Ned Kalin - US grants

In response to a stressor, activation of endocrine, autonomic, and behavioral systems occurs presumably to enhance the organism's ability to endure and survive the stress. Behavioral and psychological factors modulate the behavioral and psycholoigcal impact of exposure to a stressor. The ability to control one's exposure to aversive events is especially important. Although the behavioral impact of the control dimension has been studied extensively, few studies have explored effects on autonomic function and none have examined effects on cardiovascular system (CVS) function. Neither effects of control over an aversive event on brain neuropeptides nor the potential role of these systems in mediating the behavioral, autonomic, and CVS sequelae of aversive events differing in controllability has been explored. Our hypothesis is that the lack of control over aversive events will lead to an enhanced autonomic and CVS response and that these effects may in part be mediated by specific brain neuropeptide systems. Our objective is to determine if (i) control versus lack of control over the same aversive events is associated with differential autonomic activation and CVS response, (ii) control versus lack of control over the same aversive events is associated with differential effects on brain cortiocotropin releasing factor (CRF), vasopressin (VP), and angiotension II (AII) and in peripheral VP and AII systems, and (iii) alteration of these neuropeptide systems in the brain affects the behavioral, autonomic, and CVS outcomes associated with the lack of control over aversive events. To study (i), we will measure plasma nonrepinephrine and epinephrine and monitor blood pressure and heart rate in rats that have received physically identical electric shocks, differing only in controllability. The full timecourse of these potential changes will be explored. To study (ii), we will measure CRD, VP, and AII immunoreactivitiy using radioimmunoassay in hypothalamus, cortex, hippoampus, and brainstem, and we will measure AII and VP from plasma in rats that have received physically identical electric shocks, differing only in controllability. To study (iii), function of brain peptide systems will be altered by intracerebroventricular administration of peptide agonists and antagonists. The impact of these manipulations on the behavioral, peripheral catecholamine, and cardiovascular changes normally produced by uncontrollable and controllable aversive events will be assessed.

The specific aims of this research are to learn how blood and cerebrospinal fluid (CSF) concentrations of the anterior pituitary hormones, adrenocorticotrophin (ACTH), beta-endorphin (Beta-E), and prolactin (PRL), are related and to what extent CSF concentrations of these peptides reflect brain peptidergic activity. Recent studies suggest that measurements of peptides in CSF might be useful for the diagnosis and understanding of neurological and psychiatric illnesses. However, it is not completely understood what influences peptide concentrations in CSF and to what degree such concentrations are affected by the brain. We will simultaneously and frequently sample blood and CSF from rhesus monkeys, and measure peptide concentrations with radioimmunoassays. To study the dynamics of the relationship between blood and CSF peptide concentrations in normal monkeys, we will use various pharmacological and nonpharmacological challenges to alter peripheral peptide concentrations. We will perform similar studies in hypophysectomized monkeys to directly observe how the brain contributes to CSF peptide concentration, independent of the major peripheral source of these hormones.

Investigators have hypothesized that brain corticotropin-releasing factor (CRF) systems play an integrative role in an organism's physiological and behavioral response to a stressor. The role of CRF in hypothalamic-pituitary-adrenal (HPA) axis and autonomic function has been extensively investigated, but the data base linking CRF and stress-related behavior is only suggestive. The aim of the proposed research is to determine whether CRF brain systems are involved in the behavioral response to a stressor. To achieve this we will alter central CFR systems in the rat and measure several behavioral responses to stress of varied nature and intensity. To alter CRF systems we will administer CRF, CRF antagonist (CRFa), and CRF antiserum intracerebroventricularly (ICV). Stress will consist of varid intensities of footshock or the anticipation of footshock (i.e., conditioned effects of footshock). In this way both the intensity and nature of the stressor will be manipulated. We will also study the role of central CRF systems in the acquisition of associative learning about stressful events. Finally, we will establish whether the behavioral changes due to altered CRF systems are centrally mediated. In all of the experiments multiple dependent measures will be taken. These will be: locomotion, rearing, grooming, freezing, and antinociceptive behavior. Additionally, we will assess HPA activity with plasma levels of ACTH and corticosterone. This research should ascertain the extent to which endogenous brain CRF systems are involved in the mediation of stress-induced behavior.

We propose to more completely characterize the effects of corticotropin releasing hormone (CRH) administered intracerebroventricularly (ICV) to nonhuman primates and to determine whether endogenous CRH brain systems are involved in the primate's response to a stressor. Investigators have hypothesized that CRH systems play an integrative role in organizing the physiological and behavioral responses to a stressor. Previous work on the relationship between CRH and the stress response has primarily employed rodent models. Significant neurobiological differences exist between primates and rodents. In preliminary experiments, we found that CRH affects neuroendocrine systems, autonomic functioning, and behavior when administered centrally to rhesus monkeys; and while some differences were noted, our data were generally consistent with studies performed in rodents. Additional data are needed to define what role brain CRH plays in the response to a stressor. The use of monkeys in our studies will provide the basis for an animal model relevant to humans that will allow further exploration of the role that central CRH plays in regulating and integrating the human stress response. A comparison of rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) monkeys will establish the effects of CRH in two closely related species whose innate behavioral dispositions are, nevertheless, different. In the proposed studies, we will extend our previous work by more completely characterizing the effects of centrally administered CRH in a larger group of monkeys. Complete dose-response studies with CRH and the CRH antagonist (CRHa) will be performed, as will studies to establish the degree to which the CRH-induced effects are specific to this peptide. In Part 1, we will establish the degree to which CRH's effects are modulated by (i) species differences in behavioral disposition, (ii) sex of the animal, (iii) time of day of administration, and (iv) brain site of administration. The experiments in Part 2 will be based on data from these studies. In Part 2, CRH and CRHa will be administered to monkeys already exhibiting varying levels of behavioral and endocrine arousal. Based on the hypothesis that endogenous brain CRH systems play a role in mediating the stress response, we predict that exogenously administered CRH will enhance and CRHa will diminish the ongoing level of arousal.

We propose to more completely characterize the effects of corticotropin releasing hormone (CRH) administered intracerebroventricularly (ICV) to nonhuman primates and to determine whether endogenous CRH brain systems are involved in the primate's response to a stressor. Investigators have hypothesized that CRH systems play an integrative role in organizing the physiological and behavioral responses to a stressor. Previous work on the relationship between CRH and the stress response has primarily employed rodent models. Significant neurobiological differences exist between primates and rodents. In preliminary experiments, we found that CRH affects neuroendocrine systems, autonomic functioning, and behavior when administered centrally to rhesus monkeys; and while some differences were noted, our data were generally consistent with studies performed in rodents. Additional data are needed to define what role brain CRH plays in the response to a stressor. The use of monkeys in our studies will provide the basis for an animal model relevant to humans that will allow further exploration of the role that central CRH plays in regulating and integrating the human stress response. A comparison of rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) monkeys will establish the effects of CRH in two closely related species whose innate behavioral dispositions are, nevertheless, different. In the proposed studies, we will extend our previous work by more completely characterizing the effects of centrally administered CRH in a larger group of monkeys. Complete dose-response studies with CRH and the CRH antagonist (CRHa) will be performed, as will studies to establish the degree to which the CRH-induced effects are specific to this peptide. In Part 1, we will establish the degree to which CRH's effects are modulated by (i) species differences in behavioral disposition, (ii) sex of the animal, (iii) time of day of administration, and (iv) brain site of administration. The experiments in Part 2 will be based on data from these studies. In Part 2, CRH and CRHa will be administered to monkeys already exhibiting varying levels of behavioral and endocrine arousal. Based on the hypothesis that endogenous brain CRH systems play a role in mediating the stress response, we predict that exogenously administered CRH will enhance and CRHa will diminish the ongoing level of arousal.

