James P. Herman - US grants

DESCRIPTION (provided by applicant): Major depressive disorder is accompanied by dysfunctional cognitive and neuroendocrine processing of stressful information. The neurocircuitry underlying aberrant stress processing remains to be elucidated. Human imaging data and rodent stress studies suggest that limbic cortical regions are well-positioned to mediate hypothalamic-pituitary-adrenocortical (HPA) axis pathology seen in depression. The current proposal uses a functional/anatomical approach to test the hypothesis that limbic cortical regions, including the prelimbic and infralimbic cortices, use separate yet complementary mechanisms to integrate psychological and physiological stimuli into appropriate stress responses. The neuroendocrine and mood changes seen in depression likely reflect a failure of these cortical regions to appropriately interpret relevant stressful stimuli. This hypothesis will be tested in four Specific Aims. Aim 1 will use anatomical approaches to delineate monosynaptic and multisynaptic neural pathways connecting limbic cortices with hypothalamic stress effectors. Aim 2 will use neurocircuit targeting approaches to test the necessity and sufficiency of defined limbic cortical subregions in inhibiting responses to psychological vs. physiological stressors. Aim 3 tests the involvement of specific limbic cortical-hypothalamic and limbic cortical-hippocampal circuits in mediating responses to stress, using a targeted intervention design. Finally, Aim 4 tests for interactions between limbic cortices and other limbic stress-regulatory regions, to determine whether limbic stress integration involves convergent projections or separate ?labeled lines? to the hypothalamus. These studies are expected to identify limbic cortical circuits and mechanisms involved in stress integration, and thereby provide clear neuroanatomical targets for development of improved behavioral/pharmacological interventions for depression and other stress-related disease states.

DESCRIPTION (Investigator's Abstract): Regulation of glucocorticoid secretion is essential for maintenance of neuronal homeostasis. In normal physiology, these hormones bind to endogenous adrenocorticosteroid receptors (ACRs), through which they exert trophic actions on neurons (by way of the mineralocorticoid receptor [MR]) and serve to promote defensive responses to physiological or psychological stress (by way of the glucocorticoid receptor [GR]). The latter defensive responses are adaptive in the short run, serving to mobilize body resources. However, if release is prolonged, glucocorticoids can have multiple negative consequences for the animal. Included among these are neurotoxic effects on neurons in the hippocampus. In normal individuals, glucocorticoid release is tightly controlled, thereby maintaining subtoxic levels. Unfortunately, as a consequence of aging or Alzheimer's disease (AD), this control is lost, resulting in prolonged glucocorticoid release which has been linked with age-related hippocampal cell loss and memory deficits. Loss of the capacity to regulate glucocorticoids is a consequence of impaired stress regulation, and appears to prominently involve neuronal ACR imbalances. The present studies are designed to identify cellular mechanisms underlying ACR dysregulation in stress and aging, with the eventual goal of targeting specific pathways for prevention of glucocorticoid-related cell loss. Specific Aim 1 will address the hypothesis that stress and glucocorticoids affect ACR gene expression and protein synthesis by the same molecular mechanism, verifying the primacy of glucocorticoids in regulating receptor synthesis in vivo. Specific Aim 2 will characterize specific molecular pathways regulating GR and MR biosynthesis in neurons, identifying molecular targets for age-induced dysregulation. Specific Aim 3 will evaluate the hypothesis that stress and aging work by way of the same glucocorticoid-mediated pathway to disrupt ACR regulation. Finally, Specific Aim 4 will determine whether age- and stress-induced changes in ACR regulation specifically target the neurotrophins, glucocorticoid-responsive molecules involved in maintenance of neuronal cell viability. It is predicted that the results of this project will identify specific mechanisms responsible for impaired ACR regulation in aging and AD.

