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.

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): 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): 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): 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]

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.

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.

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.

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.

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.