Tallie Z. Baram - US grants

This project examines the expression of the neuropeptide Corticotropin-Releasing Hormone (CRH) at the mRNA and peptide levels in the developing rat brain, utilizing techniques from the realms of molecular biology, quantitative neuroanatomy and behavioral neurophysiology. CRH was first found in the hypothalamus, where its effect on ACTH and glucocorticosteroid secretion in response to stressors is now well established. The role of CRH located in specific extra- hypothalamic brain regions is less well defined. Moreover, little is known about the CRH neuronal system in perinatal and immature animals. While during late fetal life steroid levels rise with noxious stimuli, there is a hiatus in their ability to generate a stress response in the first 2 weeks of life. A transient decrease in hypothalamic CRH content during the first postnatal days has been reported. We will study CRH-mRNA expression in selected brain regions of the developing rat, using in situ hybridization; peptide content of the same discrete brain regions will be assessed by radioimmunoassay of tissue specimen microdissected ('punched') from brain slices. We will manipulate peptide and/or mRNA content by altering hormonal feedback, to gain further insight into the molecular level of control mechanisms. The significance of the perinatal perturbations of the CRH neuronal system to seizure susceptibility will be explored. CRH has been shown to be a convulsant, causing epileptiform discharges and neuronal excitation in the amygdala, hippocampus and locus ceruleus in rats. Furthermore, a seizure disorder unique to infants responds to manipulations of the CRH system, while anticonvulsants are less effective. We will use the maximal electroshock seizure model and chemically induced seizures to study whether the CRH neuronal system and its manipulation, or exogenous CRH administration alter the suspectibility of the immature brain to seizures.

This project studies the regulation of CRH gene expression by glucocorticoids (GCs) in neonatal rats. It will test the hypothesis that the negative effect of GCs on CRH gene expression (present in adult rats) is not operative in neonates. CRH, an ENDOGENOUS convulsant, is more potent and rapid-acting in neonates, when the peptide's gene expression is low, compared to adults. The project tests the hypothesis that the low CRH gene expression in the neonate contributes to the enhanced convulsant potency of CRH in this age. A specific GC-receptor antagonist (RU-38486) will be implanted adjacent to the paraventricular nucleus of neonatal rats starting on the third postnatal day. CRH-mRNA abundance in implanted rats will be compared to littermate controls using semi-quantitative in situ hybridization (ISH), and the earliest age at which GC-blockade increases CRH gene expression will be determined. The abundance of GC-receptor gene expression, and the effects of Ru-38486 will be examined (using ISH). The effects of experimentally increasing CRH gene expression, via RU- 38486 implantation, on the convulsant responses of neonatal rats to CRH will be determined. Electrographic recording using bipolar electrodes from cortex, amygdala and hippocampus will allow precise definition and localization of the neuroanatomic origin of CRH- induced seizures. These studies will yield important information relevant to human neonatal epilepsy: A better understanding of CRH gene expression and the epileptic properties of this ENDOGENOUS CONVULSANT in the neonate will help elucidate mechanisms of age-specific neonatal seizures, some of which respond to modulation of the CRH-GC feedback loop. In addition, it will provide insight into intrinsically important issues of neuropeptide regulation in the neonatal brain.

The neuropeptide, corticotropin releasing hormone (CRA), is the key coordinator of the neuroendocrine, behavioral and autonomic responses to stress. The principal investigator has shown that infusion of CRH into the cerebral ventricles of infant rats produces severe age- dependent limbic seizures. Multiple CRH-induced seizures result in selective neuronal death in amygdala and hippocampus. The seizures, like other CRH effects, are mediated via specific receptors. Two distinct members of the CRH-receptor family have been characterized CRF1 and CRF2. Both receptor types are found in amygdala, site of origin of CRH-induced seizures, and may therefore mediate this effect of CHR. The availability of ligands specific for one receptor type permits determination of the receptor mediating CRH-induced seizures. The goal of this collaborative proposal is to test the hypothesis that: 1) CRH induced seizures are mediated by CRF, and 2) that the seizures alter CRF1 (but not CRF2) gene expression. Selective non-peptide CRF1 antagonists will be administered by the P1 to infant rats prior to the infusion of CRH. Their ability to abolish the seizures induced by CRH will be compared to the established effects of non-selective peptide compounds, using both behavioral and EEG criteria. Brains of animals with CRH-induced seizures (and control brains) will be analyzed in the principal investigator's laboratory for messenger RNA levels of CRF1 and CRF2, using in situ hybridization, focusing on limbic regions. Authoradiographic studies will determine alterations in CRH-binding capacity of each receptor type in defined brain regions. This project builds on the strengths and expertise of the collaborators, it establishes CRH-induced seizures in infant rats as a reliable in vivo paradigm for testing candidate compounds as CRH receptor blockers. In essence, this Phase I project sets the stage for the commercial development of novel CRH receptor blockers as anti convulsants for selected seizure types.

