Stanley Watson - US grants

The search for biological correlates of endogenous depression (ED) has been on for several years. Anomalous regulation of the hypothalamo-pituitary-adrenal (HPA) axis has been implicated using the Dexamethasone Suppression Test (DST). While there is active debate concerning the clinical usefulness of the DST, there is general agreement that some ED patients show defective HPA regulation, especially as seen in their baseline plasma cortisol and the lack of suppression after dexamethasone (DEX). In our previous funding period, we studied the pituitary component of the HPA axis in ED patients by measuring the peptide products from the ACTH/endorphin precursor. We have shown substantial pituitary regulation defects as reflected by plasma beta-endorphin (BE) assays in ED patients when compared to psychiatric controls. These studies revealed a partial concordance between BE and cortisol dysregulation. The combination of both measures after DEX reveals abnormalities in 70% of the ED patients. Based on the peptide and steroid measures we are proposing the possible existence of 4 subgroups of ED patients with different patterns of HPA dysfunction. The present proposal is aimed at testing these notions and further exploring the nature of the HPA defect. Studies on the effect of age, sex and time of testing in normals and psychiatric subjects will further explore variables contributing to the different patterns of response post DEX. Studies with CRF-induced release of ACTH and BE, metyrapone, cortisol fast feedback and biochemical characterization of the released peptides will be conducted to elucidate the locus and nature of the dysregulation. Finally, we will study patients in remission whose DST has normalized to determine if other HPA correlates reveal continuing abnormalities.

The overall purpose of this proposal is to study the biology and regulation of a brain neuropeptide system--the pro-dynorphin neurons. We have chosen two well identified circuits: the magnocellular-posterior lobe pathway which co-synthesizes dynorphin and vasopressin, and the striato-nigral pathway whereby dynorphin projects to the dopamine rich substantia nigra. These were chosen because of the availability of pharmacological manipulations to alter their level of activity, and because of their possibly contrasting biologies as neurohormonal versus neurotransmitter systems. A number of studies will be aimed at clarifying issues related to the biology of pro-dynorphin in normal animals. These include 1) the unequivocal discrimination between pro-dynorphin and pro-enkephalin neurons, using a combination of immunohistochemistry with multiple antisera and in situ hybridization with messenger-specific cDNA probes. 2) the elucidation of the processing of pro-dynorphin into its final opioid and non-opioid products using in vivo pulse labeling. 3) the description of the anatomical relationships between pro-dynorphin products and multiple opioid receptor subtypes using combined receptor autoradiography-peptide immunohistochemistry. The regulatory cell biology of the two systems in response to changes in activity or demand will be investigated using various treatments, such as salt-loading for the magnocellular system and chronic haloperidol for the striato-nigal system. The response will be investigated at the following levels: 1) the content and ratio of the pro-dynorphin end products in cell body and terminal regions. 2) their biosynthetic rate-incorporation into precursor and conversion to products. 3) release into plasma, if technically possible. 4) changes in mRNA levels using cDNA probes in the contex of dot-blots and in situ hybridization. 5) changes in affinity, number or ratios of multiple opioid receptor subtypes. These studies will increase our understanding of regulatory neuropeptide biology. Further, the pathways under study have been implicated in functions of relevance to mental health: The magnocellular pathway modulates neuroendocrine and stress responses, whereas the striatonigro-striatal loop is critical in mediating some of the effects of antiphyschotic drugs.

