Linda Rinaman - US grants

My prior research has used neuroanatomical tracing methods and immunocytochemistry to examine central synaptic inputs to gastric vagal motor neurons in adult rats, and to examine the vagal innervation of the stomach and other digestive viscera in maturity and during fetal and postnatal development. The present application reflects my strong desire to expand this approach to include functional ontogenetic analyses of the central neural connections of the vagus that influence three critical components of homeostatic control: pituitary neuroendocrine secretion, gastric physiology, and ingestive behavior.The proposed experiments are organized into three studies. The first study will analyze the structural (anatomical) development of ascending and descending neural projections between gastric vagal regions of the nucleus of the solitary tract (NST) and the hypothalamus, focusing on ascending projections from catecholaminergic NST neurons to the paraventricular nucleus (PVN) of the hypothalamus and descending projections from PVN oxytocinergic neurons to the NST. The second study will extend these anatomical analyses by examining the functional maturation of ascending gastric vagal sensory inputs that are relayed through catecholaminergic projections from the NST to magnocellular and parvocellular PVN neurons. This study will radioimmunoassay plasma levels of oxytocin in response to gastric vagal stimulation to determine the postnatal development of vagally-mediated pituitary secretion. In addition, tract-tracing and neurotransmitter immunocytochemistry will be used in conjunction with immunocytochemical detection of cFos protein to define the subnuclear distribution, chemical phenotypes, and axonal projections of NST and hypothalamic neurons that receive gastric sensory signals at different postnatal ages. The third study will examine the functional maturation of inhibitory hypothalamic control over ingestive behavior and vagally-mediated gastric motility. This study will use physiological and behavioral methods to analyze the ontogeny of "dehydration anorexia" and inhibition of gastric emptying following stimulation of osmoreceptors in the basal forebrain that communicate through oxytocinergic projections from the PVN to the NST. The relationship between cFos expression by magnocellular neurons and pituitary secretion of OT in neonates also will be examined, as will the axonal projections and chemical phenotypes of PVN and NST neurons expressing cFos in response to osmotic stimulation. The proposed studies will further characterize the ontogeny of homeostatic responses to gastric and osmotic stimulation, and the central neural circuits that mediate these responses.

central neural pathway /tract

incretin hormone; developmental neurobiology; paraventricular nucleus; central neural pathway /tract; stress; oxytocin; synapses; cholecystokinin; brain mapping; solitary tract nucleus; chemical stimulation; neuroendocrine system; adrenocorticotropic hormone; electron microscopy; laboratory rat; mature animal; newborn animals; immunocytochemistry;

incretin hormone; developmental neurobiology; paraventricular nucleus; central neural pathway /tract; stress; oxytocin; synapses; cholecystokinin; brain mapping; solitary tract nucleus; chemical stimulation; neuroendocrine system; adrenocorticotropic hormone; electron microscopy; laboratory rat; mature animal; newborn animals; immunocytochemistry;

The Center for Neuroscience at the University of Pittsburgh (CNUP) will train eight to twelve undergraduate students each summer in its ten-week Neuroscience Research Experience for Undergraduates (Neuroscience REU). The main focus of the Neuroscience REU will be full-time student involvement in laboratory research under the guidance and supervision of CNUP faculty mentors. Students will experience the challenges and rewards of intensive, hypothesis-driven laboratory research in molecular, cellular, and systems-level neuroscience. The Program aims to encourage a broad and diversified pool of talented students to consider careers in neuroscience research, and will provide the laboratory experience that is necessary for students to compete successfully for admission to neuroscience graduate training programs. Consequently, the Program particularly seeks applicants from students at liberal arts colleges and other academic institutions with limited neuroscience research opportunities. The Neuroscience REU will officially begin in early June. A weekly "Research Breakfast" seminar series will expose students to a variety of neuroscience topics and technical approaches. During the summer, students will receive workshop training in research ethics, and will learn how to make effective oral presentations, write a research abstract, and get into graduate school. Research projects will foster a close collaboration between mentor and student, and will provide a hands-on preview of career opportunities in neuroscience research and education. Students will learn many of the skills necessary to perform research, such as proper formulation of experimental strategy and appropriate documentation and analysis of data. At the end of the 10-week Program, students will formally present the results of their training experience at a colloquium of participating students and mentors. The Neuroscience REU will accept applications from full-time undergraduate students who are enrolled at a four-year accredited college in the United States, and who have successfully completed at least one year of study in neuroscience and/or related college coursework (e.g., biology, chemistry, psychology, mathematics). Interested students should complete an application form and write a personal statement describing their interest in the Neuroscience REU. Applicants also must have two letters of recommendation sent from faculty at their home institution who are familiar with their qualifications

