Seema Bhatnagar - US grants

Chronic or repeated exposure to stress produces plasticity in a number of neural systems, including those that regulate the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis has widespread effects on physiology and behavior, including modulation of metabolism and cardiovascular function. Changes in basal activity as well as subsequent stress-induced HPA activity are seen in animals exposed to repeated stress such as cold or restraint. This project uses a more naturalistic model of stress in rodents, that of repeated exposure to social defeat. Defeat in the context of social environment, including which individual is a resident or an intruder in the local area, is a familiar situation in the natural life of many social rodents such as rats. Although a single defeat produces neuroendocrine and behavioral effects that last a few weeks, it is more common in the wild to have an individual animal regularly subjected to defeat. Therefore, repeated defeat in rats represents a potentially powerful model for the study of neural plasticity associated with chronic, repeated stress. Using a novel combination of molecular biology and behavior, the goals of this project are to 1) characterize how basal and stress-induced HPA activity change in rats subject to repeated defeat; 2) study the specific role of the paraventricular thalamus, a brain region that inhibits HPA responses in other stress situations, in the repeated defeat paradigm; and 3) identify novel brain regions in which activity is increased by repeated defeat, since these are potential sites of regulation of HPA activity.
Results will be important for identifying novel brain circuitry involved in the modulation of behavior, and with an impact on our understanding more about the role of social versus physical stress, and about the evolution of sociality. This project also fosters the career of a new investigator who combines an unusual background in molecular biology and behavior.

DESCRIPTION (provided by applicant): Chronic exposure to stress in the form of major adverse life events is associated with the development of disorders such as depression, anxiety and post-traumatic stress disorder and chronic fatigue syndrome. Changes in activity within the hypothalamic-pituitary-adrenal (HPA) axis are important features of these disorders and likely reflect plasticity in brain circuitry that coordinates these neuroendocrine responses with behavioral and autonomic function. Animals undergoing chronic stress exhibit many of the neuroendocrine autonomic and behavioral changes seen in individuals with disease. Using HPA activity as our primary endpoint, we have identified the posterior division of the paraventricular nucleus of the thalamus (pPVTh) as a critical mediator of HPA responses in chronically stressed rats though it does not seem to be functionally active in rats exposed to acute stress. Therefore, the pPVTh seems to control HPA activity specifically within the context of prior stress experience. In this proposal, we seek to characterize the neural circuits that mediate the primarily inhibitory effects of the pPVTh on HPA activity. The efferent projections of the pPVTh are limited and are primarily to limbic structures including the amygdala, prefrontal cortex and bed nucleus of the stria terminalis but also to a hypothalamic region that can more directly control HPA activity. Our general hypothesis is that the pPVTh exerts its influence through changing activity in limbic structures but not hypothalamic structures since limbic regions are more capable of evaluating sensory information within the context of past stress history. More specifically, we will determine whether the pPVTh can exert its inhibitory influence on HPA activity by acting on limbic GABA-ergic systems (Aim 1) and/or by serving as a site of negative feedback effects of glucocorticoids released by the chronic stress exposure (Aim 2). Aim 3 focuses on the pathways through which cholecystokinin released within the pPVTh alters HPA activity specifically in chronically stressed rats and Aim 4 will examine how central CRF systems interact with the pPVTh and its associated limbic circuitry. Given the specificity of pPVTh effects to the chronic stress state, characterizing this pPVTh-limbic circuitry is fundamental to understanding the association between chronic stress and changes in physiology and behavior that can lead to disease.

