Robert L Spencer, PhD - US grants

The long-term interests of this project are to determine the neurohormonal mechanisms that contribute to the ability of the HPA axis to habituate to repeated stress. Understanding the mechanisms of the habituation of the HPA axis stress response may lead to a better understanding of the mechanisms underlying dysregulation of the HPA axis that is present in a large number of individuals with clinical depression. A strong case has been made for viewing depression as a stress-related disorder, and at a physiological level depression may be a maladaptive response to repeated stress in which sensitization predominates, perhaps because of an impairment in habituation mechanisms. The operation of the HPA axis is extremely sensitive to corticosteroid feedback inhibition, and one means by which repeated stress may modify the HPA axis is by producing changes at the corticosteroid receptor level. A widely held hypothesis is that the increased level of corticosteroids present during chronic stress produces corticosteroid receptor downregulation, resulting in additional HPA axis hyperactivity. While some repeated stress studies have provided evidence for corticosteroid receptor downregulation in the hippocampus of the rat brain, other studies have failed to observe decreases in corticosteroid receptors with repeated stress, and on some occasions, increases in corticosteroid receptors were observed. The discrepancies in stress- induced corticosteroid receptor changes may pertain to whether the stress regimen is one that results in sensitization or habituation of the HPA axis stress response. For example, severe chronic stress may produce a decrease in corticosteroid receptor level and lead to increased HPA axis activity (sensitization), whereas a milder repetitive stressor may produce an increase in corticosteroid receptors and lead to decreased HPA axis activity (habituation). This latter prospect has been largely unexplored, and will be the focus of this project. Past studies examining in vivo stress effects on corticosteroid receptor levels in the brain have been hindered by the limitations of the cytosolic receptor binding assay, that until now has been the best means available for quantitatively measuring corticosteroid receptors. This homogenate based receptor binding procedure may not provide the resolution that is necessary to detect discrete changes in corticosteroid receptor levels within subregions of brain tissue. It is anticipated that studies described in this proposal will be able to overcome this limitation by the use of a radioimmunohistochemical method for measuring relative corticosteroid receptor concentrations in rat brain tissue sections. The following two specific aims are proposed: Specific Aim 1. To further validate the use of a radioimmunohistochemical procedure to measure relative changes in corticosteroid receptor levels in pituitary and brain tissue. Specific Aim 2. To use radioimmunohistochemistry to determine if there are regional changes in Type I or Type II corticosteroid receptor levels as a consequence of repeated restraint stress.

The long-term objectives of this proposal are to determine the neurohormonal mechanisms that contribute to the ability of the hypothalamic-pituitary-adrenal (HPA) axis to habituate to repeated stress. The goal of the proposed studies is to characterize the role of corticosteroid feedback inhibition in the habituation process. The overall hypothesis of these studies is that corticosteroid feedback inhibition contributes to the ability of the HPA axis to habituate to repeated stress. These studies may have relevance to understanding some of the neuropathology underlying psychiatric disorders, such as clinical depression. For example, depression has been characterized as a stress- related disorder. The HPA axis of many depressed individuals is hyperactive, and many of the physical and behavioral symptoms associated with depression resemble those present during an acute stress state. Thus, depression may be a condition in which the ability to habituate to daily stress is impaired. An understanding of the mechanisms that lead to HPA axis stress habituation may point to factors in the depressed individual that have been altered, thereby preventing adequate stress adaptation. The following 5 specific aims are proposed: Specific Aim 1. To further characterize the extent to which habituation of the HPA axis stress response to repeated restraint stress is dependent on corticosterone. Specific Aim 2. To determine if an enhanced sensitivity of the HPA axis to corticosteroid feedback inhibition contributes to habituation. Specific Aim 3. To determine if delayed corticosteroid feedback inhibition contributes to habituation. Specific Aim 4. To determine if there is an increase in corticosteroid receptor level as a consequence of exposure to repeated restraint stress. Specific Aim 5. To determine if there is an increase in corticosteroid receptor activation by corticosterone as a consequence of exposure to repeated restraint stress. A series of studies are proposed which will use the rat as an in vivo system for studying HPA axis habituation. These studies will utilize a repeated restraint stress regimen that reliably produces habituation of the HPA axis stress response. Characterization of the role of endogenous corticosteroids in the habituation process will be determined in studies in which corticosteroids are systematically manipulated by adrenalectomy and treatment with selective corticosteroid receptor agonists and antagonists Studies will also explore the possibility that a molecular mechanism leading to HPA axis stress habituation is a stress-induced change at the corticosteroid receptor level. Overall, these studies will provide important information about the mechanisms underlying an adaptive response of the HPA axis to repeated stress.