DESCRIPTION (provided by applicant): Sensory signals from within the body are delivered to brain regions that shape physiological and behavioral responses to stress and influence emotional learning. The research proposed in this R01 competing renewal application will provide new insights into the functional organization and development of viscerosensory inputs to the hypothalamus and limbic forebrain, with a focus on sensory signals from the gut. Experiments will test mechanistic hypotheses about the structure and function of noradrenergic (NA) and glucagon-like peptide-1 (GLP-1) signaling pathways that originate in the dorsal vagal complex (DVC) and target the paraventricular nucleus of the hypothalamus (PVN), lateral hypothalamic area (LHA), bed nucleus of the stria terminalis (BNST), and central nucleus of the amygdala (CeA). Hypotheses to be tested are organized into three Specific Aims. Aim 1 will use anterograde transneuronal virus tracing to label viscerosensory pathways from the stomach to the forebrain. One set of experiments will test the hypothesis that NA and non-NA DVC neurons relay gastric viscerosensory signals to discrete subregions of the PVN, LHA, BNST, and CeA. Other experiments will test the hypothesis that gastric viscerosensory inputs to the forebrain undergo significant structural maturation in rats during the first two weeks postnatal. Aim 2 will determine the necessity of DVC NA neurons for hypothalamic and limbic forebrain responses to interoceptive stress. One study will test the hypothesis that DVC NA neurons are necessary for certain interoceptive stressors [i.e., cholecystokinin (CCK), lithium chloride (LiCl), and lipopolysaccharide (LPS)] to inhibit food intake, but are unnecessary for these stressors to support conditioned taste aversion learning. A related study will test the hypothesis that DVC NA neurons are necessary for CCK, LiCl and LPS to induce cFos expression in the PVN and LHA, but are unnecessary for these stressors to induce cFos expression in the CeA. A third study will test the hypothesis that the ability of systemic CCK, LiCl, and LPS to activate cFos expression in the PVN, LHA, BNST, and CeA emerges gradually in rats during the first two weeks postnatal. Aim 3 will use electron microscopy to determine whether separate populations of NA and GLP-1-positive axon terminals converge on common postsynaptic targets in the PVN, LHA, CeA, and BNST. Experimental outcomes will advance our understanding of how viscerosensory inputs to the hypothalamus and limbic forebrain might impact diverse conditions such as anxiety disorders, visceral malaise, dysregulation of the HPA stress axis, depression, and conditioned aversions.