DESCRIPTION (adapted from applicant's abstract): The neuroendocrine stress response is a double-edged sword. Activation of glucocorticoid secretion is clearly essential for adaptation to stressful environmental or physiologic stimuli. However, stress or disease-induced glucocorticoid hypersecretion has serious deleterious consequences on metabolism, cardiovascular tone, immunity and behavior. Thus, it is critical for the organism to carefully limit the strength and duration of neuroendocrine stress activation. In recent years, it has become apparent that a sizable proportion of such stress regulation is mediated by the hippocampal formation of the brain. Integrity of the hippocampus is essential for appropriate inhibition of the stress response. Further, neuroendocrine disturbances are accompanied by hippocampal pathology in numerous stress-related disease processes, including major depression. On the basis of the available data, we have developed the hypothesis that the hippocampus serves to protect the organism from the deleterious consequences of stress by interpreting the significance of incoming stimuli with respect to previous experiences. The aims of the present proposal are designed to test this general hypothesis, and to identify specific neurocircuitry subserving translation of hippocampal information processing into inhibition of neuroendocrine stress responses. Specific Aim l tests the hypothesis that the hippocampus preferentially affects neuroendocrine responses to cognitive stimuli (novelty, conditioning) but not systemic challenge (respiratory distress, immune stimulation). Specific Aim 2 is designed to delineate neurocircuitry subserving hippocampal neuroendocrine modulation, focusing on the hypothesis that activation of the hippocampus by cognitive (but not systemic stress) is translated into inhibition of the stress axis by way of intermediary cell groups proximal to hypothalamic stress-integrative neurons. Specific Aim 3 builds on this circuit analysis by testing the hypothesis that excitatory neurotransmission in subcortical targets of hippocampal outflow leads to inhibition of hypothalamic stress outflow. Together, these studies are expected to define the raphe of the hippocampus in stress processing, and indicate that dysfunction of this structure is a likely mitigating factor in depressive disorder and stress-related disease processes.

Glucocorticoids released by the hypothalamo-pituitary-adrenocortical axis play a major role in mobilization of bodily defenses in response to physiological or psychological threat. Processes initiated by glucocorticoids can have serious deleterious consequences when prolonged; accordingly, excess exposure is normally avoided by glucocorticoid negative feedback regulation, operating at the level of the brain. Substantial evidence has indicated that this feedback mechanism is severely disrupted in affective disease, resulting in a glucocorticoid dyshomeostasis that may play a major role in determining the course and severity of illness. In the present proposal we have assembled a group of investigators to delineate mechanisms of central neurocircuit regulation of the HPA axis under normal conditions and during disease states. Evidence from several of our laboratories suggests that the HPA axis is controlled by multisynaptic limbic-hypothalamic connections that translate psychological stimuli into neuroendocrine output. The proposed network combines investigators from the disciplines of cell biology, electrophysiology, neuroanatomy, molecular biology, endocrinology, and psychiatry into an integrated research team with the central goal of determining the nature and involvement of these limbic-hypothalamic HPA-regulatory circuits in physiological and behavioral consequences of affective illness. Specific Aim 1 creates a mechanism for linking the efforts of the six component labs through a series of conferences, at which broad-based collaborative studies will be designed, results discussed and future directions determined. Specific Aim 2 provides the means for implementing the collaborative process amongst component laboratories, through pilot projects, sample exchange and active physical interaction among component labs. As a result of these collaborative efforts, this network expects to: 1) design and implement critical tests of the hypothesis that HPA dysregulation and behavioral changes seen in stress models of affective disease are fueled by limbic-hypothalamic imbalance; 2) extend testing of the limbic-hypothalamic hypothesis to functional imaging studies in human depressives; and 3) conduct multi-factorial and inter-disciplinary analyses of anatomical, physiological, pharmacological and behavioral data derived from individual experiments. These collaborative efforts should provide novel insights into integrative regulation of the HPA axis and its relationship to mental health disorders.

