Ruth Anne Eatock - US grants

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The long-term goal of this project is to show how head movement stimuli are processed by the sensory cells (hair cells) of the mammalian utricle, a vestibular organ of the inner ear. The majority of vestibular disorders, which may lead to debilitating disequilibrium, result from pathology of the vestibular organ and dysfunction of the hair cells. The proposed research will investigate normal mechanisms of stimulus processing by hair cells of the rodent utricle. These mechanisms include 1) transduction by hair cells of mechanical stimuli into an ionic current (the transduction current) and 2) voltage-dependent processing; ionic currents through voltage-dependent channels that are modulated by the voltage change evoked by the transduction current. The transduction and voltage-dependent currents of single hair cells, and a new preparation, utricles cultured from mice in the first week after birth. Preliminary results from both preparations suggest that the voltage-dependent currents of two types of hair cells (type I and II) differ sufficiently to cause substantial differences in stimulus processing. Experiments proposed here will follow up on these observations by thoroughly characterizing the voltage-dependent currents and their effects on the hair cells' voltage response. Mature hair cells can be studied with the isolated cell preparation. The cultures, while restricted to the first postnatal week, offer the advantage that the hair cells have not been subject to enzymatic dissociation procedures. The cultures are also superior for experiments on transduction, which will measure individual hair cells' transduction currents in response to a moving probe, and for investigating how transduction and voltage-dependent currents vary with region in the utricular epithelium. These electrophysiological studies will be complemented by electron microscopic studies of the anatomy of cultured and acutely excise utricles. In addition to answering questions of basic sensory processing, the proposed experiments would establish the cultured utricles as a model preparation in which to examine vestibular organ function in normal and pathological conditions.

Hair cells of the inner ear transduce the mechanical energy of sound or head movements into the electrical and chemical signals that are the currency of the nervous system. These signals arise from currents flowing through ion channels of several classes: mechanically-gated ("transduction") channels, voltage-gated channels and ligand-gated channels. It is known that hair cell signals are transformed by modulatory processes within the hair cells, sometimes activated by input from other cells, but underlying mechanisms are poorly understood. In the best-known examples, calcium ions have been implicated as intermediaries (second messenger molecules) in the transformation. This application proposes to investigate the roles of other candidate second messengers: cyclic nucleotides and nitric oxide, in modulating hair cell signals. The sensory epithelium of the mammalian utricle, a vestibular organ, will serve as the test preparation. The cyclic nucleotide experiments are motivated by the discovery in hair cells of cyclic-nucleotide-gated (CNG) channels, which are opened by the binding of cyclic nucleotides. In photoreceptors and olfactory neurons, CNG channels are the target ion channels of the stimulus, i.e., the transduction channels. In hair cells this role is fulfilled by different, mechanically-gated channels. Therefore, it is proposed here that current through the CNG channels serves to modulate, rather than initiate, the hair cell signal. This will be tested by recording hair cell signals while simultaneously stimulating them: mechanically and activating their CNG channels. Nitric oxide (NO) has recently emerged as a modulatory substance within the nervous system and elsewhere. As a short-lived gas that permeates membranes, it has the unusual property that it can influence any targets within a certain volume around its site of generation. Such a messenger could have profound impact within the networks of inner ear sensory epithelia. Preliminary data show that NO can modulate certain voltage- gated channels within hair cells and thereby affect the receptor potential. This application proposes to investigate whether NO is produced within the sensory epithelium, by hair cells or other elements, and whether it modulates hair cell transduction. These experiments may expand our understanding of complex modulatory processes that are likely to occur within hair cell sensory epithelia and to contribute substantially to the normal function of the inner ear.

The long-term goal of this work is to understand how vestibular organs, which transduce head position and movement, function and develop. Good health depends on the normal function of these organs. Damage can lead to debilitating vertigo, dizziness and an inability to maintain steady gaze. The primary afferent neurons to vestibular organs vary in the sensitivity and time course of their responses to head movement stimuli. Some of the variation correlates with region within the sensory organ. In amniotes, a further source of variation is likely to be differences between two classes of sensory hair cell, type I and II. This application proposes to take three approaches to stimulus processing by mammalian vestibular organs, using the rodent utricle as a model. The first aim is to test whether there are regional and cell-type-specific differences in the properties of the hair cell's mechanosensitive transducer conductance, which converts head movement stimuli into the receptor potential. Second, the hair cells' voltage-gated potassium conductances, which shape the receptor potential, will be characterized at the molecular level by applying probes directed at candidate proteins and messenger RNA. These conductances differ substantially between type I and II hair cells. The third aim is to characterize the normal development of hair cells from the period of peak terminal mitoses (prenatal) to birth of the animal. At birth, mouse utricular hair cells express some voltage-gated conductances and ultrastructural analysis shows that although the utricle is immature in many ways, some cells can be recognized as type I or II. The prenatal time course of acquisition of voltage-gated conductances will be determined with whole-cell recording. The expression of voltage-gated potassium channel proteins will be followed in time with molecular probes. Prenatal morphological differentiation of the utricle will be characterized. These experiments should provide insight into early differentiation of hair cells and supporting cells, as well as determine the utility of potassium channel proteins as markers of hair cell differentiation.

