Jean C. Hardwick, Ph.D. - US grants

DESCRIPTION: Control of cardiac function is a balance between sympathetic and parasympathetic branches of the autonomic nervous system. This balance can be altered under certain conditions such as pathologic activation of the immune system, e.g. allergic or local stimulation of mast cells located in the heart. Degranulation of mast cells with the release of histamine and other active molecules causes profound changes in cardiac function. The major goal of this proposal is to determine the effect of mast cell stimulation and degranulation on parasympathetic neural activity within the heart. Previous work has shown direct actions of mast cell products such as histamine and eicosanoids on cardiac myocytes, pacemaker cells and sympathetic afferent terminals, while the studies in this proposal are the first to examine effects on the parasympathetic pathway. The first specific aim is designed to determine the effect of antigen-induced mast cell degranulation on membrane properties of parasympathetic postganglionic neurons in the sensitized guinea pig heart. Tissue from sensitized animals will be used to determine the electrophysiological changes in parasympathetic neuron activity in response to antigen-induced mast cell degranulation. The parameters to be measured include membrane potential, input resistance, action potential configuration, excitability, and fast ganglionic transmission. The second aim is to determine the effect of histamine, prostaglandins and leukotrienes on the activity and membrane properties of parasympathetic neurons. The effect on membrane potential, input resistance, action potential configuration, excitability, and the function of presynaptic parasympathetic fibers will be studied. Studies will be done to correlate the types of responses recorded and either the location of the parasympathetic neurons within the cardiac ganglion, the potential peptidergic inputs (substance P, CGRP) or the proximity to cardiac mast cells.

A grant has been awarded to Dr. Jean Hardwick at Ithaca College for the acquisition of a core microscopy facility for the Biology Department. This instrumentation will be used by four separate faculty members with diverse research interests in developmental biology, comparative physiology, neurobiology and plant ecology. Specifically, these funds will be used for the acquisition of a research-quality compound microscope equipped with advanced imaging capabilities such as DIC, phase contrast and fluorescence imaging. This microscope will also be equipped with a cooled digital camera (CCD) and image analysis system for the capture of publication-quality electronic images as well as advanced image analysis. In addition, this facility will included a stereomicroscope with a epi-fluorescence for imaging of fluorescently-labeled plant and animal specimens and a dual viewing bridge for training students in various procedures.
This instrumentation will become a major component of a core departmental facility for microscopy which will complement existing equipment as well as expanding our facilities to include electronic imaging and analysis. Several faculty members use microscopy as part of their ongoing research efforts in projects including the localization of neuropeptides in the amphibian nervous system, analysis of the regulation of embryonic development in Xenopus, the physiology and biochemistry of mucus production in invertebrates, and the response of plants to injury. All of these projects require significant analysis of tissue specimens using a variety of optical techniques. For example, analysis of neuropeptide localization utilizes the technique of immunohistochemistry to determine the specific organization and function of neurotransmitters in the nervous system. The developmental studies use gene markers, such as the green fluorescent protein (GFP), to investigate the molecular signals underlying specific aspects of development. The study of mucus production requires histological analysis of the mucus producing organs themselves, and the analysis of plant response to injury will also use a GFP marker to determine the cellular responses to insect herbivore. Thus, this facility will become an integral part of the research facilities for a wide range of current faculty as well as future hires.
The Biology department at Ithaca College has a long-standing commitment to the involvement of undergraduates in research. All biology majors are required to spend at least one semester doing research in a faculty member's lab. Undergraduates are involved in every facet of faculty research and are trained in the use of all equipment associated with that work. This would mean that, with at least four different faculty members using this facility as a part of their research program, a significant number of undergraduates will also have the opportunity to use this equipment. Undergraduates at Ithaca College routinely present their research efforts at conferences and are included as co-authors on publications. Continued involvement of undergraduates in ongoing scientific research is critical in stimulating students to pursue careers in science, as well as increasing their overall scientific literacy.

neuroimmunomodulation; heart innervation; mast cell; heart pharmacology; calcitonin gene related peptide; substance P; leukotrienes; action potentials; granule; membrane potentials; prostaglandins; histamine; parasympathetic nervous system; immunocytochemistry; guinea pigs; electrophysiology;

Cell specification in eye development

Eyes in vertebrates develop from cells in two regions of the forebrain, and these cells specialize (or differentiate) to generate many cell types. The cell types include photoreceptor cells (which detect light), many types of neural cells (which integrate signals from photoreceptor cells and send signals to the brain), and several types of supporting cells. The mechanisms by which regions of the brain become specified to form eyes, and cells in these region become specified to form different cell types, are not well understood.

