Gay Holstein - US grants

The objective of the proposed research is the systematic study of morphologic and immunocytochemical changes which occur during growth, development and aging in primate brain. The model chosen for study is the substantia nigra (SN) of rhesus monkeys. The specific aims of the project are (1) to determine the qualitative changes which are apparent in the morphology of substantia nigra neurons from birth to senescence, (2) to quantify the number of nigral neurons present in this nucleus and the volume of the nucleus at each stage of life, (3) to characterize the signal ultrastructural features of developmental, adult and aged monkey SN, (4) to quantify these ultrastructural changes using an appropriate statistico-mathematical analysis and (5) to localize, characterize and quantify the immunocytochemically- identified neurotransmitter endowment of neurons, dendrites and terminals at each stage of development and aging. These aims will be accomplished at the light (LM) and electron microscopic (EM) levels, using a wide range of techniques including the classic Golgi and Nissl methods for LM, Golgi-gold toning for LM-EM correlations, quantitative analysis including ultrastructural stereology, qualitative and quantitative post-embedding, as well as single and double label pre-embedding immunocytochemistry. All of these studies will be conducted separately on material from pars compacta (SNc) and pars reticulata (SNr). The proposed studies are designed to yield normative data on the morphologic natural history of the monkey substantia nigra. Nature and time provide a potent independent variable which may operate in such a fashion that aging represents a continuum, or a reversal, of the events which characterize development. This research constitutes a first step toward understanding the results of invasive experimental manipulations, as well as interferences with normal development and aging due to traumatic, environmental and metabolic disturbances. As such, the results of this investigation will provide a basis for the interpretation of behavioral and clinical studies of normal and experimentally- altered motor system development, studies of movement disorders and nigrostriatal pathways, deficits, brain transplant studies involving implants of fetal nigral tissue, and behavioral, clinical and pharmacologic studies of normal and abnormal aging processes. Parkinson's disease, Huntington's disease, schizophrenia, the Lesch-Nyhan syndrome, Gilles de la Tourette syndrome and tardive dyskinesia have all been suspected to encompass dysfunction of the substantia nigra.

DESCRIPTION: The direct and indirect pathways of the angular vestibulo-ocular reflex (aVOR) constitute principal nervous system mechanisms for the maintenance of gaze, of normal posture, balance, and coordination, and of spatial orientation during angular locomotion. The long-term goal of this research program is to identify anatomical and neurochemical constituents of defined neuronal circuits of the central vestibular system. The objective of this current research proposal is to visualize glycinergic neurons and terminals in the vestibular nuclei, in order to further elucidate the cellular bases for inhibitory control of the velocity storage component of the aVOR. This objective will be accomplished using qualitative and quantitative immunocytochemical approaches to identify the neurotransmitter-defined neurons by light microscopy and to characterize and quantify the synaptic interactions involving these cells at the ultrastructural level. The first specific aim is to characterize the regional distribution, morphology, ultrastructural features and synaptology of glycine- immunoreactive neurons and terminals in the vestibular nuclei of normal animals. Using multiple electron-dense labels, the synaptic interactions between glycinergic and GABAergic neurons, the possible co-existence of the two inhibitory amino acids in individual vestibular cells, and the somatodendritic distribution of glutamatergic synaptic inputs to glycinergic cells will be identified and quantitated. The second specific aim is to visualize and quantify degenerating and non-degenerating glycine- immunoreactive neurons and terminals in the vestibular nuclear complex (VNC) of monkeys after midline medullary section of vestibular commissural fibers. Since the midline medullary lesion interrupts the indirect, but not the direct pathway, the degenerating elements are likely to be the neurons responsible for the production and/or maintenance of velocity storage. The third specific aim is to identify and quantify the ultrastructural features and synaptology of the central terminals of primary vestibular afferent fibers using colloidal gold-tagged lectin transport molecules. Immunocytochemical studies of this tissue will demonstrate the direct or indirect nature of the synaptic input from primary afferents to glycinergic and/or GABAergic cells in the VNC. These studies will help to specify the role of glycinergic inhibition in the aVOR, in the organization of the velocity storage mechanism, in the mediation of vestibular commissural inhibition, and in the modulation of primary vestibular afferent activity. The information generated by this research is necessary for understanding how vestibular compensation is mediated, for clarifying the neuronal substrate(s) for brainstem lesions that lead to balance disorders, and for the rational development of pharmacotherapeutics for central vestibular deficits.