Seizures induced by fever are common in the infant and young child. Whether they result in neuronal death and subsequent temporal lobe epilepsy is controversial. Experimental models for febrile seizures used to date have involved older animals, or were unsuitable for long-term studies. Work by the applicant has established an infant rat paradigm of hyperthermia-induced seizures which is age-appropriate and suitable for long-term studies. This model will be used to test the following hypotheses: (1). Hyperthermic seizures result in injury of select limbic neurons, particularly in the central and lateral amygdaloid nuclei. (2). This neuronal injury is mediated by the neuroexcitatory peptide, corticotropin releasing hormone (CRH). To define the distribution and characterize the types of neurons injured, brains will be examined at several time-points following the induction of hyperthermic seizures. Neuronal injury will be determined based on altered staining properties of affected cells. Concurrent experiments will test the hypothesis proposed for the mechanism of this neuronal injury: If neuronal injury produced by hyperthermic seizures is mediated by CRH then i). these seizures should increase the levels of CRH in the involved brain regions and ii). the injury should be prevented by the administration of CRH antagonists. Furthermore, the long-term effects of hyperthermic seizures during infancy on the development of spontaneous seizures (epilepsy) in adulthood will be determined. The proposed studies establish a long-term model for the study of febrile seizures in the infant, a model which had been pursued by investigators focusing on Developmental Epilepsy Research for close to two decades. These studies should yield fundamentally important information regarding the pathogenesis of non-genetic human developmental epilepsies, of which febrile seizures are the most prevalent. The results of the proposed studies may have significant implications for the current management of these seizures in human infants, and could lead to a more aggressive approach to febrile seizures.

DESCRIPTION (adapted from applicant's abstract): The current proposal focuses on mechanisms and consequences of early-life seizures that are provoked by proconvulsant'stressors' such as fever (hyperthermia) and hypoxia. These non-genetic 'triggered' seizures constitute the majority of developmental seizures. While the outcome of simple febrile seizures is generally benign, and the outcome of many developmental seizures depends on their etiology, emerging evidence in both human and animals indicates that prolonged febrile seizures and recurrent triggered seizures may be associated with neuronal injury. The applicant has demonstrated that the stress-activated neuropeptide, corticotropin releasing hormone (CRH), a well established key mediator of the CNS stress-response, acts as a powerful, age-specific convulsant in the developing brain and promotes injury of hippocampal neurons. Thus, CRH may play a role in the mechanisms by which triggered developmental seizures influence neuronal integrity. During the recent funding period, the applicant established the presence of a significant population of CRH-expressing interneurons in the developing hippocampus and the mechanisms by which CRH interacts with glutamate receptor activation to enhance hippocampal excitability. In addition, recent findings suggest that (1 ) pro-convulsant stressors may increase CRH expression in limbic regions and (2) excessive CRH-receptor activation during early-life may result in significant long-term consequences on hippocampal integrity and function. Therefore, the proposed research tests the hypothesis that proconvulsant stressors increase CRH levels in hippocampus resulting in augmentation by the peptide of excitotoxic mechanisms. to enhance neuronal injury and lead to long-term hippocampal dysfunction. Four experiments are proposed to test this hypothesis: 1 ) studying-using in situ hybridization and immunocytochemistry- whether proconvulsant stressors enhance CRH-expression and levels in hippocampus. Studying the unique mechanisms by which CRH leads to hippocampal neuronal death using (2) in vivo and (3) in vitro dissociated hippocampal cell approaches, and, 4) Examining the significant long-term consequences of excessive CRH-dependent excitation during early life, focusing on hippocampal neuronal loss and hippocampal-dependent cognitive dysfunction. The significance of the proposed studies derives from their focus on mechanisms and consequences of early life provoked seizures that constitute the majority of developmental seizures, but remain relatively unstudied. The proposed studies, delineating age-specific mechanisms for seizure-related neuronal injury and its consequences should lead to the development of novel, age-appropriate, specific therapies to prevent these consequences.

DESCRIPTION (provided by applicant): Early-life experience influences neuroendocrine and behavioral responses to stress long-term, with profound implications for emotional health throughout life, but the molecular mechanisms of this effect remain unclear. Studies during the current award period found that reduced expression of the stress neuromodulator corticotrophin releasing hormone (CRH) in hypothalamus was an early event in experience-evoked 'programming' of the hypothalamic pituitary adrenal (HPA) system: CRH mRNA was downregulated already on postnatal day 9 in daily handled pups, preceding the onset of attenuated stress responses and the life-long enhancement of hippocampal glucocorticoid receptor (GR) mRNA and stress-'coping' behaviors. The goal of the current proposal is to investigate the mechanisms of this stress-response neuroplasticity after handling-evoked alterations of maternal care, by testing systematically several mechanistic hypotheses: (1) An early phase of 'handling'-evoked HPA axis neuroplasticity in rat pups involves coordinated transient activation of neuronal transcription factors in stress-regulating sensory integration regions. This effect is evoked by a 'burst' of maternal care elicited by the return of 'handled' pups to the cage. (2) Recurrence of this neuronal activation, particularly in 'stress-memory storage regions' is required for enduring downregulation of hypothalamic CRH gene transcription and steady state expression. (3) Reduction of hypothalamic CRH expression is necessary and sufficient to promote the attenuated neuroendocrine stress responses and enduring elevation of hippocampal GR expression and 'coping' behaviors characteristic of high maternal care early in life. (4) CRH acts via the CRF1, receptor to 'program' HPA responses long-term, so that selective CRF1 antagonists will convert hippocampal GR expression and long-term stress responses to those seen in early-life 'handled' rats. (5) The mechanisms by which reduced CRH levels and decreased activation of CRH receptors alter hippocampal GR expression and stress responses long-term involves modulation of plasma glucocorticoid levels, so that 'clamping' these levels will prevent handling-evoked HPA axis programming. The proposed experiments will delineate the spatiotemporal onset and progression of the protein effects of early life experience (and maternal care) on the immature HPA system, and use molecular and pharmacological means to define the responsible mechanisms. These studies should help clarify the molecular and neuroanatomical basis for early-life plasticity of the HPA system. In addition, they should yield mechanistic, targeted therapy for reversing the HPA hyperactivity consequent to deficient early-life experience that characterizes, and perhaps underlies, certain human affective and anxiety disorders.

