Phyllis M. Wise - US grants

pituitary gonadal axis; neuroendocrine system; neurochemistry; aging; median eminence; prolactin; follicle stimulating hormone; luteinizing hormone; estradiol; hormone inhibitor; estrus; radiotracer; high performance liquid chromatography; blood chemistry; histochemistry /cytochemistry; radioimmunoassay; gel filtration chromatography;

The proposed studies will examine the role of brain monoaminergic and LHRH neurons, estradiol and prolactin in the age-related transition to irregular estrous cyclicity and eventual acyclicity. Experiments, using 6-17 mo. old rats as an experimental model, have been designed to determine which of the following working hypotheses is (are) the most likely explanation(s) for the onset of irregular cycles: (1) Phasic LH release (preovulatory and steroid-induced) is accompanied and presumably regulated by changes in norepinephrine (NE), dopamine (DA) and serotonin (5HT) activity. Aging may affect the pattern of neurotransmitter dynamics to inhibit LH release. (2) LH surges are regulated, in part, by the amount of LHRH released into pituitary portal blood and this release is accompanied by changes in tissue concentrations of LHRH in specific hypothalamic structures known to contain cell bodies, axons and terminal boutons of the neurosecretory neurons. Aging may affect the levels in one or more discrete hypothalamic areas, its releasability and/or concentrations in portal blood. (3) Estradiol action at the brain and pituitary are necessary for LH surges to occur. Aging may alter the responsiveness of these sites to estradiol positive feedback such that steroids become incapable of facilitating LH surges. (4) Hyperprolactinemia is often associated with acyclicity in young females. Prolactin may become elevated gradually in aging rats and feedback to alter the cyclic profile in monoamine-LHRH dynamics and cyclic LH release. These hypotheses will be tested using several methodological advances. Neuronal activity will be assessed by 4 indices: (1) turnover rates of NE, DA and 5HT and LHRH levels and (2) measurement of monoamine metabolites in microdissected brain structures thought to be important in neural control of LH and likely sites of synaptic and neurohumoral contact between NE, DA and 5HT and LHRH containing neurons, (3) measurement of LHRH and DA in portal blood and (4) use of median eminence synaptosome preparations to study compounds which modulate LHRH and DA release. Receptor assays for estradiol, progesterone and DA allow examination of changes in receptor binding in separate areas of the brain and pituitary to evaluate whether changes in responsiveness to these hormones can be correlated with changes in receptor biochemistry. These parameters will be correlated with the onset of irregular cycles in middle aged rats to evaluate the role that each plays in this age-related transition.

The proposed studies will test the hypothesis that high circulating prolactin (PRL) levels inhibit cyclic LH surges by an action on the brain. High PRL may inhibit LH surges by any of the following mechanisms. 1. Hyperprolactinemia could abolish the specific neurotransmitter signal(s) required for cyclic LH release. We will examine the pattern of turnover of changes in norepinephrine, dopamine and serotonin in discrete brain regions before and during LH surges. 2. Hyperprolactinemia could disrupt the rhythm of the "biological clock" upon which all cyclic physiological functions, including LH surges, depend. We will examine the circadian rhythms in serotonin turnover, and Alpha- and Beta-adrenergic serotonergic receptors, particularly in the suprachiasmatic nucleus, because these are indices of the biological clock which may directly or indirectly affect LH release. 3. Hyperprolactinemia could affect LHRH-containing neurons to alter the amount and/or the kinetics of LHRH release. We will study LHRH release kinetics from synaptosomes prepared from the median eminence or from medial basal hypothalamic slices. The following methods will be used. A microdissection technique allows for the removal of discrete nuclei, fiber pathways and terminal projection fields from rat brain. We will estimate norepinephrine and dopamine turnover by measuring neurotransmitter concentrations before and after synthesis inhibition. Serotonin turnover will be estimated by assessment of 5-hydroxyindoleacetic acid/serotonin ratios and 5HT accumulation after inhibition of metabolism. High pressure liquid chromatography with electrochemical detection allows for quantitation of all of these compounds within samples from individual rats. Neurotransmitter receptors will be localized and quantitated using topical quantitative autoradiography. The ability of depolarizing stimuli to evoke LHRH release from median eminence synaptosomes or medial basal hypothalamic slices will be examined. The animal model to be used is the estadiol-treated ovariectomized rat. Our previous work demonstrates that elevated PRL can abolish cyclic LH release depending upon the estradiol treatment paradigm. This animal model allows us to investigate the direct neural effects of hyperprolactinemia on cyclic LH release in the absence of confounding ovarian effects.