DESCRIPTION (provided by applicant): Glucocorticoid hormones regulate higher cognitive and emotional function in man. These hormones signal through adrenocorticosteroid receptor molecules that target DNA, acting as gene transcription factors. In brain, these factors modulate expression of genes involved in neuronal signaling and plasticity and are ultimately critical for functions such as stress integration and memory. These functions are controlled by the hippocampus, a brain region that serves as a prime target for circulating glucocorticoids. Given the potential for adrenocorticosteroid receptors to integrate hormonal signals with environmental cues, adequate maintenance of glucocorticoid responsiveness is vital for hippocampal function. Traditionally, the impact of glucocorticoids on hippocampal function was thought to be uniformly negative. However, recent data suggest that physiological levels of glucocorticoids are important for hippocampal signaling and information processing. Recent studies in our laboratory further indicate that the aged hippocampus is relatively insensitive to glucocorticoids in aging, and it is loss rather than gain of glucocorticoid action that fuels hippocampal dysfunction. This proposal therefore addresses the novel hypothesis that age-related hippocampal deficits are caused by a failure of the glucocorticoid receptor to signal in the cell nucleus, resulting in impaired DNA binding and reduced transcription of genes responsible for hippocampal signaling and synaptic plasticity. Specific Aim 1 will test the hypothesis that age-related memory impairment and stress dysregulation are fueled by reductions in nuclear GR signaling. Specific Aim 2 is designed to determine the mechanism of age-related glucocorticoid receptor translocation deficits, evaluating interaction between the glucocorticoid receptor and its nuclear chaperone complex; translocation of the receptor-chaperone complex to the nucleus; nuclear export of the glucocorticoid receptor; and nuclear degradation. Specific Aim 3 will test the hypothesis that aging decreases transcription of glucocorticoid-regulated genes in a manner predictive of reduced glucocorticoid responsiveness. Together, these studies should provide novel information [on] glucocorticoid signaling mechanisms in brain, and provide potential targets for therapies aimed at minimizing the impact of glucocorticoid-related cellular dysfunction in aging.

DESCRIPTION (provided by applicant): Stress-related diseases are accompanied by glucocorticoid and cardiovascular dyshomeostasis. In particular, glucocorticoid hypersecretion in depression is linked with both ongoing mood symptoms and with long-term physiological dysfunctions, including osteoporosis and visceral obesity. Decreased heart rate variability is associated with negative emotions in depressed patients, and contributes to the high incidence of cardiovascular pathology and mortality associated with the disorder. Thus, physiological consequences of depression contribute to both psychiatric symptoms and co-morbid disease processes. Neural and systemic effects of depression are linked to inappropriate processing of stressful information in the brain. The current proposal uses rodent chronic stress models to address mechanisms that control physiological adaptation and dysfunction following long-term stress. Our proposal tests the novel hypothesis that distinct brainstem neurons act as critical integrators of both stress habituation and sensitization, and are thereby responsible for adaptive as well as maladaptive hormonal and autonomic responses. Aim 1 will determine whether noradrenergic neurons of the nucleus of the solitary tract (NTS) are responsible for driving HPA axis hyperactivity and autonomic dysfunction following chronic stress. Aim 2 tests the hypothesis that neuroendocrine and autonomic stress pathologies are mediated through inappropriate drive of NTS noradrenergic neurons by forebrain stress-recruited pathways. Aim 3 uses a NTS gene knockdown approach (lentiviral vectors) to examine the role of NTS glucagon-like peptide 1(GLP-1) neurons in long-term stress adaptation. Aim 4 is designed to elucidate the role of NTS glucocorticoid receptor-mediated feedback in the control of stress adaptation and pathology. Understanding the role of the NTS in stress adaptation will add new insight into potential circuit pathology in affective disease, and provide novel molecular targets (GLP-1, brainstem glucocorticoid receptors) for development of therapeutic intervention strategies. PUBLIC HEALTH RELEVANCE: Depression is accompanied by major disruption of homeostatic mechanisms, including glucocorticoid hypersecretion and cardiovascular dysfunction. Importantly, physiological pathologies are involved in generation of mood symptoms, indicating a tight linkage between systemic and neural aspects of depression. This proposal seeks to identify novel neural mechanisms responsible for generation of glucocorticoid and cardiovascular dysfunction in a rat chronic stress model of depression. Our studies are expected to provide preclinical data regarding new molecular targets for promoting stress resistance and adaptation.