DESCRIPTION: (Adapted from Applicant's Abstract) Exposure to microgravity causes postural, locomotor and oculomotor modifications. In order to realize long term space flight, effective countermeasures for these abnormalities must be developed. Toward this end, it is essential to understand the cellular and biological basis underlying centrally-mediated vestibular adaptation to altered gravity conditions. The objective of the proposed research is to identify the morphologic alterations in rat cerebellar cortex that correlate with sensory and motor adaptation to microgravity. The investigators propose ground- based and space- based studies to test the hypotheses that (a) ultrastructural alterations accompany adaptation to microgravity, and (b) such alterations are pathway and neurotransmitter-specific. The merit of this idea has been emphasized in several brief communications by Krasnov and co-workers, in which ultrastructural changes in Purkinje cell synaptology have been reported in the nodulus of rats following spaceflight. These observations are of particular interest because Purkinje cells in the nodulus control habituation of the vestibulo- ocular reflex, and are likely to be critical for maintaining spatial orientation with regard to gravity. In addition, physiologic investigations have clearly indicated a role for the flocculus in controlling specific aspects of the VOR. The investigators propose to study the cerebellar cortex from (1) brain tissue already processed in the laboratory from flight and control rats of PARE.0.2 from the STS-54 shuttle mission; (2) flight and control rats from the Neurolab shuttle mission; and (3) naive laboratory rats. The tissue will be used for quantitative ultrastructural and immunocytochemical studies of synaptic circuits in the nodulus and ventral uvula, flocculus and paraflocculus, and non-vestibular cerebellar cortex. The investigators expect to obtain stereological data supporting a change in synaptology in vestibular, but not in non-vestibular cerebellum of flight rats. The qualitative and/or quantitative differences in excitatory amino acid and GABAergic neurotransmission in the nodulus and flocculus of flight rats will also be compared to controls and naive animals. The investigators expect to obtain critical information about the alterations in synaptology and neurotransmitter localization in the nodulus and flocculus that accompany adaptation to microgravity. The identification and characterization of GABAergic and GABA-receptive elements in this paradigm should lead to a greater understanding of how inhibition is modified in neuronal circuits during behavioral adaptation. Similarly, delineation of the microgravity-induced alterations in excitatory glutamatergic transmission will contribute to our basic knowledge of the morphologic basis for cerebellar-mediated motor learning. Through comparison of tissue from ground-based rats with animals sacrificed postflight and animals sacrificed during flight, it will be possible to localize, characterize and quantify the site(s) and synapses that mediate vestibular adaptation phenomena in space.

