Jamie L. Maguire, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): Catamenial epilepsy manifests at specific stages of the ovarian cycle and the pathogenesis has been attributed to fluctuations in neurosteroid levels. Neuroactive steroids, such as progesterone and its metabolites, have been shown potentiate the effects of GABA at GABAA receptors (GABAARs) and act as anticonvulsants. The pharmacology of GABAARs is dependent upon receptor subunit composition. GABAAR kinetics are conferred by the receptor subunit composition. Specifically, neurosteroid modulation of GABAARs has been shown to be dependent upon the inclusion of the GABAAR . subunit. This subunit also mediates GABAergic tonic inhibition and regulation of this subunit may have profound results on excitability. Administration of exogenous neurosteroids and withdrawal from exogenous neurosteroid administration has been shown to regulate GABAAR subunit composition. The goal of this proposal is to investigate the role of fluctuating neurosteroid levels over the estrous cycle in the regulation of GABAAR subunits. We hypothesize that endogenous neurosteroids dynamically reorganize GABARs, leading to changes in GABAergic synaptic transmission, neuronal excitability, and seizure predisposition. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): It has been known for decades that there is a co-morbidity of depression in epilepsy and recently, depression has been identified as a risk factor for epilepsy, highlighting the overlap in the pathophysiology of these diseases. However, very few studies have addressed the mechanisms mediating the co-morbidity of depression and epilepsy. Stress is a trigger for both of these disorders, and we hypothesize that dysfunction in the body's stress response, mediated by the hypothalamic-pituitary-adrenal (HPA) axis, and may play a role in the co-morbidity of depression and epilepsy. A hallmark characteristic of depression is hyperexcitability of the HPA axis and seizure activity activates the HPA axis. The output of the HPA axis is mediated by corticotrophin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN), the activity of which are under robust GABAergic control. This proposal will test the hypothesis that dysfunction in GABAergic control of the HPA axis results in hyperexcitability of the HPA axis, leading to increased seizure susceptibility. We have developed a sophisticated set of tools to test this hypothesis, including a novel, conditional knockout of one of the principal GABAARs regulating the HPA axis, the Gabrd gene. We intend to cross these mice with CRH-Cre mice to generate mice with GABAergic deficits specifically in the CRH neurons regulating the output of the HPA axis. Further, we will investigate whether an initial seizure insult alters GABAAR subunit expression in the PVN, as it does in other brain regions, thereby leading to HPA axis hyperexcitability and future seizures. Insight into the role of GABAergic control of the HPA axis in the co-morbidity of epilepsy and depression may identify novel therapeutic targets for both epilepsy and depression as well as the co-morbidity of the two, which complements the mission of the NINDS to reduce the burden of neurological diseases through research and the new strategic plan to identify new potential therapies for neurological diseases.

Project Summary It is widely-accepted by both clinicians and basic scientists that stress can trigger and worsen seizures in animal models and in patients with epilepsy through the actions of stress mediators. The body's physiological response to stress is mediated by the hypothalamic-pituitary-adrenal (HPA) axis. The majority of studies investigating the relationship between the HPA axis and epilepsy focus on the role of stress and the proconvulsant actions of corticosterone (cortisol in humans) and corticotropin-releasing hormone (CRH). Interestingly, our lab recently demonstrated that seizures themselves activate the HPA axis, forcing us to reevaluate the role of the HPA axis in epilepsy. These findings suggest that seizure-induced activation of the HPA axis may directly contribute to changes in seizure susceptibility. Further, hyperexcitability of the HPA axis is a hallmark feature of depression, implicating seizure-induced activation of the HPA axis in the comorbidity of depression and epilepsy. The overarching objective of the current study is to investigate the pathophysiological consequences of this seizure-induced activation of the HPA axis, investigating the impact on the process of epileptogenesis (Specific Aim 1), seizure activity in chronically epileptic mice (Specific Aim 2), and the role in the comorbidity with depression in epilepsy (Specific Aim 3). These Aims will determine whether seizure- induced activation of the HPA axis is culpable in worsening seizure activity, associated pathology, and depression-like behaviors in chronically epileptic mice.