DESCRIPTION (provided by applicant): This proposal focuses on the mechanisms and consequences of febrile seizures, the most prevalent seizure type in young children. An immature rat model of prolonged febrile seizures, those associated with potential development of limbic epilepsy, has been characterized, and has already shed considerable light on the neuroanatomical basis of these seizures and on their functional consequences. Importantly, it was established that experimental prolonged febrile seizures lead to long-lasting enhanced hippocampal excitability. Surprisingly, this increased excitability was associated with persistent increase in GABA-mediated inhibition of CA1 pyramidal cells. A resolution to this apparent paradox derives from preliminary data showing functional changes in the Hyperpolarization-activated Cation-Nonselective channels (HCNs) in CA1 pyramidal cells of immature rats who had experienced experimental febrile seizures: Slowed HCN channel kinetics permit increased Na+ entry, depolarizing the cell to promote action potential firing, essentially converting the potentiated inhibition to hyper-excitability. Importantly, slowing of HCN kinetics is consistent with a quantitative shift in the subunit make-up of these recently cloned channel molecules, and preliminary mRNA expression data support this notion. Therefore, this proposal will test the hypothesis that experimental prolonged febrile seizures modulate the expression of HCN channel molecules and disrupt their normal developmental expression patterns, leading to persistently enhanced excitability. Three experiments are proposed: 1) To determine the developmental spatio-temporal expression profiles of the 4 HCN subunit isoforms in defined hippocampal cell populations and single neurons, providing the foundation for probing effects of the seizures; (2) To determine the effects of the seizures on HCN expression in defined individual cells and neuronal populations in vivo; 3) To use an in vitro organotypic hippocampal culture to determine the mechapisms for seizure-induced alteration of HCN isoform expression and the consequent ?neuroplastic? changes in hippocampal excitability. The proposed studies should provide novel and important insight into the remarkable age-and seizure-specific effects of prolonged experimental febrile seizures on the developing hippocampus, changes leading to enhanced excitability long-term. In addition, these studies should contribute to our understanding of fundamental aspects of the functional anatomy of these newly characterized ion channel molecules in developing hippocampus, leading to the definition of the roles of these pacemaker channels in the development of the synchronized hippocampal network.

DESCRIPTION (provided by applicant): This proposal aims to renew support for a Training Program in Epilepsy Research. The overall goal of the program is recruitment and training of a diverse group of outstanding neuroscientists who will gain (1) sophisticated and in depth knowledge of research germane to the neurobiology, co-morbidities and cure of human epilepsy; (2) cutting edge methods and innovative experimental approaches required for successful competitive contemporary research; (3) understanding and preparation for a diverse repertoire of career choices in epilepsy research. The Training Program in Epilepsy Research accomplishes these goals via individualized, committee-supervised research training in the labs of 14 well-funded and enthusiastic preceptors. The training experience is enriched by program-specific courses, seminars and symposia that draw in the UCI Neuroscience community and potential trainees. Our success to date in achieving our ambitious training goals derives from an outstanding pool of candidates, and from synergy with the UCI Epilepsy Research Center and the UCI Clinical Epilepsy Program, which interact to provide a multifaceted training experience. These assets are complemented by an open and interactive research atmosphere and abundant collaborations among program faculty. Our approach to Epilepsy as Window to Brain Plasticity promotes both basic and translational approaches to research on epilepsy and its cognitive and emotional co-morbidities. Given the trajectories of academic careers, tangible measures of success of our training approach are becoming evident as the Training Program in Epilepsy Research is in its 9th year. Nine of eleven (82%) program alumni are in academia, all assistant professors or equivalent and 5 in tenure track, including a woman from an underrepresented minority group. The 2/11 not in Academia is involved in neuroscience as managing editor, Nature publications and college level educator. The large majority of past trainees are involved in epilepsy research, and several past and present-cycle trainees have received their own NRSA or equivalent. Our goal for the coming years of the Program-one of only two Epilepsy-focused T32s in the U.S.- are to capitalize on our success and learn from trainee and faculty input to further enhance the training experience: (1) From our 2 pre-doctoral trainees (one a minority), we learnt the beneficial effects of pre-postdoc trainee interactions to both, and request that one of our 4 yearly trainees be a senior predoctoral student; (2) We aim to increase MD recruitment (currently 10%), and that of individuals from diverse backgrounds (12.5.%; higher than in the UCI graduate pool). (3) We aim to intensify career preparation and grant writing skills via integrative efforts with the new UCI CTSA. Thus, the Training Program in Epilepsy will build on its record of launching talented neuroscientists, including minority and women into sustainable academic careers, thus enhancing the future workforce in neuroscience research.