For a long time, clinicians have had the impression that women exposed to severe stress during pregnancy are at greater risk to produce children with developmental difficulties. Although studies of the effects of prenatal stress in human offspring are very important, the necessary controlled prospective studies in human subjects are difficult or impossible to perform. Animal models provide an alternative approach. Studies in nonhuman vertebrate species have demonstrated that events occurring prenatally produce long-lasting effects of the development of brain and behavioral system. Our laboratory recently demonstrated that when pregnant rats are exposed to repeated inescapable tail shock treatments they produce offspring with increased ACTH and corticosterone concentrations. These offspring also differ behaviroally form offspring on nonstressed mothers in that they do not develop an analgesic response to stress. The proposed studies will be performed in 2 parts. Studies in Part 1 will completely characterize HPA function in prenatally stressed rat pups. Specific experiments will be performed to assess mechanisms underlying activation and inhibition of the HPA system. Studies in Part 2 will assess factors involved in transmitting the effects of prenatal stress from mother to offspring. Emphasis will be placed on characterizing fetal and maternal hormonal changes induced by prenatal stress as well as examining the effects of the postnatal rearing environment. An additional focus of studies in Part 2 will be to test the hypothesis that prenatal stress-induced alterations in glucocorticoids play an important role in HPA and behavioral alterations in offspring. These studies will establish important principles regarding the mediation of the effects of prenatal stress on the development of the HPA system and stress related behavior in offspring. Ultimately such principles will aid in our understanding of the effects of prenatal stress on human development.

DESCRIPTION: (Adapted from applicant's abstract) Previous studies from our laboratory characterized a fearful/anxious endophenotype in rhesus monkeys that is. relevant to understanding normal human anxiety, as well as anxiety disorders. During the last funding period, we established a technique to selectively and reliably lesion the primate amygdala with ibotenic acid. The investigators' data are among the first to use modern lesioning techniques with sophisticated behavioral measures to address mechanisms underlying emotional processing in primates. Results demonstrate an important role for the amygdala in the processing of acutely fearful stimuli but a lack of amygdala involvement in mediating the dispositional behavioral, emotional, and physiological characteristics associated with fearful/anxious temperament. Orbitofrontal regions are bi-directional linked with the amygdala and the proposed studies will examine the role of orbitofrontal cortex in emotional processing, as well as the functional interaction between the amygdala and orbitofrontal cortex. Using ibotenic acid lesions and positron emission tomography, the investigators will explore the hypothesis that right orbitofrontal regions are primarily involved in mediating the behavioral and emotional responses associated with fearful/anxious temperament. The findings from these studies will be highly relevant to humans, addressing the role of amygdala orbitofrontal interactions in mediating normal emotion and psychopathology.

Investigators have hypothesized that brain corticotropin-releasing factor (CRF) systems play an integrative role in an organism's physiological and behavioral response to a stressor. The role of CRF in hypothalamic-pituitary-adrenal (HPA) axis and autonomic function has been extensively investigated, but the data base linking CRF and stress-related behavior is only suggestive. The aim of the proposed research is to determine whether CRF brain systems are involved in the behavioral response to a stressor. To achieve this we will alter central CFR systems in the rat and measure several behavioral responses to stress of varied nature and intensity. To alter CRF systems we will administer CRF, CRF antagonist (CRFa), and CRF antiserum intracerebroventricularly (ICV). Stress will consist of varid intensities of footshock or the anticipation of footshock (i.e., conditioned effects of footshock). In this way both the intensity and nature of the stressor will be manipulated. We will also study the role of central CRF systems in the acquisition of associative learning about stressful events. Finally, we will establish whether the behavioral changes due to altered CRF systems are centrally mediated. In all of the experiments multiple dependent measures will be taken. These will be: locomotion, rearing, grooming, freezing, and antinociceptive behavior. Additionally, we will assess HPA activity with plasma levels of ACTH and corticosterone. This research should ascertain the extent to which endogenous brain CRF systems are involved in the mediation of stress-induced behavior.

psychopharmacology; personality; fear; benzodiazepines; psychophysiology; restraint; conditioning; hypothalamic pituitary axis; neuroanatomy; heart rate; cortisol; diazepam; amygdala; frontal lobe /cortex; brain electrical activity; cerebral dominance; biological models; pituitary adrenal axis; adrenocorticotropic hormone; psychological stressor; benzodiazepine receptor; autoradiography; Macaca mulatta; electroencephalography; behavior test;

OBJECTIVE To use the rhesus monkey as a model to further characterize the behavioral and physiological correlates of extreme behavioral inhibition and to investigate the brain mechanisms underlying this trait. RESULTS Behavioral inhibition (BI) or, in its extreme form, freezing, is an adaptive response which individuals engage in when confronted with threatening situations. Clinical research has demonstrated that in its excessive form BI is a trait marker for children who are very shy and develop into overly fearful adolescents and adults. In addition, these individuals have increased adrenal-cortical and autonomic activity when tested in nonstressful conditions. Later in life, these individuals have an increased likelihood of developing clinically significant anxiety and depression suggesting that extreme behavioral inhibition may be an early marker for later development of psychopathology. Because of its potential clinical importance, we have developed a paradigm in rhesus monkeys to assess BI. As in human children, we have found that there is marked individual variability in the tendency to engage in BI and that this is a stable trait. We have characterized the biological correlates of e xtreme BI and found that these animals have relative right frontal electrical activity of elevated basal cortisol levels. The degree of frontal EEG asymmetry is stable within animals and is positively correlated with cortisol levels. These findings further characterize the endocrinology phenotype of fearful temperaments and point to mechanistic studies to further understand the biology of fear-related psychopathology. FUTURE DIRECTIONS Future studies are planned to further assay the role of neurochemical systems within the amygdala in mediating these responses KEY WORDS benzodiazepine, neurobiology, development, fear, anxiety, cortisol, defense, behavior, brain, psychopathology FUNDING NIMH MH52354 PUBLICATIONS Kalin NH, Shelton SE, Rickman M, Davidson RJ Individual differences in freezing and cortisol in infant and mother rhesus monkeys. Behav Neurosci. 122(1):251-254, 1998. [J] Kalin NH, Larson C, Shelton SE, Davidson RJ Asymmetric frontal brain activity, cortisol, and behavior associated with fearful temperaments in rhesus monkeys. Behav Neurosci. 112(2):286-292, 1998.[J] Kalin NH, Shelton SE Ontogeny and stability of separation and threat-induced defensive behaviors in rhesus monkeys during the first year of life. Am J Primatology 44:125-135, 1998. [J]