DESCRIPTION (provided by applicant): This is an application for an Independent Scientist Award (K02) that would be essential in allowing the PI to devote at least 75 percent of his effort to his research program and research career development. For the past 6 years the PI has systematically studied a stress habituation paradigm in rats and found that the expression of habituation depended on corticosteroid negative feedback, especially as a result of corticosteroid actions mediated by mineralocorticoid receptors (MR). This work was primarily supported by NIH B-Start (MH54742) and R29 (DK49143) awards. Recently, the PI has been awarded a 5-year R01 award (MH62456) to pursue further these studies. The funded research project entails an extensive range of studies that will 1) determine the brain regions critical to MR-mediated modulation of stress habituation, 2) characterization of CRH and AVP gene expression and modulation with habituation and altered corticosteroid negative feedback, 3) examination of the extent to which HPA axis stress habituation is associated with a generalized shift in MR-mediated corticosteroid negative feedback, and 4) determine if HPA axis stress habituation is associated with changes in corticosteroid receptor expression. The PI's immediate career objectives are to extend his research program in terms of its quality, productivity and breadth of technical expertise. The PI's long-term career objective is to make a substantial scientific contribution to the understanding of the neural adaptive processes that impact on stress responses. A Career Development Plan is proposed that will be instrumental in helping the PI achieve these career objectives by extending the PI's collaborations and training in the areas of neuroanatomy, cell biology, molecular biology, and systems physiology. Increased knowledge, technical expertise and interaction with specialists in each of these areas will be vital for ongoing and future studies in the PI's laboratory designed to decisively determine underlying mechanisms of stress adaptation. The PI's Department and Institution are committed to supporting the PI's research program and have provided extensive laboratory space and physical resources. Through Department approved teaching reduction, the K02 award will increase substantially the amount of time and coherent blocks of time that the PI can devote to his research and career development, thereby assuring the success of these endeavors.

DESCRIPTION (provided by applicant): Glucocorticoid hormones have powerful multifactorial regulatory effects on every physiological system. Dysregulated patterns of glucocorticoid hormone secretion, often as a result of chronic stress, have adverse effects on physical and mental health. Dysregulation of glucocorticoid secretion is strongly associated with some psychological disorders (e.g. depression and posttraumatic stress disorder) and other biomedical disorders (e.g. Type II diabetes, chronic fatigue syndrome, fibromyalgia). Glucocorticoid secretion is controlled by the neuroendocrine hypothalamic-pituitary-adrenal (HPA) axis system, and the principal factor that has regulatory control over HPA axis activity is glucocorticoid negative feedback. Understanding of the mechanisms responsible for glucocorticoid negative feedback is limited. To address that gap in knowledge, this ongoing research project will determine the molecular, cellular and systems level mechanisms of glucocorticoid negative feedback. This knowledge is necessary to discern how chronic stress leads to altered glucocorticoid negative feedback function, and it will point to new strategies for targeted interventions that will prevent and perhaps reverse alteration of glucocorticoid negative feedback function associated with chronic stress. The guiding hypothesis of this project is that glucocorticoids produce multiple effects within the intrinsic anatomical elements of the HPA axis as well as effects on neural circuits that dictate the moment-to-moment HPA axis activity. These glucocorticoid effects have distinct time frames of onset and expression, as well as separate underlying molecular mechanisms. By establishing these cellular sites of action and temporal patterns of expression this project will determine specific mechanisms of glucocorticoid negative feedback. Previous work on this project identified independent actions of glucocorticoids that are evident within different time intervals after a phasic increase in glucocorticoids that can be distinguished as fast (<15 min), short-term (~ 1 hr), and delayed (~3 hr) negative feedback actions. Four Specific Aims organized around this conceptual framework will address the following: Aim 1] To determine the temporal requirements and receptor mechanisms by which glucocorticoids produce fast negative feedback at the hypothalamic paraventricular nucleus. Aim 2] To use hypothalamic organotypic cultures to determine cellular and molecular mechanisms by which glucocorticoids produce intrinsic negative feedback on CRH neurons. Aim 3] To determine in vivo mechanisms of glucocorticoid short-term and delayed negative feedback. Aim 4] To determine the relationship between chronic stress adaptation and glucocorticoid negative feedback function. The new information derived from these proposed studies can then be applied to 1) the design of better (more sensitive or revealing) HPA axis related measures and challenge conditions in patient populations, 2) identification of new candidate risk genes associated with HPA axis dysregulation, and 3) development of new treatments that may selectively normalize HPA axis function without disturbing appropriate glucocorticoid signaling throughout the body.

