Suzanne M. Moenter, PhD - US grants

DESCRIPTION (provided by applicant): Episodic release of gonadotropin-releasing hormone (GnRH) forms the final common pathway for the central regulation of reproduction. As such, understanding the mechanisms underlying this release has direct benefits to human health by providing new strategies for fertility and contraception in humans and in important food and fiber producing species. State-of-the-are electrophysiological approaches will be used to investigate the intrinsic and network properties that underlie episodic GnRH release in four specific aims. Aim 1 will examine the ionic conductances intrinsic to individual GnRH neurons that underlie initiation of action potential generation. How these conductances are modified over time in an individual neuron and how they change between reproductive state will be determined. Aim 2 will examine the voltage- and calcium-activated conductances induced by action potential firing in GnRH neurons. Differences in these conductances between action potential spikes and following the final action potential in a burst will be determined to address the question of how conductances change to stop firing of these cells. Aim 3 will test how GnRH neurons communicate with one another. Previous work shows that these neurons can use GnRH and glutamate as transmitters. The response to GnRH is dose-dependent with low doses inhibiting and high doses stimulating GnRH neuron activity. The signaling pathways underlying these differential responses will be tested with ligands that signal through specific GnRH receptor-Gprotein combinations. Chemical synaptic and electortonic coupling between GnRH neurons will be examined with dual recordings and the site of coordination explored. The fourth aim will explore interactions between GnRH neurons the local network in which they reside. Recent data indicate GnRH neurons not only release hormone to contol the downstream anterior pituitary, but also locally to regulate their GABAergic afferents. We wll explore other local network interactions with GABA and glutamate neurons, and with the astrocytes that ensheath GnRH neurons. A possible role for astrocytes in coordinating GnRH neuron activity will be addressed. Together these studies will provide insight into both the intrinsic and network properties leading to episodic GnRH release.

DESCRIPTION (provided by applicant): Estradiol affects many central neural functions but the mechanisms are not well understood. Gonadotropin- releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. GnRH neurons are output neurons of the central nervous system and the effects of estradiol on fertility in the whole animal are well understood. By determining the mechanisms by which estradiol alters neural networks using GnRH neurons as a model, we will provide information vital for human health and designing new strategies for fertility and contraception. The work will also be relevant to estradiol action on more complex neural systems that are not as approachable for study and for which the output is not well defined, such as those governing cognition and memory formation. State-of-the-art electrophysiological approaches will be used to investigate the neurobiological mechanisms underlying estradiol feedback on GnRH neurons. The increase in estradiol that occurs during the female reproductive cycle has a biphasic effect on GnRH release. Initially estradiol inhibits GnRH release via negative feedback. With sustained exposure to elevated estradiol, however, the response to this steroid switches from negative to positive. Positive feedback induces a surge in GnRH release that is the neural prerequisite for ovulation. Using a mouse model that generates daily transitions between negative and positive feedback upon exposure to constant physiological estradiol levels, three specific aims concerning the actions of estradiol will be addressed. Comparisons will be made with data from animals in which the feedback loop was opened by castration. In Aim 1, the effects of estradiol on fast synaptic transmission to GnRH neurons will be studied, including activity of afferent neurons and responsiveness of GnRH neurons. In Aim 2, the effects of estradiol on intrinsic conductances of GnRH neurons will be explored, including whether estradiol-induced changes are due to changes in phosphorylation of ion channels. In Aim 3, the effects of three selected neuromodulators hypothesized to mediate estradiol feedback will be examined. Together these studies will provide insight into the direct and transsynaptic mechanisms utilized by estradiol to bring about negative and positive feedback regulation of GnRH release.