OBJECTIVE To demonstrate that individuals with positive disposition compared to those with negative dispositions have lower levels of stress-related hormones, better immune function, and are more likely to survive stressful social and physiological insults. RESULTS Evidence for a link between negative affect and disease progression exists. However, there is almost no scientific evidence examining the effects of positive affect on disease progression. In humans, social stress is ubiquitous and often leads to psychological and physical disability. Numerous studies have demonstrated that loss of a significant other is associated with depression and anxiety, as well as increased medical illnesses and increased mortality. While the mechanisms that underlie these effects are not well understood, recent studies have demonstrated linkages between the brain and a variety of physiological systems, including the immune system. These anatomical studies suggest a pathway by which stress and emotions may regulate the immune system. Different individuals experience the same stressful life events in very different ways. However, little is known about the factors that increase or decrease an individualUs vulnerability to stress and its effects on health. Individual responses to stressful events appear to be more complexly related to disease progression than was earlier thought. It appears that it is the interaction between stressful events and an individualUs temperament that determines how individuals perceive and cope with stress. Failures in the ability to cope result in negative affective states that may increase oneUs vulnerability to certain disease processes. For example, a recent study demonstrated that the presence of depression greatly increased the risk of death in individuals who recently suffered heart attacks. To begin to understand the interaction among the factors involved in survival, researchers from the WRPRC have combined efforts and expertise with researchers at the Caribbean Primate Center in Cayo Santiago Puerto Rico to exploit the unique opportunity provided by the free ranging colony and its inherent naturalistic stressors. Affiliative behaviors have been quantified and individual differences in those measures appear to be trait-like. Among four year old males, those that were most avoidant had higher cortisol levels. In contrast, those animals that gave the most aggression had lower levels of cortisol. KEY WORDS emotions, development, defense, behavior, brain, affect, longevity, immune, stress, health, HPA axis, hormones FUNDING HealthEmotions Research Foundation PUBLICATIONS Shelton SE, Berard JD, Kalin NH Individual differences in aggression and avoidance are associated with HPA activity. Presented at the XVIIth Congress of the International Primatological Society, Antananarivo, Madagascar, August 1998. [A]

OBJECTIVE To understand the neural system underlying the expression of emotion in primates. RESULTS We have focused on elucidating mechanisms underlying the expression of fear and anxiety. This is an area of great importance because, when appropriate, fear-motivated behaviors are adaptive; however, when overly intense or expressed out of context, extreme fear is characteristic of many forms of psychopathology. We are using state-of-the-art techniques to understand the role of the amygdala and its nuclei in mediating fearful states. We will assess the role of the amygdala in mediating 1) classically conditioned autonomic responses, 2) ethologically relevant threatening stimuli, and 3) affiliative and defensive behavior expressed in social situations. This year fifteen monkeys have been lesioned using ibotenic acid. These axon sparing lesions are resulting in more subtle alterations then those reported with more destructive lesioning. Specifically, the data suggests that amygdala lesions disrupt the ability to express learned fears. Eventually, these studies will define biolog ical mechanisms regulating the expression of the primateUs adaptive responding to threatening stimuli. These findings will provide insight into mechanisms mediating maladaptive responding in humans, which is characteristic of fear-related psychopathology. FUTURE DIRECTIONS Histological analysis of all lesions are being performed to correlate site and extent of lesions with effects KEY WORDS neurobiology, amygdala, defense, behavior, brain, psychopathology, conditioning FUNDING NIMH MH46729 PUBLICATIONS Kalin NH, Shelton SE, Rickman M, Davidson RJ Individual differences in freezing and cortisol in infant and mother rhesus monkeys. Behav Neurosci. 122(1):251-254, 1998. [J] Kalin NH, Larson C, Shelton SE, Davidson RJ Asymmetric frontal brain activity, cortisol, and behavior associated with fearful temperaments in rhesus monkeys. Behav Neurosci. 112(2):286-292, 1998.[J] Kalin NH, Shelton SE Ontogeny and stability of separation and threat-induced defensive behaviors in rhesus monkeys during the first year of life. Am J Primatology 44:125-135, 1998. [J] SE Shelton, RJ Davidson, NH Kalin Extreme right frontal EEG activity is associated with elevated CRH levels. Presented at the Society for Neuroscience, Los Angeles, CA, November 1998. [A]

DESCRIPTION (adapted from applicant's abstract): Brain CRF systems are important in integrating endocrine, autonomic, and behavioral responses to stress. Preclinical and clinical research suggests an important involvement of CRF systems and the amygdala in mediating symptoms associated with anxiety disorders and depression. CRF containing cell bodies and fibers, as well as the CRF binding protein and CRF receptors are found throughout the amygdala. The investigators have found that the major site of outflow from the amygdala, the central nucleus (CeA), is a brain site where endogenous CRF systems mediate stress-induced behavioral responses. Using site specific administration of a CRF antagonist (CRFa), they demonstrated that CRF receptors in the region of the CeA mediate acute and conditioned freezing behavior. Complementing these studies, they demonstrated that acute stress exposure results in increased amygdala CRF mRNA concentrations. This is the first direct evidence demonstrating that stress activates amygdaloid CRF systems. The overall aim of this proposal is to continue studies examining the role of amygdaloid CRF systems in mediating behavioral responses to stress. The investigators will characterize the effects of stress on the activation of the genes for CRF, its receptor, and its binding protein. The studies will provide important hypotheses regarding the role of CRF systems in mediating pathological anxiety, and other forms of human psychopathology.

OBJECTIVE: To understand the neural system underlying the expression of emotion in primates. RESULTS We have focused on elucidating mechanisms underlying the expression of fear and anxiety. This is an area of great importance because, when appropriate, fear-motivated behaviors are adaptive; however, when overly intense or expressed out of context, extreme fear is characteristic of many forms of psychopathology. We are using state-of-the-art techniques to understand the role of the amygdala and its nuclei in mediating fearful states. We will assess the role of the amygdala in mediating 1) classically conditioned autonomic responses, 2) ethologically relevant threatening stimuli, and 3) affiliative and defensive behavior expressed in social situations. This year fifteen monkeys have been lesioned using ibotenic acid. These axon sparing lesions are resulting in more subtle alterations then those reported with more destructive lesioning. Specifically, the data suggests that amygdala lesions disrupt the ability to express learned fears. Eventually, these studies will define biological mechanisms regulating the expression of the primate's adaptive responding to threatening stimuli. These findings will provide insight into mechanisms mediating maladaptive responding in humans, which is characteristic of fear-related psychopathology. FUTURE DIRECTIONS Histological analysis of all lesions will be performed to correlate site and extent of lesions with effects. Other studies will examine the effects of reversible inactivation of the amygdala with lidocaine. KEY WORDS neurobiology, amygdala, defense, behavior, brain, psychopathology, conditioning

OBJECTIVE: To use the rhesus monkey as a model to further characterize the behavioral and physiological correlates of extreme behvioral inhibition and to investigate the brain mechanisms underlying this trait. RESULTS Behavioral inhibition (BI) or, in its extreme form, freezing, is an adaptive response which individuals engage in when confronted with threatening situations. Clinical research has demonstrated that in its excessive form BI is a trait marker for children who are very shy and develop into overly fearful adolescents and adults. In addition, these individuals have increased adrenal-cortical and autonomic activity when tested in nonstressful conditions. Later in life, these individuals have an increased likelihood of developing clinically significant anxiety and depression suggesting that extreme behavioral inhibition may be an early marker for later development of psychopathology. Because of its potential clinical importance, we have developed a paradigm in rhesus monkeys to assess BI. As in human children, we have found that there is marked individual variability in the tendency to engage in BI and that this is a stable trait. We have characterized the biological correlates of extreme BI and found that these animals have relative right frontal electrical activity of elevated basal cortisol levels. The degree of frontal EEG asymmetry is stable within animals and is positively correlated with cortisol levels. These findings further characterize the endocrinology phenotype of fearful temperaments and point to mechanistic studies to further understand the biology of fear-related psychopathology. FUTURE DIRECTIONS Future studies are planned to further assay the role of neurochemical systems within the amygdala in mediating these responses. A continuation of the funding is currently being applied for. KEY WORDS benzodiazepine, neurobiology, development, fear, anxiety, cortisol, defense, behavior, brain, psychopathology