DESCRIPTION (provided by applicant): Stress is a leading presenting, precipitating and exacerbating factor for a wide array of biomedical pathological conditions, with an especially strong etiological link with mental disorders. Researchers have made significant recent advances in understanding the neurobiology of stress. Determining the molecular, cellular and neurosystems mechanisms through which stress alters brain function is essential to understanding the mechanistic relationship between stress and mental disorders. A Neurobiology of Stress Workshop is being organized to be held June 15-18, 2010 at Boulder Colorado. This rigorous scientific meeting will bring together preclinical and clinical researchers who study stress-brain interactions and their impact on mental and physical health. This Workshop addresses an important need to strengthen the community of stress researchers in a manner that will maximize the productivity and clinical benefit of future stress research. Thus, the Workshop will provide a unique opportunity for researchers to participate in face-to-face examination of recent research advances, to share perspectives, identify relevant issues, debate controversies and exchange diverse expertise. Five sessions are planned in which invited speakers will present new research work, novel ideas, and examination of clinically relevant issues. Session themes are organized around specific stress-related factors and their impact on mental health and disease. Specifically, these stress-related factors of focus are 1) energy metabolism, 2) lifespan epochs, 3) risk factors, 4) resistance/resilience factors, and 5) cognitive and emotional factors. One of the five sessions is a targeted multidisciplinary cross-fertilization session featuring metabolism/energy balance and its relationship with stress neurobiology. In addition to the discussion time within each session, the Workshop features extensive time for interaction among all attendees at the opening data blitz reception, shared daily lunch period, Poster Session, and a social hour that will feature an informal discussion of the future of stress research. A priority of the Workshop is to foster the professional development of new investigators and women by including them at all levels of meeting organization and Program participation. Further the Workshop will nurture career development of graduate students and postdoctoral researchers by giving them ample opportunity to participate in the Workshop via the data blitz session, Poster Session, discussion sessions, "Meet the Speaker" luncheon roundtables, and an on-line Meeting Issues Blog. Travel Grants will be made available to select trainees through a merit based application process, with a detailed plan in place to recruit applications from interested minority candidates. Funds are requested in this application to support Trainee Travel Grants, Poster Session costs, and meeting related costs for new investigators, women and minorities that are participating in the Scientific Program. This Workshop will address critical needs in the stress research community that are ongoing, and consequently we envision this meeting as serving as a model for a recurring series of Stress Workshops. PUBLIC HEALTH RELEVANCE: The adverse effects of stress on mental and physical health has come to the fore as one of the most pressing biomedical problems in our society. The proposed Neurobiology of Stress Workshop to be held June 15-18, 2010 in Boulder Colorado will bring together basic, preclinical and clinical researchers and affiliated trainees in order to significantly enhance the productivity and clinical benefit of future stress research.

Optimal brain function depends on a daily cycle of fluctuating operation (a circadian rhythm) and synchronization of that rhythm to the environmental day-night cycle. A specific region of the mammalian brain, the suprachiasmatic nucleus, is responsible for coordinating the brain's circadian rhythms. Little is known about how this coordination happens. This project examines the circadian function of the prefrontal cortex, a brain region involved in complex thinking, such as planning and decision-making, and the control of mood and emotions. The project will test the innovative hypothesis that a specific hormone (glucocorticoid hormone) serves as communicator between the suprachiasmatic nucleus and the prefrontal cortex. The project will use a systems biology approach to determine how glucocorticoid hormones regulate prefrontal cortex circadian rhythms in rats. The project also will use cutting-edge manipulations of prefrontal cortex gene expression to discern the mechanisms by which circadian rhythms contribute to optimal prefrontal cortex function. The studies will result in new understanding of glucocorticoid hormone physiology and its role in the coordination of brain circadian function. This new understanding could have significant benefit for society by promoting new strategies to combat disturbed circadian function, such as occurs with night-shift work, jet lag, seasonal extremes, aging, and certain disorders. The research will provide excellent research opportunities for high school, undergraduate, and graduate students from diverse backgrounds through an ongoing partnership with the University of Colorado's STEM Center for Learning and Diversity. Findings from this research will be disseminated to the scientific community and the general public through research team presentations on campus, at regional and national scientific meetings and community forums, and postings on a public lab web-site.