DESCRIPTION (provided by applicant): The seventh biennial Gordon Research Conference on Gravitational Effects on Living Systems will be held July 1-6, 2001 at Connecticut College. The theme of the meeting is mechanosensing, an ancient sensory capability. Approximately 100 participants are expected, including 23-25 speakers and 13 discussion leaders. The participation of trainees will be actively sought. While gravity affects living systems in many ways, mechanosensing is an appropriate focus for the meeting. Gravity is detected by mechanosen-sors, but most importantly for the meeting's success, this is an exciting and critical time for research on mechanosensors. The crystal structure of a prokaryotic mechanosensitive channel has recently been elucidated. Behavioral screens of eukaryotic genetic model animals, the yeast, roundworm and fruitfly, have yielded different mechanosensory cascades, including two putative classes of mechanosensory ion channels. Strong functional parallels have recently been shown between the mechanosensory chordotonal organs of flies and the sensory hair cells of the vertebrate inner ear. Intense genetic scrutiny of the mammalian inner ear has produced several pivotal successes in identifying genetic mutations underlying inner ear pathology in mice and humans. In addition to such work on specialized mechanosensors, there have been major recent advances in understanding the responses of nonmechanosensory tissues (muscle, bone, shoots and roots of plants) to mechanical input. The proposed meeting will bring together scientists who don't normally have a chance to meet either because they study different kinds of organisms (microorganisms, plants or animals), or different kinds of mechanical responses (mechanically evoked cascades in specialized mechanosensors vs. tissues that re- model in response to mechanical inputs, including gravity), or because they take widely different approaches (physiology vs. genetics). Moreover, the Gordon Conference format facilitates informal communications and discussions of controversial issues. These factors will promote inter-disciplinary transfer of ideas and approaches and the generation of new strategies for the study of mechanotransduction.

DESCRIPTION: (provided by applicant) The sensory hair cells in vestibular organs provide information about head movements to reflexes that control eye, head and body position. Damage to these cells as a result of age, disease or trauma leads to impaired mobility and quality of life. This proposal focuses on the contributions of various classes of ion channel protein to the normal function of hair cells in mammalian vestibular organs. Each of three specific aims is directed at a different stage in stimulus processing: mechanoelectrical transduction, shaping of the receptor potentia1 by voltage-gated ion channels, and afferent transmission. Results will be examined for variation with hair cell type (I vs. II), hair cell location within the sensory epithelium, and developmental stage. Mechanoelectrical transduction is the process by which hair bundle deflection gates mechanosensitive ion channels, producing a transduction current. It has been measured under in vitro conditions that are likely to have changed its properties. One series of experiments will make recording conditions more physiological. The influence of hair bundle morphology on transduction and bundle stiffness is poorly understood and will be examined. The transduction current initiates a voltage change (receptor potential), which activates voltage-gated ion channels. Potassium (K+) channels help set resting potential and provide negative feedback on receptor potentials. The complement of K+ channels in a hair cell varies with cell type and location in the sensory epithelium. K+ channel subunits expressed by single hair cells will be identified by expression profiling: RNA amplified from cDNA from single cells is used to probe a panel of candidate cDNAs. Candidate subunits will be followed up with immunocytochemistry to localize the protein. The voltage-gated sodium conductance (gNa) in mammalian vestibular hair cells inactivates at very negative potentials, raising questions about its function. Mechanisrns controlling the inactivahon range, the effect of gNa on the receptor potential, and the identity of the gNa protein will be investigated. Afferent neurons make large cup-shaped (calyx) endings on type I hair cells, in contrast to the small (bouton) endings they form on other hair cells. Afferent transmission from type I hair cells is therefore likely to have unusual properties. One set of experiments will describe synaptic vesicle exocytosis: Is it different in type I hair cells relative to type II hair cells? Do its properties in mammalian vestibular hair cells differ from those in cochlear hair cells? Another set will test whether the type-I-specific conductance, gK,L, is inhibited by the afferent transmitter present in the synaptic cleft. Such inhibition would produce strong positive feedback on afferent transmission. Ion channels in the postsynaptic calyx ending, which initiate the afferent response to the hair cell transmitter(s), will be characterized.

