Steven F. Maier - US grants

One of the most important factors in determining an individual's reaction to a stressful event is the expectation about that event: Does the individual have control over the stressor, or does he not? When humans or animals are exposed to stressful events over which they have no control, they experience a number of behavioral and physiological disturbances which do not occur if they DO have control, even if the stressors are physically identical. Such disturbances, and the theoretical entities thought to produce them, are widely believed to be important (and alterable) causes of depression, anxiety, urban stress, etc. It is therefore important to understand the mechanisms by which the controllability vs. uncontrollability of stressors alters both behavior and physiological functioning. Using a variety of experimental approaches, Dr. Maier has already produced an extensive behavioral groundwork for the current studies. He is now pursuing several major lines of research, all directed at answering this fundamental question: What determines the expectation of controllability, and its attendant consequences? Dr. Maier is carrying out four related sets of studies: 1) The role of the neurotransmitter norepinephrine in controllability; 2) the relationship between stressor controllability and spinal cord processes that regulate pain perception; 3) the role of stressor controllability in altering daily activity patterns, and the sensitivity of these activity patterns to pharmacological manipulation (e.g., with antidepressants); and 4) the nature of the cognitive changes produced by stressors, and the role of fear and anxiety in mediating these cognitive changes. Recent research by Dr. Maier and others has made it clear that the stress response is not simply the result of exposure to a particular physical stimulus or condition, but rather is the result of a complex interaction between the event itself and the organism's expectations, about that event. The results of the current studies will broaden our understanding of the role of "stressor controllability" in the development of "coping" behaviors, and in the ability of stressed individuals to adapt to their environment.

The organism's degree of behavioral control over stressors is among the most powerful factors in determining the individual's reaction to an encounter with such an event. Organisms that have no control over aversive events to which they are exposed reveal a variety of behavioral and physiological disturbances that do not occur if the organism does have control, even though the stressors are physically identical. Effects that depend on the controllability of the events experienced have been called "learned helplessness" effects. These effects and the theoretical entities said to produce them have been proposed to be important causes of reactive depression, certain instances of failure in school, some psychological aspects of aging, some of the effects of crowding, urban stress, anxiety, etc. Thus it is important to understand the mechanisms by which the controllability/uncontrollability of stesssors alters behavior and physiological functioning. The research proposed here is directed at such an understanding. Moreover, the controllability dimension exerts effects at many different levels and affects numerous systems (behavioral, endocrine, immune, neurochemical). A multidisciplinary approach designed to understand learned helplessness at each of these levels is proposed here, with the long-term goal of a truly integrated understanding of this set of phenomena. Three specific lines of research are proposed. The first centers on the role of controllability in modulating the impact of stressors on the immune system. Here the focus will be on three interrelated questions: a) What is the critical behavioral/psychological dimension of the stress situation that leads to changes in immune function?; b) What is the mechanisms by which this behavioral event(s) alters immune function?; c) What aspects of immune function are actually altered? The second line of research explores the role of the hypothalamo-pituitary-adrenal (HPA) system and central peptide hormones in learned helplessness. Two major questions will be posed. a) Is shock of differing controllability associated with differential activation of central nervous system CRF, beta-endorphin, and ACTH, and peripheral HPA activation?; b) Do the behavioral and neurochemical outcomes of exposure to uncontrollable shock depend on intact HPA function? The final line of research continues the prior work of this laboratory and examines the nature of the behavioral changes produced by uncontrollable stressors, and the involvement of opioid and aminergic mechanisms in these changes.

There is considerable recent interest in potential relationships between exposure to stressors and the functioning of the immune system. This is because alterations in immune function might mediate the impact of stressors on the development of diseases ranging from AIDS to cancer. The proposed research has 3 general goals. 1) Most studies in the literature have used a single set of stressor parameters and measured a single aspect of immunity. Thus the immune changes which occur have not been well characterized. This has led to great difficulty in assessing the reliability and nature of immunologic change produced by stressors, and also makes it difficult to conduct research that goes beyond demonstration and explores mechanism. It is not currently possible to even ascertain what measures of immune function might be most sensitive to stress-induced change. Thus a major goal is to determine the pattern of in vivo and in vitro changes in immune function that are produced by a variety of systematically varied stressor conditions. 2) There has been very little effort directed towards determining behavioral factors which might modulate the impact of stressors on immunity and determine whether effects do or do not occur. Here 3 factors which have proven to be important in modulating other stressor effects will be studied. These are the degree of behavioral control which the organism has over the stressor, defeat, and dominance. Moreover, the relationship between these will be studied as well as the role of fear/anxiety in mediating the effects obtained. 3) The neural and neuroendocrine mechanisms which mediate the effects obtained in 1) and 2) will begin to be explored. The focus will be on changes in hypothalamic norepinephrine and pituitary-adrenal function as mediators of in vivo changes in specific antibody formation produced by stressors.

