Anna Lysakowski - US grants

This Small Grant will be used to gather preliminary data on the efferent innervation of the peripheral vestibular apparatus. Recent work has suggested that this population may be heterogeneous in its neurotransmitter neurochemistry and in its peripheral targets. This suggestion, if confirmed, would change our view that the efferents are a nonspecific system. The goal of this study is to define potential subpopulations of efferent neurons in the chinchilla based on their brain stem locations, their projections to one, the other or both ears, their neurochemical identities and their peripheral terminations on hair cells, calyces and other afferent processes. The specific aims are: 1) to define separate populations of efferent neurons in the brain stem based on current knowledge of possible transmitters and efferent anatomy and 2) to correlate this knowledge with an analysis of the regional synaptic relations of chemically-defined efferent boutons and fibers in the periphery. Efferent neurons in the brain stem will be identified by means of fluorescent retrograde tracer injections into the peripheral vestibular apparatus and will be simultaneously characterized immunohistochemically by the use of antibodies to several putative neurotransmitters (acetylcholine, gamma-aminobutyric acid, calcitonin gene-related peptide, met-enkephalin and others). The analysis will be extended to the periphery where electron microscopic immunohistochemical methods will be used to characterize neurochemically distinctive efferent boutons in terms of their terminations in the sensory epithelium. The significance of this project is that a precise knowledge of the different elements comprising the population of efferents will further our understanding of brain stem control of peripheral sensory processing.

The purpose of this grant is to investigate the efferent innervation of the peripheral vestibular apparatus. Knowledge of the neurotransmitters and the projections from the brainstem efferent neurons to the peripheral labyrinth will help us understand the role of the vestibular efferent system in health and disease. Recent studies have suggested that neurons in the vestibular efferent nucleus are heterogeneous in their transmitter composition and in their peripheral targets. Such heterogeneity changes our long-held view that vestibular efferents are a nonspecific system. The goal of this study is to define subpopulations of efferent neurons in the chinchilla based upon their brain stem locations, their projection patterns, their peripheral endings, their neurotransmitters, and their receptor subtypes. Specific aims are: 1) to characterize anatomical and neurochemical subpopulations of brainstem efferent neurons by combining retrograde and anterograde tracers with transmitter immunohistochemistry, and 2) to determine details of the morphological terminations of efferents in the periphery, at both the light and electron microscopic levels, and details of their neurochemistry at the molecular level by determining their transmitter receptor subtypes. Efferent neurons in the brainstem are identified by means of retrograde tracer injections into the peripheral vestibular apparatus. They are simultaneously characterized immunohistochemically with antibodies to several putative neurotransmitters (acetylcholine, calcitonin gene- related peptide, nitric oxide synthase, met-enkephalin, adenosine triphosphate, and others). In the periphery, the distribution of receptor sub-types for peptidergic, purinergic and muscarinic transmission will be examined in an attempt to specifically understand the slow response of afferents to efferent stimulation. Vestibular afferents are identified by means of extracellular horseradish peroxidase injections or calretinin immunohistochemistry. The innervation patterns of efferent terminals in the periphery is determined by injections of biotinylated dextran amine centrally. Electron microscopic immunohistochemical methods are used to characterize chemically distinct efferent boutons according to their terminations in the sensory epithelium, that is, by the class of afferents (calyx, dimorphic or bouton) and by the region (central or peripheral zones) that they innervate. The intent is to produce a body of knowledge about the structural basis of the efferents, and their relationship to the afferents, from which physiologically and pharmacologically testable hypotheses can be derived.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In the mammalian vestibular labyrinth, hair cells are capable of generating axial deformations in response to variations in transmembrane potential. Our understanding of electromechanical transduction in the hair cells depends on precise knowledge of intracellular structure, and efforts to model hair cell physiology are likewise limited to the fidelity of known intracellular geometry. This collaboration focuses on the ultrastructure of vestibular hair cells. More precisely, we are looking at the subcuticular region (below the cuticular plate), using electron tomography to visualize and analyze rootlet architecture at the junction of the stereocilia bundle and apical region of the cell. Our main interest is the striated organelle (SO), a unique structure located at the apical end of hair cells (auditory and vestibular sensory cells), just below the cuticular plate. It is particularly prominent in mammalian type I vestibular hair cells. The central hypothesis we are investigating with aid from the NCMIR is that striated organelles provide structural and functional connections between the apical and basal parts of the hair cell. The goals of this joint effort are to describe the three-dimensional structure of this particular organelle and to identify its components (or at least, some of the components). Our study should appeal to: 1) sensory physiologists interested in mechanisms of sensory transduction and mechanotransduction, 2) neuroscientists interested in feedback from afferents to the transduction apparatus, 3) protein chemists interested in potential new motor proteins, contractile elements, calcium dynamics, and the molecular architecture of these mechanisms, 4) molecular and cell biologists interested in actin, molecular motors, the cytoskeleton and mitochondria, and 5) microscopists and morphologists interested in a new and elegant structural apparatus.