OBJECTIVE: To perform preclinical studies to examine neural circuits and mechanisms underlying primate sleep. RESULTS Increased daytime emotionality, anxiety, and depression are commonly associated with sleep alterations. Since the amygdala plays an important role in emotional processing and is connected to sleep regulating regions, we are in the process of examining the role of the primate amygdala in regulating sleep. These studies are of particular relevance since sleep patterns in rhesus monkeys are similar to those in humans. Using highly selective techniques, ibotenic acid lesions of the amygdala were performed in 17 rhesus monkeys. The effects of these lesions on frontal EEG asymmetric activity and sleep are currently being studied. All regional EEG data have been collected and are awaiting analysis. Sleep studies have been completed in 4 lesioned and 2 control animals. Overall, the data demonstrate that chair adapted rhesus monkeys exhibit relatively normal patterns of sleep as defined by stages 1-4 and REM. Preliminary data suggests that amygdala lesioned animals may be engaging in sleep associated with less awakenings and longer REM periods. Heart rate data has also been collected and will be of interest since autonomic instability occurs during REM. FUTURE DIRECTIONS Another 12 animals will be studied this year to more completely characterize the role of the amygdala. In addition, histological analysis will be performed on the brains of these animals to correlate sleep changes with site and extent of the lesion. KEY WORDS emotion, anxiety, sleep, REM, amygdala, lesion and Department of Psychiatry

psychological stressor; psychosocial separation; ethology; psychophysiology; emotional adjustment; psychoneuroimmunology; longitudinal animal study; neuroendocrine system; behavioral /social science research tag; Macaca mulatta;

DESCRIPTION (adapted from applicant's abstract): Brain CRF systems are important in integrating endocrine, autonomic, and behavioral responses to stress. Preclinical and clinical research suggests an important involvement of CRF systems and the amygdala in mediating symptoms associated with anxiety disorders and depression. CRF containing cell bodies and fibers, as well as the CRF binding protein and CRF receptors are found throughout the amygdala. The investigators have found that the major site of outflow from the amygdala, the central nucleus (CeA), is a brain site where endogenous CRF systems mediate stress-induced behavioral responses. Using site specific administration of a CRF antagonist (CRFa), they demonstrated that CRF receptors in the region of the CeA mediate acute and conditioned freezing behavior. Complementing these studies, they demonstrated that acute stress exposure results in increased amygdala CRF mRNA concentrations. This is the first direct evidence demonstrating that stress activates amygdaloid CRF systems. The overall aim of this proposal is to continue studies examining the role of amygdaloid CRF systems in mediating behavioral responses to stress. The investigators will characterize the effects of stress on the activation of the genes for CRF, its receptor, and its binding protein. The studies will provide important hypotheses regarding the role of CRF systems in mediating pathological anxiety, and other forms of human psychopathology.

OBJECTIVE To perform preclinical studies to examine neural circuits and mechanisms underlying primate sleep RESULTS Increased daytime emotionality, anxiety, and depression are commonly associated with sleep alterations. Since the amygdala plays an important role in emotional processing and is connected to sleep regulating regions, we are in the process of examining the role of the primate amygdala in regulating sleep. These studies are of particular relevance since sleep patterns in rhesus monkeys are similar to those in humans. Using highly selective techniques, ibotenic acid lesions of the amygdala were performed in 17 rhesus monkeys. The effects of these lesions on frontal EEG asymmetric activity and sleep are currently being studied. All regional EEG data have been collected and are awaiting analysis. Sleep studies have been completed in 4 lesioned and 2 control animals. Overall, the data demonstrate that chair adapted rhesus monkeys exhibit relatively normal patterns of sleep as defined by stages 1-4 and REM. Preliminary data suggests that amygdala lesioned animals may be engaging in s leep associated with less awakenings and longer REM periods. Heart rate data has also been collected and will be of interest since autonomic instability occurs during REM. FUTURE DIRECTIONS Another 12 animals will be studied this year to more completely characterize the role of the amygdala. In addition, histological analysis will be performed on the brains of these animals to correlate sleep changes with site and extent of the lesion. KEY WORDS emotion, anxiety, sleep, REM, amygdala, lesion FUNDING P01-RR00167

OBJECTIVE To demonstrate that individuals with positive disposition compared to those with negative dispositions have lower levels of stress-related hormones, better immune function, and are more likely to survive stressful social and physiological insults. RESULTS Evidence for a link between negative affect and disease progression exists. However, there is almost no scientific evidence examining the effects of positive affect on disease progression. In humans, social stress is ubiquitous and often leads to psychological and physical disability. Numerous studies have demonstrated that loss of a significant other is associated with depression and anxiety, as well as increased medical illnesses and increased mortality. While the mechanisms that underlie these effects are not well understood, recent studies have demonstrated linkages between the brain and a variety of physiological systems, including the immune system. These anatomical studies suggest a pathway by which stress and emotions may regulate the immune system. Different individuals experience the same stressful life events in very different ways. However, little is known about the factors that increase or decrease an individualUs vulnerability to stress and its effects on health. Individual responses to stressful events appear to be more complexly related to disease progression than was earlier thought. It appears that it is the interaction between stressful events and an individualUs temperament that determines how individuals perceive and cope with stress. Failures in the ability to cope result in negative affective states that may increase oneUs vulnerability to certain disease processes. For example, a recent study demonstrated that the presence of depression greatly increased the risk of death in individuals who recently suffered heart attacks. To begin to understand the interaction among the factors involved in survival, researchers from the WRPRC have combined efforts and expertise with researchers at the Caribbean Primate Center in Cayo Santiago Puerto Rico to exploit the unique opportunity provided by the free ranging colony and its inherent naturalistic stressors. Affiliative behaviors have been quantified and individual differences in those measures appear to be trait-like. Among four year old males, those that were most avoidant had higher cortisol levels. In contrast, those animals that gave the most aggression had lower levels of cortisol. KEY WORDS emotions, development, defense, behavior, brain, affect, longevity, immune, stress, health, HPA axis, hormones FUNDING HealthEmotions Research Foundation PUBLICATIONS Shelton SE, Berard JD, Kalin NH Individual differences in aggression and avoidance are associated with HPA activity. Presented at the XVIIth Congress of the International Primatological Society, Antananarivo, Madagascar, August 1998. [A]