This program project is concerned with the study of the biology and regulation of the hypothalamo-pituitary-adrenal (HPA) axis, with particular emphasis on peptide biology. The peptides of interest are the hypothalamic neuropeptides, arginine vasopressin (AVP), pro-dynorphin and corticotropin releasing factor (CRF), and the anteror pituitary peptide family, pro- opiomelanocortin (POMC). The four projects involved in this program range from basic animal studies of the dynorphin- vasopressin systems, to clinical human studies of the HPA dysregulation in major depression. Nevertheless, these projects are closely interrelated in terms of attempting to derive a basic understanding of the regulation and dynamics of the HPA axis. This is predicated on the notion that such as improved knowledge base will be useful in elucidating the function of the peptides in this stress-related system, and the responsiveness of the various elements to changing demands, either under normal or psychopathological conditions. Specifically, Project 1 focuses on the opioid peptide family pro- dynorphin, which is co-stored with AVP in hypothalamic magnocellular neurons. Since AVP is a known ACTH/B- Endorphin secretagogue, a potential role of dynorphin in this stress-related function will be explored. In addition the posterior lobe and the anterior lobe dynorphin systems will serve as valuable models for studying its biosynthesis and regulation. Project 2 is a preclinical proposal looking at the effect of variables such as age, sex and tricyclic treatment on the cellular regulation of POMC in the anterior lobe. Project 3 is a clinical project exploring the HPA axis in major depression with basal measures and challenge studies using corticosteroids and CRF. Project 4 is a collaborative inter-university effort focused on CRF challenges in depression. The multidisciplinary approach involves the use of molecular biology immunohistochemistry, peptide measurements and characterization, biosynthesis and release studies and behavioral and pharmacological challenges. Information derived from each of the projects will be considered in the interpretation of results from the other projects. The net result should be increased insight in the biology of stress and depression.

This program project is concerned with the study of the biology and regulation of the hypothalamo-pituitary-adrenal (HPA) axis, with particular emphasis on peptide biology. The peptides of interest are the hypothalamic neuropeptides, arginine vasopressin (AVP), pro-dynorphin and corticotropin releasing factor (CRF), and the anteror pituitary peptide family, pro- opiomelanocortin (POMC). The four projects involved in this program range from basic animal studies of the dynorphin- vasopressin systems, to clinical human studies of the HPA dysregulation in major depression. Nevertheless, these projects are closely interrelated in terms of attempting to derive a basic understanding of the regulation and dynamics of the HPA axis. This is predicated on the notion that such as improved knowledge base will be useful in elucidating the function of the peptides in this stress-related system, and the responsiveness of the various elements to changing demands, either under normal or psychopathological conditions. Specifically, Project 1 focuses on the opioid peptide family pro- dynorphin, which is co-stored with AVP in hypothalamic magnocellular neurons. Since AVP is a known ACTH/B- Endorphin secretagogue, a potential role of dynorphin in this stress-related function will be explored. In addition the posterior lobe and the anterior lobe dynorphin systems will serve as valuable models for studying its biosynthesis and regulation. Project 2 is a preclinical proposal looking at the effect of variables such as age, sex and tricyclic treatment on the cellular regulation of POMC in the anterior lobe. Project 3 is a clinical project exploring the HPA axis in major depression with basal measures and challenge studies using corticosteroids and CRF. Project 4 is a collaborative inter-university effort focused on CRF challenges in depression. The multidisciplinary approach involves the use of molecular biology immunohistochemistry, peptide measurements and characterization, biosynthesis and release studies and behavioral and pharmacological challenges. Information derived from each of the projects will be considered in the interpretation of results from the other projects. The net result should be increased insight in the biology of stress and depression.

This Program Project application represents our continued efforts to understand, at a molecular, cellular and integrative level, key neurons systems which are both intrinsically important and have direct relevance to the study of two major psychiatric disorders-depression and schizophrenia. The four individual proposals represent a basic to clinical progression beginning with molecular biological studies of two neurocircuits-the brain component of the stress axis, and the dopaminergic motor and limbic pathways, moving to the study of these same systems in post-mortem human brains, and ending with neuroendocrine challenge studies in normal controls and subjects suffering from major depression. Thus, Project I investigates basic mechanisms which are critical to controlling stress responsiveness by examining its key elements at a molecular level and describing the anatomical organization of circuits which either initiate or limit the stress response. Project II studies, also at a molecular and anatomical level, two receptor systems relevant to schizophrenia and depression, specifically the dopaminergic and the serotonergic receptor families. Project III extends the study of the elements of the stress axis and of the two receptor families to post-mortem human brain, attempting to delineate their gene expression and their alterations in the brains of suicide victims and of schizophrenics. Finally, Project IV uses novel challenge paradigms to increase our understanding of the dysregulation of the stress axis in depression, both at the pituitary and the brain levels.