DESCRIPTION (provided by applicant): The proposed research aims to validate a developmental model of epileptogenesis, and delineate surrogate markers for the epileptogenic process and for associated neuronal dysfunction. A major goal of Epilepsy Research, considered an Epilepsy Benchmark, is to intervene in the epileptogenic process and abort the development of epilepsy and of related cognitive dysfunction. This goal requires: (1) validating models leading to epilepsy (spontaneous recurrent seizures) and (2) having reliable criteria or "markers" for progressive epileptogenesis. The applicants have focused on the epileptogenic process culminating in temporal lobe epilepsy (TLE), and specifically on the potential causative role of prolonged febrile seizures (FS). An immature rodent model of prolonged FS has been characterized and has led to the discovery of molecular changes that promote hyperexcitability in the limbic circuit. Recent findings, using nocturnal video-EEG monitoring, indicate that a subset of adult animals that had experienced experimental prolonged FS early in life develop classical limbic seizures, with behavioral and EEG correlates. Therefore, the proposed research will test the hypotheses that the prolonged FS model can be used to predict TLE and hippocampal cognitive deficits, and that MRI lesions will serve as surrogate markers for epileptogenesis and/or hippocampal cognitive deficits. The project aims to (1) Determine the nature, distribution and time-course of abnormal MRI signals, (2) determine the value of MRI in predicting epileptogenesis, (3) determine the value of MRI in predicting the development of hippocampal neuronal dysfunction at the single cell and system levels. Based on exciting preliminary data, it is strongly believed that the outcome of this work will provide important data for future interventional strategies targeting mechanisms of disease processes - a major goal for this RFA.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this proposal is to determine the role of the stress-neuromodulator corticotropin releasing hormone (CRH) in early-life neuroplasticity that contributes to neurological disease. Studies during previous funding periods have demonstrated that central administration of synthetic CRH to infant rats led to long-term deficits of hippocampus mediated cognitive function, similar to those described after severe early-life stress in human infants. We have also demonstrated the presence of the 'cellular machinery' for endogenous CRH in immature hippocampus, i.e., substantial populations of CRH-expressing interneurons and abundant CRH receptors on hippocampal pyramidal cells innervated by these interneurons. Therefore, the current proposal aims to definitively test the hypothesis that enduring upregulation and excessive release of endogenous hippocampal CRH contribute crucially to the impaired functional integrity of the hippocampus following early-life adverse events such as chronic psychological stress or recurrent seizures. The driving hypothesis of this research is that certain stresses occurring during a period of heightened hippocampal plasticity elevate the 'set-point' of hippocampal CRH expression, increasing the peptide's levels in hippocampus longterm. Therefore, subsequent, acute stress at any point in life will evoke 'excessive' release of CRH from hippocampal interneurons. Pathological levels of CRH provoke acute and long-term hippocampal neuronal dysfunction, associated with synaptic reorganization. This research proposal focuses on understanding this process at the synaptic, cellular and system levels, defining the means for interrupting the deleterious actions of excessive endogenous CRH on hippocampal function and integrity. Therefore, the proposal aims to: (1) Investigate the effects of early-life (P2-P9) chronic 'psychological1 stress on hippocampal CRH expression long-term. (2) Delineate the unique synaptic machinery by which CRH (an excitatory neuropeptide), is stored in inhibitory interneurons, define the site of the postsynaptic elements mediating the peptide's actions, and determine whether stress activates post-synaptic hippocampal pyramidal cells via the release of endogenous CRH. (3) Characterize the functional, electrophysiological and structural hippocampal changes induced by early-life chronic psychological stress in a novel model. (4) Determine whether these effects of early-life stress are abrogated by blocking the actions of endogenous, stress-released CRH; in other words, whether the endogenous peptide is a required, crucial mediator of the enduring, life-long adverse effects of early-life stress on hippocampal functional integrity. These studies address the impact of early-life chronic psychological stress, such as child abuse and neglect (and perhaps recurrent seizures), on critical learning & memory functions. By defining the role of the stress-activated neuropeptide, CRH, in the mechanisms of the established adverse effects of these early-life events, these studies will provide truly innovative and exciting targets for prevention of a tremendous loss of human potential. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This revised competitive renewal proposal focuses on the mechanisms by which febrile seizures (FS), the most prevalent seizure type in infants and children, may lead to Epilepsy. The applicant has characterized an immature rat model of prolonged FS, those associated with subsequent development of limbic, 'temporal lobe' epilepsy (TLE) in human studies. During the current award period, it was discovered that experimental prolonged FS lead to limbic epilepsy in approximately 35% of animals, establishing the paradigm as a valid model of developmental epileptogenesis. In addition, these seizures induced changes in the expression and co- assembly of specific ion channels, the hyperpolarization-activated, cyclic nucleotide gated (HCN) channels, that promoted hippocampal hyper-excitability. Remarkably, changes in the expression and in the relative abundance of the same members of the HCN channel family were found also in human hippocampus from individuals with TLE and a history of early-life seizures, confirming the relevance of molecular changes in these channels to clinical epileptogenesis. The current proposal aims to address important gaps in our understanding of the epileptogenic process bridging prolonged 'FS' and limbic epilepsy: 1) First, in vitro systems will be used to determine how these developmental seizures lead to down-regulation of the HCN1 isoform expression and increased expression of HCN2, by testing hypotheses about seizure-evoked, calcium-mediated cellular cascades influencing coordinate HCN isoform expression; 2) To determine how seizures evoke re-arrangements of HCN channel molecules into heteromeric channels that promote hyper-excitability: the role of post-translational glycosylation of the channels in the expression of heteromeric channels will be studied in vitro; 3) Finally, in vivo experiments will investigate the parameters of the inciting FS that govern epileptogenesis, aiming to generate a more powerful model, where seizures elicit epilepsy in the majority of rats. Public health relevance (lay language): Epilepsy is the most common chronic brain disorder in young individuals. Temporal lobe epilepsy is the most refractory epilepsy, and is associated with early-life prolonged febrile seizures. Understanding the steps and the mechanisms by which febrile seizures convert a 'normal' hippocampus to an epileptic one is a critical first step in intercepting the epilepsy-generating process resulting in temporal lobe epilepsy. [unreadable] [unreadable] [unreadable]

