Eric G. Krause - US grants

DESCRIPTION (provided by applicant): Body fluid homeostasis is maintained by integrated hormonal and behavioral mechanisms, including water intake. The majority of research on this topic has been done in male animal models. However, it is becoming increasingly clear that females respond differently to dehydration than do males. In fact, there is general agreement that estrogen modulates water intake in response to dehydration. Despite this general agreement, there are conflicting findings about the role of estrogen in the responses to specific dehydrational challenges. In addition, methodological differences in hormonal manipulations make these studies difficult to assess. More importantly, very little is known about the neural mechanisms and central pathways underlying the effect of estrogen on water intake. Most studies that have examined this issue have used central administration of angiotensin II to stimulate water intake. Although results from these studies provide compelling evidence for a central estrogen/angiotensin II interaction in the control of water intake, it is unclear whether such an interaction is involved in more physiological stimuli for water intake. Accordingly, the goals of the present proposal are to distinguish estrogen effects on drinking behavior and ascertain the mechanism by which the hormone affects the thirst of female rodents. By doing so, we will test the overall hypothesis that central interactions between estrogen and angiotensin II underlie the effect of estrogen on physiologically stimulated water intake.

DESCRIPTION (provided by applicant): Hypertension affects approximately 50 million Americans and progression of this disease significantly increases risks for heart and renal failure. The development of hypertension is believed to be due, in part, to overactivity of the Renin-Angiotensin-System (RAS), an endocrine system critical for the regulation of cardiovascular function and hydromineral balance. The effector peptide of the RAS, angiotensin II (ANGII), mediates compensatory responses to blood loss, sodium depletion, and hypotension. In the periphery, ANGII elicits vasoconstriction and promotes Na+ reabsorption by binding to angiotensin type 1 receptors (AT1R) on blood vessels. In the brain, ANGII binds to AT1R in circumventricular organs (CVOs) to initiate changes in hormone release, sympathetic outflow, and increased water and sodium consumption. While much is known about the effects of ANGII on vasculature reactivity and renal Na+ handling, the central pathways governing responses to ANGII remain unclear. Previous studies have used AT1R antagonists or brain lesions to examine central responses to ANGII, but these approaches have limitations. Lesion studies allow for the evaluation of the function of brain regions, but lack the resolution to provide information about specific neuronal phenotypes. Conversely, pharmacological manipulations allow evaluation of neuronal phenotypes, but limiting the administration of the drug to discrete brain regions is problematic. An alternative method is to use antisense methodology to inhibit the expression of target genes in discrete brain regions. Administration of antisense alters specific neuronal phenotypes within individual brain regions, thereby allowing evaluation of the function of neurons within this discrete population. For the proposed experiments, antisense will be used to inhibit the expression of the AT1R in specific brain nuclei, thereby allowing evaluation of the role of these receptors in mediating responses to circulating ANGII. Specifically, antisense targeted against the AT1R will be injected into specific CVOs of rats. Subsequently, I will examine behavioral, endocrine, and neural responses to treatments that differentially increase circulating ANGII. The results will provide valuable insight to the function of AT1R within specific brain regions and the role they play in mediating responses to circulating ANGII, while also serving as a foundation for future studies employing central genetic manipulations.