DESCRIPTION (provided by applicant): Gender differences in rates of affective and anxiety-related disorders are well-established and these differences emerge in adolescence. In both adolescents and adults, there is a strong link between depression and stressful life events with a stressful life event often preceding an episode of depression, for example. So, it is not surprising that the maturation and sexual differentiation of stress-responsive systems such as the locus coeruleus-norepinephrine (LC-NE) and hypothalamic-pituitary-adrenal (HPA) axis coincides with the emergence of gender differences in rates of affective and anxiety disorders in adolescence. Adolescent girls are more sensitive to peer stress and this increased sensitivity may make females more vulnerable to developing affective disorders as they age. However, little is known about how social stressors in adolescence can impact stress-regulatory systems that are altered in affective and anxiety disorders. In preliminary data, we observed that adult female rats exhibited neuroendocrine hyperresponsiveness to stress in adulthood after adolescent social isolation and there were concomitant changes in specific neural substrates. These data suggest that social isolation during adolescence has enduring and sex-specific effects on adult stress reactivity and on stress-related neural substrates. Here, we propose to study the effects of adolescent social isolation stress on adult LC-NE function in female rats. The LC-NE system mediates arousal and attentional processes that are fundamentally disrupted in affective and anxiety disorders. Therefore, an enduring effect of adolescent stress on this system could contribute to psychiatric disorders that are prevalent in females in adulthood. The central hypothesis of this application is that the stress of social isolation during adolescence will produce an enduring change in the LC-NE system such that it is tonically activated (Specific Aim 1) and/or hyperresponsive to stress (Specific Aim 2) in adult female rats. In these Specific Aims, we will record LC activity in unanesthetized freely moving adult female rats. We further hypothesize a key role for CRF in regulating LC activity. In Specific Aim 3, we will examine CRF mRNA in brain regions providing CRF afferent inputs to the LC and CRF receptor protein in the LC to determine how isolation during adolescence changes CRF afferents and receptors in the LC in adult females. Thus, the proposed experiments will identify potential mechanisms by which LC responsivity to stress are regulated by events in adolescence in females thereby enhancing our understanding of the neural mechanisms underlying arousal and attentional processes that are key components of stress-related psychiatric illness that are exhibited in higher rates by females. PUBLIC HEALTH RELEVANCE: Women have higher rates of depression and anxiety than men and these higher rates are first seen in adolescence and both depression and anxiety can be precipitated by stress. In this application, we will use female rats to examine how social stress in adolescence changes activity and function in a neural system that has been implicated in stress-related affective disorders. The results of these studies will help our understanding of the relationship between stress and depression and anxiety in women.

Project Summary/Abstract Social stress has adverse consequences for physical and mental health throughout life and its impact may be particularly relevant during adolescence as this is a time of substantial growth and reorganization of brain circuits. Social stress also has a differential impact in females compared to males though most research has focused on males. The proposed research uses the resident intruder model of social stress that we refined to understand how individuals cope during repeated social stress and how this determines individual consequences of repeated social stress for behaviors associated with depression and impaired cognitive function. Our research focuses on the locus coeruleus (LC) norepinephrine system, a major stress response system that mediates arousal and cognition in response to stress. The working hypothesis that frames this research is that individual differences in stress vulnerability result from differences in the circuitry that is engaged to regulate LC activity during stress. Specifically, excitatory regulation of the LC by corticotropin-releasing factor (CRF) and orexinergic afferents is hypothesized to underlie a passive coping style and vulnerability to stress-related disorders that are characterized by hyperarousal. In contrast, inhibitory enkephalin inputs to the LC are counterregulatory and are associated with a more active coping strategy and decreased vulnerability to those disorders. We hypothesize that LC-activating inputs and LC- inhibiting inputs are differentially altered in development and between sexes. This hypothesis is tested in 3 specific aims using technical approaches that include cellular measurements of receptor expression and trafficking, in vivo electrophysiology in behaving rats, chemogenetic manipulation of circuits using DREADDs and assessment of cognitive function and behavior. Aim 1 will identify stress-induced plasticity in circuits that regulate LC activity and how this is dependent on age and sex. Aim 2 will use DREADDs and neuropharmacological techniques to directly test the role of LC activity and its CRF, orexin A and enkephalin afferents in coping strategy in response to social stress. Aim 3 will test the role of the LC and its afferents on the pathological consequences of social defeat, including anhedonia, morphine conditioned place preference and cognitive function in the attention set-shifting test. Because coping strategy is an important determinant of stress resilience, the results of the proposed studies will significantly increase our understanding of how coping strategy can optimize activity of the LC-NE arousal system. The results will inform therapies designed to modify coping strategy in an effort to promote resilience and whether these therapies should be different depending on age and on sex.  