DESCRIPTION (Adapted from the Applicant's Abstract): Dr. Maier plans an extensive and broad research program which will attempt to provide an integrated understanding of how stress impacts on measures of the immune system at the behavioral, physiological and immunological levels. Two stressors, inescapable shock and defeat, will be utilized and in vivo changes in immune function will be studied. The first set of studies will focus on the behavioral factors which are important in determining whether and how stressor exposure will alter the development of antibodies and antibody (Ab) secreting cells to the antigen (Ag) KLH. The research will focus on behavioral controllability of the stressor and dominance. The second set of studies will focus on the neural and neuroendocrine mediators by which the stressors alter Ab levels. This will include noradrenergic processes associated with the paraventricular nucleus (PVN). The applicant hypothesizes that activation of this area is central to the alteration of Ab levels to Ag administered before the stressor. In addition, he will consider peripheral neuroendocrine mediators and focus on corticosteroids and catecholamines. The third set of studies are focussed on on immunology with a consideration of neural and neuroendocrine effects of the stressors on alterations in cell trafficking between immune compartments leading to changes in cell types in the lymphoid compartments which in turn lead to Ab alterations. These studies will involve the use of about 2,000 male Holtzman Sprague-Dawley rats per year.

There is considerable recent interest in potential relationships between exposure to stressors and the functioning of the immune system. This is because alterations in immune function might mediate the impact of stressors on the development of diseases ranging from AIDS to cancer. Indeed, it has been argued that individual differences concerning the development and expression of AIDS symptoms from infection with HIV might be partially accounted for by the occurrence of psychological or physical stress at a critical stage in the cascade of immune reactions to the virus. This proposal focuses on in vivo changes in immune function. The specific aims of the proposal are: 1) To study behavioral factors which are important in determining whether and how stressor exposure will alter the formation of specific antibody (Ab) and Ab secreting cells (ASCs) to the antigen (Ag) KLH. Although there have been numerous demonstrations that exposure to painful or aversive events can alter some aspect of immune function, there has been very little analytic work directed at isolating important behavioral modulating factors. Here we wish to focus on 2 factors: a) Behavioral controllability of the stressor, both in the shock and defeat paradigms, b) Dominance. 2) To study neural and neuroendocrine mediators by which the stress conditions studied alter Ab levels. Focus is on 2 different but related issues. a) The central nervous system processes that mediate between the stressor and the peripheral changes that could contact immune organs and cells. Noradrenergic (NE) processes associated with the paraventricular nucleus (PVN) of the hypothalamus will receive particular attention. The PVN is a key structure in controlling both pituitary-adrenal and sympathoadrenomedullary activity, and the NE input to the PVN is a major stimulatory factor. b) The peripheral neuroendocrine mediators that are responsible for the stress-produced Ab changes. Here emphasis will be on elucidating the roles of adrenal corticosteroids and plasma catecholamines released by the adrenal medulla and sympathetic terminals. 3) To study immunologic changes produced by stressors and their neural and neuroendocrin sequelae that might be responsible for the reduced Ab formation, the focus will be on stressor produced alterations in cell trafficking between immune compartments, resulting in changed ratios of lymphocyte phenotypes.

The degree of behavioral control which an organism has over a stressor (ability to alter the onset, termination, duration, intensity, or temporal pattern of the event) is an important determinant of the behavioral and physiological impact of the stressor. Numerous behavioral, neurochemical, hormonal, and immunological changes follow exposure to a stressor if it is uncontrollable, but not if the identical stressor is controllable. Effects such as these which depend on the uncontrollability of a stressor have been called "learned helplessness effects". They have played an important role in psychological theory, in understanding the physiology of stress, in understanding the environmental regulation of endogenous pain modulation mechanisms and immune function, and have been proposed to be involved in the etiology of numerous human disorders. Depression, anxiety disorders, and post- traumatic stress disorder are but examples. Despite the seeming importance of stressor controllability and the considerable amount of research that has been directed at explaining its operation, there is still no adequate general explanation at either the behavioral or physiological level. The present proposal develops an integrated behavioral and physiological explanation of learned helplessness effects. It also gives special attention to an exploration of the proximate causes of the behavioral changes at the time of test. At a behavioral level the critical issue to be tested is whether uncontrollable stressors might produce intense "anxiety" that dissipates over a 3-5 day period, and whether some of all of the behavioral sequelae of IS might reflect this state of anxiety. At a physiological level the proposed research will explore the possibility that this state of anxiety involves a temporarily hyper-responsive serotonergic (5-HT) system originating from the dorsal raphe nucleus (DRN). The proposed research will also determine the cause of this hyper-responsivity, with a focus being on GABAergic mechanisms. Finally it will explore whether the diverse behavioral outcomes of exposure to uncontrollable stressors are produced by the 5-HT projections of the DRN.

