Elisabeth Glowatzki, Ph.D. - US grants

DESCRIPTION (provided by applicant): This proposal seeks to investigate mechanisms of synaptic transmission at the inner hair cell (IHC) afferent synapse in the mammalian cochlea. In the inner ear, sound signals are converted into hair cell receptor potentials, and subsequently are translated at the afferent synapse into firing rates in auditory nerve fibers. Coding of sound critically depends on the diverse firing properties of auditory nerve fibers. Important features of auditory nerve fibers are their spontaneous firing rates, their thresholds of activation and their `rate level functions', describing the changes in firing rates in response to different sound pressure levels. Three main `sites' in cochlear transmission have been suggested to determine auditory nerve fiber properties: 1) basilar membrane mechanics; 2) IHC receptor potential; 3) IHC afferent synaptic transmission. Convincing arguments have been made that auditory nerve fibers with distinct properties (low versus high spontaneous rates; high versus low thresholds of activation) innervate the same IHCs. However, all 10-20 afferent fibers contacting one IHC `sense' the same receptor potential and therefore it is likely that differences in afferent fiber properties must arise at single synapses. The aim of this study is to ask which properties of auditory nerve fibers arise at the IHC afferent synapse, and how pre- and/or postsynaptic components specifically determine these properties. Excised cochlear tissue from 2-4 week old rodents will be used to perform simultaneous whole cell recordings from IHCs and afferent fiber terminals directly where they contact the IHCs. The transfer function at the IHC afferent synapse will be measured directly by controlling the IHC membrane potential and monitoring postsynaptic activity in the afferent terminal. Extracellular recordings will be used to monitor the rate of action potentials at the afferent terminal. Key to this approach is that it allows recordings after hearing onset, when the diverse properties of auditory nerve fibers have developed. Afferent fiber spontaneous rates, thresholds and transfer functions will be determined and afferent fiber adaptation will be measured. Simultaneous recordings from pairs of afferent fibers will provide a direct test of whether fibers with different properties do innervate a single IHC. Hypotheses based on in vivo experiments that have suggested specific loci as the origin for specific auditory nerve fiber properties will be reevaluated. The specific pre-or postsynaptic mechanisms responsible for specific firing patterns will be further investigated. A better understanding of the mechanisms that underlie the generation of diverse auditory nerve fiber firing properties will provide the basis for an improvement in cochlear implant design. The studies outlined in this proposal seek to understand the mechanisms that underlie synaptic transmission at the first synapse in the auditory pathway, the synapse between hair cells and auditory nerve fibers. The conversion of the hair cell receptor potential into a firing rate in the auditory nerve fibers is an important step in coding the sound signal for transmission to the brain. Our results will support studies that aim to model how auditory nerve activity is generated. These approaches can provide a future basis for better cochlear implant design.

DESCRIPTION (provided by applicant): At the inner hair cell (IHC) ribbon synapse, the first synapse in the auditory pathway, 'analog' sound information is converted into a 'digital' pattern of action potentials at auditory nerve fibers and transmitted to the brain. The relative time of each spike within a train of action potentials carries important information that needs to be faithfully represented in the auditory pathway. A well characterized form of temporal coding is phase locking: auditory neurons are capable of firing at a particular time within each cycle of a low-frequency stimulus. This phenomenon is required for localizing a sound source by computing the small difference in time at which the wave arrives at the two ears. Interaural delays can be as small as 10 microseconds, emphasizing the precision of temporal coding by the auditory periphery. The goal of this proposal is to investigate the mechanisms that allow the IHC ribbon synapse to release neurotransmitter with high precision and over long periods of time. In acutely excised rat cochlear preparations, simultaneous patch-clamp recordings will be performed from IHCs and postsynaptic terminals of auditory nerve fibers. Recently, we have shown that short-term facilitation occurs at this synapse, producing not only an increase in release but also a reduction in latency. Our first aim is to investigate the mechanisms underlying this phenomenon. We will study the role of the residual intracellular Ca2+ concentration by controlling its spread with specific buffers, and by monitoring its decay time course with fluorescent dyes. Facilitation will also be studied by uncaging Ca2+ in the cytosolic space. Secondly, the underlying mechanisms of phase-locked synaptic responses will be investigated. Preliminary experiments show that synaptic responses at the IHC ribbon synapse phase-lock to periodic stimuli. This feature will be further explored by applying stimuli with variable amplitude and by testing whether the preferred phase is conserved. These experiments will be compared with responses to single step depolarizations. We will evaluate the role of short-term facilitation and depression in establishing phase-locking. Finally, the ability of the IHC ribbon synapse to signal continuously with high precision will be studied. It has been shown that in response to steady IHC depolarization, this synapse exhibits short-term depression. The time course of recovery of synaptic responsiveness following a depleting stimulus will be evaluated. Given that neural synchrony is required for complex tasks such as speech intelligibility, the outcome of this study will hopefully provide the basis for potential improvements in cochlear implant design and a better understanding of hearing deficits that originate at the IHC afferent synapse. This research will be done primarily in Argentina, at the Instituto de Investigaciones en Ingenier¿a Gen¿tica y Biolog¿a Molecular (INGEBI) in collaboration with Dr. Juan Goutman, with the companion grant being R01 DC006476, 01-01-2004 to 11-29-2013. PUBLIC HEALTH RELEVANCE: The inner hair cell ribbon synapse is the first synapse in the auditory pathway and is responsible for transmitting information about the acoustic environment to the brain. This study focuses on identifying cellular mechanisms that allow this synapse to perform this task continuously and with highest temporal precision. Facilitation of the synaptic signal and the involvement of calciumdynamics in this process are being studied in depth.