This workshop on the Neurobiology of Stress, to be held in Philadelphia, PA June 12-15th, brings together researchers (principal investigators and trainees) who study stress-brain interactions and the basic neurobiology of these interactions. In recent years there have been significant advances in the neurobiology of stress research. Those advances include new understanding of the neurosystems, neurochemistry, and cellular biology that supports stress responses and stress adaptation. Session topics and speakers for this Workshop have been selected to cover many of those exciting new advances. The Workshop will provide an important forum where new advances can be critically evaluated and integrated into the collective knowledge and perspectives of active basic stress researchers. Moreover, it is expected that the intensive scientific exchange will generate new ideas and collaborations that support future advances. There are significant broader impacts that will emerge from this meeting. The results will be widely disseminated in two premier journals. Assessment tools will be developed to determine the extent to which scientific exchange occurred, to assess the value of the scientific knowledge and content acquired, and any resultant collaborations or exchanges. These multifactorial tools can be broadly disseminated to organizers of other conferences. Finally, multiple opportunities exist for trainees to become engaged in the scientific program, including a specific plan to recruit female and minority students to attend the meeting. A significant portion of the requested funds will be used to fund meritorious trainees. This will help expand diversity and encourage trainees to stay in science careers and this is important for generating new ideas and new approaches towards solving problems that have plagued American society for decades.

DESCRIPTION (provided by applicant): Chronic exposure to stress in the form of major life events such as bereavement, prolonged conflict or low socioeconomic status is associated with increased incidence of depression, post-traumatic stress disorder and chronic fatigue syndrome. However, some individuals are resilient to the effects of stress while others are more vulnerable. Identifying the substrates underlying resilience and/or vulnerability could lead to novel individualized treatments for enhancing resilience or mitigating vulnerability. Our preliminary work has identified a model of repeated social defeat in adult male rats in which two distinct subpopulations emerge with different coping strategies, one that is resilient and one that is vulnerable to the behavioral and neuroendocrine consequences of repeated social defeat. Our preliminary data also show that these two subpopulations differ in the expression of orexins, peptides that are key for arousal, wakefulness and vigilance. The resilient population exhibits lower orexin expression. These and other data lead to the central hypothesis that dampened orexin system function is associated with resilience to the effects of repeated defeat. Two Specific Aims are proposed to test the central hypothesis. In Specific Aim 1, we will determine the effects of inhibition or stimulation of orexin release on behavioral and neuroendocrine outcomes produced by social defeat in the vulnerable and resilient populations. We hypothesize that inhibition of orexin release during defeat will decrease anxiety- and depressive-like behaviors and alter neuroendocrine function shifting the vulnerable subpopulation towards a resilient phenotype. Specific Aim 2 will determine the potential sites of actions of orexins using combination of functional neuroanatomical and whole cell electrophysiological approaches. To modulate orexin release, an emerging technology, DREADDs (designer receptors exclusively activated or inhibited by designer drugs) will be used. DREADDs is a directed pharmacological approach that allows minimally invasive chronic inhibition of stimulation of endogenous orexin release, preliminary data demonstrate the feasibility of this approach in our lab. Together, the results from the proposed experiments will provide novel insights into the specific involvement of orexins in resilience or vulnerability to the effects of repeated stress potentially highlighting teir key role in mediating resilience to the effects of stress.