DESCRIPTION (provided by applicant): Clinical research has increasingly emphasized the importance of ascending viscerosensory pathways from the caudal brainstem, including noradrenergic (NA) pathways, in stress responses and affective/emotional state. Dysregulated NA signaling is implicated in the pathophysiology of stress-related psychiatric illnesses, including depression and anxiety disorders. The proposed research will test hypotheses about the structure and function of ascending NA pathways that modulate neural activity within interconnected regions of the paraventricular nucleus of the hypothalamus (PVN), central nucleus of the amygdala (CeA), and anterolateral bed nucleus of the stria terminalis (alBST). These pathways arise from NA neurons in viscerosensory regions of the nucleus of the solitary tract (NST) and ventrolateral medulla (VLM), with little or no direct contribution from the pontine locus coeruleus. Revealing the functional organization of these systems in rats has clinical relevance, and may contribute to the development of new therapeutic options for treating stress-related emotional dysregulation. Our working hypothesis is that both physiological (interoceptive) and cognitive/emotional stressors alter viscerosensory signals that are relayed by medullary NA neurons to the hypothalamus and limbic forebrain, and that these signals are critical for shaping emotional state as evidenced by stress responsiveness, motivated behavior, and emotional learning. NA neurons within the NST and VLM have branching axons that target more than one hypothalamic and limbic forebrain target. Retrograde tract-tracing experiments in Aim 1 will simultaneously examine NA axonal collateralization and ascending pathway recruitment by a malaise- inducing agent, lithium chloride (LiCl). The anatomical data obtained in Aim 1 will facilitate interpretation of functional data obtained in Aim 2, in which NA inputs to the PVN, CeA, and/or alBST will be selectively destroyed before rats are assayed for behavioral and physiological responses to three distinct challenges: (1) LiCl, (2) exposure to a fear- and anxiety-inducing predator odor, trimethylthiazoline (TMT), or (3) systemic yohimbine (YO), a pharmacological agent that robustly increases NA signaling throughout the brain. Parallel experiments in Aim 3 will test the hypothesis that direct communication between the CeA and alBST is necessary for behavioral and physiological responses to LiCl, TMT, and YO in rats with otherwise intact central NA circuitry. The proposed research will reveal new aspects of the functional organization of viscerosensory NA inputs to the hypothalamus and limbic forebrain that play a critical role in mediating physiological and behavioral responses to emotionally significant events. Dysregulated noradrenergic signaling in the brain is implicated in stress-related psychiatric illnesses, including depression and anxiety disorders. The proposed research will test hypotheses about the structure and function of noradrenergic pathways that modulate neural activity within interconnected regions of the hypothalamus and limbic forebrain. Experimental outcomes could lead to the development of new therapeutic options for treating stress-related emotional pathologies.

DESCRIPTION (provided by applicant): Early life experience can alter adult emotionality and stress responsiveness, but only limited research has examined how experience shapes the development of central neural circuits. We predict that manipulation of early life experience will alter the functional organization of central visceral circuits, and that altered neuroanatomy will correspond with altered behavioral, physiological, and neural responses to stressful events. The proposed research will test this prediction by performing experiments in adult male and female rats with a developmental history of having been handled briefly for daily maternal separation of either 15 min (MS15) or 3 hr (MS180) during the first two postnatal weeks. Non-separated (MS0) rats will serve as controls. As young adults, all rats will be screened for behavioral differences in the elevated plus maze, and blood samples will be collected to document restraint stress-evoked excursions in plasma corticosterone. Subsequent experiments in Aims 1 and 2 will be performed in behaviorally- and hormonally-screened adult rats. Aim 1 experiments will test the hypothesis that early maternal care (manipulated via MS15 and MS180) interacts with sex to differentially alter the anatomical features of central visceral circuits. Retrograde transneuronal transport of pseudorabies virus will be used to probe for differences in central autonomic circuits in rats with different developmental histories. In the second experiment, experience-dependent alterations in noradrenergic (NA) sensory pathways will be examined. For this, a unique lentivirus vector that expresses enhanced green fluorescent protein under the control of a dopamine beta hydroxylase promoter will be microinjected into the caudal medulla to label the axonal arbors of transfected NA neurons that project to the hypothalamus and limbic forebrain. Aim 2 experiments will test the hypothesis that early maternal care interacts with sex to differentially alter stressor-induced neural Fos activation in central visceral circuit nodes. Rats will be perfused with fixative after restraint, LiCl treatment, predator odor exposure, or matched control treatment for analyses of stimulus-induced Fos expression in medullary NA neurons and in their central projection fields. NA terminal immunolabeling density and CRF/CRH labeling also will be quantified to determine whether sex and/or MS group differences interact. Data will be analyzed and interpreted within the context of behavioral and hormonal responses in the screening tests, with attention paid to predicted effects of early postnatal experience and sex on anatomical and physiological outcomes. The proposed work will advance our understanding of how early maternal care can alter the developmental trajectory of central visceral circuits in males and females, and will provide new insights regarding the impact of early experience on adult emotionality and stress responsiveness. Interactions between infants and their mother (or primary caregiver) are critical for normal growth and development, and perturbations can disrupt physiological and behavioral functions in the offspring. The proposed research will use anatomical and physiological methods in rats to test the hypothesis that the influence of early life events on later responses to stress and emotional events is linked to developmental plasticity of circuits that provide visceral sensory feedback to the brain and generate emotional expression.