The longterm objective of this research is to evaluate the neuroendocrine mechanisms by which hyperprolactinemia (hyperPRL) suppresses female reproductive function. The specific aims of this proposal are to test the working hypothesis that hyperPRL suppresses pulsatile and cyclic LH release by modulating opiatergic neuronal activity. We will assess whether this, in turn, influences the activity of catecholamine- containing neurons resulting in an altered pattern of GnRH release or whether opiates directly affect GnRH neurons. Furthermore, we will test two corollary hypotheses: (1) when hyperPRL does not affect LH, it is because the opiatergic/catecholamine/GnRH link is not functional in that endocrine milieu; and (b) if we restore the opiatergic/catecholamine/GnRH pattern, we can restore normal LH secretion, despite the presence of hyperPRL. Finally, we will test the hypothesis that hyperPRL compromises pituitary responsiveness. To test these hypotheses, we will (1) differentiate the circumstances under which hyperPRL does and does not suppress LH release; (2) examine how hyperPRL alters opioid neuronal tone; (3) assess whether hyperPRL- induced changes in opiate and catecholamine activity determine the efficacy of hyperPRL to suppress LH; and (4) determine whether hyperPRL decreases pituitary responsiveness to GnRH. We will assess opiate tone using 4 indices of activity: quantitation of opiate peptide concentrations in microdissected brain regions, monitoring of patterns of opiate peptide release into microdialysis probes placed in critical brain regions, measurement of opiate mRNA expression assessed using in situ hybridization, and autoradiographic quantitation of opiate receptor densities in key terminal areas. Three indices of catecholamine activity will be autoradiographic quantitation of adrenergic receptor densities. Patterns of GnRH release will be measured at the pituitary gland using dialysis methodology. Pituitary responsiveness will be measured in vivo and in vitro using the reverse hemolytic plaque assay in combination with in situ hybridization. Using this multifaceted approach, we should deepen the understanding of the neuroendocrine factors that regulate the pulsatile and cyclic release of LH and how hyperPRL perturbs this balance resulting in a different pattern of LH secretion.

The objectives of this research are to better understand the neural mechanisms that regulate the age-related deterioration of the female reproductive system. Cyclic reproductive function in rodents depends upon the integrity of the "biological clock" and normal circadian rhythms. The rhythmic occurrence of several neurochemical events regulates the release of LHRH and gonadotropins. Experiments have been designed to test the hypothesis that the ability of the biological clock to maintain the circadian rhythmicity of several neurochemical and neuroendocrine events, which are critical to cyclic LH release, deteriorates during middle-age. This causes the gradual disappearance of regular estrous cycles and the transition to age-related infertility. The significance of the working hypothesis is that the experimental results will be the first evidence to document that changes in the biological clock, or its ability to entrain rhythms, occur early during the aging process. They will be the first data to suggest that changes in the biological clock may cause, and not merely be the result of age-related deterioration. The specific aims of this proposal are to answer the following questions: 1. Do norepinephrine and serotonin activities exhibit circadian rhythms? 2. Do the receptors which bind to norepinephrine (alpha1-, alpha2-, Beta1- and Beta2-adrenergic) and serotonin (5HT1 and 5HT2) exhibit circadian rhythms? 3. Do pharmacological manipulations which alter neurotransmitter activity rhythms to mimic those of middle-aged rats cause aging-like LH surges? The following methods will be used: (1) A microdissection technique will discriminate nuclei, fiber pathways and terminal projection fields from the rat brain. (2) Norepinephrine and serotonin turnover rates will be determined using high pressure liquid chromatograhic methods. (3) Neurotransmitter receptors will be localized and quantitated using topical quantitative autoradiography. Thus, we will monitor the rhythmicity of two neurotransmitters and their receptors under controlled endocrine conditions to determine the relationship between these and the endocrine milieu and the time of day. We will compare these rhythms with those in aging rats under similar conditions and correlate age-related changes with the disappearance of regular LH surges.