? DESCRIPTION (provided by applicant): Stress increases the risk of psychiatric disorders such as post-traumatic stress disorders and depression. Women are twice as likely to suffer from these disorders as men. We do not fully understand the neurobiology underlying the sex difference in these stress- related disorders. The hypothalamic neuropeptides orexins are important for attention and arousal and regulate responses to stress, depression- and anxiety-related behaviors. However, little is known about sex differences in the expression of orexins or in the physiological and behavioral processes that they regulate. In preliminary studies in adult rats, females exhibited higher orexin mRNA, activation of orexin cells, and orexin concentrations in the cerebrospinal fluid compared to males under non-stressed conditions. In response to repeated restraint, females showed impaired habituation of behavioral and hypothalamic-pituitary-adrenal (HPA) responses and deficits in the cortically-mediated attention set-shifting task, a test of cognitive flexibility, compared to males. Based on these preliminary data, the central hypothesis of this application is that elevations in the expression and activation of orexins in females underlie sex differences in habituation to stress and in attention-related cognitive function. Four aims are designed to test this hypothesis at levels of analysis ranging from whole cell electrophysiology to behavior. In Specific Aim 1, we will test sex differences in orexin functions at the single cell level by using whole cell electrophysiology to assess whether differences in excitatory or inhibitory inputs to orexin neurons contribute to the observed sex differences. In Specific Aim 2, we will test whether circulating estrogens regulate orexin expression and electrophysiological activity and/or whether they drive the prepro-orexin promoter. We demonstrate sex differences in habituation of the HPA axis and struggling behavior and in cognitive performance in the cortically-mediated attentional set shifting task. In Specific Aims 3 and 4, we will test whether the higher orexin expression in females leads to these disruptions by stimulating or inhibiting orexins using DREADDs. Using multiple levels of analyses, the proposed research will provide the first comprehensive analysis of how orexins regulate sex differences in habituation and cognitive consequences of stress. With a better understanding of an important neural substrate underlying sex differences in stress responses, we may be able to develop improved, sex-specific, targets for stress-related psychiatric illnesses.

Project Summary/Abstract Stress is implicated in cognitive dysfunctions that characterize many psychiatric diseases, including depression, substance abuse and post-traumatic stress disorder. One mechanism by which stressors impact on cognition is through their engagement of monoamine systems that regulate activity of the prefrontal cortex (PFC) that governs executive functions. The locus-coeruleus (LC)-norepinephrine (NE) system, is a major stress response system that initiates arousal and modulates cognitive flexibility in response to stressors through its regulation of the PFC. Stress-induced engagement of the LC-NE system is mediated by CRF neurotransmission. It has been hypothesized that stress-induced impairments in executive function arise from excessive LC-NE drive to the medial PFC (mPFC) and resulting inhibition of mPFC function. This important hypothesis has never been directly tested. Although the activating effects of stressors and CRF on activity of single LC neurons have been well described, a major gap exists in our understanding of how these single cellular events translate to changes in cortical activity that then govern executive functions such as cognitive flexibility. To address this question, this research will quantify network activity in response to CRF or stress in a circuit linking the LC with the mPFC and the orbitofrontal cortex (OFC), PFC subregions that have been implicated in different cognitive processes. Network activity is the synchronization of neural oscillations that supports communication between brain regions and is recorded by local field potentials (LFPs). The following Aims will quantify LC-PFC network dynamics, including their strength, connectivity and directionality by recording LFPs in these regions in response to stress and CRF and determining how these effects translate to changes in cognitive flexibility. Because stress-related psychiatric diseases are more prevalent in females and female LC neurons are more sensitive to CRF compared to males, this research will also reveal the role of sex as a determinant of stress effects. Aim 1 will identify the effects of activating CRF1 or Gs-protein-related signaling in LC neurons on network activity within the LC-PFC circuit. These effects will be examined at rest and during performance of tasks that test cognitive flexibility. Aim 2 will identify the effects of acute and repeated social stress (resident-intruder stress) on LC-PFC network activity, and will dissect the role of CRF in the LC in these effects. Aim 3 will determine whether repeated social stress has enduring effects on LC-PFC network activity that translate to changes in cognitive flexibility. Together these studies take a network approach to elucidate how cellular effects of stress on LC neurons are amplified to cortical circuits to affect cognitive flexibility, an important attribute of executive function that is impaired in multiple stress-related psychiatric disorders.