The proposed research will examine eye development in a model laboratory organism, the frog Xenopus laevis. Cells within the developing eyes communicate by releasing specific signaling proteins; these proteins bind to receptors on other cells, influencing cell determination. One group of signaling proteins, called FGFs, is used in several processes during eye development. FGFs can bind to, and activate, any of four different FGF receptors (FGFRs), but it is not clear whether activation of specific FGFRs affects cell differentiation in the eye.

Experiments will be conducted to test the hypothesis that activation of different FGFRs causes eye cells to differentiate in different ways. Inhibitory FGFRs will be expressed in the developing eye; it will then be possible to compare the effects of blocking signaling through, say, FGFR-1 with the effects of blocking other FGFRs. This analysis will show whether different FGFRs play distinct roles in eye development. The experiments will utilize recently developed transgenic technology in frogs, which makes it possible to introduce specific genes into frog embryos. The introduced genes will consist of DNA that directs the gene to be expressed only in the eye, coupled to DNA encoding an inhibitory FGFR. .

These studies will lead to a better understanding of the mechanisms by which vertebrate eyes develop, and which processes may be disrupted when eyes develop abnormally. Finally, comparison of eye development in different organisms will contribute to a better understanding of how eyes have evolved.

Undergraduate students will be strongly involved in the proposed research. This research will thus serve, in part, to train the next generation of scientists.

DESCRIPTION (provided by applicant): The control of cardiac function is achieved primarily through a balance between the parasympathetic and sympathetic branches of the autonomic nervous system. When this balance is altered, there can be profound and life threatening alterations in the control of the heart. One potential factor that may impact this balance is the activation of the immune system in response to inflammatory stimuli. Localized activation of the immune system initially occurs via the stimulation of mast cells located within the tissue, which release potent bioactive mediators. One consequence of this mast cell activation may be a change in the production of the substance nitric oxide (NO) within the tissue. NO is also known to be an important regulator of cardiac function. In addition, activation of mast cells with inflammation may increase the ability of tissues to produce NO by increasing the levels of the synthetic enzyme, nitric oxide synthase (NOS) in cells. The major goal of this study is to determine how NO affects the function of neurons within the heart that help control cardiac function and to examine whether activation of mast cells in the heart can alter the levels of NOS in the heart. Isolated tissue from guinea pig hearts, containing the neurons under study, will be used to record the electrical responses from individual neurons and determine the effects of NO on their activity. To study the effect of mast cell stimulation on NOS expression levels, tissues will be exposed to mast cell stimulators and then analyzed using microscopy to monitor changes in enzyme localization. In addition, the tissue will be analyzed at the molecular level to look for changes in enzyme expression and mRNA levels. The results of this study will provide new information on the interaction between the immune system and the nervous system in the control of cardiac function. In particular, it will provide important information concerning the regulation and of NO in the control of cardiac function.