The goal of this project is two-fold: 1) to increase our understanding of the neuronal circuitry critical to stress activation, stress termination, and the differential stress responsiveness across the circadian rhythm of the rat; and 2) to understand how this circuitry may be differentially activated as a function of the nature of the stressor (i.e., the ability of the animal to cope with it) and as a function of individual differences. Thus, we plan to examine, as a prototypical activational pathway, the catecholaminergic inputs which impinge upon the final common path of stress integration in the brain--the hypothalamic neurons which express corticotropin releasing hormone (CRH). We plan to ask whether the CRH neurons have compartments which can be distinguished based on gene expression, and whether these compartments are differentially responsive to differentially responsive to different stressors. In terms of stress inhibition, we shall focus on the role of the inhibitory neurotransmitter, gamma-amino-butyric acid (GABA) as a mediator of both glucocorticoid-dependent and independent negative feedback. Finally, in the anatomical studies, we shall study the role of the suprachiasmatic nucleus (SCN) as a structure which may adjust the "gain" on the stress response as a function of the circadian rhythm, regulating not only the basal tone, but also stress responsiveness and stress termination. At the more behavioral level, we shall use a number of well-established models to look at how brain circuits, or specific modules within them, may become differentially activated as a function of the degree of control that the animal has over the stressor (executive vs. yoked model), or the outcome of a social stress (victory vs. defeat following an agonistic encounter). Finally, we shall ask whether animals which exhibit individual differences in stress responsiveness either at the endocrine level (Fisher vs. Lewis rats) or at the behavioral level (High responders vs. Low responders) do so because they are activating different modules of these stress-responsive neuronal circuits. Together, this set of studies should increase our understanding of the neuronal underpinning of stress responsiveness and coping with stress.

The goal of the present application is to provide continued support for training postdoctoral fellows in neurobiology and biological psychiatry with particular emphasis on the biology of mood disorders. This program exists in the context of the Mental Health Research Institute (MHRI) of the University of Michigan Medical Center, and has been funded by the NIMH for over a quarter of a century. The present application represents a refocusing of the program to emphasize training in modern methods of neurobiology-including genetics, molecular biology, neuroanatomy, signal transduction, neuroendocrinology, neuroimaging-and their relevance to depression and anxiety disorders. This plan represents a continuation of our past efforts at fostering close interactions between physicians and basic scientists in arenas of relevance to understanding brain function and dysfunction. However, it has been sharpened in a number of ways to strengthen this interface and provide our trainees with a coherent and focused program: 1) Research and training efforts will be focused on a shared strength between the MHRI and the Psychiatry Department-i.e., the biology of stress and mood disorders. 2) We have expanded our faculty to 29 members, including a number of young, well-trained neuroscientists and biological psychiatrists. 3) Particular emphasis is placed on giving the physician trainees the opportunity to acquire both a conceptual and working knowledge of modern molecular approaches. This will be achieved through laboratory exposure, and through a working seminar series focused on in-depth discussion of studies ranging from the molecule to the clinic. 4) Our basic science trainees will be required to attend a course in biological psychiatry with the psychiatry residents, to get a sense of the current critical questions in this area. Support for 5 trainees per year is requested. All trainees will hold either the MD degree of the PhD degree. All would be tasked to selected mentors among the faculty and define a specific and coherent research proposal. Trainees will have a primary research mentor to direct research efforts and a secondary mentor to expand their knowledge in needed fields. In cases where the selected mentor is young and has limited training experience, an appropriate senior co-mentor will be chosen to ensure excellent support for the trainee. This program has graduated a significant number of excellent trainees, several of them physicians, who have gone on to research careers in academic institutions. It has also trained several members of disadvantaged minorities who have been very successful. This NIMH Training Grant will be administered by the Program Director who is the Co-Director of the MHRI and Associate Chair for Research in Psychiatry. He will be assisted by an Executive Committee, which will help in the selection of fellows, will share in the distribution of slots, and will oversee the courses relevant to this training grant, including the ethics series. The trainees will have access to world- class facilities and laboratories, in the context of a rich and supportive environment.

psychopharmacology; depression; hypothalamic pituitary axis; cortisol; hormone regulation /control mechanism; metyrapone; corticotropin releasing factor; arginine vasopressin; clinical research; human subject;