Project Summary The vestibular organs of the inner ear convey signals about head motions to the brain, resulting in motor reflexes that maintain gaze and balance as well as the perception of balance and orientation. Dysfunction of the vestibular system can therefore substantially affect the ability to lead our everyday lives. Peripheral vestibular dysfunctions, like benign paroxysmal positional vertigo (BPPV) and Meniere's disease, lead to disabling episodes of vertigo and other symptoms. To analyze the pathophysiology of such diseases, it is crucial to understand how head motion signals are processed in the vestibular peripheral organs. In the crista, the sensory organ of the semicircular canals, the sensory hair cells, respond to head rotations with a deflection of their hair bundles, activating hair cell receptor potentials. Type I hair cells are close to completely ensheathed by a postsynaptic calyx ending of the afferent vestibular nerve fiber, a unique feature of the vestibular periphery, and type II hair cells are contacted by fibers with the more conventional bouton endings. The innervation pattern of these hair cell types is quite complex, yet follows a specific morpho- physiological pattern, and results in afferent fibers with differences in their response properties, for example in their regularity of resting discharge, their response properties to external stimuli and efferent inputs. Here we investigate synaptic transmission at the highly specialized type I hair cell/calyx synapse with the aim to understand the mechanisms that underlie firing patterns of the calyx afferent fibers. We have developed a preparation of excised cristae from 2-4 week old rodents to perform electrophysiological recordings from type I hair cells and calyx afferents, for some questions simultaneously. Using confocal analysis, we also characterize the morphological features of calyx afferents and assess the localization of specific synaptic proteins using antibody labeling or live imaging with fluorescently coupled markers. In Aim 1, we characterize the relation of hair cell membrane potential and afferent firing rate. We have found that glutamate accumulation and spillover in the synaptic cleft induces slow membrane potential changes and subsequent modulation of the afferent firing rate. We investigate the contribution of release properties and glutamatergic synaptic transmission to shaping the postsynaptic response pattern. Aim 2 investigates whether a cholinergic feedback loop from the calyx to the type I hair cell exists that may modulate afferent transmission. Here we put forward a new concept, including a calyx to hair cell feedback loop that may explain some of the in vivo recorded response patterns of calyx afferent firing. In Summary, we investigate the cellular mechanisms underlying calyx afferent firing properties. These studies are designed to gain a better understanding of possible vestibular peripheral dysfunctions, a prerequisite for developing treatments for such impairments.

This proposal aims to test the hypothesis that type II afferents serve as cochlear nociceptors. Taking cues from the human complaint of hyperacusis after hearing loss, we will examine the structure and function of type II afferents in normal and post?trauma cochleas. The working hypothesis is that painful hyperacusis, noxacusis, includes hyperactivity of type II afferents, by analogy to hyperalgesia of somatic nociceptive C?fibers. Thus we will examine type II structure and function in normal and post?trauma cochleas of rats and mice. In parallel we will investigate the properties of surviving outer hair cells in post? trauma cochleas. Our methods include: ex vivo electrophysiology, light and electron microscopy, utilization of optogenetic and chemogenetic tools,and validation and quantification of mouse models in which type II specific bio?markers are expressed. A necessary first step is to extend our ex vivo experimental approach to older cochleas so that changes wrought by acoustic trauma can be compared to the normal condition. We will compare damaging sound, ototoxic antibiotics and genetically encoded biotoxins to produce experimentally tractable effects on tissue for ex vivo experiments. The properties and synaptic connections of type II afferents and outer hair cells will be examined in the excised cochlear tissue of these animals. We will continue to explore type II specific genetic mouse models. Genetically?encoded reporter proteins, voltage? and calcium?sensitive indicators, biotoxins, and opto? and chemo?genetic modulators have become highly informative tools in neurobiology generally and for the inner ear specifically. Our ongoing work has characterized one mouse line, tyrosine hydroxylase promoter driven Cre?recombinase expression. Three other candidate type II specific Cre lines will be validated and quantified. With such transgenic models it becomes possible to study innervation patterns by expression of fluorescent reporter proteins, and to activate, eliminate, or modulate type II activity for anatomical and physiological studies. Cre?dependent expression of genetically?modified G?protein?coupled receptors (DREADDS) will provide mice in which type II activity can be increased or decreased by injection of a novel synthetic ligand, depending on the specific construct. Varying combinations of systemic and round window drug delivery will be employed to increase the specificity of experimental manipulations. The over?arching goal of this program of experiments is to complete the description of type II afferents, a still?unresolved component of cochlear innervation. The working hypothesis is that these serve as cochlear nociceptors. If correct these are a likely neurobiological substrate for noxacusis (painful hyperacusis). By defining the basic cellular and molecular mechanisms of type II function and plasticity, future therapeutic targets can be identified to ameliorate or prevent noxacusis.