DESCRIPTION (provided by applicant): Obesity is a major contributor to serious health problems. The incidence of obesity within the US has soared during the last 30-40 years, with a contributing cause being the increased consumption of high-fat foods. Diets high in fat can result in overeating (hyperphagia), which promotes obesity in susceptible individuals. There is evidence in both humans and rats that hyperphagia is related to reduce sensitivity to the satiating effect of dietary fat, due to reduced engagement of brainstem satiety circuits. The proposed research will further elucidate the functional organization of these circuits, highlightin a potentially critical role for hindbrain noradrenergic neurons that co-express prolactin-releasing peptide (PrRP). The proposed work is consistent with the NIH Strategic Plan for Obesity Research by its focus on physiological neural mechanisms that regulate food intake and body weight. Animal models can provide critical insights into physiological and behavioral factors that predispose humans to become obese. Further, PrRP neurons are located within human caudal brainstem in a distribution similar to that in rodent species. Thus, experimental outcomes will have translational implications for understanding how dietary fat promotes overeating in humans who are susceptible to diet-induced hyperphagia, while others exposed to the same diet remain relatively resistant. We propose that behavioral satiety is generated, at least in part, by recruitment of PrRP-positive neurons in the caudal visceral portion of the nucleus of the solitary tract, and that these neurons are polysynaptically linked to brainstem oral ingestive control motor neurons. PrRP neurons receive direct visceral sensory input from gastrointestinal vagal afferents, and central PrRP signaling is implicated in the homeostatic control of food intake in rats and mice. The proposed research will use adult male rats to challenge the overarching hypothesis that satiety signals recruit brainstem PrRP signaling pathways that limit meal size. In addition, we will test the hypothesis that a high-fat diet attenuates this natural PrRP-mediated satiety process in individual rats that develop hyperphagia, but not in resistant rats. We propose that increased consummatory responses to high fat diet are due, at least in part, to attenuated satiety signal-induced recruitment of brainstem PrRP neurons that act to limit food intake. Outbred Sprague-Dawley rats are an ideal experimental model for the proposed research, because approximately 50% develop behavioral hyperphagia (i.e., increased meal size and daily food intake) that promotes increased body weight gain during high fat diet exposure, whereas the remainder are resistant, and do not increase their daily intake or BW more than they do on normal control diet.

PROJECT SUMMARY/ABSTRACT Affective anxiety is characterized by behavioral inhibition accompanied by cognitive arousal and vigilance, reflecting a future-oriented emotional state. Anxiety is an adaptive response to perceived threat; however, chronic anxiety is debilitating, and anxiety disorders are the most common type of mental illness in the United States. Identification of neural circuits that underlie behavioral responses to threat in animal models is essential for understanding neurobiological mechanisms that contribute to normative and pathological anxiety in humans. Basic and clinical research has emphasized the importance of the central nucleus of the amygdala (CEA) and the anterolateral bed nucleus of stria terminalis (alBST) in regulating affective and physiological components of anxiety. The CEA and alBST are heavily interconnected and share many common sources of input, including input from caudal brainstem neurons that convey sensory feedback from body to brain; this feedback strongly modulates emotional state, including threat responses. The proposed research will test new hypotheses regarding the organization and behavioral role of CEA/alBST-projecting brainstem neurons that express glucagon-like peptide-1 (GLP1). GLP1 neurons are activated to express cFos in rats after acute threat, and GLP1 receptor signaling in the CEA/alBST increases arousal/vigilance and behavioral inhibition/avoidance in rats, akin to anxiety in humans. We recently developed a transgenic Sprague Dawley rat (Gcg-Cre) in which Cre is efficiently and selectively expressed by GLP1 neurons in the caudal nucleus of the solitary tract and intermediate reticular nucleus (i.e., NTS/IRtGLP1 neurons). Using validated viral tools and behavioral assays in this new model organism, we will conduct comparative analyses of the synaptic connectivity and functional role of NTS/IRtGLP1à CEA/alBST circuits in male and female Gcg-Cre rats, including documentation of potential sex differences. Since the distribution of GLP1 neurons in brainstem and GLP1 receptors in limbic forebrain appears similar in rats and humans, results from this basic science project have potential translational relevance for understanding neurobiological bases of normal and pathological symptoms of anxiety in humans.