Chronic stress early in life (ES), including neglect, abuse, loss of parent and severe poverty, affects the majority of the world's children (UNESCO report, 2004). This is of major clinical importance because chronic childhood stress is associated with cognitive (and psychiatric) disorders later in life. Because elimination of global ES is not feasible, effective therapies that can be given post hoc to prevent the effects of ES on mid- life cognitive decline are necessary. Having defined a rodent model of ES which results in enduring deficits of hippocampus-dependent cognitive function and LTP, together with dendritic atrophy, we found that post hoc blocking of the receptor (CRFR1) of the stress-activated neuropeptide, corticotropin releasing hormone (CRH) immediately after the ES period, abrogated these deficits. Whereas these data are encouraging, major gaps in our knowledge require study in order to translate these experimental findings into therapies for children. In this revised continuation proposal, we propose (1) to test if pathological activation of central or of peripheral CRFR1 is responsible for ES-provoked learning and memory defects and dendritic atrophy; (2) to distinguish between the hypothesis that ES leads to enduring changes in hippocampal structure and function that are irreversible after a critical period of development, and the possibility that ES initiates hippocampal derangements that progress throughout life. In the latter case, therapeutic interventions in young adult ES graduates will still prevent the cognitive and structural deficits; (3) Because the structural changes provoked by ES-dendritic atrophy and synapse / spine loss--underlie the cognitive deficits, the mechanisms of dendritic atrophy will be studied, focusing ontheroleof hippocampal CRH-CRFR1 signaling; (4)Becausedendriticatrophyderivesfromchroniclossofdendriticspines,themechanismsbywhichstress, via CRFR1 activation, provokes dendritic spine collapse will be examined. The proposed studies, spanning in vivo and in vitro systems, will provide insight into the mechanisms by whichESimpactsneuronalintegrity,synapticplasticityandcognitivefunctionlong-term.BecauseESaffects the majority of the world's children, these studies address a problem of paramount importance, which is strikingly understudied. The proposed studies will identify a novel mechanism, CRH-CRFR1 signaling, as pivotal in the disturbances provoked by ES. Because the proposed studies will demonstrate the potential for post hoc intervention, and because compounds targeting CRFR1 are under clinical development, the results of these studies have tremendous translational potential.

DESCRIPTION (provided by applicant): This proposal focuses on understanding the neurobiological mechanisms of resilience to stress-related disorders. Because stress-related disorders--including depression, post-traumatic stress disorder and childhood cognitive and emotional defects that follow neglect and abuse--affect a significant proportion of children and adults and exact a huge human potential and financial toll, understanding the processes that engender resilience to these disorders carries profound impact. Vulnerability and resilience to disease are governed by the interaction of genes and environment / experience. Importantly, experience during early postnatal life influences the expression of stress-related genes, which may promote resilience or vulnerability to stress-related disorders. However, little is known about how these important effects take place. Augmenting early life experience by enhancing maternal care in the rat results in persistent alterations of stress-related genes: reduced expression of hypothalamic corticotropin releasing hormone (CRH) was found by the applicant already by the end of the enriched-experience period on postnatal day 9, preceding attenuation of stress responses and increased expression of hippocampal glucocorticoid receptor (GR). In addition, reducing CRH-CRH-receptor signaling in immature non-enriched rats was sufficient to up-regulate hippocampal GR persistently and to confer enduring resilience to stress. These findings indicate that repression of hypothalamic Crh gene expression is an early and crucial step in the molecular cascade bridging enriched maternal-derived early-life experience and the enduring neuroplasticity of the stress system that promotes resilience to stress-related disorders. The current proposal addresses important gaps in our understanding of the process by which augmented sensory input from maternal care promotes resilience to stress-related disorders: how are maternal-derived signals converted to information that 'commands'hypothalamic neurons to repress Crh gene expression? What molecular mechanisms initiate this repression? Maintain it throughout life? The aims of the proposed research are to (1) Test the hypothesis that augmented early-life maternal-care initiates epigenetic programming of the Crh gene via increased expression of the transcriptional repressor Neural Restrictive Silencing Factor (NRSF) and increased NRSF binding to a cognate binding site (NRSE) in the regulatory region of Crh. (2) Test the hypothesis that NRSF binding to NRSE is required for repression of Crh gene expression in hypothalamic neurons from experience-enriched rats. (3) Employ controlled, in vitro systems to test the hypothesis that NRSF upregulation and Crh repression in hypothalamic neurons are a result of reduced excitatory synaptic input to these neurons. (4) Examine the mechanisms that may be responsible for the maintenance of the suppressed expression of CRH and consequent attenuated stress responses and resilience to stress-related disorders. In summary, resilience to stress-related disorders is of major clinical significance. The paramount contribution of early-life experience to this resilience can be studied in suitable animal models, and information is now available regarding the molecular and functional changes of the 'stress system'that contribute to resilience. The proposed research will define how these molecular changes are initiated and maintained, and identify potential targets for translational clinical use, with profound impact on human health. PUBLIC HEALTH RELEVANCE: Some people are more resilient to stressful situations that can provoke depression or learning and memory problems in other individuals. There is some evidence that having a good experience early in life might promote this resilience. Here we hope to use animal models to discover exactly how this resilience happens. The resulting information would enable us to use the same mechanisms as a basis for therapy in people who have not had the same type of optimal childhood experience.