The medial prefrontal cortex (mPFC) is central to the advanced cognitive function in mammalian species. Strong diurnal variation in cognitive function suggests that mPFC operation is under circadian control. The mPFC lacks direct neural connection with the suprachiasmatic nucleus (SCN), the master clock in the brain, but has high expression of glucocorticoid receptors, supporting the prospect that corticosterone (CORT) participates in the entrainment of mPFC rhythmic clock gene expression. Preliminary data show dynamic modulation of mPFC clock gene expression that depends on the prior daily profile of diurnal circulating patterns of CORT. Moreover, a mPFC-dependent memory task, conditioned fear extinction, displays a diurnal variation in expression that is absent in adrenalectomized rats. Based on these supporting preliminary data, the project will determine whether: 1) CORT contributes to entrainment of mPFC rhythmic clock gene expression, 2) an appropriately timed daily CORT pulse is necessary for normal entrainment of diurnal variation in mPFC-dependent conditioned fear extinction, and 3) CORT entrainment of mPFC-mediated conditioned fear extinction depends on CORT activation of mPFC glucocorticoid receptors and rhythmic mPFC clock gene expression. The proposed research will provide excellent scientific training opportunities for a diverse group of high school, undergraduate, and graduate students. A range of avenues will be used for dissemination of the findings of this project and their value to society.

Psychiatric disorders, especially those marked by dysregulated mood and emotional control, such as depression, bipolar disorder, post traumatic stress disorder (PTSD) and schizophrenia, are associated with physiological and cognitive features of disrupted circadian function. Circadian regulation is necessary for appropriate anchoring of the optimal performance of virtually every cell and system of the body to fluctuating daily demands. Although the suprachiasmatic nucleus (SCN) of the hypothalamus serves as the body?s master circadian pacemaker, cells in other brain regions and in peripheral tissues express many of the same molecular clock elements (i.e. clock genes) as those found in the SCN. Recent advances have been made in determining the functional role and regulation of cellular clocks in peripheral tissues. Those studies demonstrate an important circadian entraining influence of the endogenous glucocorticoid hormones (CORT) on peripheral tissue cellular clock function. However, there is very little understanding of the function and regulatory processes of cellular clocks in extra-SCN brain regions. The prefrontal cortex (PFC) is a brain region that plays a central role in organizing and coordinating physiological, behavioral and emotional responses. Animal and human studies show that there is rhythmic clock gene expression in the PFC. Recent studies have found that normal clock gene expression in the PFC of rats depends on appropriate profiles of CORT secretion. Moreover, disruption of CORT-entrained PFC clock gene expression results in impaired diurnal patterns of conditioned fear extinction memory. PTSD is associated with impaired circadian function, compromised PFC function and dysregulation of CORT secretion. In addition, individuals with PTSD suffer from persistent conditioned fear responses. Improving conditioned fear extinction learning is a primary therapeutic objective for treating PTSD. Consequently, this project will use a rat animal model to determine the mechanistic basis by which PFC clock gene expression and CORT interdependently regulate conditioned fear extinction memory. The project is organized around 3 specific aims: Aim 1] To determine how time of day, circadian CORT and ventral medial PFC (vmPFC) clock gene expression modulate the activity of neuronal projections from the vmPFC to the basal medial amygdala (BMA) during conditioned fear extinction training and recall. Aim 2] To determine how time of day, circadian CORT and vmPFC clock gene expression modulate neuroplasticity- related processes in the vmPFC that support conditioned fear extinction recall. Aim 3] To test the necessity and sufficiency of vmPFC to BMA projections for mediating time of day, circadian CORT and vmPFC clock gene expression regulation of conditioned fear extinction recall. The proposed studies will provide new understanding of how circadian and CORT factors dynamically interact to regulate PFC function. These studies will also lead to better understanding of the underlying mechanisms of conditioned fear extinction memory, a neuroprocess that has important clinical relevance for circadian and stress-related disorders.