DESCRIPTION (Adapted from applicant's abstract): Despite the growing body of research concerning interactions between stress and immunity, there is relatively little understanding of the neural, endocrine, and immunologic mechanisms by which an environmental stressor ultimately alters some measure of immune function. Stressors do not have a pathway directly to immune cells and organs. Rather, they alter neural activity, which in turn regulates neuronendocrine and autonomic processes, which in turn impact on organs andcells of the immune system. During the previous grant period the research has indicated that a variety of stressors interfere with the generation of antibody to an antigen (Ag). The research further suggested a hypothesis to explain these findings. The hypothesis is that stressors can induce IL-1b in brain which both downregulates Type I glucocorticoid receptor function in the hippocampus and in addition leads to an outflow of products from brain that activate a classic acute phase response in the periphery. Thus macrophages became activated, the liver shifts towards the production of acute phase proteins (positive reactants) and away from albumin and carrier proteins (negative reactants), etc. The downregulation of Type I glucocorticoid receptors in the hippocampus elevates basal levels of plasma corticosteroids. This effect, in combination with the reduced corticosteroid binding globulin (a carrier protin) production by the liver, leads to large increases in free corticosterone for a 2-3 day period following IS, which in turn produces a reduction in the Th1-like "subpopulation" of T-helper cell during the time period in which Ag-specific T abd B cells develop (4 days after Ag and IS). The activated macrophage also is seen as suppressing T cell function. Thus developing T and B-cells receive insufficient Th cytokines to develop adequately, and this is hypothesized to be the ultimate cause of the eventual reduction in Ig to Ag. The proposed research is designed to test each step of this hypothesis.

DESCRIPTION (Adapted from the Applicant's Abstract): Dr. Maier plans an extensive and broad research program which will attempt to provide an integrated understanding of how stress impacts on measures of the immune system at the behavioral, physiological and immunological levels. Two stressors, inescapable shock and defeat, will be utilized and in vivo changes in immune function will be studied. The first set of studies will focus on the behavioral factors which are important in determining whether and how stressor exposure will alter the development of antibodies and antibody (Ab) secreting cells to the antigen (Ag) KLH. The research will focus on behavioral controllability of the stressor and dominance. The second set of studies will focus on the neural and neuroendocrine mediators by which the stressors alter Ab levels. This will include noradrenergic processes associated with the paraventricular nucleus (PVN). The applicant hypothesizes that activation of this area is central to the alteration of Ab levels to Ag administered before the stressor. In addition, he will consider peripheral neuroendocrine mediators and focus on corticosteroids and catecholamines. The third set of studies are focussed on on immunology with a consideration of neural and neuroendocrine effects of the stressors on alterations in cell trafficking between immune compartments leading to changes in cell types in the lymphoid compartments which in turn lead to Ab alterations. These studies will involve the use of about 2,000 male Holtzman Sprague-Dawley rats per year.

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of the research program has been to understand "stress"--what is it, what modulates it, and how it impacts different levels of organismic functioning. All of the seemingly diverse work of the PI's laboratory flows from this fundamental interest. The PI's early work was behavioral. He gradually shifted to an interest in neural mechanisms, and more recently has added work on endocrine and immune consequences of exposure to stressors. He now studies behavioral, neurochemical, neuroendocrine, and immune changes induced by stressors with the long-term goal of understanding how these different levels communicate, interact, determine, and modulate each other. There are numerous studies of stress at each of these levels of function, but relatively few multidisciplinary efforts to understand how the levels are integrated. His long-term goal is to arrive at such an integrated understanding. The specific work of the next 5 years will focus on 3 major issues: 1) neural, endocrine, and immune mechanisms by which stressors modulate in vivo immune function; 2) the role of peripheral vagus nerve in mediating cytokine-to-brain communication; 3) the neural mechanisms which mediate the behavioral consequences of stressors; and 4) the role of central nervous system cytokines in integrating these phenomena.