OBJECTIVE To use the rhesus monkey as a model to further characterize the behavioral and physiological correlates of extreme behavioral inhibition and to investigate the brain mechanisms underlying this trait. RESULTS Behavioral inhibition (BI) or, in its extreme form, freezing, is an adaptive response which individuals engage in when confronted with threatening situations. Clinical research has demonstrated that in its excessive form BI is a trait marker for children who are very shy and develop into overly fearful adolescents and adults. In addition, these individuals have increased adrenal-cortical and autonomic activity when tested in nonstressful conditions. Later in life, these individuals have an increased likelihood of developing clinically significant anxiety and depression suggesting that extreme behavioral inhibition may be an early marker for later development of psychopathology. Because of its potential clinical importance, we have developed a paradigm in rhesus monkeys to assess BI. As in human children, we have found that there is marked individual variability in the tendency to engage in BI and that this is a stable trait. We have characterized the biological correlates of e xtreme BI and found that these animals have relative right frontal electrical activity of elevated basal cortisol levels. The degree of frontal EEG asymmetry is stable within animals and is positively correlated with cortisol levels. These findings further characterize the endocrinology phenotype of fearful temperaments and point to mechanistic studies to further understand the biology of fear-related psychopathology. FUTURE DIRECTIONS Future studies are planned to further assay the role of neurochemical systems within the amygdala in mediating these responses KEY WORDS benzodiazepine, neurobiology, development, fear, anxiety, cortisol, defense, behavior, brain, psychopathology FUNDING NIMH MH52354 PUBLICATIONS Kalin NH, Shelton SE, Rickman M, Davidson RJ Individual differences in freezing and cortisol in infant and mother rhesus monkeys. Behav Neurosci. 122(1):251-254, 1998. [J] Kalin NH, Larson C, Shelton SE, Davidson RJ Asymmetric frontal brain activity, cortisol, and behavior associated with fearful temperaments in rhesus monkeys. Behav Neurosci. 112(2):286-292, 1998.[J] Kalin NH, Shelton SE Ontogeny and stability of separation and threat-induced defensive behaviors in rhesus monkeys during the first year of life. Am J Primatology 44:125-135, 1998. [J]

The UW is a world leader in the emerging field of Affective Neuroscience, the multi-disciplinary approach to understanding the neurobiological bases of emotion. In addition, the UW excels in the imaging sciences and is preeminent in the use of nonhuman primates to study and model human disease. UW researchers have a long tradition of using Positron Emission Tomography (PET) to study nonhuman primates. However, the current human scanners only allow for the resolution of large brain regions (>5mm). In addition, to studies in rhesus monkeys, we have important projects in marmosets, a primate species that is much too small to image with the current scanners. Therefore, we are requesting funds for a high resolution animal PET scanner that will bring state-of-the-art scanning to our primate studies. These studies will investigate the function of neural circuits and require the ability to image small and discrete brain regions. At UW several major research programs have been established to focus efforts in brain and behavior research. These efforts uniquely integrate preclinical and clinical approaches to explore basic brain mechanisms. The HealthEmotions Research Institute (HERI) was recently established at UW to organize and extend these efforts. HERI's mission is to develop state-of-the-art scientific methods to support interdisciplinary efforts to explore brain function as it relates to human emotion, health, and disease. The Wisconsin Regional Primate Center and the Harlow Primate Laboratory are also located on the UW campus. A critical aspect of our work is the ability to perform mechanistic studies linking nonhuman primate studies to clinical research. Dedicated to understanding brain development as it relates to learning and behavior, the Waisman Center is another valuable resource. The Keck Foundation recognized the excellence of the Wisconsin programs in brain and emotion research by recently joining with the UW to establish the Keck Laboratory for Functional Brain Imaging and Behavior. This world class functional imaging laboratory, housed at the Waisman Center, contains clinical PET and a 3T MRI, and importantly facilities for studying nonhuman primates. The funds requested will purchase a high sensitivity, high resolution (2mm cubic) MicroPET scanner to support and extend ongoing animal PET imaging research programs while complementing human imaging studies. This dedicated animal research scanner will be jointly administered through HERI and Waisman Center and will be a core component of the Keck Lab. This scanner will also enhance research efforts in such wide-ranging areas as cardiology, oncology neurology, radiology and medical physics.

DESCRIPTION (provided by applicant): The brain CRH system plays a prominent role in integrating the responses to stress. Dysregulation of the CRH system has been implicated in stress-related psychopathologies such as depression and anxiety. CRH and related ligands mediate their effects through at least two receptors, R1 and R2a. While R1 has been clearly linked to stress and psychopathology, little is known regarding the function of R2a. The proposed studies will extend our earlier work implicating the amygdala CRH system in regulating stress-related effects. We will now explore the contributions of CRH receptor subtypes in mediating stress-induced responses. Our preliminary data strongly suggest that lateral septum (LS) R2a mediates fear-related behavior. We will therefore explore the hypothesis that R2a in the LS and R1 in the central nucleus of the amygdala work together to mediate adaptive fear-related behaviors. Little is known about the mechanisms by which stress influences critical intracellular signaling pathways. Because phosphorylation of the transcription factor CREB is a common step in many intracellular signaling pathways, we will use an ethologically relevant psychological stressor to determine the extent to which stress-induced R1 and R2a activation mediates effects on CREB phosphorylation. Additionally, using specific protein kinase inhibitors, we will determine which protein kinases are involved in mediating the acute behavioral effects of CRH. To link these data to psychopathology, we will characterize changes in CRH receptors and CREB and pCREB in rats that fail to adapt behaviorally to chronic stress exposure. In addition, to explore new treatment strategies for stress-related psychopathology, rats that display maladaptive behavioral responses will be treated with viral vectors expressing R1 or R2a antisense constructs to continuously reduce either R1 or R2a. It is predicted that antisense producing viral vectors will reduce stress-like responses in these rats. Finally, to directly explore changes in CRH and CREB in relation to human psychopathology, we will characterize CRH receptors and CREB mRNA in postmortem amygdala samples from unipolar depressed, bipolar, and schizophrenic subjects. Overall, these studies will provide important new insights into the role of R2a, interactions between R1 and R2a in adaptive and maladaptive fear responses, intracellular changes underlying these responses, and alterations in the CRH system and CREB associated with human psychopathology.

Our work demonstrates that the rhesus monkey provides an excellent model to study mechanisms underlying human anxiety and fear. In our studies with rhesus monkeys, we found stable, brain (right frontal EEG asymmetry), endocrine (increased cortisol and CRH), and behavioral (prolonged dispositional freezing) characteristics related to anxiety that we termed the anxious endophenotype. These findings are particularly relevant to understanding the development of human psychopathology, since children with extremely inhibited temperament and similar biological features are at increased risk to develop anxiety disorders. One of our most pertinent findings in the monkeys was the relation between extreme right frontal EEG activity and extreme anxiety. These findings are consistent with human studies demonstrating an association between asymmetric right prefrontal brain electrical activity and negative affect. It is likely that different aspects of the anxious endophenotype are mediated by the interactions of limbic, brain stem, and cortical regions. In previous work, we systematically investigated the role of the amygdala in mediating the anxious endophenotype. Using selective ibotenic acid lesions, we found an important role for the amygdala in mediating acute fear-related responses to novel stimuli; however the data suggested that the amygdala does not play a major role in mediating the stable biological and behavioral traits associated with the anxious endophenotype. While lateral prefrontal cortex (LPFC) has been implicated in cognitive processes such as attention, anticipation, and working memory, recent studies suggest a role for LPFC in integrating emotional and cognitive information important in directing goal-related behavior. Evidence from our, and other, laboratories supports LPFC involvement in emotion processing and points to hemispheric specialization. We believe that studies examining the role of LPFC in emotion processing will provide new and important insights into prefrontal cortical mechanisms underlying the anxious endophenotype and will provide an opportunity to integrate findings related to the cognitive functions of this region with emotion regulation. Therefore, in rhesus monkeys, we will use a variety of techniques (emotion probes, selective ibotenic acid lesions, PET imaging, and intra-cerebral site specific administration pharmacology strategies) to systematically evaluate the contributions of LPFC in emotion processing.