DESCRIPTION (provided by applicant): Hypertension affects approximately 50 million Americans and progression of this disease significantly increases risk for cardiovascular disease. Recent clinical studies demonstrate, that hypertension also is associated with mental health disorders. Specifically, hypertension increases the risk of anxiety disorders, and inversely, anxiety disorders predict the risk and severity of hypertension and cardiovascular disease. While the onset of hypertension is attributed to over activity of the renin-angiotensin-system (RAS), the development of anxiety is associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. The effector peptide of the RAS, angiotensin II (ANGII), exerts the majority of its biological effects via activation of angiotensin type 1 receptors (ATI R). Interestingly, ATI R are expressed throughout the HPA axis and emerging evidence has implicated ANGII as an important mediator of the stress response. Chronic exposure to stress up-regulates the HPA axis and RAS, thereby negatively affecting cardiovascular and mental health. Given that chronic stress similarly up-regulates the HPA axis and the RAS, it is likely that an interaction between these systems underlies the effects that chronic stress has on mental health and cardiovascular function. In this regard, our recent study found that circulating ANGII drives activation of the HPA axis by stimulating AT1R in the subfornical, a specialized forebrain structure that binds blood-borne hormones. Because repeated stress increases ATI R expression in the subfornical organ, it is likely that chronic stress potentiates the influence of circulating ANGII on the HPA axis, thereby inducing cardiovascular and affective disorders. Collectively, these studies suggest that therapies targeting the RAS may influence the onset of brain-based disorders like anxiety, and therefore, it is important to understand the central angiotensinergic pathways that influence responses to stress. Accordingly, the proposed experiments will utilize antisense oligodeoxynucleotides to inhibit AT1R expression in the subfornical organ of laboratory rats to determine the contribution of these receptors to the cardiovascular, HPA and behavioral responses to acute and chronic exposure to stress. These studies are designed to test the overall hypothesis that inhibition of AT1R in the subfornical organ will limit stress responding and alleviate the deleterious consequences of chronic stress exposure.

Hypertension affects approximately 50 million Americans and progression of this disease significantly increases risk for cardiovascular disease. Recent clinical studies demonstrate that hypertension also is associated with mental health disorders. Specifically, hypertension increases the risk of anxiety disorders, and inversely, anxiety disorders predict the risk and severity of hypertension and cardiovascuiar disease. While the onset of hypertension is attributed to over activity ofthe renin-angiotensin-system (RAS), the development of anxiety is associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. The effector peptide ofthe RAS, angiotensin II (ANGII), exerts the majority of its biological effects via activation of angiotensin type 1 receptors (AT1R). Interestingly, AT1R are expressed throughout the HPA axis and emerging evidence has implicated ANGII as an important mediator of the stress response. Chronic exposure to stress up-regulates the HPA axis and RAS, thereby negatively affecting cardiovascular and mental health. Given that chronic stress similarly up-regulates the HPA axis and the RAS, it is likely that an Interaction between these systems underlies the effects that chronic stress has on mental health and cardiovascular function. Because repeated stress increases AT1R expression in the brain, it is likely that chronic stress potentiates the influence of ANGII on the HPA axis, thereby inducing cardiovascular and affective disorders. Collectively, these studies suggest that therapies targeting the RAS may influence the onset of brain-basied disorders like anxiety, and therefore, it is important to understand the central angiotensinergic pathways that influence responses to stress. Accordingly, the proposed experiments will to inhibit ATI R in the brains of laboratory rats and mice to detemriine the contribution of these receptors to the cardiovascular, HPA and behavioral responses to chronic exposure to stress. These studies are designed to test the overall hypothesis that inhibition of AT1R in the brain will limit stress responding and alleviate the deleterious consequences of stress related illnesses.