The degree of behavioral control which an organism has over a stressor (ability to alter the onset, termination, duration, intensity, or temporal pattern of the event) is an important determinant of the behavioral and physiological impact of the stressor. Numerous behavioral, neurochemical, hormonal, and immunological changes follow exposure to a stressor if it is uncontrollable, but not if the identical stressor is controllable. Effects such as these which depend on the uncontrollability of a stressor have been called "learned helplessness effects." They have played an important role in psychological theory, in understanding the physiology of stress, in understanding the environmental regulation of endogenous pain modulation mechanisms and immune function, and have been proposed to be involved in the etiology of numerous human disorders. Depression, anxiety disorders, and post-traumatic stress disorder are but examples. Despite the seeming importance of stressor controllability and the considerable amount of research that has ben directed at explaining its operation, there is still no adequate general explanation at either the behavioral or physiological level. The present proposal is designed to directly test an integrated behavioral and physiological explanation of learned helplessness effects that has emerged during the previous grant period. This hypothesis includes both a description of the immediate neurochemical effects of uncontrollable stressors as well as a putative set of mechanisms for how the behavioral sequalae of such stressors are produced. At a behavioral level the critical argument is that uncontrollable stressors produce intense "anxiety" that dissipates over a 3-5 day period, and that many of the behavioral consequences of these stressors reflect this state of anxiety. At a physiological level it is argued that this state of anxiety involves a temporarily hyper-responsive serotonergic (5-HT) system originating from the dorsal raphe nucleus (DRN) and that the diverse behavioral outcomes of exposure to uncontrollable stressors are produced by excessive 5-HT released in projections of the DRN.

[unreadable] DESCRIPTION (provided by applicant): Stress is a major factor that interacts with genetic makeup & drug experience to determine vulnerability to addiction & relapse. The degree of behavioral control that an organism (rat to human) has over a stressor is arguably the most potent modulator of the behavioral & physiological impact of stressors yet discovered, & a lack of control over stressors has been found to be a predisposing factor to addiction at the human level. The research to be conducted focuses on the role of behavioral control & lack of control over stress in determining the impact of stress on behavioral and neurochemical responses tro drugs of abuse. Uncontrollable, relative to controllable stress, has been shown to sensitize serotonergic (5-HT) neurons within the dorsal raphe nucleus (DRN) so that for a period of days these neurons respond to input in an exaggerated fashion, releasing excessive amounts of 5-HT in projection regions. Research conducted during the past grant period has shown that uncontrollable stress potentiates morphine-induced conditioned place preference, psychomotor responses, nucleus accumbens dopamine release, and plasma corticosterone (CORT) for a period of many days, whereas exactly equal controllable stress does not. Furthermore, uncontrollable stress-induced sensitization of DRN 5-HT neurons & release of excessive 5-HT was shown to be necessary to produce these phenomena, as was the potentiated CORT increase. Moreover, preliminary data suggest that it is 5-HT released in the medial prefrontal cortex (mPFC) that is critical to the augmented behavioral and neurochemical responses to morphine produced by prior uncontrollable stress. The Aims of the new proposed work are to determine a) whether 5-HT and CORT act within the mPFC to produce exaggerated responses to morphine & how this occurs; b) whether uncontrollable stress would potentiate behavioral & neurochemical responses to drugs other than opiates if they are experienced in a way that activates DRN 5-HT neurons; and c) how control over stress prevents stressors from increasing reactivity to drugs of abuse & confers resilience. This research should provide insights into the neural mechanisms that produce vulnerability & resilience to the modulatory effects of stress on reactions to drugs of abuse & suggest methods to combat the deleterious effects of stress. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Gradual cognitive decline does develop with senescence. However, it has frequently been noted that cognitive declines in older individuals often occur precipitously, & that these drops are typically preceded by events (surgery, viral or bacterial infection, injury) that involve peripheral inflammation/innate immune cell activation. Importantly, even when there is recovery from such declines, their occurrence is a predisposing factor to the development of long-term dementia. The cause(s) of this type of aging-related cognitive decline are unknown, & the long-term goal is to understand the mechanisms involved & develop appropriate therapies. During the past grant period we validated an animal model of this process, developed a mechanistic set of hypotheses to account for this phenomenon, & provided preliminary evidence in support. Work conducted during the past grant period & the further work here proposed is directed at understanding this phenomenon & its causes, as well as the discovery of therapeutic interventions. The hypothesis that has been developed involves several steps: 1) Peripheral inflammatory events signal the brain. 2) Microglia in specific brain regions become activated as part of the cascade of events in the brain induced by the I am sick/injured signal from the periphery, & the microglia produce inflammatory mediators, such as interleukin-1 (IL-1). Thus, peripheral inflammation leads to neuroinflammation. 3) Inflammatory mediators, particularly IL-1, can interfere with neural plasticity (e.g., long-term potentiation, LP) in regions such as the hippocampus, & therefore disrupt processes such as hippocampal long-term memory formation. IL-1 can do so directly & by interfering with other processes known to be critical for neural plasticity & memory formation. We have, & continue to test the hypothesis that large & prolonged elevations of IL-1 interfere with brain derived neurotrophic factor (BDNF) transcription & post-translational processing, & BDNF is well accepted as a critical mediator of synaptic plasticity & memory. 4) Aging primes or sensitizes microglia. This is the key assertion with regard to aging. During neurodegenerative disease microglia are overtly inflammatory in that their phenotype has shifted to ongoing production of inflammatory molecules. During pre-senescent aging, microglia show upregulated markers of activation, but they do not typically produce increased ongoing levels of inflammatory products such as IL-1. However, if stimulated they produce exaggerated quantities of inflammatory products, & do so for prolonged periods. 5) Thus, peripheral inflammation should lead to an exaggerated neuroinflammatory response in aging individuals, & we have demonstrated that this is the case. 6) The exaggerated amount & duration of the brain IL-1 increase in aging subjects during peripheral inflammation should interfere with cognitive processes such as memory for a prolonged period of time. Here these will all be tested. Here we explore the nature of microglia sensitization with age, its causes, and its cures. We also determine whether these cures prevent IL-1, BDNF, and memory deterioration.