psychological stressor; ethology; Primates; animal colony; behavioral /social science research tag;

neuropsychology; sleep; amygdala; Primates; animal colony;

social psychology; psychobiology; Primates; animal colony; behavioral /social science research tag;

Affective; Animal Testing; Animals; Behavior; Behavioral; Behavioral inhibition; Brain; CRISP; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Emotions; Encephalon; Encephalons; Exhibits; Freezing; Funding; Grant; IT Systems; Information Systems; Information Technology Systems; Institution; Investigators; Lateral; Libraries; MR Imaging; MR Tomography; MRI; Macaca mulatta; Magnetic Resonance Imaging; Magnetic Resonance Imaging Scan; Measures; Mediating; Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance; Medical Imaging, Positron Emission Tomography; NIH; NMR Imaging; NMR Tomography; National Institutes of Health; National Institutes of Health (U.S.); Nervous; Nervous System, Brain; Nuclear Magnetic Resonance Imaging; Operation; Operative Procedures; Operative Surgical Procedures; PET; PET Scan; PET imaging; PETSCAN; PETT; Physiologic; Physiological; Positron Emission Tomography Scan; Positron-Emission Tomography; Prefrontal Cortex; Process; Proton Magnetic Resonance Spectroscopic Imaging; Rad.-PET; Research; Research Personnel; Research Resources; Researchers; Resources; Rhesus; Rhesus Macaque; Rhesus Monkey; Role; Services; Source; Structure; Surgical; Surgical Interventions; Surgical Procedure; Systems, Data; Technology; United States National Institutes of Health; Zeugmatography; endophenotype; neural; relating to nervous system; social role; surgery; trait

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To understand the neural system underlying the expression of emotion in primates. Baseline brain metabolism, behavior, and physiological measures were characterized in preparation for surgery on subgenual Area 25, an area that has been implicated in depression. The findings from these studies will be highly relevant to humans, addressing the role of amygdala-orbitofrontal interactions in mediating normal emotion and psychopathology. This work used WNPRC Animal Services, surgery, pathology, operational services, and library and information services.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To evaluate the role of the lateral prefrontal cortex in modulating emotion and in mediating the behavioral and [unreadable] physiological characteristics of the anxious endophenotype.[unreadable] [unreadable] High levels of freezing (behavioral inhibition) are traits associated with the anxious endophenotype. We have [unreadable] defined three groups of rhesus monkeys exhibiting low, medium, and high levels of freezing behavior. We are [unreadable] further testing these animals on other behavioral and physiological measures. We are using PET and MRI [unreadable] technology to examine brain activity and structure. These measures will help us evaluate specific contributions of [unreadable] the lateral prefrontal cortex (LPFC) in emotion processing in rhesus monkeys. This research used WNPRC Animal [unreadable] Services, Surgery, and Library and Information Systems Services.

DESCRIPTION (provided by applicant): Anxiety and affective disorders constitute a group of heterogeneous illnesses that are common and show significant heritability. Considerable work has focused on identifying the genes involved in anxiety and affective disorders providing interesting leads, but no definitive answers. One of the most prominent childhood risk factors for the development of these illnesses is behavioral inhibition (BI), a temperamental disposition characterized by extreme shyness and inhibition in response to novel situations or strangers. Using young rhesus monkeys, we have developed a model that is analogous to childhood BI and have demonstrated that individual differences in monkey BI are significantly heritable. In addition, we have used functional imaging to identify the brain regions associated with BI. In this project we will measure BI, associated physiological parameters, and functional brain activity in a large multi-generation pedigree of rhesus monkeys. These phenotypes will be used in whole genome linkage analyses to investigate the genetic basis of brain mechanisms underlying anxiety and depression. By combining proven approaches to the study of the genetics of complex disease with simultaneous analysis of intermediate brain reactivity phenotypes, our novel strategy using young rhesus monkeys will: 1) identify novel genes that influence BI, 2) quantitate the influence of genetic variation on individual differences in reactivity of the neurocircuitry underlying emotion, and 3) determine specific genes that are involved in mediating both individual differences in BI and increased reactivity of emotion-related brain circuits. These studies are not feasible in rodent species or humans. The proposed experiments provide an invaluable opportunity to identify novel genetic factors that play a major role in the development of human anxiety and affective disorders, results that will be immediately relevant to children at risk for the development of psychopathology. PUBLIC HEALTH RELEVANCE Anxiety and affective disorders constitute a group of mental illnesses that can be inherited. Using young rhesus monkeys as subjects, this study will: 1) identify genes that influence behavioral inhibition, 2) quantitate the influence of genetic variation on individual differences in reactivity of emotion-related brain circuits, and 3) determine specific genes that are involved in mediating both individual differences in behavioral inhibition and increased reactivity of emotion-related brain circuits. The proposed experiments provide an invaluable opportunity to identify genetic factors that play a major role in the development of human anxiety and affective disorders, results that will be immediately relevant to children at risk for the development of psychopathology.

Anxiety, depression and substance abuse are among the most common types of psychopathology that have their onset during adolescence. Anxious temperament identified during childhood is a significant risk factor for the later development of these illnesses. Since mechanistic and controlled stress exposure experiments cannot be performed in children, the proposed studies will use an extensively validated non human primate model with state of the art functional imaging and molecular methods to understand the neural circuits and molecular mechanisms underlying the adolescent vulnerability to develop stress-induced psychopathology. An important component of these studies is that they will, in young non human primates, use methods of assessment similar to those in the human studies, including functional and structural imaging, to prospectively study the interaction between increased childhood amygdala reactivity and the development of stress-induced psychopathology during adolescence. Importantly, brain tissue will be collected to assess the extent to which dysregulated amygdala corticotropin releasing factor systems underlie the adolescent vulnerability to develop anxiety and depression. It is expected that these studies will find that: 1) increased amygdala reactivity in childhood is associated with anxious temperament and predicts later risk, 2) maladaptive responses to stress that are associated with adolescent psychopathology involve failure of adaptive prefrontal-amygdala interactions, and 3) increased amygdala CRF activity underlies the vulnerability to develop stress-induced psychopathology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To understand the neural circuits and molecular mechanisms underlying the adolescent vulnerability to develop stress-induced psychopathology. The full title of this project is, "The Neurobehavioral Bases of Emotion Regulation and Dysregulation in Adolescence: Developmental Mechanisms Underlying the Risk to Develop Anxiety and Depression." Using microPET images, we defined groups based on high, middle and low amygdala reactivity during a behavioral challenge. These subjects have been further characterized behaviorally and physiologically both prior to and following relocation stress. These data will be used to define the parameters that engender vulnerability to psychopathology. This research used WNPRC Animal Services, Pathology, Operational Services, and Library &Information Services. PUBLICATIONS: None.