DESCRIPTION: (Applicant's abstract) Many physiological processes and behaviors in virtually all eukaryotes exhibit endogenous circadian rhythmicity. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus drives these rhythms and serves as the critical neural pacemaker. During aging, many rhythms exhibit shorter periods, phase advances, attenuated amplitudes and temporal desynchronization and fragmentation. In elderly humans, this results in fragmentation of sleep-activity rhythms and desynchronization of endocrine rhythms which may lead to poor health. The long-term objectives of this proposal are to understand (1) the critical cellular and molecular mechanisms that translate the oscillations of individual neurochemicals and cells into the coordinated, synchronized and precise rhythms characteristic of the SCN and (2) how changes in these mechanisms influence declining pacemaker function during aging. Vasoactive intestinal peptide (VIP) and somatostatin (SRIF) may play key roles as communicators of differential afferent input, broadcasters of efferent messages of circadian time or modulators of entrainment. Therefore, the applicant will use cellular and molecular methods to address the following specific aims: (1) characterize the temporal pattern of VIP and SRIF (a) secretion and cellular peptide expression, and (b) levels of mRNA and primary transcript in organotypic cultures containing the SCN. (2) assess the ability of 5HT to phase shift the rhythms of VIP and SRIF secretion in organotypic slices and compare this to its ability to phase shift in dispersed cell cultures. (3) mimic the effects of age on VIP and SRIF by targeted suppression of VIP and/or SRIF via antisense oligonucleotides administered to organotypic slices or dispersed cell cultures and determine the effect on the pattern of expression of the other peptide. The applicant will assess whether 5HT can still phase shift the rhythms of these peptides when VIP or SRIF is suppressed. The applicant states that forthcoming information will deepen our understanding of the cellular and molecular mechanisms that underlie rhythmic activity of the SCN and how changes in these mechanisms may lead to aging-like desynchronization of circadian function. Together with the IRPGs submitted by Drs. Duncan and McMahon, they will focus on this area using a broad array of methods and approaches that is not possible with any single investigator.

The goal of this Training Program is to prepare promising graduate students and postdoctoral fellows for successful careers in the neurobiology of aging. We will provide broad-based training in modern concepts in the neurobiology of aging with specific emphasis on changing cellular and molecular interactions that occur in the aging brain. The unifying focus of the training faculty is our interest in understanding the mechanisms by which the nervous system responds to changes that occur with age under normal and pathological conditions. This Training Program provides the formal framework for faculty in different departments, who share a common interest in the molecular and cellular basis of brain aging, to provide in depth training in aging. The emphasis of the Training Faculty complement each other: some focus on processes that occur in the brain during normal aging, others emphasize the neuropathological diseases that predominate in the aging brain, and still others use in vivo and in vitro systems to model the aging brain. Thus, trainees will learn from faculty who utilize a broad spectrum of state-of-the-art methodological approaches to probe critical questions in aging research. During the previous granting period, our record of recruiting promising pre- and post-doctoral fellows, providing state-of- the-art training, and placing them in excellent positions is outstanding. Our success attests to our ability to identify, attract, train and place promising young investigators in the area of aging is clear due to our changing demography and the increasing average lifespan. Normal aging or pathology of the nervous system account for a large percent of deaths in the elderly. Only if we better understand the basic mechanisms that regulate the aging of the brain and apply this to the treatment of the elderly, can we hope to improve the quality of life during the latter half of the lifespan. Our ability to attract and train graduate students and postdoctoral fellows to meet the challenges in the area of neurobiology of aging will be greatly enhanced by this Training Program.