DESCRIPTION (provided by applicant): RELEVANCE TO PUBLIC HEALTH: The experience of stressful events is an etiological factor in the development of numerous mental disorders, yet some individuals are resilient in the face of adversity. An understanding of factors that promote resilience is critical to the development of more effective therapies. An extensive literature suggests that coping processes are key, and that the perceived ability to exert control over negative circumstances is a key coping process. The research to be conducted is directed at understanding the neural mechanisms that produce the stress-buffering effects of control/coping. The degree of behavioral control that an organism has over a stressor modulates the behavioral and physiological impact of a stressor, with uncontrollable stressors producing sequelae that do not occur following physically identical controllable stressors. In addition, an initial experience with controllable stress blocks later uncontrollable stress from exerting its usual effects ("behavioral immunization".) Prior research has focused on understanding the mechanisms by which uncontrollable stress produces its effects, but here the focus will be on understanding how it is that having control prevents stressors from producing negative outcomes. The basic hypothesis to be explored is that a) the presence of control activates medial prefrontal cortex (mPFC) inhibition of brainstem (dorsal raphe nucleus) and limbic (amygdala) structures that are activated by stressors and that play important roles in mediaitign the effects of stress., and b) the experience of controlling a stressor "ties" mPFC activation to the stressor, or some aspect of the stress experience (e.g., fear), so that the later occurrence of stressors, even if they are uncontrollable, now activates the mPFC and thus inhibition of brainstem and limbic structures nortmally activated by uncontrolalble stress. This process is expected to provide protection and produce resilience. This overall rationale leads to 3 Specific Aims. SPECIFIC AIM I: How does a prior experience with controllable stress (escapable shock, ES) block the subsequent behavioral impact of uncontrollable stress (inescapable shock, IS)? SPECIFIC AIM II: Are the protective effects of prior ES general, so that the impact of stressors other than tailshock is blunted? Are the protective effects of mPFC activation general, so that mPFC activation during defeat rather than IS will also be protective SPECIFIC AIM III: Do the resilience-producing effects of ES extend beyond behaviors &processes regulated by the DRN (e.g.. escape behavior) to processes regulated by other structures also innervated bv the PFC?