DESCRIPTION (provided by applicant): Stressful life events promote anxiety and hypertension and significantly increase the risk for cardiovascular disease which the leading cause of death in the U.S. Developing strategies that limit stress responding to prevent affective and cardiovascular disorders is the long-term goal of this project. Anxiety, hypothalamic pituitary adrenal (HPA) axis dysfunction and enhanced cardiovascular reactivity to stress contribute to the onset of affective and cardiovascular disorders and limiting these indices of stress responding may be therapeutic. Oxytocin (OT) is being used to treat stress-related disorders; however, activation of oxytocin receptors (OTRs) can have diverse effects, and consequently, identifying specific OTRs regulating stress responding may improve treatments. Magnocellular OT neurons are excited by hypernatremia, increases in the plasma sodium concentration (pNa+), and this results in long-lasting increases in central levels of OT. Driving endogenous OT release by elevating the pNa+ in rodents causes activation of OTRs that inhibit corticotropin-releasing-factor (CRF) neurons in the central amygdala (CeA) and paraventricular nucleus of the hypothalamus (PVN). These neurons are implicated in the etiology of anxiety and hypertension, and intriguingly, OTR mediated inhibition of CRF neurons decreased anxiety-like behavior, blunted HPA activation and attenuated cardiovascular responses to stress. Determining the neural mechanism(s) by which this occurs is the main objective of this proposal. In this regard, hypernatremia interacts with stress to increase neuronal activation in the bed nucleus of the stria terminalis (BNST) and the lateral ventral septum (LVS), which express OTRs and connect to brain regions mediating stress responding. Ongoing studies suggest that OTR expressing neurons in the BNST and LVS project to the CeA or function as GABAergic interneurons. These results suggest that hypernatremia activates magnocellular oxytocinergic neurons that release OT in the BNST and LVS, stimulating OTRs expressed on GABAergic neurons that inhibit CRF neurons, thereby attenuating anxiety, HPA activation and cardiovascular responses to stress. Aim 1 will use mice with OTR-specific Cre expression, neuroanatomical tract-tracing and in vitro patch-clamp electrophysiology to test the hypothesis that magnocellular OT neurons send axons that depolarize neurons in the BNST and LVS by activating OTRs. Aim 2 will utilize Cre-inducible adenoassociated virus (AAV) and optogenetics to test the hypothesis that neurons expressing OTRs in the BNST and LVS have GABAergic efferents that inhibit CRF neurons in the CeA and PVN. Aim 3 will delete OTRs in the BNST and LVS of mice to test the hypothesis that these receptors mediate the stress dampening effects of hypernatremia. Completion of these experiments will determine the specific population of OTRs that are activated by endogenously released OT to limit stress responding, thereby producing important information that may guide the development of therapeutics for comorbid affective and cardiovascular disorders.

Project Summary Intranasal oxytocin (OT) decreases alcohol craving and affective dysfunction in abstinent alcoholics, providing a potential pharmacotherapy for alcoholism. Excessive habitual ethanol use dysregulates mesocorticolimbic reward circuitry which may include decreased OT activation of neurons in the ventral tegmental area (VTA). Here, we propose that ethanol dysregulation of OT signaling specifically alters oxytocinergic innervation and OT receptor (OTR) expression within the VTA, which in turn, facilitates ethanol-seeking in an effort to maintain VTA activation. Recently, we discovered that VTA neurons that synthesize glutamate (GLU) and dopamine (DA) express OTRs indicating the complexity of OT regulation of VTA function and reward. Our use of advanced neuroanatomical techniques combined with in vivo optogenetics provides a unique opportunity to quantify ethanol-induced plasticity in OT signaling in the VTA as well as the consequences of this plasticity on reward seeking behavior. Specifically, we will use genetically altered mice, directed viral gene transfer, and in vivo optogenetics to identify and manipulate pre- and post-synaptic OT signaling in the VTA after daily binge ethanol intoxication. These studies will test the overall hypothesis that manipulations of pre- and post-synaptic OT signaling in the VTA alters reward states and that OT regulation of VTA circuitry and positive reinforcement is impaired by daily binge ethanol intoxication. Aim 1 will quantify the impact of daily binge ethanol intoxication on the number and phenotype of OTR-expressing neurons in the VTA by delivering Cre- inducible adenoassociated virus to mice that have Cre recombinase directed to the OTR gene (OTR-Cre). The effects of daily binge ethanol on hypothalamic OT mRNA and oxytocinergic innervation of the VTA will be quantified using amplified in situ hybridization and mice with Cre recombinase directed to the OT gene. Aim 2, will reveal functional consequences of ethanol-induced structural plasticity by evaluating how daily ethanol intoxication affects operant responding for stimulation of presynaptic or post-synaptic OT signaling within the VTA, which will be recapitulated using in vivo optogenetics. These experiments will determine whether daily binge ethanol decreases OT reward signaling by altering OT fibers and/or OTR expression by DA and GLU neurons in the VTA. Elucidating the impact of daily binge ethanol intake on endogenous OT regulation of VTA neurons will inform pathophysiologic mechanisms of alcohol use disorder and guide the development of novel therapeutic interventions for alcohol use disorders, particularly as they might differ according to sex.