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway regulating reproduction. Pulsatile release of GnRH stimulates secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitary gonadotropes and is absolutely required for fertility. In female mammals, shifts in GnRH pulse frequencies help drive the preferential release of LH or FSH at specific times of the cycle to create appropriate hormone milieux for ovarian follicle maturation. GnRH pulse patterns are largely regulated by negative feedback from the ovarian steroids progesterone and estradiol. Although this feedback is well characterized in vivo, the underlying cellular mechanisms and neural pathways have yet to be elucidated. This has precluded understanding the neural components of common forms of hypothalamic infertility, such as polycystic ovarian syndrome (PCOS), in which elevated circulating androgen levels are accompanied by a persistent high frequency of LH (and presumably GnRH) release. The latter appears to be due in part to androgens interfering with the efficacy of progesterone feedback. Considerable evidence suggests one mechanism of steroid feedback regulation of GnRH release is transsynaptic. In particular, anatomical and physiological data support a role for gamma-aminobutyric acid (GABA)- and opiate peptide-producing neurons in this communication. Four Specific Aims are proposed to investigate the cellular mechanisms of progesterone feedback, and how androgens might alter the efficacy of progesterone feedback. The primary methodology will be electrophysiological recordings of green-fluorescent protein-identified GnRH neurons in acute brain slices. Aim 1 will investigate the effects of steroid and neurotransmitter milieux on the firing properties and firing patterns of GnRH neurons. Aim 2 will examine how steroids and neurotransmitters alter GABAergic drive to GnRH neurons. Aims 3 and 4 will study the effects of steroids and neurotransmitters on potassium and calcium currents, respectively, as these play major roles in setting firing properties of neurons as well as their ability to respond to synaptic input. These studies will help us understand GnRH neuron physiology in both healthy and diseased states, knowledge paramount for improving treatments for hypothalamic fertility disorders, developing novel contraceptive methods, ensuring effective reproduction in endangered and food-producing species, and understanding other similar neuronal systems.

insulin sensitivity /resistance; pathologic process

Gonadotropin-releasing hormone (GnRH) neurons are the final common pathway by which the brain controls fertility. In the common reproductive disorder polycystic ovary syndrome (PCOS), GnRH neurons are hyperactive, leading to increased frequency of luteinizing hormone (LH) release from the pituitary, driving increased levels of testosterone production. Androgens have a stimulatory role centrally, forming a positive feedback loop to increase GnRH neuron activity. Androgens also interfer with the normal negative feedback actions of progesterone, which typically reduce activity of this system. In addition to infertility, PCOS predisposes women to many metabolic disorders, including insulin resistance, central adiposity and dyslipidemia. Elevated insulin can also act to increase testosterone production, further stimulating the above positive feedback loop. In this proposal, we will examine neurobiological mechanisms underlying the effects of androgens, progesterone and metabolic cues in regulating GnRH neurons, as well as how these factors interact at the central level using state-of-the-art electrophysiological approaches combined with careful animal model development. This main thrust will be complemented by electrophysiological and limited metabolic studies of a mouse model that has many of the phenotypic characteristics of women with PCOS. In Aim 1, the effects of steroids and metabolic cues on the intrinsic properties of GnRH neurons will be studied, including how these factors interact to alter ion channel function. Aim 2 will examine the changes in GABAergic fast synaptic transmission to GnRH neurons that are brought about by steroids and metabolic cues. In Aim 3, we will continue characterization of prenatally androgenized mice as a preclinical model for PCOS. Mice treated prenatally with dihydrotestosterone exhibit disrupted estrous cycles, and elevated levels of testosterone and LH similar to women with PCOS. Preliminary data indicate these mice are glucose intolerant independent of altered adiposity. We will further characterize the metabolic phenotype and examine the causal relationships between reproductive and metabolic aspects of this disorder. Together these studies will provide novel information about the basic biology of GnRH neurons and the pathological state of PCOS that will lead to preclinical and clinical trials of new therapies