[unreadable] DESCRIPTION (provided by applicant): The search for the origins of diversity in primary vestibular afferent response dynamics has been a dominant theme of vestibular research and is the major subject of the present proposal. The long-term goal of the work is to study the role of transmitter systems in shaping afferent responses, and the immediate objective is to identify the impact of GABA and glutamate as hair cell transmitters. The central hypothesis of the research is that the crista performs a mathematical differentiation (differential calculus) of some velocity-sensitive input signals in processing convergent excitatory (glutamatergic) and inhibitory (GABAergic) hair cell synapses onto dendrites of single afferent neurons and that this push-pull input accounts, at least in part, for the wide range of observed afferent responses. Three specific aims will be pursued. These aims embrace a multidisciplinary approach utilizing techniques and experimental models selected for their particular suitability for the experiments, the research experience of the investigators, and the applicability of the results to understanding human vestibular disorders. Anatomical, physiological, pharmacological and mathematical tools are employed concomitantly to address the aims and test hypotheses from multiple perspectives. The first aim is to relate the chemical anatomy of the hair cells in the horizontal canal crista ampullaris to primary afferent response dynamics. The second aim is to identify the relationship between hair cell transmitter phenotype, hair cell morphology, and hair cell activation and response kinetics. The third specific aim is to evaluate the effects of pharmacological manipulation of GABA transmission on the horizontal canal nerve and on individual afferent fibers. The role of transmitter phenotype in shaping primary afferent activity is a fundamentally and clinically important aspect of vestibular function that is poorly understood. Most studies will be conducted in toadfish, which are experimentally advantageous because of their availability, the ease of exposing and extracting their labyrinths, and the similarity of vestibular endorgans throughout the vertebrate phyla. Additional studies in mouse are proposed in order to relate findings in toadfish to a mammalian vestibular system, thus establishing the generality and human health relevance of the results. The proposed research is unique, and will have direct bearing on the development of more effective therapeutic interventions, both medicinal and device-oriented, for peripheral vestibular disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The existence of a functional link between the vestibular system and blood pressure control has been known for nearly a century. It is currently thought that arterial baroreceptors participate in a regulatory circuit that maintains sympathetic tone through the baroreflex while signals from the vestibular end organs drive a faster mechanism that counteracts the effects of a change in posture. This latter circuit is often called the vestibulo-sympathetic reflex (VSR). Primary afferents of this pathway terminate on cells in the caudal vestibular nuclear complex (VNCc). These second order neurons, in turn, project to brainstem sites involved in cardiovascular regulation such as the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). Cells in the RVLM are thought to integrate the vestibular input with baroreceptor and other sensory afferents and send excitatory projections to preganglionic sympathetic neurons in the intermediolateral cell column of the spinal cord. While the principal neurotransmitter of these presympathetic vasomotor RVLM cells is likely to be glutamate, numerous neuroactive molecules have been co-localized in these cells, including catecholamines of the C1 cell group. In addition, bulbospinal vasomotor RVLM cells receive monosynaptic GABAergic projections from the CVLM, which tonically inhibit the RVLM neurons. As a result, CVLM cells can be viewed as sympathoinhibitory interneurons in the vasomotor pathway. The long-term goal of our research program is to identify the neurotransmitters, receptors, and signaling pathways that participate in vestibulo- autonomic projections so they can be targeted specifically by pharmacotherapeutics to ameliorate vestibulo- autonomic disorders. The specific objective of this research project is to identify the structural and chemical anatomy of vestibular pathways that contribute to blood pressure regulation. The project has two aims that will be pursued using rats as the experimental model, bilateral sinusoidal galvanic vestibular stimulation to activate the vestibular nuclei, telemetric detection of blood pressure, anterograde and retrograde tract-tracing, and immunofluorescence detection of the immediate early gene protein product c-Fos together with pathway- specific neurotransmitters and modulators. Aim 1 will identify the sensitivity, topography, cytology, neurotransmitter(s), and modulator(s) of vestibular neurons of the VSR. This aim will test the overall hypothesis that VNCc cells of the VSR pathway have a morphological, hodological and/or chemoanatomical phenotype that is distinct from other VNCc neurons and the baroreflex pathway. Aim 2 will identify the innervation pattern(s), synaptology and postsynaptic partners of vestibulo-sympathetic axons in RVLM and CVLM. This aim will address our over-arching hypothesis by determining the neuronal and synaptic specificity of vestibular input to pre-sympathetic vasomotor circuitry. These hypotheses are fundamental to our long-term goal, since regions or cells where the VSR pathway is morphologically or chemoanatomically segregated from the baroreflex pathway are candidate sites for specific pharmacological intervention into the VSR.

DESCRIPTION (provided by applicant): Changes in body position and posture are detected by the vestibular system and are normally accompanied by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by functional integration of the vestibular system and blood pressure control mechanisms in the caudal medullary reticular formation: signals from the vestibular otolith organs provide the primary source of input to the shorter latency vestibulo-sympathetic reflex (VSR), while arterial baroreceptors regulate sympathetic tone via the relatively long- latency baroreflex. Although the clinical literature has consistently reported a decline in vestibulo-autonomic function with age, and this decline is associated with increased risk of falling and increased mortality in older people, the cellular mechanisms underlying this loss of function are not known. The overall goal of this two-year exploratory project is to identify chemoanatomic alterations in key caudal brainstem sites of aged rodents, correlated with assessment of VSR and baroreflex function. The two specific aims are (1) to obtain physiological measures of autonomic activity, particularly heart rate and blood pressure (a) under baseline conditions, (b) in response to baroreflex activation and (c) in response to vestibular stimulation in young-adult and aged rats and mice;and (2) to visualize Fos protein, and transmitter and modulator expression in medullary regions activated by the three conditions of Aim 1, in young adult and aged rats and mice. This research will evaluate the overall hypothesis that specific alterations in the chemical anatomy of the caudal medulla parallel an age-dependent decline in VSR and baroreflex control of blood pressure. The project will also assess the utility of two common rodent models of aging for studies of vestibulo-autonomic structure and function. PUBLIC HEALTH RELEVANCE: Rapid adjustments in blood pressure normally accompany movements and changes in body position and posture;these compensatory adjustments allow humans to stand up without fainting. Orthostatic hypotension, which is highly prevalent in the elderly, occurs when the vestibulo-sympathetic reflex fails to mediate such compensatory adjustments. This project will evaluate the hypothesis that specific alterations in the chemical anatomy of the caudal medulla parallel an age-dependent decline in vestibulo-sympathetic- and baroreflex control of blood pressure, thereby suggesting new pharmacotherapies to ameliorate orthostatic hypotension and more specific anti-hypertensive medications that do not elicit disabling vestibular side effects such as dizziness and vertigo.