Project Summary It is widely-accepted by both clinicians and basic scientists that stress can trigger and worsen seizures in animal models and in patients with epilepsy through the actions of stress mediators. The body's physiological response to stress is mediated by the hypothalamic-pituitary-adrenal (HPA) axis. The majority of studies investigating the relationship between the HPA axis and epilepsy focus on the role of stress and the proconvulsant actions of corticosterone (cortisol in humans) and corticotropin-releasing hormone (CRH). Interestingly, our lab recently demonstrated that seizures themselves activate the HPA axis, forcing us to reevaluate the role of the HPA axis in epilepsy. These findings suggest that seizure-induced activation of the HPA axis may directly contribute to changes in seizure susceptibility. Further, hyperexcitability of the HPA axis is a hallmark feature of depression, implicating seizure-induced activation of the HPA axis in the comorbidity of depression and epilepsy. The overarching objective of the current study is to investigate the pathophysiological consequences of this seizure-induced activation of the HPA axis, investigating the impact on the process of epileptogenesis (Specific Aim 1), seizure activity in chronically epileptic mice (Specific Aim 2), and the role in the comorbid depression (Specific Aim 3). These Aims will determine whether seizure-induced activation of the HPA axis is culpable in worsening seizure activity, associated pathology, and depression-like behaviors in chronically epileptic mice.

Project Summary Excessive alcohol consumption leads to alcohol use disorders in approximately 7% of individuals and is associated with numerous health conditions, substantial economic burden, and is the third leading cause of preventable deaths in the United States. Despite the fact that alcohol misuse is a serious health and economic concern worldwide, we still do not fully understand the basic mechanisms contributing to alcohol consumption. One primary factor thought to drive consumption is the anxiolytic effects of alcohol. However, the specific cell types and networks mediating the anxiolytic effects of alcohol are unknown. Recent studies have elucidated connections between the basolateral amygdala (BLA) and the medial prefrontal cortex (mPFC) in the network communication of anxiety. Oscillations within the BLA and mPFC and the coupling between these regions is thought to be driven by parvalbumin (PV) interneurons in the BLA. PV interneurons in the BLA express the GABAAR ? subunit at a high density, which is thought to be a target of action for alcohol. We hypothesize that alcohol acts preferentially on PV interneurons in the BLA, modulating local oscillations and frequency coupling between the BLA and mPFC, thereby mediating the anxiolytic effects. This proposal represents the first attempt to examine the cell type-specific effects of alcohol on the network communication of anxiety. The current application will explore the cell type-specific targets of alcohol in the BLA and determine whether the GABAAR ? subunit on PV interneurons plays a role in mediating the anxiolytic effects of low dose alcohol (Specific Aim 1). This application will determine whether the anxiolytic effects of alcohol involve modulation of the local oscillations in the BLA and mPFC as well as the frequency coupling between the mPFC and BLA (Specific Aim 2). Further, we will determine whether driving the network communication of safety recapitulates the anxiolytic effects of alcohol and whether disruption of the pro-safety communication reduces the anxiolytic effects of alcohol (Specific Aim 3). This proposal will define specific cell types and associated neural pathways that are most sensitive to alcohol and how these circuits orchestrate sensitivity to alcohol. This application will determine whether the anxiolytic effects of alcohol are mediated by GABAAR ? subunit containing receptors on PV interneurons in the BLA through the coordination of activity between the mPFC and BLA.

Project Summary Epilepsy is highly comorbid with anxiety (Gaitatzis, 2005), which negatively impacts the quality of life of these patients (Johnson, 2004). To-date, we have little knowledge of the mechanisms underlying this comorbidity. Recent studies provide strong evidence for a role of network communciation between the basolateral amygdala (BLA) and the medial prefrontal cortex (mPFC) in mediating the expression of fear and anxiety (Stujenske et al., 2014;Felix-Ortiz et al., 2016) (for review see (Tovote et al., 2015)). A recent collaborative effort between the Maguire and Reijmers' labs demonstrates a role for parvalbumin (PV) interneurons in mediating the transition between the network communication of fear and the behavioral expression of fear (Davis, 2017). Specifically, we demonstrate that silencing PV interneurons in the BLA increase the reactivation of fear neurons following extinction and are required to suppress the network communication of fear, findings which are correlated with an increase in the behavioral expression of fear. These studies have largely focused on transitions between states of safety and states of fear and anxiety under physiological conditions. Few studies have investigated how this neural circuit communication may become dysregulation or corrupted under pathological conditions. Here we propose to investigate whether dysregulation in the network communication of anxiety may play a role in comorbid anxiety in epilepsy. Our preliminary data demonstrates a loss of PV interneurons in the BLA of chronically epileptic mice which we hypothesize facilitates the reactivation of anxiety neurons, promoting the network communication and the behavior expression of anxiety.