DESCRIPTION (provided by applicant): This application addresses Challenge Area 15 (Translational Science). Specific Challenge Topic 15-MH-109 Prefrontal cortex regulation of higher brain functions and complex behavior. There is growing emphasis on understanding what might produce resilience to the behavioral and neural impact of stress, and coping factors are thought to play a pivotal role. Behavioral control over the stressor is a key aspect of coping, and the presence of control not only blunts the effects of the stressor being controlled (Maier et al., 2006), but also reduces and even eliminates the behavioral and neurochemical changes produced by subsequent stressors, even if the subsequent stressor is not behaviorally controllable (Amat et al.,2006). That is, the experience of controlling a potent acute stressor appears to "immunize" the organism against the effects of later (acute) stress. Importantly, the ventral medial prefrontal cortex (vmPFC) has been shown to mediate both the immediate and proactive protective effects of behavioral control. The presence of control has been shown to activate top-down vmPFC inhibitory control over stress-responsive brainstem and limbic structures, thereby reducing the neurochemical impact of the stressor, as well as the behavioral sequelae of the activation of these structures (Amat et al.,2005). Furthermore, the experience of control over a potent stressor alters the vmPFC in such a way that subsequent stressors that are uncontrollable now activate vmPFC inhibitory control, thereby leading to protection against subsequent stressor exposure (Amat et al., 2006;Baratta et al, 2008). The research that has explored the resilience-inducing properties of control, as well as the role of the vmPFC in stress-resilience, has focused entirely on acute stress exposure. However, it is chronic stress that is most often implicated as an etiological factor in the development of a broad spectrum of psychiatric and somatic disorders. There has been virtually no study of experiential factors and mechanisms that might be important in producing resistance to the behavioral and neurochemical impact of chronic stress, and the present proposal is addressed at these issues. The research determines whether a) an experience with control over an acute stressor will prevent and/or reverse the impact of a chronic social stressor, adult social isolation, and b) the vmPFC is a critical mediator of these protective effects. Chronic stress is an etiological factor in the development of numerous disorders. Not all individuals exposed to chronic stress are severely impacted, and an understanding of factors that make individuals resilient are likely to lead to new treatments. The proposed research will determine whether a) the experience of behavioral control over a potent acute stressor before exposure to a different chronic stressor will prevent, and/or the experience of control after a chronic stressor reverse, the behavioral and neurochemical effects of the chronic stressor, and b) the protection is mediated by activation of the medial prefrontal cortex by control.

DESCRIPTION (provided by applicant): While there has been increasing recognition of the importance of microglial and astrocyte activation in the creation & maintenance of diverse enhanced pain states, an important aspect of glial functioning that has not yet been explored in the context of pain enhancement is the effect of a sensitized, or primed, microglial response. Evidence has accrued from outside of the pain field that the past history of microglial activation can dramatically alter their response to new challenges. Microglia can reach a primed state via a variety of challenges, including peripheral or central trauma/inflammation, stress, prior pain, and exposure to opioids, which strikingly are known co-morbidities for the transition of acute to chronic pain, including neuropathic pain. While in such a primed state, microglia now dramatically over-respond to new challenges, stronger and longer than before. We believe such prior challenges that result in glial priming can set the stage for the transition of acute to chronic pain following peripheral & central neural damage, resulting in chronic neuropathic pain. Re-activation of primed microglia may lead to a transition from acute pain to chronic pain as a result of a neuroinflammatory response that is greatly amplified in both magnitude and duration. Goals. (1) In accordance with the specified goals of this RFA, develop new rat models to study the transition from acute to chronic neuropathic pain, based on the premise that a first challenge (Hit 1: peripheral or central trauma/inflammation, stress, prior pain, exposure to opioids) will markedly enhance pain induced by a subsequent (second) challenge (Hit 2: peripheral or central neural inflammation/injury). (2) Utilize the refined robust models to test the potential of non-opioid, non-addictive blood-brain barrier permeable glial activation inhibitors & resolvins to prevent the transition of acute to chronic pain. (3) Given the remarkably powerful positive effects produced by chronic voluntary exercise in creating resiliency to a multitude of negative outcomes (including constraining glial/immune reactivity), chronic voluntary exercise will also be tested for its ability to prevent the transition from acute to chronic pain, an approach enabled by teaming with an expert from outside the pain field (M. Fleshner). (4) Discover intracellular changes that differentiate rats which do vs. do not transition from acute to chronic neuropathic pain, & define how these potential cellular markers of impending chronic pain are affected by successful interventions (glial inhibitors, voluntary exercise). This will lay the groundwork for identifying and targeting changes reliably predictive of the transition of acute to chronic neuropathic pain.