The cognitive defects provoked by short-term stress are now emerging as a major clinical problem. The impact of severe, hours-long stresses-such as incurred on a battlefield or during emotional or physical trauma--is profound, because these stresses are frequent and unavoidable in modern life. Remarkably, relatively little is known about the underlying mechanisms. In a novel rodent model of a physical / psychological short-term stress, we observed major memory impairments associated with selective disturbances of hippocampal LTP and loss of dendritic spines. Thus, disturbances to synaptic plasticity and spine integrity, both involving dynamic actin organization, are shared among stress and several disorders discussed elsewhere in this application, and may share common mechanisms. Stress involves steroid, monoamine and peptide mediators; we have shown that the neuropeptide CRH (corticotropin releasing hormone) is released in hippocampus during stress and can impair dendritic spines in domains that are impaired after stress, suggesting involvement of CRH in stress-induced defects of spine integrity and synapse function. Therefore, this project will test the hypothesis that acting via CRH-CRH receptor (CRFR{1}) signaling, stress leads to disturbances of dendritic spine actin organization: (1) Stress Interferes with basal actin assembly resulting In spine loss; (2) In common with other disorders discussed in this program, stress deranges activity-driven assembly and stabilization of the spine actin-skeleton. Four Specific Aim are (1) To test if short stress reduces the number and actin-assembly of mature spines, and if this takes place via mechanisms involving endogenous CRH-CRFR{1} signaling; (2) To test if short stress disrupts activity-induced spine actin assembly and stabilization by mechanisms involving CRH-CRFR{1} signaling; (3) To examine the mechanisms downstream of CRFR{1}, specifically if the actin-regulating GTPase RhoA is involved in CRH-provoked spine loss, and if this spine loss is prevented by BDNF; and (4) To translate the results of the above In vitro studies into potential therapies, by testing if stress-induced deficits of spines, LTP and memory are abrogated by blocking CRFR{1}, augmenting BDNF, or combining the two approaches. Collectively, these studies will transform our understanding of how stress provokes memory deficits. Stress is the most common complaint of individuals in the modern world, and its effects on human function are inestimable. Uncovering the mechanisms by which stress impairs cognitive function and deriving targeted therapies based on these mechanisms will therefore have a profound impact.

DESCRIPTION (provided by applicant): Febrile seizures are the most common type of seizure seen in young children. Unfortunately, some children with prolonged febrile seizures appear to be at risk for long-term cognitive disturbances. Identifying those individuals at risk for cognitive impairment and discovering the responsible mechanisms would provide opportunities for therapeutic intervention. In preliminary studies through an R21 funding mechanism, we used an immature rat model of long experimental febrile seizures (EFS) and established that a subgroup of rats experiencing these seizures developed spatial memory impairment and aberrant place cell function during adulthood. These deficits in hippocampal-dependent spatial cognition were accompanied by elevated MRI T2 signals in the hippocampus one month after the seizures. These findings demonstrate for the first time a direct causal effect of EFS on function of specific neurons that govern memory performance suggesting that MRI might be a potentially predictive biomarker for individuals at risk for cognitive disturbances. Furthermore, we have found that MRI T2 signals in hippocampus one month after the seizures are associated with inflammatory activation but no overt cell death indicating that inflammatory mediators might contribute to both MRI abnormalities and neuronal dysfunction, providing a target for selective intervention. However, prior to the application of these findings to the management of children with FSE additional critical information is needed. It is not known if hippocampal MRI changes take place early enough to be predictive of cognitive defects, and thus useful for potential intervention or whether the observed cellular and cognitive impairments are result of the FSE or of ensuing epileptogenesis. Determining if hippocampal levels of the inflammatory cytokine interleukin (IL)-12 distinguish FSE rats that became epileptic from those who did not and if this cytokine is involved in the cognitive defects provoked by EFS is necessary. In this proposal we will assess whether the cognitive defects in a subset of rats experiencing FSE are predicted by selective MRI changes that are visible early after the seizures and whether the cognitive and place-cell defects after FSE emerge prior to, and independent from, the epileptogenic process and spontaneous seizures, and define the underlying mechanisms of such deficits. To ascertain whether inflammatory cytokine expression distinguishes rats with cognitive defects after FSE from those with intact hippocampus-dependent memory we will compare rats with and without cytokine changes after EFS. The results of this study will set the stage for therapeutic intervention in children at risk for cognitive problems following febrile seizures.

The Administrative Core will function to enable and coordinate efficiently a rich repertoire of Center activities. It will work closely with the PI, Executive Committee and the two other Cores to implement the goals of the proposed Center. It will also interface with the NIMH, the UCI and Southern California communities and resources. The aims of the Administrative Core are: (A) To coordinate the logistics of the activities of the Center and provide an effective management structure; (B) To coordinate and implement the training / educational activities of the Center, including attracting new investigators to the field of developmental vulnerabilities and mental health; (C) To augment the 'value-added' by the Center by interfacing and interacting with existing University resources, enhancing the research, education and community outreach goals of the Center. The Administrative Core will be led by the Center Director, and will include a Website / Computer manager and an Administrator. It will be the logistics hub of the Center, facilitating the following activities: Executive Committee communications (weekly), Research meetings (monthly), Seminar series (monthly), Annual Conference, Internal Advisory Board meetings, and External Advisory Board meetings (twice a year). It will implement trainee recruitment, interacting with UCI graduate programs and Minority/ diversity programs. The center will administer seed grants (pathway to independence) for trainees, and school / community outreach efforts. The Core will manage the Center finances and regulatory functions (IACUC, IRB). It will interface with University Resources to leverage Center resources, engaging ICTS, and existing mentorship programs. The Core will house the Center's Website, a venue for educational and data-sharing efforts, that will benefit both professional and lay communities