DESCRIPTION (provided by applicant): Continuing support is requested for an interdisciplinary program that provides broad and fundamental predoctoral training in the neurosciences. Trainees are recruited nationally and competitively selected from among the applicants to the existing Neuroscience Graduate Program at the University of Cincinnati, as well as from other graduate programs at the College of Medicine. The proposed training grant supports entering students during their first two years of training, prior to the beginning of dissertation work. Trainees receive didactic instruction in basic neuroscience and cell/molecular biology through a series of existing courses, journal clubs and seminar series, and research training through rotations in the laboratories of participating faculty. The core of the proposed training program rests upon growing and vigorous interdepartmental Neuroscience Program. The University of Cincinnati is strongly committed to the growth of neuroscience, and the Neuroscience Graduate Program receives an administrative budget and a limited amount of stipend and tuition support from the Dean of the College of Medicine. Participating faculty have active and productive research programs reflecting a diversity of approaches toward understanding nervous system function. The program sponsors a number of activities that foster cohesiveness and a sense of identity for students in the program and encourages student-faculty interactions. These include the Cincinnati Neurofest, an international Neuroscience symposium; bimonthly student luncheons with the Program Director and other faculty; an annual recruitment weekend; a welcome reception for new students in early fall; a gathering for students and faculty at the annual meeting of the Society for Neuroscience; and an annual student-faculty retreat. During the last grant period, this training grant has allowed us to significantly expand our recruitment of high quality applicants, and further develop the training program and its curriculum. In addition, by increasing our visibility at both local and national levels, this training grant has provided the key stimulus for positive changes that broadly affect our neuroscience community. These have included the recruitment of additional neuroscience faculty, the development of new interdisciplinary research groups and training grants in specialized areas of neuroscience, and initiation of the process by which the Cincinnati Neuroscience Graduate Program will attain formal State of Ohio designation as an independent graduate degree (Ph.D.) program.

DESCRIPTION (provided by applicant): Dysfunction of the hypothalamo-pituitary-adrenocortical (HPA) axis is a common feature of major affective illnesses. Neuroendocrine disturbances are typically manifest as cortisol hypersecretion and glucocorticoid negative feedback resistance, both of which expose individuals to excessive levels of stress hormones and their deleterious sequelae. The mechanism underlying glucocorticoid hypersecretion is currently ill-defined. Work from our laboratories as well as others indicate that pathologically elevated glucocorticoids are likely due to hyperactivity of central stress-integrative neurons in the parvocellular PVN, which represent the final common pathway for HPA axis activation. In this proposal, we use a rat chronic stress model of depression to test the novel hypothesis that stress-related illnesses frequently characterized by sustained activation of HPA outflow result from the induction of biochemical and/or structural neuroplastic changes in hypophysiotropic regions of the paraventricular nucleus. This hypothesis will be tested in three Specific Aims. The first Aim will test the hypothesis that chronic stress induces functional plasticity of post-synaptic receptor expression in the HPA effector neurons in the paraventricular nucleus. Experiments will evaluate the prediction that chronic stress reconfigures receptor populations to favor excitatory neurotransmission over inhibition, test for chronic stress enhancement of excitatory neurotransmitter actions on glucocorticoid secretion, and use a genomics-guided approach to provide an integrated analysis of stress-induced receptor changes in paraventricular nucleus neurons. The second Aim will test the hypothesis that chronic stress disrupts glucocorticoid feedback sensitivity in paraventricular nucleus neurons controlling HPA axis responses. These experiments will determine if chronic stress reduces the capacity for nuclear glucocorticoid receptor signaling, and assess stress effects on feedback efficacy at the level of the PVN. The third Aim will test the hypothesis that chronic stress induces morphological changes in the parvocellular PVN that predict enhanced excitability. These studies will determine whether chronic stress enhances excitatory vs. inhibitory neurotransmitter innervation of paraventricular CRH neurons, assess the ability of stress to affect paraventricular neuronal morphology, and use a genomics-guided approach to probe for possible molecular mechanisms underlying stress plasticity. Overall, this project will provide critical new information on hypothalamic mechanisms mediating neuroendocrine dysfunction in affective disease states.