The overall purpose of this Program Project competing renewal application is to continue to investigate the neurobiology of stress and depression, using a combination of anatomical, molecular biological endocrine, and behavioral tools, in the context of four individual projects. The main focus is to understand the limbic neuronal circuits which participate in the evaluation of stressors, and transduce the activation and termination of stress responses. A particular interest is to link this anatomical information with a more functional perspective, in an attempt to understand the neuronal basis of individual differences in stress responsiveness, as well as differences which accompany coping and social control (P1). While this level of analysis captures the system at one point in time, a main interest is to understand the plasticity of the system - i.e., how this complex circuitry is laid down during development; how differences in stress responsiveness may emerge and affect function later on in life, including old age; and how repeated or multiple stress alters the organism's response to subsequent stressors (P2). These basic studies form the foundation for studies in human postmortem brain which investigate the elements of stress circuitry in humans, its interface with systems which are likely altered by antidepressant drugs, and its alteration by the processes that result in suicide (P3). Finally, the combination of these basic and pre-clinical studies offer a rich framework in which to address some questions regarding the neuronal basis of stress and depression in humans. This latter set of studies draws on almost all the elements above to frame its central question: is the neurobiology of stress a key to understanding the biological basis of depression? (P4)

Studying the neuroanatomical substrates of stress, suicide and depression in human brain is the central theme of this project. While these are also major foci of this entire Program Project application, this project specifically emphasizes postmortem human brain in an effort to understand the fundamental anatomy of stress-related circuits in man, and the possible changes in gene expression within these circuits which may accompany suicide and depression; in addition, however, this project includes some pre-clinical studies in the rodent specifically focused on the interface between stress circuits and the sites of action of antidepressant drugs, as these studies will help us interpret the human findings. Therefore, this project comprises 3 Specific Aims. The first Specific Aim involves the study of serotonergic (5HT) and nor-adrenergic (NA) systems as they respond to stress and antidepressants in rodent models. These paradigms will allow us to establish a clearer view of the functional links between the stress axis and monoamine systems, thereby clarifying the impact of monoamine-altering compounds on depression and on the endocrine disturbances associated with it. The second Specific Aim directly analyzes postmortem human brain from suicide victims, suicide victims also having a documented history of affective disease, and controls. The same circuits and systems laid out in the rodent will now be analyzed inhuman brain (prefrontal cortex, limbic circuits associated with the stress axis, 5HT and NA nuclei and their receptors). The third Specific Aim is designed to allow the three-dimensional (3D) analysis of individual human brain nuclei at cellular resolution using mRNA levels. This method will substantially increase the power and value of human postmortem studies by combining the regulatory information found in mRNA levels with anatomical precision and with high resolution statistical tools. In sum, at the end of the requested 5 years of this proposal we expect that we will have made very substantial gains in appreciating the circuitry of the stress axis in rodent and man, and begun to establish the relationship between the stress axis and affective disease at the level of biochemically specific brain nuclei and structures.

DESCRIPTION: (Provided by Applicant): This is a proposal for a competing renewal of a postdoctoral training grant located in a multidisciplinary setting at the University of Michigan. The 13 faculty are all NIDA grantees (or Co-Pls) and have expertise in the neurobiology of substance abuse, with particular emphasis on the area of opioid and psychostimulant drugs. The focus of the proposal is the training of Ph.D. and M.D. postdoctoral fellows in state-of-the-art approaches for studying mechanisms underlying abuse of psychoactive drugs. This includes studying the genetic, developmental and environmental factors that lead to vulnerability to substance abuse: the mode of action of drugs of abuse at the molecular, cellular, anatomical and behavioral levels; and the long-term consequences of pschoactive drugs on the brain, as mediated through mechanisms of neural plasticity. The working assumption is that the functional and structural brain remodeling associated with chronic drug use lies at the basis of tolerance, sensitization, physical dependence, and psychological addiction to these drugs. The drug abuse research community at the University of Michigan is of high quality and has a long history in the field. Beyond their individual strengths, the training faculty members have long-term scientific and training relationships with each other. These historical strengths have recently been significantly enhanced with a number of new initiatives' at the University of Michigan, designed to facilitate life sciences research in general, and neuroscience research in particular. They include state-of-the-art tools for mouse and rat genetics, genomics, proteomics, and informatics. Thus, our trainees will profit from a highly sophisticated, yet extremely supportive, research and training environment.