DESCRIPTION (provided by applicant): The present proposal explores a novel conceptualization of glucocorticoid (GC) action in the stress response during a fight/flight emergency. This is that stress-induced GCs function to alert the organism's CNS innate immune system to potential danger signals that include 1) exogenous pathogens and/or pathogen associated molecular patterns (PAMPs), & 2) endogenous danger signals or alarmins, which can be released in response to a wide array of stimuli including sterile injury. Here, GCs prime or sensitize the CNS innate immune/inflammatory response to subsequent stimulation, such as occurs upon exposure to infectious agents or injury. The idea is that stress-induced GCs can function as an endocrine warning signal to the CNS innate immune system to induce a preparatory response (microglial sensitization) to subsequent proinflammatory stimuli. Neuroinflammatory processes are a focus here given the emerging roles of CNS pro-inflammatory cytokines in the etiology of psychiatric disorders in which stress & GCs are key etiologic factors. The primary objectives are: 1) to explore how stress sensitizes microglia to pro-inflammatory challenges & 2) examine whether GCs mediate stress-induced sensitization of microglia. The general hypotheses to be tested are that A) stress sensitizes microglia to pro-inflammatory stimuli by upregulating pattern recognition receptors (PRRs) on microglia. The PRRs Toll-Like receptor (TLR) 2 and/or 4 transduce the pro-inflammatory cytokine effects of endogenous danger signals. We propose that stress primes microglia by upregulating TLR 2 and/or 4 on microglia for a period of days post-stress. Subsequently, upregulated TLRs expressed by microglia sense endogenous danger signals released upon exposure to a pro- inflammatory stimulus. B) TLR mediation requires that the later pro-inflammatory stimulus increase an endogenous molecule in the brain that acts at the TLRs. High Mobility Group Box1 (HMGB1), an alarmin released in response to danger, is an endogenous protein in the CNS that acts at TLRs & we suggest that HMGB1 is increased by a pro-inflammatory stimulus & then acts at the stress-upregulated TLRs, thereby inducing a potentiated pro-inflammatory cytokine response. C) GCs mediate the stress-induced sensitization of microglia (i.e. upregulation of TLR 2/4). Clinically, stress and neuroinflammatory processes are emerging as important factors in the etiology of psychiatric disorders. Also, an extensive body of evidence implicates GCs in the etiology of such disorders. The proposed work may provide valuable insight into how stress/GCs regulate neuroinflammatory processes in psychiatric disorders. Thus, the present research may lead to a reconceptualization of stress/GCs as neuroinflammatory predisposing factors in the etiology of psychiatric disorders (i.e. major depression, PTSD).

? DESCRIPTION (provided by applicant): The experience of adverse events is an etiological factor in the development of numerous mental disorders, yet some individuals are resilient in the face of adversity. An understanding of factors that promote resilience is critical to the development of more effective therapies. Human studies have suggested that coping factors are key, and that the actual or perceived ability to exert control over adverse events is an important coping process. The degree of behavioral control that an organism has over a stressor potently modulates the impact of the stressor, with uncontrollable stressors producing neurochemical and behavioral sequelae that do not occur if the stressor is controllable. Furthermore, an initial experience of controllable stress protects or immunizes against the typical negative outcomes of subsequent uncontrollable stress. The research to be conducted in this proposal will be directed at understanding the neural mechanisms that mediate the stress-buffering effects of coping/control. Prior work has identified a role for the medial prefrontal cortex (mPFC) as a structure that is sensitive to the dimension of control and here we will use genetic targeting strategies for monitoring and subsequently manipulating subcircuits embedded within the mPFC that are critical for immunization. Specific Aim I will determine if mPFC cells that are activated by an initial experience of control are also engaged during later adversity. Specific Aim II will determine if the mPFC is involved in mediating the effects of control because it participates in the corticostriatal action-outcome system, a circuit thought to be important for organisms to encode and use contingency information about the relationship between its actions and outcomes. The proposed research is the first of its kind, and will lay the groundwork for understanding how mPFC activity during coping behavior produces resilience to future challenge.

? DESCRIPTION (provided by applicant): Neuroinflammation is now regarded to be a contributing factor in the etiology of a wide range of psychiatric disorders and a potential mediator of the extensive co-morbidities that are present between these disorders. It has been suggested that psychiatric disorders may result when normal neuroinflammatory responses to stimuli that induce these responses become exaggerated. The experience of both acute and more chronic stressors also are associated with the development of a number of disorders. It has been tempting to suppose that these two processes are related, but neither acute nor chronic stress have proved to produce persistent neuroinflammation beyond the stressor exposure. However, we have recently found that both acute and chronic stressors, even though they do not produce either large or long-lasting neuroinflammation, potently exaggerate neuroinflammatory responses to both peripheral and central inflammatory stimuli that are administered later. Importantly, this sensitized neuroinflammatory reaction persists for many days after stressor exposure. However, the mechanisms that lead stressors to sensitize subsequent neuroinflammation remain largely unknown. Within the past decade there has been a revolution in understanding the mechanisms involved in mediating peripheral innate immunity/inflammation. These new mechanisms and processes have been studied almost exclusively in the periphery, and whether or not they occur in the CNS is unknown. Our Preliminary Studies strongly encourage the possibility that these are present in CNS innate immune cells (microglia) and that they are involved in mediating stress-induced sensitization of neuroinflammatory responses to subsequent inflammatory challenges. The global goals of the proposed research are to a) firmly establish the presence of these processes, heretofore unstudied in brain, in brain, and b) explore the role of these processes in stress- induced sensitization of neuroinflammation, as well as the behavioral changes that typically induced by the activation of innate immune cells in the brain.