DESCRIPTION (provided by applicant): The medial prefrontal cortex is a primary brain mediator of stress and mood. In humans and in animal models, medial prefrontal cortical dysfunction is associated with emotional disturbances, impaired fear extinction and inefficient termination of physiological stress responses. Medial prefrontal cortex dysfunction is linked to numerous mental illnesses, the most prominent being depression and post-traumatic stress disorder, diseases that are triggered by life stress and result in long-term inappropriate stress responding. Notably, gene expression in the prefrontal cortex is exquisitely sensitive to stress exposure, with the vast majority of regulated mRNAs showing pronounced down-regulation. Recent studies have convincingly demonstrated that non-coding RNAs, including microRNAs and alternatively expressed 3'-unstranslated (3'-UTR) mRNA sequences, play a major role in mRNA down-regulation in numerous tissues, including brain. This Exploratory Proposal is designed to perform detailed analysis of prefrontal cortical non-coding RNAs (microRNAs) and 3'UTRs (mRNAs) using newly-developed deep sequencing technology, affording a heretofore unprecedented assessment of miRNA and 3-UTR regulation by chronic unpredictable stress in rat. The unpredictable stress regimen reliably models physiological and behavioral symptoms of depression, allowing for extrapolation of preclinical findings to putative mechanisms of functional dysregulation in human cortex. Aim 1 will use deep sequencing methods to provide a comprehensive and quantitative analysis of existing as well as novel chronic stress-regulated miRNAs in the prefrontal cortex of C57BL6 mice. Aim 2 will apply the deep sequencing methods to identification of chronic stress-regulated 3'-UTR sequences. In both Aims, follow-up studies will verify specific regulation of targeted miRNAs and 3'-UTR sequences in the prefrontal cortex, and use anatomical methods to localize expression to distinct cortical subregions and cell types. Identification of novel stress-regulated miRNAs and 3'-UTRs in mouse will inform our understanding of mechanisms underlying human stress-related disease, and provide possible future targets for intervention in disease processes. PUBLIC HEALTH RELEVANCE: This proposal provides an exploratory assessment of regulation of stress-regulated non-coding RNAs (i.e. microRNAs) and 3'UTRs (mRNAs) in the mouse prefrontal cortex using deep sequencing technologies, to provide insight into novel mechanisms of stress-related diseases associated with cortical dysfunction (e.g., depression). At the conclusion of these studies, we anticipate development of a heretofore unprecedented database of stress-regulated non-coding RNA regulation that can be mined to identify novel processes endangering cortical integrity in the face of prolonged stress. Identification of novel stress-regulated miRNAs and 3'-UTRs will inform our understanding of mechanisms underlying human stress-related disease, and define new avenues for intervention in cortically-mediated mental illness.