The genetic, molecular, and biochemical causes of mood disorders are still poorly understood. Disturbances in the limbic-hypothalamic- pituitary-adrenal (LHPA) stress axis and the serotonin system are commonly found in depression. However, the pathophysiology of mood disorders and the effectiveness (or lack thereof) of various antidepressants suggest that other neural systems may contribute to the primary or secondary changes in this disorder. The availability of brains from control and depressed subjects provides us with the opportunity to examine the molecular changes in depression in the context of known circuits. As described in the Center Overview, we will address the central hypothesis using two major approaches to the study of alterations in gene expression: 1) The "expression candidate approach" will rely on classical tools such as in situ hybridization to characterize known gene products in the context of circuits proposed to be involved in the regulation of stress emotional responsiveness and emotional executive function. It will also include preliminary genetic characterization of the DNA from control and depressed subjects for markers with known polymorphisms relevant to psychiatric diseases (i.e. serotonin transporter) (Specific Aim I). 2) The "expression array approach" will rely on the new microarray and DNA chip technologies to screen for known and unknown genes whose expression may be altered (either induced or repressed) in depression (see Dr. Cox's proposal). The opportunity to scan the expression levels of thousands of mRNA species for regulatory changes in important brain regions will clearly enhance our understanding of both stress and monoamine systems, and will also identify regulated genes in new neural pathways that are relevant to mood disorders. We may also detect highly regulated mRNA sequences from genes with unidentified functions, leading to new areas of study. The post-array analyses will include studies for validation of the regulation changes detected by microarray and DNA chip technologies using additional control and depressed brains. Validation studies will include comparison of mRNA values (and ratios) with those obtained via in situ hybridization of known mRNAs. Biological validation will include in situ hybridization analysis to identify the anatomical sties of expression of altered mRNAs and regulatory studies with rodent models (Specific Aim II). This work will rely on the effects of the Stanford site, and will integrate with the findings from UC site, in order to yield a more comprehensive view of the biological underpinnings of major depressive disorders.

Cocaine Impact on Neural Plasticity: Modulation by Genetic Vulnerability &Social Stress. This project tests the hypothesis that a major impact of cocaine on the brain is to alter the expression of genes relating to neural plasticity in reward and stress-related circuits, and to affect hippocampal morphology and neurogenesis. The proposed studies will use two lines of animals selectively bred for twelve generations for the novelty-seeking trait (high responders-HR versus low responders-LR). This trait relates to general reactivity to the environment including "novelty-seeking" behavior, and predicts initial likelihood to self-administer drugs. The selectively bred animals will be exposed to cocaine, followed by various periods of abstinence using two methods of cocaine delivery: Self-Administration and Experimenter Administration. They will be tested either under control conditions or following a social stressor. The differential impact of these manipulations will be assessed by measuring altered expression of a panel of growth factor genes, stress genes and synaptic plasticity genes in two regions of the hippocampus (HC) and in the core and shell of the nucleus accumbens (NAcc). The choice of the target genes was based on a combination of existing literature findings and our own data based on microarray profiling after social stress and drug self-administration. Associated changes in hippocampal morphology and neurogenesis will be studied at each of'these phases. Of particular interest will be those drug-related genes whose alterations are sustained long after drug administration has ceased, as they may point to mechanisms of enduring effects of substance abuse that would lead to relapse. Promising genes will be functionally characterized by protein analysis and pharmacological administration of agonists, antagonists or analogues to assess their potential role in modulating the sequelae of exposure to cocaine in animals with differing susceptibility to the drug. This work should lead to new discoveries regarding the molecular mechanisms of addiction and provide novel targets for its treatment.