DESCRIPTION (provided by applicant): One of the most promising predictors of the development of human anxiety disorders and depression is extreme behavioral and social inhibition (BI) which is evident prior to adulthood. Studies in human and nonhuman primates demonstrate that individuals with extreme BI commonly have increased reactivity of stress-related systems such as the hypothalamic-pituitary-adrenal axis and the amygdala. While significant advances have been made in understanding BI, its specific pathophysiological mechanisms remain unknown. Studies using non-human primate models of BI are valuable because monkeys display a temperamental disposition that closely maps onto human behavior. However, for various reasons, primate models are not ideal for performing in-depth mechanistic studies. In this regard, rodent models are advantageous as molecular and genetic approaches can be used to establish putative novel mechanisms which can be tested by manipulating specific brain regions with the administration of selective pharmacological agents. It is our contention that further progress in understanding the molecular basis of extreme BI can only be achieved by using an integrative, cross species approach that combines the advantages of both the rodent and non-human primate models to identify brain systems and molecular mechanisms involved in mediating this early risk factor for the development of anxiety and depression. Our laboratory has extensive experience with both non-human primate and rodent models of BI. In our rhesus monkey model there is a physiological correlate of BI as evidenced by a trait-like elevated pattern of brain circuit activity that includes the amygdala that is predictive of a highly anxious temperament. Our rodent studies selecting rats and mice that display a trait-like disposition to engage in extreme BI in response to predator exposure, provide evidence for a phenotype that is analogous to that associated with the human risk to develop anxiety and depression. In this proposal, we will bring together results from both model species using microarray technology to identify genes that are associated with the extreme BI temperament that are shared between rhesus monkeys and mice. The power of this approach is that it will allow us to exploit the relative ease of using mice to identify genes of interest and to perform mechanistic studies while at the same time using data from primates to verify that the genes identified in mice are relevant to primate and human BI. It is important to underscore that while extreme BI is a risk factor for the development of stress-associated psychopathology, only a subset of individuals with this temperament will develop psychiatric illnesses. Therefore, we will also use mice to begin to understand how the genes identified as being associated with extreme BI are related to the vulnerability of extreme BI individuals to develop psychopathology when exposed to chronic social stress. Ultimately the proposed studies will provide insight into the mechanisms that confer susceptibility to psychopathology in at-risk individuals. PUBLIC HEALTH RELEVANCE: Human studies have demonstrated the importance of extreme behavioral inhibition as a temperamental disposition that serves as an early vulnerability marker for the development of anxiety disorders and depression. The proposed experiments will identify novel genes systems that mediate this extreme temperament by combining molecular studies on our adolescent mouse and monkey models of behavioral inhibition. Ultimately, these studies will provide new insights into brain mechanisms mediating the risk to develop anxiety and depressive disorders.

DESCRIPTION (provided by applicant): Anxiety disorders in children are prevalent and are increasingly recognized as a major public health concern. In addition to the psychological suffering and disability associated with childhood anxiety disorders, these symptoms are commonly associated with comorbid depressive symptoms, exacerbate other medical illnesses, and increase the later risk to develop anxiety disorders, depression and comorbid drug and alcohol abuse. Despite the importance of generalized anxiety disorder (GAD) occurring during childhood, little is known about the brain alterations that mediate its development and pathophysiology. The overall aim of this proposal is to build on our work in young nonhuman primates by identifying intermediate brain phenotypes that are linked to early human childhood GAD. The hypothesis that altered prefrontal-amygdala connectivity and altered amygdala chronometry is associated with symptoms of GAD will be evaluated with structural and functional MRI, in conjunction with relevant physiological and behavioral measures. While these methods have been commonly used in adults and less frequently in adolescents, very few studies have examined these measures in relation to clinically significant anxiety in pre-adolescent children. This is particularly important since anxiety and depression in adults and adolescents frequently begins as clinically significant anxiety earlier in life. Understanding alterations in the neural circuitry that underlie the expression of GAD in preadolescent children will provide a rationale for using biomarkers aimed at early detection. Specifically, these data will lay the foundation for prospective longitudinal studies examining the utility of assessing amygdala chronometry and prefrontal-amygdala connectivity in relation to the early detection and treatment of these childhood illnesses. This proposal is intended to fund the initial stages of this research with the long-term plan to obtain a very large sample of children with GAD. Because of the heterogeneity of comorbid symptoms, obtaining a large sample will allow us to parse GAD and associated symptoms in relation to distinct underlying neural alterations. This has the potential to provide new insight into the current conceptualization of GAD and will set the stage for novel brain based classification of symptom patterns in children with clinically significant anxiety. The opportunities from performing these studies in GAD children are numerous. They will provide a developmental framework for understanding the structure and function of brain circuits that are associated with the earliest expression of psychopathology. In addition, this knowledge may provide a rationale for the development of psychosocial and pharmacologic interventions that target key components of the involved neural circuit and take into account the plasticity of the developing child's brain. Early more effective interventions tha are based on a sound neurodevelopmental rationale hold the promise for the prevention of later adolescent and adult anxiety, depression and substance abuse. PUBLIC HEALTH RELEVANCE: Anxiety disorders in children are prevalent and are increasingly recognized as a major public health concern. In addition to the psychological suffering and disability associated with childhood generalized anxiety disorder (GAD), these symptoms are commonly associated with depressive symptoms, exacerbate comorbid medical illnesses, and increase the risk to develop drug and alcohol abuse. The proposed studies are aimed at understanding the brain alterations associated with childhood GAD so that early diagnosis can be improved and more effective interventions can be developed.

PROJECT SUMMARY: Childhood anxious temperament (AT) is a key risk factor for developing anxiety and comorbid depression. In primates, AT is evident early in life, stable, and associated with increased threat reactivity. Early adversity is known to increase the risk of developing extreme AT. While adversity is common, the neural mechanisms linking it to AT are not understood. This understanding would permit identification of novel therapeutic targets with the potential for developing neuroscientifically-informed interventions. This proposal builds on work validating the AT phenotype and identifying the neural circuit underlying AT. Recent microarray and RNA sequencing (RNA-seq) work suggests the hypothesis that extreme AT reflects neuroplasticity deficits in the lateral division of the central nucleus of the amygdala (CeL), a key regulator of amygdalar outflow to regions that give rise to signs of anxiety, and the region most predictive of AT in our imaging work. This proposal aims to understand how peer rearing (PR), a controlled early adversity manipulation, causes extreme AT in primates, something not possible in human studies. The use of primates increases the likelihood that discoveries will translate to at-risk children. PR and maternally reared (MR) animals will be longitudinally assessed, testing adversity's impact on the development of AT. Repeated multimodal imaging will assess adversity's impact on the development of amygdala reactivity and prefrontal-amygdala anxiety regulation circuits. Importantly, the primate model affords an opportunity to test whether adversity's harmful effects on AT are mediated by alterations in CeL neuroplasticity pathways. Immunohistochemical and RNA-seq analyses will be performed on CeL microdissected neurons. This novel synthesis of tools promises new insights into how adversity-induced molecular alterations manifest in brain function, connectivity, and structure, and how these macroscopic changes contribute to extreme AT? insights not readily available from molecular-level rodent or systems-level human studies. Further, a stem cell model of CeL GABAergic neurons will be created and compared to neurons from CeL. A valid stem cell model would enhance understanding of AT's molecular bases and accelerate the screening of new therapeutics.