Project Summary/Abstract The degree of behavioral control that an organism (rodent to human) can exert over an adverse event is one of the most potent variables yet discovered that modulates the behavioral and neurochemical impact of that event. When the organism does have an element of control, the behavioral and neurochemical sequelae of the adverse event are blunted or eliminated. Importantly, the experience of control not only blunts the impact of the stressor being controlled, but also blunts the impact of stressors experienced much later (at least two months in rat), that is, control produces future resilience in the face of adversity. The research to be conducted in this proposal will be directed at investigating the neural circuits by which the prefrontal cortex mediates the stress-buffering effects of coping/control. Specific Aims will examine the precise prefrontal circuits involved in the separable features of behavioral control: (a) the detection of control and (b) the subsequent use of that information to regulate stress-responsive systems accordingly. In addition, over the last grant period we have extended the study of controllability phenomena to females, and surprisingly, here control does not blunt the impact of stress. The proposed research focuses on the roles of specific prefrontal cortex circuits in mediating the effects of control on stress resilience, as well as a determination of exactly how critical prefrontal circuits may respond to stressors differently in males and females.

Project Summary Immunoregulation, indicated by a balanced expansion of effector T-cell populations and regulatory T cells (Treg), is known to be driven by microbial signals, mainly by organisms with which mammals coevolved, including: (i) the commensal microbiota, which have been altered by the Western lifestyle, including a diet that is commonly low in microbiota-accessible carbohydrates; (ii), pathogens associated with the ?old infections? that were present throughout life in evolving human hunter-gatherer populations; and (iii) organisms from the natural environment with which humans were inevitably in daily contact (and so had to be tolerated by the immune system). Immunoregulation is thought to be compromised in modern high-income settings due to reduced contact with these three categories of organisms. A failure of immunoregulation, attributable to reduced exposure to the microbial environment within which the mammalian immune system evolved, is thought to be one factor contributing to recent increases in stress-related and chronic inflammatory disorders in high-income countries. Immunization with one of these ?old friends?, in the form of a heat-killed preparation of Mycobacterium vaccae, a nonpathogenic, environmental saprophyte with anti-inflammatory and immunoregulatory properties, has been shown to increase stress resilience in mice, as measured by prevention of stress-induced increases in anxiety, prevention of stress-induced exaggeration of spontaneous colitis and prevention of chemically induced colitis in a model of inflammatory bowel disease. However, the mechanisms through which M. vaccae mediates is stress protective effects on behavior, at the level of the central nervous system, are not known. Our preliminary data demonstrate that immunization with M. vaccae increases expression of the anti-inflammatory cytokine, interleukin (IL)-4, in the rat brain, prevents stress- induced priming of hippocampal microglia, and prevents stress-induced decreases in juvenile social investigation in rats. In this R21 proposal, we propose to determine, using male and female rats, if the stress- protective effects of immunization with M. vaccae, as measured by prevention of inescapable stress (IS)- induced increases in anxiety, conditioned fear, and escape deficits, and prevention of priming of hippocampal microglia, are dependent on increases in IL-4. We will also determine, for the first time, if IL-4 is sufficient to protect against IS-induced behavioral deficits. Finally, we will determine if the stress-protective effects of immunization with M. vaccae involve inhibition of specific neural circuits involved in mediating the behavioral sequelae of IS, including locus coeruleus, lateral habenula, and bed nucleus of the stria terminalis projections to the serotonergic dorsal raphe nucleus. Together, the proposed studies will provide the first detailed investigation of neural mechanisms through which immunization with M. vaccae promotes stress resilience.

Project Summary/Abstract The degree of behavioral control that an organism (rodent to human) can exert over an adverse event is arguably the most potent variable yet discovered that modulates the behavioral and neurochemical impact of that event. When the organism does have an element of control, the behavioral and neurochemical sequelae of the adverse event are blunted or eliminated. Importantly, the experience of control not only blunts the impact of the stressor being controlled, but also blunts the impact of stressors experienced much later (at least one month), that is, control produces future resilience in the face of adversity. Recent research has extended the study of the impact of experiencing control to determining whether future fear processes might be altered. Indeed, the experience of control reduces future fear conditioning, facilitates future fear extinction and prevents the spontaneous recovery of fear. In addition, we have extended the study of controllability phenomena to females, and surprisingly, here control does not blunt the impact of stressors or alter later fear conditioning. The proposed research focuses on the roles of specific prefrontal cortex circuits in mediating the effects of control on fear processes, as well as a determination of exactly how critical prefrontal circuits may respond to stressors differently in males and females.