[unreadable] DESCRIPTION (provided by applicant): MicroRNAs (miRNAs), small (~22nt non-coding RNAs) function as sequence-specific post-transcriptional regulators of gene expression, by reducing target mRNA levels and/or inhibiting protein translation. Schratt et.al. (2006) have shown a link between miRNA function, neuronal spine development and the action of at least one growth factor that may play a role in neuroplasticity. Yet, little is known about miRNA expression patterns in the adult brain, or whether changes in miRNA expression occur following either acute or repeated drug use or whether any miRNA might underlie any known messenger RNA (or protein) changes known to occur following drugs use (or abuse). We hypothesize that miRNA expression alterations will be distinct across various stages of substance abuse and that drug exposure will result in distinctive miRNA expression patterns (anatomical and/or temporal) in two distinct groups of rodents selectively-bred based upon the novelty seeking trait (High Responders (HR) vs Low Responders (LR)), as these two groups of rodents have unique patterns of gene expression basally, after various types of challenges, and display individual differences in the propensity for substance abuse. In Aim 1, we will use laser capture microscopy to isolate specific brain regions known to be direct targets of cocaine and/or undergo drug-induced changes in synaptic plasticity prior to broad-scale miRNA profiling. We will use tissues derived from both HR and LR rodents under basal conditions; conditions where gene expression differences exist. miRNA expression differences will be validated by miRNA qPCR and spatial patterns of expression analyzed by miRNA in situ hybridization (ISH) methods. In aim two, we will use a cocaine sensitization paradigm (where drug exposure can be held constant) in both HR and LR rodents to examine miRNA regulation by cocaine at two time points (immediately following cocaine and following a period of abstinence) isolated and monitored as in aim 1. In aim 3, we will use cocaine self-administration procedures in HR and LR rodents, collect tissues at two time points (acute and abstinence) to examine miRNA regulation. In Aim 4, we will use antagomirs to block basal or cocaine regulated HR/LR miRNA differences to determine if miRNA blockade alters drug-induced behaviors. Together, these findings will shed light on whether miRNAs contribute to the initial propensity to seek drugs of abuse or play an important role in the induction and/or maintenance of cocaine addiction. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Our project evaluates small but novel genetic elements called microRNAs (miRNAs) that are thought to have broad influences on the types or amounts of proteins cells produce. In this proposal, we plan to examine whether there are miRNA changes in two different rodent lines that vary in their propensity to seek drugs under basal conditions. Further, we plan to examine in each rodent line how different miRNA levels vary in multiple brain regions as a function of cocaine use both acutely as well as long after the drug treatment has ceased. These findings will shed light on whether miRNAs contribute to the initial propensity to seek drugs of abuse or play an important role in the induction and/or maintenance of cocaine addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to study the neurobiological mechanisms of affective resilience to anxiety and stress, and to identify strategies for enhancing resilience specifically in highly vulnerable individuals. Our general hypothesis is that epigenetic mechanisms are critical predisposing factors that shape responsiveness to negative valence and impact vulnerability to chronic anxiety disorders. More specifically, we focus on the Fibroblast Growth Factor 2 (FGF2) which serves as a master molecular organizer that is critical during development and continues to be active in shaping affective responsiveness throughout life. This proposal tests the hypothesis that FGF2 is an endogenous resilience molecule that is not only a target of epigenetic mechanisms but is itself a modifier of these mechanisms that, in turn, fine-tune affective responsiveness. The proposed series of studies relies on two lines of rats that we have genetically selected based on their propensity to explore a mildly stressful novel environment. Our breeding strategy over 40 generations has resulted in contrasting behavioral phenotypes that capture a stable bias towards negative valence responsiveness versus positive valence responsiveness. In particular, bred Low-Responder rats (bLRs) exhibit greater basal anxiety and depression behaviors, greater fear conditioning, greater responsiveness to chronic and social defeat stress, lower levels of hippocampal FGF2 and a distinct epigenetic profile when compared to bred High Responders (bHRs) that are significantly more resilient. Thus bLRs are a model of vulnerability to negative affective disorders and a target for resilience enhancement. Aim 1 uses direct administration of FGF2 to promote resilience during early life and in adolescence. It also investigates environmental complexity (EC) as a strategy for promoting resilience during adolescence. It studies the impact of these interventions on two molecular master organizer genes in hippocampus- FGF2 itself, which reduces anxiety and the glucocorticoid receptor GR, which enhances anxiety behavior. Aim 2 characterizes the basal epigenetic profiles associated with the bHR/bLR phenotypes and the impact of resilience induction on these profiles both at the global level and in association with FGF2 and GR promoters. These studies will be extended to anatomical analyses using a range of tools including Clarity. Aim 3 addresses at a mechanistic level the functional bidirectional relationship between FGF2 and epigenetic mechanisms and their impact on resilience--- be it basal resilience (in bHRs) or induced (in bLRs). It uses virally-mediated, targeted RNA intervention strategies to knockdown either FGF2 or histone modifying enzymes in the hippocampus and determine whether they play an essential role in the induction of resilience. Together, these studies will provide a highly targeted approach to understanding and harnessing epigenetics as key factors in affect regulation.