Project Summary Hypertension is the most important risk factor for the development of cardiovascular disease, the leading cause of death in the U.S. Despite therapeutic advancements, nearly 20-30% of hypertensive patients have uncontrolled high blood pressure. This resistant hypertension is associated with elevated sympathetic activity and abnormal baroreflex reflex control and thus is termed neurogenic hypertension. Animal models of neurogenic hypertension consistently implicate augmented release of GABA within the intermediate nucleus of the solitary tract (intNTS) as a contributing pathophysiological mechanism. Correcting this pathophysiological mechanism is a logical step towards decreasing high blood pressure of neurogenic origin. However, GABA and its receptors are poor therapeutic targets because GABA is the predominant inhibitory neurotransmitter in the CNS. To circumvent this impediment we began investigating whether neurons in the intNTS that express angiotensin type 2 receptors (AT2R) may serve as an access point for therapeutic interventions that relieve neurogenic hypertension. Using a novel transgenic mouse model (AT2R-eGFP reporter mouse) we discovered that GABA neurons in the intNTS robustly express AT2R and optogenetic excitation of these neurons significantly increases blood pressure. Intriguingly, DOCA-salt hypertension in mice increased indices of GABA synthesis in the intNTS but central delivery of the AT2R agonist, Compound 21 (C21), abrogated this hypertension and downregulated indices of GABA synthesis in the intNTS. Relevant to the proposed research, the antihypertensive effects of brain AT2R activation were abolished by deleting AT2R from GABA neurons. Based on these results we hypothesize that GABA neurons in the intNTS that express AT2R may be manipulated to reverse the onset of neurogenic hypertension. Two Specific Aims are proposed to substantiate or refute this hypothesis. Aim 1 combines genetic and pharmacological approaches to evaluate the consequences of deleting or stimulating AT2R on GABAergic neurons in the intNTS. Aim 1 tests the hypothesis that activation of AT2R expressed on GABAergic neurons in the intNTS alleviates DOCA-salt hypertension in mice by decreasing GABA synthetic enzymes within these neurons, which consequently decreases sympatho- excitation and improves baroreflex function. Aim 2 utilizes in vitro optogenetic and in vivo chemogenetic approaches to evaluate how DOCA-salt hypertension with or without C21 affects GABA neurotransmission within baroreflex circuits mediating cardiovascular function. Aim 2 tests the hypothesis that the activity of neurons within the intNTS that express AT2R control baroreflex sensitivity and sympathetic outflow and their selective inhibition mediates the reversal of hypertension. Execution of the proposed experiments will identify a discrete population of neurons that can be targeted to control blood pressure and provide preclinical evidence for the development of novel approaches for alleviating resistant hypertension.