DESCRIPTION (provided by applicant): In humans, onset of depressive illness frequently occurs in the late adolescent period, often in conjunction with significant life stress. In additio, adolescent stress or trauma are frequently linked to later development of PTSD. Both disorders affect nearly twice as many women as men, suggesting a sexually dimorphic course of disease development. Notably, the adolescent period coincides with the terminal development of prefrontal cortex connectivity, which comprises the key cognitive-emotional circuit affected in both depressive illness and PTSD. The prefrontal cortex and its targets undergo aberrant structural rearrangement as a consequence of stress or stress hormone exposure in adults. Prior studies indicate that glucocorticoid stress hormone responses are exaggerated in adolescence, predicting that these groups may be selectively vulnerable to negative effects of stress on brain structure. Thus, adversity during adolescence may compromise the final development of this key emotional regulatory pathway, resulting in inappropriate mood regulation. This proposal uses a rat model to test the hypothesis that adolescent chronic stress produces short- and long-term reorganization of prefrontal cortical emotional control circuits, resulting in pronounced impairments in cortical regulation of mood, impulsivity, and stress reactivity. Aim 1 uses a battery of behavioral and physiological tests to establish the impact of adolescent chronic stress and sex on depression-like behavior, extinction of fear conditioning, delayed response learning, and glucocorticoid homeostasis, functions controlled by the prefrontal cortex and known to be disrupted in depression and PTSD. Aim 2 uses anatomical methods to test the integrity of prefrontal projection pathways, focusing on changes in synaptology of the prefrontal cortex and downstream targets and, using a novel viral tracing approach, the development of prefrontal efferent connectivity over time, again focusing on both males and females. Aim 3 tests the role of glucocorticoid receptor signaling in immediate and lasting behavioral and endocrine effects of adolescent stress in males and females, using time-constrained knockdown of receptor synthesis during stress exposure. Together, these studies are expected to clearly identify a role for adolescent stress in prefrontal cortical pathologies in both males and females, and provide insight into possible interventions for minimizing development of stress-related diseases in both sexes.

Project Summary Engaging in pleasurable pastimes (e.g., hobbies, sports, and other leisure activities) can improve mood and reduce perceived stress, suggesting that these activities are an effective means to confer stress resilience. Chronic stress is often unavoidable, making the development of strategies to enhance stress resilience a clear priority for the prevention or amelioration of stress-related diseases. Since beneficial behaviors likely promote stress buffering via activation of brain pleasure and reward circuitry, we have developed and characterized a rat model of stress buffering using intermittent access to a natural reward, limited sucrose intake (LSI). LSI reduces the adverse behavioral effects of chronic stress (e.g., diminished sociability and threat appraisal) and decreases hypothalamic-pituitary-adrenocortical axis reactivity. The stress-buffering provided by LSI is reproduced by a noncaloric sweetener and other naturally rewarding behaviors (sexual activity), but not by intragastric gavage of sucrose, supporting that the stress-protective effects of LSI are primarily due to its rewarding properties. Our preliminary data suggest that LSI acts by altering top-down regulation of the basolateral amygdala (BLA) by the prelimbic medial prefrontal cortex (PL mPFC). In addition, BLA projection neurons can be divided into multiple subsets based on their distinct efferent projection sites, and can play distinct roles in BLA-related behaviors. Thus, while LSI reduces total stress-induced neuronal activation (cFos) in the BLA, the impact on distinct BLA PN populations will likely underlie its role in stress resilience. In support of this idea, LSI reduces post-stress cFos expression in the ventral hippocampus (vHPC) and increases it in the anterodorsal bed nucleus of the stria terminalis (adBST) ? two regions that have receive direct BLA input and exert opposing effects on stress-related behaviors. This suggests that LSI may provide stress resilience by reducing the activity of direct BLA-vHPC projections, and increasing the activity of direct BLA-adBST projections. This proposal therefore uses the LSI model to test the hypothesis that chronic engagement in naturally rewarding experiences promotes behavioral resilience to chronic stress by altering a stress-reward neurocircuitry linking the mPFC, BLA, vHPC and adBST. The first aim tests the contribution of PL top-down regulation of the BLA, while the second aim tests the contribution of specific BLA projections to the vHPC and adBST. Chemogenetic (DREADD) technology is combined with a retrograde viral approach to obtain circuit- specific modulation of neural activity. The effects of circuit manipulation (activation and inhibition) on sociability and threat appraisal behaviors is assessed in the context of chronic stress and/or reward (LSI). This work has important implications, suggesting the presence of endogenous neurocircuits for stress buffering that can be recruited by engaging in naturally-rewarding behaviors. An improved understanding of these neurocircuit mechanisms may be leveraged to develop therapeutic strategies that minimize the adverse effects of chronic stress on mental health, and may guide the optimization of alternative interventions for stress relief.