions on the stress-circuitry. Our studies focus predominantly on estrogen receptor beta (ER?). Rodent studies show that the alpha form of ER (ER?) increases adrenal corticosterone (CORT) and the pituitary adrenocorticotropic hormone (ACTH) response to stressors whereas activation of ER? inhibits HPA activity. Importantly, ER? is highly expressed in neurons of the PVN of both male and female mice to allow integration of gonadal hormone levels with stress-related inputs. Using novel transgenic mouse models, we will identify stress responsive ER?-ergic neural circuitry of the mouse hypothalamus and determine how activation of PVN ER? reduces HPA drive and energy balance. Specific aim 1 will determine if OT is required for ER? regulation of PVN function using a novel Oxytocin:cre recombinase mouse line and an ER?-cre mouse line to genetically manipulate OT and ER? neurons. Aim 2 will assess the function of ER? neurons that are incorporated into the stress circuitry of the mouse brain following multimodal stress and chronic unpredictable mild stressors. Aim 3 will elucidate molecular changes and sex differences that occur in PVN ER? neurons in response to MMS and to glucocorticoids. In all cases we are highly cognizant of the presence of sex differences in these physiological pathways and will explore the role that estradiol or 5?-androstan 3?,17? diol (3? diol), a metabolite of the androgen, dihydrotestosterone that binds and activates ER?, have on HPA axis activation and feeding behaviors. The results of these studies will provide novel insight into the role played by PVN ER? neurons in controlling hypophysiotrophic function and metabolism with hopes of identifying novel targets for therapeutic approaches to treating stress and associated neurological deficits.

PROJECT SUMMARY / ABSTRACT The overarching goal, and ultimate impact, of this project is to identify the cellular and physiological pathways whereby developmental overexposure to glucocorticoids cause long-term, sex-selective programming of adult anxiety- and depressive-like behaviors, neuroendocrine function and autonomic nervous system responses to stress. Developmental programming, the permanent adaptation of the fetus to maternal environmental signals can elevate fetal glucocorticoids to program neurodevelopment. Preliminary data in mice and rats show sex- biased changes following late-gestation exposure to the synthetic glucocorticoid, dexamethasone. These include increased anxiety- and depressive-like behaviors, and hyperreactive neuroendocrine and autonomic nervous system responses to stress and changes in gene expression in the hypothalamus. In all cases, adult females showing a greater change than males. We hypothesize that these adult dysfunctions have a common mechanism related to programmed changes in the hypothalamic paraventricular n. (PVN), a brain region that has been shown to influence all of these physiological endpoints. Our studies show similar responses in both rats and mice allowing these studies to exploit the well-described physiology of the rat as well as the power of mouse genetic approaches. 3 specific aims are described. Aim 1 will determine the causal factors for long- term alterations in anxiety-and depressive-like behaviors, neuroendocrine response to stress and an imbalance of the autonomic nervous system. Moreover, this aim will examine the central renin-angiotensin system as a central mediator of the observed changes in brain function following prenatal glucocorticoid overexposure. Aim 2 will determine the impact of prenatal glucocorticoid exposure on connectivity between hypothalamic preautonomic and brainstem autonomic nuclei and use mouse genetic approaches and viral vectors to specifically activate neuron populations in the PVN. Aim 3 will use transcutaneous vagal nerve stimulation to modulate autonomic nervous system balance and reverse the effects of prenatal glucocorticoid exposure on adult behaviors, neuroendocrine, and autonomic responses to stress. The results of these studies will identify, not only the brain circuitry underlying the sex-biased developmental programming of behavioral, endocrine and autonomic responses in adulthood, but also reveal potential therapeutic mechanisms whereby these changes can be reversed in adulthood.

OVERALL SUMMARY. Major depressive disorder (MDD) topped heart disease as the number one cause of disability worldwide, and women have twice the risk of men. MDD is associated with abnormalities in the stress response circuitry, areas that are among the most sexually dimorphic in the brain. These areas are dense in sex steroid and glucocorticoid receptors coupled with cytokine receptors. Further, activity in these areas has been associated with cortisol response, autonomic dysfunction, and immune responses, which we showed differed by sex. This is important since autonomic dysregulation is significantly associated with cardiovascular disease. In fact, women are at twice the risk for the co-occurrence of MDD, autonomic dysregulation and heart disease, leading to a 3-5-fold risk of death in women from heart disease, often with unrecognized and untreated MDD. Thus, understanding early biomarkers for sex differences in MDD and autonomic dysregulation will provide knowledge for early intervention, attenuating later life disability, in particular for women who are at higher risk. The scientific mission of this SCORE is to identify stress-immune pathway abnormalities, beginning in fetal development, that have shared consequences for sex differences in brain circuitry regulating mood and lifelong recurrent MDD and dysregulation of hormone and immune responses to stress, and autonomic and neurovascular dysfunction in early midlife. We aim to facilitate transdisciplinary, translational collaboration among basic and clinical investigators to enhance our understanding of the impact of sex on MDD and central and peripheral autonomic function and provide the groundwork for translating this knowledge into sex-selective therapeutics. Further, we aim to serve as an interdisciplinary resource to train and disseminate findings about sex differences in MDD and autonomic dysregulation to the scientific and medical communities, policy makers, and the public. To accomplish this, three integrated studies are proposed: 1) a clinical population neuroscience study relating prenatal risk biomarkers to sex differences in brain circuitry and physiologic deficits in response to stress in MDD in early midlife; 2) clinical study using direct transcutaneous neuromodulatory stimulation of the vagus nerve, auricular branch (or taVNS) to target the circuitry associated with stress-immune function and map its neuroanatomic, physiologic and clinical effects in MDD by sex, in the same subjects as in project 1; and 3) rodent model studies that will map out the central mechanistic pathways involved in projects 1 and 2. In addition, three cores will contribute to the success of this SCORE: 1) Leadership Administration Core to administer and oversee the administrative integration of the studies and cores; 2) Resources Core to provide shared technical expertise across studies; and 3) Career Enhancement Core, to supplement the training of junior faculty and others on the topic of our SCORE, and become pedagogical ambassadors to the scientific, medical and public communities about sex differences in depression and comorbidities with general medicine, a topic with global public health implications.