The Building Diversity in Biomedical Sciences Program responds to RFA-HL-16-008 and seeks to continue its nearly 25-year tradition of excellence initiated under the aegis of the original NHLBI T35-sponsored short term minority training programs. In the past fifteen years, our program has trained 237 undergraduate students who are members of groups recognized by NIH as underrepresented in biomedical science. All of these trainees have completed their undergraduate degrees or remain enrolled. Continued contact with nearly 97% of these alumni reveals that after graduation 70% have entered graduate health science degree programs and a total of 90% are engaged in the biomedical workforce. In the coming period, we will build on this record of success by providing an intensive 10-week mentored summer research experience with a focus on cardiovascular and pulmonary diseases and issues of direct concern to NHLBI. The training program will be informed by educational scholarship as well as biomedical science. Research will be supplemented by training in oral and written communication through experiences that build skills and enhance self-confidence. Trainees will be exposed to the ways basic science can directly impact health through a Clinical Connections Workshop and learn about the breadth of biomedical science careers through sessions on IDPs and career planning. A group of 40 faculty members whose research is aligned with the NHLBI mission will train 15 undergraduates each summer. A formative and summative evaluation plan will support the program and be used by the Directors and the Program Steering Committee to modify the program as appropriate.

Project Summary Many psychiatric illnesses are characterized by episodes of behavioral disruption. The current project attempts to understand the mechanisms mediating transitions between healthy and unhealthy brain and behavioral states. Stress is a major risk factor for psychiatric illnesses and is routinely employed to alter behavioral states in preclinical models. This application will utilize chronic and postpartum stress to facilitate the transition to the unhealthy network and behavioral state and investigate the mechanisms mediating these transitions. Neuroactive steroids (NAS) exert robust anxiolytic and antidepressant effects and a NAS-based treatment, brexanolone/Zulresso®, recently received FDA approval as the first antidepressant treatment for postpartum depression. This project will utilize these clinically effective NAS to investigate the impact on network and behavioral states. Our preliminary data demonstrates that chronic unpredictable stress and inappropriate postpartum stress can corrupt network activity in the basolateral amygdala (BLA) and that NAS can restore healthy network and behavioral states. Further, we demonstrate the ability of NAS to alter network activity across species, including in humans (Conte Center Overview preliminary data), highlighting the translational relevance of this approach. Despite these provocative preliminary findings, we still lack an understanding of the mechanisms mediating transitions between network and, therefore, behavioral states. The prolonged antidepressant effects of allopregnanolone are not easily explained by the known mechanism of action as positive allosteric modulators (PAMs) at GABAA receptors. In collaboration with our Conte Center collaborators and the Chemistry Core, we will investigate which of the diverse properties of NAS are capable of restoring healthy network and behavioral states with the goal of gaining a better understanding of their therapeutic properties.

Project Summary There is extensive preclinical evidence for anticonvulsant effects of neuroactive steroids, which are thought to be mediated through their actions as positive allosteric modulators (PAMs) on GABAA receptors (GABAARs). These studies have relied on the administration of exogenous neuroactive steroids, although, neurosteroids can also be synthesized in the brain. However, we know very little about the function of endogenous neurosteroids due to the lack of tools necessary to investigate their function. The rate-limiting enzymes involved in neurosteroid synthesis, 5?-reductase 1 or 2, are expressed in the hippocampus and given their actions as PAMs at GABAARs, they are well-suited to constrain network excitability in this region. To investigate the function of endogenous neurosteroids, we generated novel mouse models (floxed Srd5a1- IRES-GFP and floxed Srd5a2-IRES-tdTomato) enabling visualization and quantification of changes in the expression of these enzymes associated as well as the ability to knockout these enzymes in a cell type- and brain region-specific manner. Here we propose to utilize these novel tools to test the hypothesis that endogenous neurosteroids constrain neuronal excitability and that deficits in 5?-reductase 1 and 2 expression in the hippocampus contributes to seizure susceptibility. We will determine whether 5?-reductase 1 and/or 2 expression is altered in chronically epileptic mice (Specific Aim 1) and if loss of 5?-reductase 1 and/or 2 expression in the hippocampus increases network excitability and acute seizure susceptibility (Specific Aim 2) or increases seizure frequency in chronically epileptic mice (Specific Aim 3).