DESCRIPTION (provided by applicant): Previous studies have shown that an imbalance in autonomic efferent neuronal tone, with reduced parasympathetic activity coupled with increased and heterogeneous sympathetic outflow, increases the risk of cardiac arrhythmias and sudden death. While the progression of cardiac disease affects multiple aspects of cardiac control, it is those changes in peripheral autonomic neuronal processing and their projections that ultimately determine the neuronal coordination of the heart. The intrinsic cardiac nervous system (ICN), the final common pathway for such neural control, integrates information from multiple inputs and mediates short- loop reflex control of regional cardiac indices. Although multiple studies have focused on cardiac stress- induced changes in post-ganglionic innervation patterns to the heart, little attention has been paid to the critical role of information processing within autonomic ganglia and how they remodel/adapt to imposed stress. It is our hypothesis that cardiac stress-induced adaptations within the ICN facilitate coordination of efferent parasympathetic output and that these changes are reflected in functional and phenotypic alterations in select intrinsic cardiac neuronal populations. These cardioprotective adaptations within the ICN could counteract, in part, the maladaptive effects of excessive sympatho-excitation associated cardiac stress. Numerous studies have identified molecular mechanisms associated with cardiac remodeling, including increased activity of the renin-angiotensin system, changes in nitric oxide (NO) production, and alterations in end-organ sensitivity to neurotransmitters. For each of these factors, while their direct effects on myocyte function are well established, recent data indicates that many of their cardiac effects are mediated via alterations in function within the cardiac nervous system. To specifically address these points this proposal will evaluate how the elements of the ICN adapt to chronic disease using two different animal models of heart disease: myocardial infarction (MI) and chronic pressure overload (PO). The proposed experiments will focus on two specific adaptations of the ICN: (1) changes in neuronal responses to neuromodulators and (2) changes in ICN network efficiency. We will also evaluate the efficacy of targeted pharmacologic therapy to mitigate adverse remodeling of the ICN. Using a whole mount preparation of the guinea pig cardiac plexus, we will evaluate the physiological responses of individual intrinsic cardiac neurons to autonomic neurotransmitters with and without potential neuromodulators, such as angiotensin II and NO in tissues from control, MI and PO animals to characterize stress-induced changes in neuronal responses. In addition, we will evaluate the output of individual neurons to stimulation of vagal and intraganglionic fiber inputs to evaluate integrated network function. Changes in neuronal activity will then be compared between untreated disease models and disease models treated with standard therapeutics such as 2-receptor blockage, AT receptor inhibition, or inhibition of NO generation, to determine if these therapies modulate the ICN function. PUBLIC HEALTH RELEVANCE: This proposal will examine how the autonomic nervous system adapts to chronic heart disease, with the goal of developing better therapeutics for the treatment of chronic disease. The project will use animal models of chronic heart disease to examine the changes that occur within the cardiac autonomic nervous system with disease as well as evaluating how standard therapies for cardiac disease affect these processes.

? DESCRIPTION (provided by applicant): Chronic heart disease induces functional and phenotypic changes within cardiac tissues and the neuronal systems that control the heart. An imbalance in autonomic neuronal control that results in reduced parasympathetic activity coupled with increased and heterogeneous sympathetic activity increases the risk of cardiac arrhythmias and sudden death. The intrinsic cardiac nervous system (ICN), the final common pathway for parasympathetic autonomic control to the heart, integrates information from multiple inputs and can produce short-loop reflex control of cardiac function on a beat-to-beat basis. While multiple studies have focused on cardiac stress-induced changes in sympathetic neuronal control, much less attention has been paid to the critical role of information processing within peripheral parasympathetic autonomic ganglia and how they remodel/adapt to imposed stress. It is our hypothesis that cardiac stress-induced adaptations within the intrinsic cardiac nervous system represent efforts to maintain parasympathetic efferent output via functional and phenotypic alterations in select intrinsic cardiac neuronal populations. These adaptations within the ICN could counteract, in part, the maladaptive effects of excessive and heterogeneous sympathetic excitation associated with progressive cardiac disease. Increased sympathetic efferent activity can alter cardiac function through increased release of norepinephrine and neuropeptide Y (NPY) from sympathetic efferent fibers, along with sympathetic-induced increases in the production of the peptide hormone angiotensin II (AngII) and its metabolite, Ang(1-7). To specifically address the importance of altered peptide levels on the development of cardiac pathology, this proposal will evaluate how the intrinsic cardiac nervous system responds to elevated levels of AngII and NPY following myocardial infarction (MI) in the guinea pig. Using a whole mount preparation of the guinea pig cardiac plexus, we will evaluate the physiological responses of individual intrinsic cardiac neurons to AngII and NPY in tissues from control and MI animals. Our primary specific aims are (1) to determine the role of Angiotensin II receptors (AT1R, AT2R, MasR) in the modulation of ICN output with MI and (2) to determine the effects of increased release of NPY on the ICN following MI. We will use a multidisciplinary approach, which includes sharp electrode voltage recordings from individual neurons in the whole mount preparation, whole cell voltage clamp recordings from neurons within the intact network, immunohistochemistry, and biochemical measurements of protein and mRNA expression levels. Combined, the results from these studies will aid in determining the mechanisms by which these peptides can modulate neuronal function. This information can be used to develop better pharmacotherapies to treat chronic heart disease.