DESCRIPTION (provided by applicant): This is a continuing study of synaptic transmission in vestibular organs with emphasis on the type I hair cell and its calyx ending, phylogenetically recent acquisitions only found in reptiles, birds and mammals. These structures are restricted to central/striolar zones in reptiles and birds. Their increasing importance in mammals is suggested by their distribution throughout the neuroepithelium of all organs. In addition, while type I and type II hair cells occur in approximately equal numbers in rodent cristae, type I hair cells predominate in the cristae of monkeys and possibly of humans. The peculiar structure and distinctive physiology of these structures raise problems as to how synaptic transmission is accomplished. 1) Type I hair cells have a distinctive basolateral current that may compromise synaptic transmission; 2) Housekeeping functions cannot be done by supporting cells; and 3) The geometry and electrophysiology of the calyx ending place unusual demands on the flow of synaptic currents to the spike encoder. We have only fragmentary knowledge as to how these problems are solved. Yet, because these structures become of increasing importance in mammals (including humans), such knowledge is crucial to our understanding as to how vestibular organs process information. Of clinical interest, the type I hair cell and/or its calyx ending are especially sensitive to aminoglycoside ototoxicity and age-related degeneration. Physiological studies will be done in the turtle posterior crista and will be integrated with morphological studies to be done in rats and turtles. There are three specific aims. 1) Hair cells: We will characterize synaptic transmission from type I hair cells by making whole- cell recordings from the calyx ending. The hypothesis is that neurotransmitter release from type I hair in low- frequency vestibular hair cells may differ from high-frequency auditory and vibratory organs. 2) Homeostasis: Both K+ ions and glutamate neurotransmitter are released from hair cells during transduction. In the case of type II hair cells, supporting cells serve to clear these substances. The presence of the calyx ending precludes supporting cells from acting in the same way for type I hair cells. We hypothesize that the type I hair cell and/or its ending subsume these housekeeping functions. 3) Postsynaptic mechanisms: We will explore how various ion channels and neurotransmitter receptors shape synaptic and spiking activity. The hypothesis to be tested is that the molecular organization of the calyx creates separate microdomains with discrete functions: synaptic transmission, spike initiation, and discharge regularity.

Abstract There are at least a dozen forms of mitochondrial-related deafness (both non-syndromic and syndromic forms, as well as aminoglycoside-induced) and associated vestibular disorders. After connexin-related deafness, mitochondrial-related deafness is the second most common form, responsible for 5% of all post-lingual deafness. Just one form of mitochondrial-related deafness (the A1555G mutation) is present in over half of all cochlear implant patients who become deaf due to aminoglycosides. Yet, most mitochondrial diseases are considered rare or orphan diseases, and have been largely ignored by the scientific community. Mitochondrial forms of deafness, however, are as common as several well-known neurological diseases, e.g., Hunting- ton's, ALS, or certain forms of muscular dystrophy. In other organs in the body, the structure of mitochondria and their role in apoptosis and cell death is a topic of intense research interest. Such a framework of structural studies is missing in the inner ear. This proposal aims to fill the gap by studying hair cell mitochondria in both whole animal and isolated mitochondrial prepara- tions for the purpose of addressing hair cell damage and cell death in mitochondrial-associated forms of deafness. Besides the emphasis on mitochondrial-related deafness, we think that this proposal merits consideration as an R21 proposal because we will be using imaging approach- es relatively new to the inner ear field to study mitochondrial fine structure in inner ear cells. We will also relate these findings to isolated sub-populations of inner ear mitochondria that we pre- dict will respond differently to ototoxic insults such as aminoglycoside or cisplatin exposure. With information gained from this proposal, therapeutics can be more precisely targeted toward specific parts of the mitochondrial respiratory chain that are compromised in ailing hair cells and can thus rescue those that have been exposed to environmental assaults (noise, chemothera- py, ototoxic antibiotics) or aging.

Project Summary/Abstract The vestibular inner ear supplies information about head motion and position to the brain, driving powerful reflexes that stabilize gaze and posture during head motions, and contributing to our sense of heading and orientation as we move through the world. Although we are not normally aware of these functions, their loss severely affects mobility by destabilizes vision and causes vertigo. Loss of vestibular function often originates in damage to hair cells and their synapses with the afferent vestibular nerve fibers that project to the brain. These hair cells, synapses, and afferent fibers have striking properties that are only partly understood. The longterm goal of this program of research is to build a comprehensive understanding of how vestibular information is generated and encoded in the inner ear. The current proposal focuses on the synaptic transfer of head motion signals from hair cells to primary vestibular neurons (Aim 1) and the subsequent initiation of action potentials (spikes) (Aim 2) in the mouse utricle, a model preparation for genetic, developmental and physiological studies. Principal methods are whole-cell patch clamping of hair cells and afferent neurons; immunolocalization of voltage-gated ion channels, pumps and synaptic markers; and computational modeling of the hair cells, synapses and afferent nerve fibers, incorporating current information on ion channels, pumps, and morphology. Vestibular afferent neurons make conventional bouton synaptic terminals on type II hair cells and unique calyceal contacts on type I hair cells. At both boutons and calyces, hair cells release vesicles of glutamate (?quantal? synaptic transmission) into the synaptic cleft, activating glutamate receptor-channels in the postsynaptic membrane to produce excitatory postsynaptic potentials and initiate spikes. At calyceal contacts, an additional ?non-quantal? transmission mechanism depends not on vesicular release or gap junctions, but rather on flow of ions from the hair cell through ion channels into the synaptic cleft and into the calyx through different ion channels. Postsynaptic responses to controlled stimulation of individual hair bundles show that quantal and non-quantal transmission modes can occur at the same calyceal synapse and that the non-quantal mode provides a fast signal that may be important for high-speed vestibular reflexes. Proposed experiments and modeling will investigate the impact of key hair cell ion channels on non-quantal transmission and delineate how quantal and non-quantal transmission are integrated in individual calyces and afferent nerve fibers. Other experiments will test how specific voltage-gated potassium and sodium channels in calyces and boutons shape the postsynaptic voltage response and spikes in the axonal initial segment. Immunolocalization has revealed remarkable concentrations of ion channels in microdomains of the calyx ending and nearby spike initiation zone. Experiments focus on channels with the potential to shape salient differences in response dynamics and spike timing between afferents of different connectivity (hair cell inputs) and different zones of the sensory epithelium.