Description (provided by applicant): The general purpose of this proposal is to understand how precise neural connections are achieved during the development of the central nervous system (CNS). Precise connections between axons and their postsynaptic targets are essential for the proper functions of the central nervous system. These connections are roughly established by guidance cues during early development and are subsequently fine-tuned in an activity-dependent manner. The latter fine-tuning process involves stabilization and elimination of synaptic connections. It is generally accepted that competition between converging inputs plays an important role in this process although the cellular basis of this competition is poorly understood in the CNS. In addition, while it is generally agreed that synaptic modifications may underlie neuronal remodeling, whether a long-term depression (LTD)-like form of plasticity is involved in the elimination of synapses is unclear. To address these two critical issues, we will use one well-studied model of activity-dependent fine-tuning: the segregation of two eyes inputs to the lateral geniculate nucleus (LGN). Initially LGN neurons receive converging functional synaptic inputs from both contralateral and ipsilateral eyes. While the contralateral eye inputs are retained, the ipsilateral inputs are subsequently withdrawn and their synapses are eliminated. This segregation process is activity-dependent and requires competition between two eye inputs. In this proposal, we will answer two important questions: (1) how does converging inputs compete? (2) Does synaptic depression occur prior to synapse elimination and withdrawal of ipsilateral eye inputs? These questions will be addressed using a unique in vitro intact rat LGN preparation in which two eye inputs are intact and can be electrically stimulated independently. The locations and properties of individual synapses will be examined using a combination of recording and imaging methods. Insights obtained from this work will form the foundation for future examination of synapse elimination in the CNS and to understand the relationship of input competition, synaptic modification and structural remodeling. In addition, the obtained results may also shed light on various diseases associated with the disorders of neural development. PUBLIC HEALTH RELEVANCE Insights obtained from this proposal will form the foundation for future examination of synapse elimination in the central nervous system and to understand the relationship of input competition, synaptic modification and structural remodeling. In addition, the obtained results will also shed light on various diseases associated with the disorders of neural development.

? DESCRIPTION (provided by applicant): In humans, orthostatic hypotension (OH) is defined as a fall in blood pressure of at least 20/10 (systolic/diastolic) mmHg within three minutes of standing or during head-up tilt. It occurs in a wide variety of medical conditions, including neurologic disorders. Neurogenic OH is common in patients with dizziness, vertigo, brainstem lesions, and diseases such as multiple system atrophy and Parkinson's disease. Clinical studies indicate that one cause of neurogenic OH may be vestibular system hypofunction, particularly involving vestibulo-sympathetic reflex pathways. Support for this derives from numerous studies in humans and experimental animals that provide clear evidence for a functional link between the vestibular system and blood pressure control. Both vestibular system disorders (e.g. balance) and OH are more prevalent and severe in the elderly. This exploratory project uses an aged rat model to study one putative cellular alteration underlying OH. Pituitary adenylate cyclase-activating peptide (PACAP) is a neuromodulator involved in a wide range of physiological functions including cardiovascular control and neuroprotection. Recent preliminary studies indicate that PACAP is normally present in rat vestibular neurons, and that this expression is severely altered in aged animals with behavioral signs of OH. The present proposal seeks to determine, in both sexes, whether OH in aged rats is associated with degeneration of PACAP-containing neurons of the vestibulo-sympathetic reflex pathway. Aim 1 is to assess blood pressure changes in young-adult and aged male and female rats in response to sinusoidal galvanic vestibular stimulation and nose-up tilt, both of which activate the vestibulo- sympathetic reflex. We hypothesize that blood pressure will decrease in most aged rats, but few young-adults, establishing a rodent model of orthostatic hypotension. Aim 2 is to compare FluoroJade C labeling, which identifies degenerating neurons, in PACAP-positive vestibulo-sympathetic reflex projection neurons in tissue from the physiologically characterized rats studied in Aim 1. The retrograde tracer FluoroGold will be used to identify the projections, and cFos labeling will be used to verify that the neurons are activated by the stimulus. We hypothesize that OH correlates with degeneration of PACAP-containing vestibular neurons of the vestibulo-sympathetic reflex pathway. If successful, this project will provide the foundation for a comprehensive study addressing the cellular bases for the decline in vestibulo-sympathetic function in the elderly.