The University of Kentucky and the Sanders-Brown Center on Aging are recognized for their research on the causes and repercussions of Alzheimer's Disease. I will use the Academic Leadership Award to expand this focus of excellence to encompass a broader spectrum in the biomedical sciences. The program objectives are to: enhance and coordinate research, training and curriculum development in the normal aging of the brain and nervous system, the biology of the menopause and the aging of the cardiovascular system. I will focus on these three specific areas of biomedical science since new knowledge in these areas is essential if we are to improve the quality of life of the elderly. In addition, we have the greatest opportunity to markedly improve research and training in these areas because of the coming together of several coincident circumstances. This award will serve as a catalyst to achieve 3 major goals: (1) enhance research efforts in the aging of the brain and nervous system, the biology of the menopause and the aging of the cardiovascular biology. (2) improve the quality of training in gerontology by accelerating the development of innovative courses in the doctoral program in Gerontology focused on biomedical gerontology and providing training opportunities to clinical fellows. (3) integrate the foci of research and amplify the impact of enhanced research and education initiatives. This award will greatly increase the research potential and academic capacity for the study of aging at the University of Kentucky. I have received commitments from several administrative offices to match some of the funds dedicated to specific goals. Therefore, an Academic Leadership Award will allow me to leverage internal resources and thereby amplify the impact of this funding. I anticipate that this Award will enable the University of Kentucky to be a truly national model for comprehensive and integrated centers of excellence in biomedical research and training in aging.

The objectives of the Center of Biomedical Research Excellence in Women's Health (COBREWH) are to deepen our understanding of the unique role of gender, female reproductive hormones, and selective estrogen modulators (SERMs) in the manifestation of health and disease and use this focus as a platform to develop promising junior investigators and enhance their success at competing for NIH grant support. We will achieve these objectives by (1) assembling a cadre of established NIH- funded scientists to examine timely and important questions related to gender, female reproductive hormones and hormone action, (2) mentoring junior faculty with state-of-the-art multi-disciplinary training in concepts relevant to women's health, (3) mentoring junior faculty in research projects focused on women's health that will insure that they successfully establish independent NIH-funded research careers, (4) developing and supporting shared core facilities to optimize use by COBREWH faculty in cutting edge methods, (5) facilitating pilot projects and collaborations in complementary research areas that are essential to improving women's health, and (6) strategically hiring promising new faculty to develop a critical mass of funded, productive and well-respected investigators in women's health research. A critical challenge for the COBREWH is to provide junior investigators with focused in-depth research experiences that enhance their chances to obtain funding in a highly competitive funding environment, while providing the inter-disciplinary framework within which they can grow and thrive over the long-term. To meet this challenge we have divided the research in this COBREWH into 3 broad overlapping themes which will umbrella 5 research projects that interact with each other in terms of concepts and methods. These research projects are included in the COBREWH because they attack critical questions in women's health and because the University of Kentucky is fortunate to have senior, well-funded faculty with extensive experience in mentoring junior investigators in each of these areas. Thus, junior investigators will learn from established NIH-funded faculty who utilize a broad spectrum of state-of-the-art integrative, clinical, cellular and molecular approaches to probe critical questions in women's health research. This structure will insure that the proposed COBREWH program will meet the challenge of exponentially increasing the NIH- funded research in the Commonwealth of Kentucky.