DESCRIPTION (provided by applicant): Between 15 and 20% of couples have difficulty conceiving; failures of the reproductive system thus affect many individuals. In females, understanding the control of ovulation is critical for helping those with infertility conceive singe, as opposed to multiple, births, and for developing novel methods to prevent unwanted pregnancy in manners that are consistent with the acceptable social mores of most of the population, while minimizing side effects. The goal of this proposal is to increase our understanding of the generation of the central neural signal that ultimately leads to ovulation. This signal is provided by a shift in output of gonadotropin-releasing hormone (GnRH) neurons from one that is strictly episodic, producing on/off GnRH pulses that drive pituitary hormone release, to one in which GnRH release is continuously elevated for several hours. Estradiol initiates this GnRH surge, which induces the luteinizing hormone (LH) surge that subsequently triggers ovulation. To induce the GnRH surge, central estradiol action switches from negative feedback to positive feedback. Ovariectomized (OVX) mice treated with constant physiological levels of estradiol (OVX+E) undergo daily shifts from negative to positive feedback that are timed to the light-dark cycle, allowing mechanistic studies in a reduced variable model. In ovary-intact mice, this switch in estradiol feedback mode occurs on proestrus. Previous work in the daily surge model established several mechanisms engaged by estradiol that would lead to suppression of GnRH neurons during negative feedback and activation of these cells during positive feedback. In the proposed work, these findings will be extended with experiments that range from reductionist investigation of neurobiological mechanisms to whole animal studies, all aimed at elucidating the upstream neuronal networks engaged by estradiol to regulate GnRH neurons and surge generation. In Aim 1, we will study kisspeptin neurons in the anteroventral periventricular (AVPV) region, postulated to mediate estradiol positive feedback. We will determine how their inputs and intrinsic properties change with estradiol and time of day. We will also study how estradiol feedback alters functional connectivity between kisspeptin and GnRH neurons using paired recordings in brain slices. Preliminary data indicate firing pattern, intrinsic properties and neurotransmission to AVPV kisspeptin neurons are altered both by estradiol and/or time of day. In Aim 2, we will study the mechanisms by which an acute stress disrupts the LH surge. This aim will test the neurobiological mechanisms that are disrupted by stress, and determine effector cells using genetic and surgical approaches. This aim will also expand our knowledge of mechanisms underlying the surge to the natural cycle. Preliminary data indicate a diurnal pattern to stress inhibition of surge generation, that the stress peptide corticotropin- releasing hormone inhibits GnRH neurons and that this is exacerbated by gonadal factors. Integration of the data resulting from the study of an excitatory and an inhibitory afferet network into existing knowledge will increase our understanding of the central neuronal control of ovulation by estradiol.

Abstract Reproductive health is the window to overall health and offers enormous opportunities and challenges for high- quality interdisciplinary research and careers. The goal of this renewal is to continue the efforts of the Career Training in Reproductive Biology (CTRB) Program to train exceptional predoctoral students for diverse scientific careers in the reproductive sciences. Funds are requested to train five predoctoral students per year in a dual program consisting of two main parts. First, to prepare for a research career, students will engage in rigorous, hypothesis-based scientific laboratory work in reproductive biology. Second, to prepare students for varied career paths, they will complete a University of Michigan-sponsored Certificate program or other structured career development activities to hone skills in teaching, translational research, public policy, biotechnology, entrepreneurship, biotechnology or other areas of their choosing. These career development programs were specifically designed to dovetail with graduate work to prepare students with the qualitative and quantitative skills needed for careers in specific disciplines. This innovative program addresses the needs of reproductive biology trainees in a way unlike any other program at the University of Michigan. Fourteen select faculty mentors, who are highly recognized scientists with a passion for and extensive combined experience in predoctoral education, enthusiastically comprise the CTRB Program faculty. Trainees are drawn from exceptionally strong graduate programs in biomedical sciences, biomedical engineering and environmental health sciences. Specific training activities include formal courses in Mammalian Reproductive Physiology, Scientific Communication and Responsible Conduct of Research, and statistical analyses/experimental design, participation in a mentoring workshop with their preceptor, monthly mentored trainee research presentations, group discussions of laboratory management, expanded ethics training in reproductive research, two annual symposia in reproductive sciences and twice-yearly mentoring meeting. Trainee input has and will continue to actively shape the training program. Trainees and mentors submit formal competitive applications that are reviewed and required for appointment to the CTRB Program. The CTRB Program is monitored through active engagement of faculty and trainees in program development, and by an experienced internal advisory committee. These mechanisms ensure program responsiveness to demands of a continuously evolving research environment and changing trainee needs.