Project Summary Stressful life events contribute to the etiology of anxiety and hypertension and increase the risk for cardiovascular disease, which is the leading cause of death in the U.S. Despite a myriad of research, nearly one-third of patients with anxiety and/or hypertension are resistant to current treatments and understanding the pathophysiology underlying these disorders is necessary to identify novel therapeutics. Stressors perceived in the environment or those arising from the internal milieu create neural signals that converge on the paraventricular nucleus of the hypothalamus (PVN), which integrates these signals and transduces them into cardiovascular, neuroendocrine and behavioral responses. Chronic unpredictable stress elicits gene x environment interactions that promote neurochemical plasticity within the PVN that heighten stress responsiveness and promote affective and cardiovascular disorders. A premise of this proposal is that angiotensin receptor signaling within the PVN is a key mediator of gene x environment interactions that control cardiovascular reactivity, neuroendocrine axes and anxiety subsequent to chronic stress. Traditionally, the RAS is considered an endocrine system that elevates blood pressure by increasing the binding of angiotensin II (Ang-II) to its angiotensin type-1 receptor (AT1R). However, we recently found that optogenetic activation of neurons in the PVN that express AT1R(s) augment, but optogenetic inhibition or selective deletion of AT1R(s) from the PVN dampen stress responding in mice. Concomitantly, we evaluated the influence of brain angiotensin converting enzyme 2 (ACE2) on stress responding. Angiotensin converting enzyme 2 metabolizes Ang-II into angiotensin 1-7 which promotes cardio- protection, in part, by activating Mas receptors. Interestingly, we discovered that up-regulating ACE2 activity in the brain potently dampens stress responding in mice. Collectively, these observations have led to our overall hypothesis that balance between AT1R stimulation and ACE2 activity dictates excitation or inhibition of specific neuronal phenotypes within the PVN to promote susceptibility or resiliency to stress-related disease. We propose the following specific aims to substantiate or refute this hypothesis. Aim 1 uses mice with Cre recombinase directed to AT1R(s) and in vivo optogenetics to test the hypothesis that chronic excitation of AT1R-expressing neurons in the PVN recapitulates the pathophysiology that follows chronic stress. Aim 2 uses mice with AT1R selectively deleted from the PVN to test the hypothesis that such receptors mediate gene x environment interactions that exaggerate stress responding subsequent to chronic stress. Aim 3 uses mice with ACE2 overexpression directed to neurons that synthesize corticotrophin-releasing-hormone (CRH) to test the hypothesis that CRH-ACE2 interactions relieve chronic stress-induced pathophysiology. Collectively, the proposed research will reveal, at a very detailed and mechanistic level, how brain angiotensin signaling contributes to the etiology of stress-related disease and will inform on novel therapeutics.

Project Summary Stressful life events are linked to the etiology of cardiovascular disease (CVD), which is the leading cause of death in the U.S. The mechanisms by which stress causes pathophysiology contributing to CVD are poorly understood and effective therapeutics that relieve stress and improve cardiovascular health are lacking. A premise of this proposal is that exploration of the neural circuits controlling the perception of stress may provide insight towards mechanisms underlying CVD and interventions aimed at its reversal. Causally-linking patterns of neural activity to stress and the development of CVD in humans is challenging. However, preclinical studies using laboratory mice that implement modern neuroscience and genetic technologies to excite or inhibit specific neural circuits make causally-linking neural activity and indices of stress responsiveness achievable. Using genetically-modified mice, we revealed that the activity of neurons that express genes encoding particular angiotensin receptor subtypes is coupled to cardiovascular, neuroendocrine and behavioral responses to stress. Specifically, we discovered that neurons expressing the angiotensin type-2 (AT2R) and Mas receptor (MasR) densely populate cortical and limbic brain regions controlling the perception of psychological stress and that excitation of these neurons decreases blood pressure, heart rate, circulating levels of corticosterone and anxiety- like behavior. In the periphery, we discovered that the nodose ganglion is densely populated by neurons expressing the angiotensin type 1a receptor (AT1R). These neurons function as primary baroreceptor afferents and excitation of these neurons lowers blood pressure, heart rate and energy expenditure. Collectively, these observations have led to the overall hypothesis that excitation of particular neuronal populations that express the AT1R, AT2R or MasR alters the perception of stress to protect against CVD. Experiments will use the Cre-LoxP system in mice with a cadre of modern neuroscience techniques and classical systems physiology to confirm or refute this hypothesis. Initial experiments utilize Cre-diver mice with virally-mediated gene transfer and in vivo optogenetics to determine whether the excitation or inhibition of neurons that express AT1R, AT2R, or MasR attenuates or exacerbates stress responding. Subsequent experiments use a model of stress-induced pathophysiology to evaluate how the structure and function of neurons that express the AT1R, AT2R or MasR is altered by disease. The final experiments attempt to alleviate stress-induced pathophysiology with optogenetic, genetic or pharmacological manipulations that alter the excitability of neurons that express the AT1R, AT2R or MasR. We anticipate that the proposed research will reveal, at a detailed and mechanistic level, neural circuits that provide stress relief, thereby guiding development of novel therapeutics for CVD.