The cessation of reproductive function is one of the most dramatic and fairly rapid endocrine changes in the life of a female and carries profound and broad repercussions. At one time, it was thought that decreasing estradiol concentration, resulting from exhaustion of ovarian follicles, caused the menopause. Now, it is clear that many factors interact to bring about declining reproductive function and the cessation of reproductive cycles. Furthermore, we are beginning to appreciate the most- reproductive state affects multiple functions that extend beyond the traditional reproductive axis. An increasing number and an increasing proportion of women will liver a larger fraction of their lives in the postmenopausal state. Therefore, it is critical that we deepened our understanding of the factors that regulate the transition to acyclicity and, conversely, the impact of transition to acyclicity on the function of multiple systems. The program is organized into five research projects that are supported by one Core. The projects are integrated and coordinated to test the following hypotheses (1) changing estrogen responsiveness governs the health and function of ovarian follicles during aging, (2) age-related and estradiol-dependent alterations in the pattern of expression in the ovarian metalloproteinases and their inhibitors result in abnormalities in follicular development and ovulation, (3) aging and/or the cumulative exposure to estradiol regulate changes in somatotroph and lactotroph function, (4) alterations in glutamatergic and adrenergic regulation of GnRH neurons cause changes in their ability to induce LH surges; and (5) estradiol plays a critical protective role in the brain; therefore, declining estradiol concentrations lead to an increased vulnerability to brain injury and neurodegeneration. An Administrative/Animal ore supports all of the projects, providing organizational planning, integrating results from each project into a more comprehensive overview, and maintaining all animal records and coordinating their optical endocrine treatments and use. We submit this program project proposal because we share interests in interdependent aspects of reproductive aging. A common threat that weaves through all of the projects is our interests in the role of estradiol in aging. Finally, this mechanism of support will permit us to use aging animals in the most optimal manner, allowing us to compare data and economize on the costs of aging animals.

The cessation of reproductive function is one of the most dramatic and fairly rapid endocrine changes in the life of a female and carries profound and broad repercussions. At one time, it was thought that decreasing estradiol concentration, resulting from exhaustion of ovarian follicles, caused the menopause. Now, it is clear that many factors interact to bring about declining reproductive function and the cessation of reproductive cycles. Furthermore, we are beginning to appreciate the most- reproductive state affects multiple functions that extend beyond the traditional reproductive axis. An increasing number and an increasing proportion of women will liver a larger fraction of their lives in the postmenopausal state. Therefore, it is critical that we deepened our understanding of the factors that regulate the transition to acyclicity and, conversely, the impact of transition to acyclicity on the function of multiple systems. The program is organized into five research projects that are supported by one Core. The projects are integrated and coordinated to test the following hypotheses (1) changing estrogen responsiveness governs the health and function of ovarian follicles during aging, (2) age-related and estradiol-dependent alterations in the pattern of expression in the ovarian metalloproteinases and their inhibitors result in abnormalities in follicular development and ovulation, (3) aging and/or the cumulative exposure to estradiol regulate changes in somatotroph and lactotroph function, (4) alterations in glutamatergic and adrenergic regulation of GnRH neurons cause changes in their ability to induce LH surges; and (5) estradiol plays a critical protective role in the brain; therefore, declining estradiol concentrations lead to an increased vulnerability to brain injury and neurodegeneration. An Administrative/Animal ore supports all of the projects, providing organizational planning, integrating results from each project into a more comprehensive overview, and maintaining all animal records and coordinating their optical endocrine treatments and use. We submit this program project proposal because we share interests in interdependent aspects of reproductive aging. A common threat that weaves through all of the projects is our interests in the role of estradiol in aging. Finally, this mechanism of support will permit us to use aging animals in the most optimal manner, allowing us to compare data and economize on the costs of aging animals.

DESCRIPTION (provided by applicant): Funds are requested to equip animal research space in the University of California Davis Genome and Biomedical Sciences Facility (GBSF). The addition of fixed cage racks will transform this space into a stateof- the-art rodent barrier facility to serve all of campus and will provide critical facilities needed to underpin our internationally respected Mouse Biology Program. Completion of this project will allow us to fulfill our longterm objective to use rodents as a platform to attack critical questions in biomedical sciences in multiple research programs. As a recipient of a "Center of Excellence in Nutritional Genomics" and, therefore, a designated institution of emerging excellence, these funds will accelerate our growth in research in genomics in the broadest sense of the word. With its modern animal research space, the GBSF will allow us to combine forces of researchers in basic genomics and proteomics, molecular medicine, biomedical engineering, bioinformatics, and biostatistics. These cross-disciplinary programs will bring together established investigators, new investigators, research fellows and visiting scientists who use rodents as animal models in a centrally located research.