DESCRIPTION (provided by applicant): Anxious temperament (AT) is a critical childhood risk factor for the development of anxiety and depression. Human and monkey AT is identifiable early in life, stable across development, and heritable. Children with extreme AT often meet diagnostic criteria for social anxiety disorder, demonstrating the blurred boundary between extreme temperamental anxiety and what is traditionally considered psychopathology. Elucidating the molecular mechanisms underlying extreme AT is essential for efforts aimed at altering the development of anxiety and depressive disorders. We validated a primate model of AT that is remarkably similar to childhood AT. Evidence supports this developmental rhesus monkey model as being the optimal model for establishing mechanisms underlying pathological childhood anxiety. The primate model enables mechanistic hypothesis testing that cannot be performed in humans. Furthermore, the primate model is readily translatable as it uses the same imaging, behavioral, and endocrine measures routinely used in human research. Our prior lesion and imaging studies in a large sample of juvenile macaques identified 3 key nodes in the neural circuit mediating AT: the central nucleus of the amygdala (Ce), anterior hippocampus (aHip), and orbitofrontal cortex (OFC). Our data demonstrates that the OFC has a regulatory role in relation to AT's altered Ce and aHip metabolism. Using tissue from the Ce region predictive of AT, our preliminary microarray work identified transcripts whose expression levels predicted AT and its underlying Ce activity. Some of the more exciting findings implicate alterations in the expression of neuroplasticity genes to be associated with AT, supporting a hypothesis that AT is associated with altered early neurodevelopmental processes. The proposed work will significantly extend this line of research. We will combine functionally- guided laser capture microdissection (LCM) of Ce, aHip, and OFC neurons with both RNA sequencing (RNAseq) and behavioral/neuroimaging techniques (metabolic activity and functional connectivity). This will allow us to characterize molecular neuronal mechanisms in the regions that underlie AT and its neural substrate. By using OFC lesions to permanently reduce AT and activity in its neural substrate, we will characterize the top-down influences of OFC on the regulation of Ce/aHip genes in neuronal populations relevant to AT and its long-term maintenance. Finally, we will use RNAseq to assess the utility of using transcripts measured in blood as peripheral biomarkers of AT and its associated altered neural substrate. HEALTH/NIMH MISSION RELEVANCE: Childhood AT confers marked risk for the development of anxiety and depression. These disorders are associated with substantial morbidity, mortality, and socioeconomic burdens. Data from the proposed studies should substantially increase our understanding of the molecular mechanisms underlying the expression and top-down regulation of AT and its neural substrate. Deep RNA sequencing in neurons from within the altered brain regions underlying AT will provide a detailed and comprehensive view of the genetic regulation of the neural circuitry that mediates and regulates childhood AT. These studies cannot be performed in humans but can be accomplished in young primates and will facilitate the identification of novel targets for interventions aimed at ameliorating anxiety and depression early in life.

? DESCRIPTION (provided by applicant): Persistent and high levels of sustained anxiety during childhood are a strong predictor of the development of anxiety and depressive disorders during adolescence. This is particularly relevant to females because after puberty girls are twice as likely to develop these disorders. The goal of this proposal is to understand the biological mechanisms of sustained anxiety in highly anxious girls, how it changes over time, and how it can transform into psychopathology. Capitalizing on our unique experience with studies of anxiety in both human and nonhuman primates, we will use a translational neuroscience approach to understand the neurobiology of sustained anxiety in highly anxious girls and young anxious female rhesus monkeys. Neuroimaging studies will focus on the bed nucleus of the stria terminalis (BST) because it is thought to be involved in sustained anxiety and prolonged threat preparedness. Similar paradigms will be used in humans and monkeys to: 1) characterize developmental trajectories of brain function in highly anxious girls; 2) examine the relevance of altered BST function in relation to the onset of anxiety and depressive disorders; 3) test the causal role of the BST in anxiety as young anxious female monkeys mature into adolescence; and 4) define BST molecular alterations that are linked to altered BST function and sustained anxiety. Although sustained anxiety responses can be adaptive, many patients with stress-related psychopathology experience extreme levels of maladaptive sustained anxiety, especially under conditions of uncertainty. To understand the neural underpinnings of sustained anxiety in anxious girls across the transition to adolescence, 170 highly anxious girls will be followed from age 10/11 to age 13/14 with clinical assessments, multimodal neuroimaging, and behavioral and hormonal measures of sustained anxiety. Based on previous work, it is expected that 40% of these girls will maintain high levels of anxiety into adolescence, with up to half of these stably anxious girls developing bona fide anxiety and/or depressive disorders. Parallel functional neuroimaging tasks will be used with anxious girls and anxious female nonhuman primates to assess brain activation associated with sustained anxiety during exposure to prolonged and uncertain threat. Mechanistic studies in young anxious female monkeys will use precise MRI-guided lesions of BST neurons to test the causal role of the BST in anxious behavioral and to identify, for the first time in primates, regions that are functionally modulated by BST input. Finally, BST neurons will be harvested from monkey brains using laser capture microdissection, and RNA deep sequencing will be used to link variations in gene expression to anxiety severity and BST metabolism. These translational studies provide the opportunity to test hypotheses about the clinical relevance and causal role of BST function. In addition, examining the molecular composition of BST neurons provides an invaluable opportunity to identify novel molecular mechanisms that contribute to the at-risk child phenotype. This combination of modalities and methods has high translational potential for developing novel, BST-focused, anti-anxiety treatments.

Project Summary Anxiety disorders (ADs) are the most prevalent psychiatric ailments in children and adolescents. Childhood ADs substantially impair development and confer high risk for later psychopathology including persistent anxiety, depression and comorbid substance abuse. Given the high prevalence and long-term negative impact of childhood ADs, it is critical to successfully treat ADs early in development. Unfortunately, many children with these disorders either fail to fully respond to treatment or relapse. Understanding the biology of childhood ADs is crucial for developing early interventions with the potential to reduce the chronicity and comorbidity associated childhood onset. Cross-species conservation of brain-behavior relationships provides a unique opportunity to link basic neuroscience in nonhuman primates with clinical neuroscience in childhood ADs to identify the neural substrates of childhood anxiety. The proposed project will capitalize on this exceptional advantage by using two integrated approaches: 1) a multisite multimodal imaging study that extends promising basic science findings in nonhuman primates to a large sample of preadolescents with ADs and 2) a molecular study that translates neural findings in childhood ADs to gene expression studies in nonhuman primates using our fully-phenotyped nonhuman primate brain bank focused on alterations in neuroplasticity transcripts. Findings from our nonhuman primate studies highlight the importance of identifying neural substrates that contribute to shared variance across ADs, as well as specific neural substrates that are associated with particular anxiety phenotypes. This approach is consistent with evidence of shared and specific features of childhood ADs: most children with ADs present with an admixture of symptoms and treatment responses are similar across diagnoses suggesting shared neural substrates; however, substantial variation in symptoms and presentation suggest heterogeneous neural substrates. The study will focus on three specific aims: (1) Identify neural alterations that are shared among childhood generalized anxiety disorder, social anxiety disorder, and separation anxiety disorder; (2) determine neural alterations linked to specific anxiety-relevant RDoC constructs (i.e. acute, potential, and sustained threat, and generalization of conditioned fear); and (3) Investigate molecular alterations in brain regions central to childhood ADs, guided by the childhood AD studies in Aims 1 and 2, with a particular focus on neuroplasticity-related alterations. Studying a large sample of AD children is critical to parse the heterogeneity common in childhood ADs; to achieve this goal, we have assembled three sites with expertise in anxiety (nonhuman primate models of anxiety, childhood anxiety, developmental psychology) and advanced methods (gene expression, neuroimaging, and statistics). Ultimately we aim to elucidate the neurobiological mechanisms underlying childhood ADs to identify novel brain targets for treatment, including shared and phenotype-specific neural correlates of childhood ADs.