DESCRIPTION (provided by applicant): This is a revised application for a Conte Center addressing the complex developmental mechanisms contributing to adolescent vulnerabilities to mental illnesses. These are generally believed to arise from an interaction of genetic and environmental influences during sensitive developmental epochs, yet there are major gaps in our knowledge regarding the nature of these signals, and the mechanisms by which they promote disease. The innovative unifying hypothesis guiding this revised application is that disturbed patterns of maternal signals early in life, especially their fragmentation and unpredictability, contribute greatly to adolescent emotional and cognitive vulnerabilities. Guided by constructive suggestions of the original Reviewers, strong preliminary data now support this hypothesis. The overarching goal of the proposed Center is to execute a multi-dimensional and integrated plan that brings this exceedingly important clinical problem into both animal and human research laboratories. Capitalizing on their complementary strengths and employing cross-species platforms and sophisticated coordinated analytical methods, the Center aims to translate the resulting discoveries into clinically significant predictive models. The Centers approach benefits from a unique resource, a large well-characterized human cohort, to perform longitudinal and cross-sectional studies from fetal life to adolescence. Center research will focus on advanced structural and functional brain imaging in this cohort and in cognate animal models, combined with state-of-the-art molecular approaches to gene regulation in time and space. Center Aims will (1) characterize patterns of pre- and postnatal maternal signals that influence adolescent vulnerabilities across species, using conventional and innovative methods, and generate trajectories of altered behaviors in infancy, childhood and adolescence; (2) address the potential underlying mechanisms using common platforms in human and rodents, including structural and functional imaging, and mechanistic studies in rodents; 3) through a strong Statistics / Computational approach, combine behavioral and imaging outcomes along time to generate potentially predictive models for adolescent behaviors that augur pathology. The Center's significance derives from addressing crucial clinical questions via robust, cross-species multidisciplinary, state-of-the-art approaches. The Center tests a highly innovative hypothesis that builds on and provides a theoretical unifying framework for a large existing human and experimental literature. Major assets of the Center approach include a large longitudinal human cohort, predictive animal models, common methodological platforms across species, strong preliminary data, improved integration of Projects' design and data flow and rigorous and innovative analyses of the large and interdependent data sets and their integration into predictive models. Thus, the impact of the information generated by the Center will be substantial, aiming to transform our understanding of how early life environment contributes to disorders that affect 20% of adolescents.

Improving our understanding of the mechanisms by which environmental signals during sensitive early-life periods contribute to mental illness will have profound impact on human health. In concert with the Center approach. Project 1 will test the overarching hypothesis that exposure to fragmented / unpredictable patterns of maternal signals promotes adolescent emotional and cognitive vulnerabilities that can be predicted and detected early in their trajectory. In accord with the constructive suggestions of the Reviewers of the original submission and supported by strong preliminary data, the studies proposed for the revised Project 1 will employ rodent models and viral / genetic and epigenetic tools to address the underlying mechanisms at molecular, cellular and network levels. These studies will inform and be shaped by Projects 2.3. and carried out in tight integration with Project 4 and the imaging and computational Cores Project 1 will aim to: (1) analyze patterns of maternal behaviors in an established model of fragmented care, and, building on new data showing vulnerabilities to emotional and cognitive problems already In adolescent rats. identify the onset and trajectories of these vulnerabilities to adulthood; (2) In concert with Project 4. test the hypothesis that the basis of altered emotional and cognitive functions resulting from fragmented patterns of maternal signals derives from shifts in the functional connectivity of the involved brain networks; (3) examine for sex dependence of the consequences of fragmented patterns of maternal care, guided by human data. The results, in turn will influence timing and types of human testing in Projects 3.4: (4) Probe causal molecular mechanisms in defined brain regions that may underlie the emotional and cognitive consequences of fragmented maternal care patterns. Building on new data and novel technologies, we will look for mechanisms of broad enduring gene expression changes, using Crh as a marker qene. Regulating expression in space and time, we will also test if enhanced CRH expression is necessary and sufficient to mediate the effects of fragmented care; finally, we will determine identities of other involved genes, and the mechanisms regulating enduring changes in gene expression at the chromatin level. In summary. Project 1 will focus on a crucial clinical problem and will test an innovative concept, that fragmented patterns of maternal signals might be a common denominator of the established, profound influence of the mother on the emotional and cognitive outcome of her progeny. It will use innovative viral-genetic methods and epigenetic tools to probe the underlying mechanisms, and integrate single-neuron discoveries with structural and network analyses of Projects 2. 3 and 4. The resulting discoveries will inform and guide behavioral and imaging studies in the other projects, contributing to identification of potential biomarkers and generation of predictive models for adolescent vulnerabilities. Hence, the potential impact of the proposed studies is tremendous.

DESCRIPTION (provided by applicant): Epilepsy is the third most common chronic neurological disorder, affecting 2.5 million persons in the USA. Temporal lobe epilepsy (TLE), with seizures involving the hippocampal circuit, is one of the most severe epilepsies in adults, and is refractory to medical treatment in >30% of individuals. TLE is commonly preceded by febrile status epilepticus (FSE). Whereas the relationship of FSE to human TLE is not fully understood, experimental FSE provokes TLE in animal models, suggesting that FSE might contribute to epileptogenesis in both normal and predisposed brain. Importantly, the contribution of FSE to TLE (as a 1st or 2nd hit) may be predictable and preventable. TLE commonly affects the young, exacting a tremendous toll on human potential. However, there are major challenges in preventing or aborting TLE: (1) We do not know the mechanisms for the contribution of FSE to TLE; (2) We do not have predictive markers to identify the 30-40% of those experiencing FSE who will proceed to develop TLE; (3) We do not have mechanism-based drugs to abort the epileptogenic process that bridges FSE and TLE. The three specific aims of this competing renewal proposal address these major challenges. We capitalize on results obtained during the current award period, on our published and novel findings regarding the molecular, cellular and network changes that follow FSE and contribute to epileptogenesis, and on exciting data supporting clinically-translatable predictive markers. We employ innovative methods in in vitro and in vivo systems, and integrate hypothesis-driven and large scale gene expression analyses. We devise innovative decoy and mimic approaches for both probing mechanistic questions and for potential translation. Our work to date has revolutionized the scientific approach to the study of febrile seizures and their consequences, has been published in high-impact journals and has been cited > 4000 times. In this renewal application, we embark on highly novel, bench-derived yet translatable solutions to a major human health problem.