Gonadotropin-releasing hormone (GnRH) neurons form the final common central pathway regulating fertility. Properly patterned GnRH release is absolutely required for fertility, but is often disrupted in women with polycystic ovary syndrome (PCOS). PCOS affects - 8% of women. In most women with PCOS, there is a persistent high frequency of LH release reflecting high frequency GnRH release. Prenatally androgenized (PNA) mice have neuroendocrine phenotypes similar to women with PCOS, including increased GnRH neuron activity, allowing mechanisms of this increase to be studied. PCOS is being detected at younger ages, suggesting the antecedents of this disorder may be developmentally programmed. The neurobiological changes underlying increased GnRH activity in this disorder and when during development changes occur are largely unknown. The working hypothesis is that GnRH neuron activity during the prepubertal period is critical for attracting appropriate afferent inputs and that alterations in GnRH neuron activity during this time period that are induced by PNA alter the adult wiring and function of the GnRH neuronal circuitry. We will test this hypothesis in two aims using state-of-the-art electrophysiology, defined animal models, and cell-targeted genetic tools to elucidate the molecular and physiologic changes that occur. Aim 1 will characterize postnatal development of the GnRH neuronal network and the effects of prenatal androgen exposure. Aim 2 will determine the role of GnRH neuron activity before puberty in establishing the adult network. Preliminary data indicate that GnRH neuron action potential firing and GnRH release are increased during the juvenile period in PNA vs. control mice. Neuronal activity during development is important for establishing appropriate synaptic interactions. Consistent with this, there is increased fast synaptic input to GnRH neurons at two weeks of age in PNA mice. We will characterize the normal development of GnRH neuron activity, release, pattern of fast synaptic input, response to inhibitory and excitatory neuromodulators, and pituitary responsiveness. We will then manipulate GnRH neuron activity in vivo during the neonatal/juvenile period by targeting inhibitory designer receptors exclusively activated by designer drugs (DREADDs) to GnRH neurons using cre-lox technology to determine the role of GnRH neuron activity in establishing these parameters. To complement the electrophysiology studies, we have adapted an innovative method for celltype- specific translating mRNA enrichment to examine the molecular underpinnings of the functional changes observed in PNA mice at the level of the GnRH neuron.

Project Summary Gonadotropin-releasing hormone (GnRH) neurons form the final common central pathway regulating fertility. Properly patterned GnRH release is required for fertility and is often disrupted in women with polycystic ovary syndrome (PCOS). Hyperandrogenic PCOS affects ~8-10% of women. In these women, there is a persistent high frequency of luteinizing hormone (LH), and likely GnRH, release. Prenatally androgenized (PNA) mice have neuroendocrine phenotypes similar to women with PCOS, including high LH pulse frequency, and can be used to study mechanisms of this increase. Pathophysiology similar to PCOS is being detected at younger ages, suggesting the antecedents of this disorder may be developmentally programmed. In the previous work from a different funding mechanism (NCTRI), we characterized the development of GnRH neuron activity and GABA transmission to these cells, showing that PNA disrupts both parameters before puberty, and that PNA- induced changes before and after puberty are different. The neurobiological mechanisms underlying these observations are largely unknown. Our working model to explain these findings is that 1) PNA alters the biophysical properties of GnRH neurons and their afferents; 2) altered epigenetic programing at least in part underlies these changes; 3) PNA increases excitatory GABA synaptic drive to GnRH neurons before puberty and this increase continues in adults; 4) before puberty in PNA mice, GnRH neurons initiate intrinsic changes to adapt to the increased GABA drive, and firing output is reduced; 5) developmental changes in PNA mice lead to failure of these GnRH neuron adaptations, so that in PNA adults, increased GABA drive contributes to increased GnRH neuron firing; 6) the increased neuroendocrine drive increases androgens, which are critical to maintain neuroendocrine PNA phenotypes in adults. We will test this model in two aims. Aim 1 will identify the mechanisms underlying prepubertal adaptation of GnRH neurons in PNA mice to increased GABA drive. Aim 2 will characterize the epigenetic landscape in GnRH neurons during development, and changes induced by PNA. The role of the ovary and androgen replacement in establishing and maintaining epigenetic changes will also be assessed. Preliminary data indicate that GnRH neuron action potential firing, calcium currents and potassium currents are all differentially regulated in prepubertal vs adult PNA mice compared to controls. To complement the electrophysiology studies, we have adapted epigenetic profiling to libraries made from a few hundred neurons and established fluorescent cell sorting protocols that yield sufficient numbers of enriched GnRH neurons for these analyses. We are thus positioned to examine the molecular and biophysical underpinnings of the functional changes of GnRH neurons observed in PNA mice. This work will provide mechanistic insight currently lacking on the typical functional development of GnRH neurons through the pubertal process, associated epigenetic changes, and how these parameters may be altered in hyperandrogenic disorders; is not possible to obtain these insights from human studies.