Colleges and universities in the three-state region of Washington, Oregon and Idaho are proposing to form the Pacific Northwest Louis Stokes Alliance for Minority Participation (PNW LSAMP). This Alliance will enable educators and advocates from science centers, pre-college programs, community colleges and four-year institutions to leverage their resources and existing relationships to increase minority participation in science, technology, engineering and mathematics (STEM) majors. The projected demographic shifts in these three states of Idaho, Oregon and Washington present an extraordinary opportunity for colleges and universities to greatly increase the diversity of students earning STEM degrees. Each state has committed substantial resources to efforts to increase degrees granted in STEM as part of economic development initiatives.

Intellectual Merit
To take advantage of this opportunity and reach its goal of doubling the number of STEM bachelor degrees granted to underrepresented students, the Pacific Northwest Alliance will develop and/or strengthen programs and services related to the following priorities:
1. Increase pathways to college for URM students while strengthening their interest in pursuing STEM careers;
2. Create a culture and community of success for URM STEM majors on four-year college campuses to improve retention and graduation rates; and
3. Expand opportunities for URM STEM majors to participate in campus-based and national undergraduate research experiences.

PNW LSAMP partners will implement best practices in STEM diversity initiatives that have been disseminated by existing LSAMP programs around the country including early outreach and recruitment, community college transfer assistance, supplemental instruction, mentoring, summer bridge programs, and undergraduate research experiences. In addition to participation in a joint annual research conference, each of the community college and 4-year institutions has committed to establishing PNW LSAMP centers on their campus that will serve as visible and accessible support systems for underrepresented students with an interest in STEM

Broader Impacts

The Alliance will address the national and regional imperative to increase the number of degrees granted in STEM by connecting institutions of higher education with precollege and science advocacy programs. The Alliance partnership will provide seamless transitions for students as they proceed on the pathway from high school to college and from community colleges to 4-year colleges. The diversity of institution type in the Alliance will supplement student access to summer bridge and orientation programs, tutoring and mentoring relationships, and undergraduate research experiences. The Alliance will further leverage its efforts through the inclusion of science centers that serve as important portals to the public and already have a number of established stellar programs targeted to raise student interest in STEM.
The Alliance will also encourage collaboration between the many broadening participation programs funded by the National Science Foundation that exist on member campuses, including Noyce Fellowships, SSTEM/CSEMS, STEP, and ADVANCE. The synergy created by bringing all of these diversity-related programs together will augment institutional transformation on each campus.

The project proposes to integrate NSF-funded efforts at the University of Washington (UW) in an innovative way to improve the experiences of underrepresented undergraduate minorities, women and students with disabilities in the College of Engineering. A primary catalyst for this synergy is the use of the UW's PEERs project (Promoting Equity in Engineering Relationships), which seeks to positively impact the climate of engineering through a cadre of change agents who create and encourage improved and more equitable relationships. The four institutional partners for the proposed collaboration are the institution's:
1. ADVANCE Center for Institutional Change;
2. Center for Workforce Development;
3. Center for Engineering Learning and Teaching (CELT); and
4. Disabilities, Opportunities, Internetworking, and Technology (DO-IT).

These institutional partners will use the PEERs model to enhance the goals of three NSF-funded projects and to leverage lessons learned from the existing awards to work toward improving the climate for and the participation of underrepresented minority, female and disabled engineering students; and provide a foundation for campus-wide replication. The three NSF awards upon which the I3 project will build include:
1. Collaborative Research - Northwest Engineering Talent Expansion Partnership: A Coordinated Regional Recruitment and Retention Effort (DUE-0431659);
2. CCLI: Developing Engineering Lifelong Learners Through Freshman Seminars and Faculty Development Workshops (DUE-0737535); and
3. Northwest Alliance for Access to Science, Technology, Engineering, and Mathematics (HRD-0227995).

Four primary objectives will be achieved by this project
1. Raise awareness of unconscious and implicit biases toward underrepresented minority, women and disabled students;
2. Promote actions both majority and underrepresented minority, women and disabled students and faculty can take to counteract these biases to cultivate a more welcoming and success-promoting climate;
3. Cultivate change agents among both student and faculty bodies; and
4. Build a foundation, and collaboration mechanisms, for future efforts to make STEM and other programs campus-wide welcoming and accessible to underrepresented minority, women and disabled students.