The goals of this four-day workshop on the Neurobiology of Stress are to bring together researchers from diverse disciplines and at all professional levels (undergraduate student to full professor) to share and discuss recent advances in the neurobiology of stress research, promote intensive exchange of ideas across disciplines, foster community among stress researchers, and nurture professional development for trainees and junior faculty participants. This workshop, which takes place in Newport Beach, CA, is the 4th of a biannual workshop series that offers the only small forum focused on the neurobiology of stress. The award provides support to junior faculty, trainees, and high school science teachers to attend this workshop. The intellectual merit of the workshop derives from its relatively small size, which promotes interactions and extensive exchange of ideas among participants, the assembly of top neuroscientists in the field of stress, and the multidisciplinary nature of the plenary session topics. With a substantial fraction of the speakers being women and about half of the participants being trainees, including trainees recruited through national minority scientist networks, this workshop reaches out to and offers opportunities for groups underrepresented in science. The inclusion of local science high school teachers in this workshop seeds dissemination of insights in classrooms.

The topics of this workshop on the Neurobiology of Stress range from epigenetics and stress to evolutionary perspectives on stress, and incorporate recent advances in molecular biology, genetics, microbiotics, imaging, and systems biology. The format entails 5 plenary sessions with 4 speakers each; 2 roundtable sessions, one of which is dedicated to mentoring of trainees in small informal groups; poster sessions (trainee presenters only); and one keynote lecture. Workshop participants will be invited to contribute manuscripts reporting findings related to workshop topics to the open access journal Neurobiology of Stress. The broader impacts of this workshop include broadening participation, extensive opportunity for exchange across professional levels, and public outreach to the local schools. These aspects, combined with the workshop's central goal of exchanging and advancing promising new ideas in stress research, constitute long-term benefits for the larger stress neurobiology community.

This is a resubmitted renewal proposal for a Conte Center focused on the contribution of early-life experiences, especially unpredictable and fragmented maternal and environmental signals, to adolescent vulnerabilities and adult mental illness via mechanisms involving disruption of the maturation of cognitive and emotional brain circuits. Complex behaviors involve coordinated activities of brain circuits. During development, environment- derived sensory signals influence circuit maturation (e.g., visual, auditory) and may drive aberrant circuit maturation that can promote emotional and cognitive problems. Yet the nature of the signals that contribute to vulnerabilities to mental illness, and how they disrupt brain circuit maturation is unclear. Among environmental influences, early-life adversity is an established risk factor for mental illness, and aspects of adversity (e.g., maternal depression, poverty) explain a significant portion of mental problems later in life. Yet there are serious gaps in our ability to identify early vulnerability to mental illness. Here, we posit that unpredictable, fragmented sensory signals (FRAG) from the mother and environment constitute a previously unrecognized indicator of early-life adversity. This hypothesis originated from mechanistic animal studies where consistent, predictable patterns of maternal-derived signals promote resilience by modulating excitatory synapse number and function of specific cell populations. By contrast, FRAG promotes aberrant maturation of brain circuits involved in emotion and cognition, with commensurate behavioral deficits. During the original award we focused on several cognitive and emotional vulnerabilities and these remain outcomes in this proposal. Additionally, we identified anhedonia as a robust direct consequence of early-life FRAG in experimental systems, associated with evidence of aberrant pleasure / reward circuit maturation. Anhedonia, a dimensional (RDoC) entity linked to multiple mental disorders, is a recently-identified core feature of PTSD. We emphasize anhedonia in the proposed renewal because we find that it follows FRAG in children, adolescents and young adults and predicts risk for post-combat mental illness in a vulnerable population of Marines. Thus, supported by compelling recently-published and preliminary data and guided by the reviews of the original renewal proposal, we test the Center?s overarching hypothesis: It states that, in concert with established types of early-life adversity, fragmented and unpredictable maternal and environmental signals contribute to vulnerabilities to mental illness via mechanisms involving disruption of the maturation of cognitive and emotional brain circuits. The proposed Center will aim to: 1) Test the relative contribution of FRAG, along with other early-life risk factors, to mental health outcomes including anhedonia, considering sex and using tools enabling assessments across diverse cohorts. 2) Test the mechanisms underlying the effects of FRAG on the developing brain, with sensitivity to age- and sex-specific vulnerabilities and age-appropriate assessment tools. We will employ imaging and computational models focusing on brain circuit disruption; we will employ molecular and viral-genetic tools and capitalize on experimental animal systems to identify the underlying neurobiological mechanisms. 3) Create behavioral and neuroimaging sex-specific developmental trajectories from infancy to adulthood using our 3 prospective, well-characterized cohorts and repeated intra-individual measurements; generate predictive models for risk of anhedonia and vulnerability to mental illness; supported by preliminary data, identify intra-individual epigenetic signatures of FRAG in children as potential biomarkers. 4) Serve as a training forum and a magnet for the study and improved understanding of how early life experiences influence emotional and cognitive outcomes. In summary, guided by the reviews of the original, this resubmitted renewal proposal identifies FRAG as a novel source of aberrant brain circuit development that portends vulnerability to mental illness; it integrates FRAG within existing frameworks and offers novel, age-and sex-specific predictive markers of vulnerability to mental illness, and hence experiment-based, transformative paths for preventative interventions.