DESCRIPTION (provided by applicant): Vestibular afferent nerve fibers convey head position and motion signals from the inner ear to the brain, where they drive reflexes controlling gaze, posture and balance and contribute to perceptions of orientation and self-motion. We propose to study how electrical signals are generated in primary vestibular afferents, focusing on the contributions of intrinsic ion channels and calcium binding proteins. We will examine the origins of specific patterns of activity (firing patterns of action potentials, also called spikes) in vestibular afferents. In recordings made in vivo, vestibular afferents vary greatly in the regularity of inter-spike intervals;this variation is highly systematic, co-varying with many morphological and physiological properties. Thus, highly regular afferents tend to carry tonic signals, to have small diameters and extended dendritic arbors, and to innervate peripheral zones of the sensory epithelia. In contrast, highly irregular afferents tend to have phasic signals, to have large diameters and compact dendritic arbors with large, calyceal afferent terminals, to innervate central zones and to express particular Ca2+ binding proteins (CBPs). High regularity may enhance the information content and temporal encoding for head movement frequencies below ~20 Hz;high irregularity may reflect high sensitivity to synaptic currents. Over two decades ago, investigators exploring possible factors in setting firing patterns suggested that intrinsic ion channels in the afferent fibers were likely to be important. We now know that ion channel complements in vestibular ganglion neurons (VGNs), as in many brain neurons, are much more complex than previously imagined, and we have better tools to study this issue. We propose to use the whole-cell patch clamp method on VGNs from rodents to characterize firing patterns and the underlying voltage- and calcium-gated ion channels. VGNs will be studied in vitro as isolated somata and also within a semi-intact preparation that includes sensory epithelia and the ganglion. The two preparations have different advantages: isolated VGNs provide superior voltage clamp, while VGNs in the semi-intact preparation receive synaptic input from hair cells. Preliminary data suggest that VGNs express specific complements of ion channels, giving rise to distinct endogenous firing patterns in response to injected currents. We will test the hypothesis that the endogenous ion channels help establish different patterns of spike regularity in vivo, and the involvement of specific ion channels in setting firing patterns. We will use RT-PCR to screen for candidate ion channels and immunocytochemistry to localize channels according to afferent type, zone within the sensory epithelium, and cellular location (soma vs. afferent terminal). We will investigate whether specific CBPs help set firing patterns by modulating calcium-dependent potassium channels. We will develop computational models to test our comprehensive understanding of how ion channels contribute to firing patterns.

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.

DESCRIPTION (provided by applicant): About 35% of Americans experience balance problems such as vertigo, lack of coordinated movements, and dizziness. Balance problems can severely reduce quality of life, add to high healthcare costs, and lead to premature death due to falls. A major cause of balance disorders is the loss of vestibular hair cells in the inner ear, which convert head movements into electrical signals that are sent to the brain via the vestibular nerve. Hair cell death has many causes, including gene mutation, injury, infection, therapeutic surgery and drug treatments, and aging. The proposed project brings together the expertise of two investigators who share the following goals: to understand the fundamental mechanisms underlying vestibular hair cell function and to develop ways to treat balance disorders resulting from hair cell injury or loss. The Stone lab at the University of Washington (UW) recently discovered that one type of vestibular hair cell - type II - in adult rodents has a feature that has never been described: one or more extensions of cytoplasm (or processes) that project laterally from the base of the cell, sometimes over several cell lengths. These processes have a variety of shapes and seem to contact other cells in the epithelium, including other type II hair cells. This latter observation raises the novel idea that direct communication between type II hair cells could modulate vestibular signaling. In addition, the Stone lab found that type II-lie hair cells with processes are the only hair cell type that is regenerated spontaneously in adult mouse vestibular epithelia after damage (Golub et al., 2012). In order to develop treatments for individuals with balance disorders, it is critical to define both the identity of spontaneously regenerated hair cells and the role that they play in vestibular processing. For this project, the Stone lab at UW and the Eatock lab at Harvard University propose to characterize the hair cell processes, to identify the hair cell type that bears them, and to begin to examine how these processes affect the coding of head movements and the transmission of sensory information from the inner ear to the brain. For both normal and regenerated states, the Stone lab will analyze morphological and molecular properties of hair cells with processes, while the Eatock lab will examine their physiological properties and define the types of connections that the processes make with nerves and other hair cells. Proposed studies in normal vestibular organs are essential steps in defining the structure of the novel processes and their functions in vestibular processing. Confirmation of hair cell-hair cell communication would transform our understanding of hair cell biology by allowing the possibility of lateral interactions as documented in the retina. Studies of damaged vestibular organs will help uncover the relationship between normal and regenerated hair cells with processes and the potential of regenerated hair cells to restore function in balance and hearing disorders.

DESCRIPTION (provided by applicant): The Auditory System Gordon Research Conference (Auditory GRC) will convene ~125-150 scientists of diverse expertise at Bates College on July 13-18, 2014, to discuss and debate new findings in hearing research and related fields, under the theme of Encoding hearing: From Genes to Behavior. The Auditory GRC is preceded by a smaller Gordon Research Seminar (Auditory GRS, July 12-13) comprising talks and posters by 40-50 investigators-in-training and serving as a gateway to the GRC. Together the Auditory GRC & GRS offer a unique combination of features, including: breadth of research; cutting-edge emphasis; mingling of investigators from all ranks and diverse sub-fields and locales; and intimate size and extended discussion time, allowing for close and sustained interactions. Some of these features arise from unique qualities of the hearing research field and others from the Gordon Conferences' established nature. Since the 1930s, the GRC organization has supported meetings that are famous for their unfettered debate and discussion of emerging results. The program of the 2014 Auditory GRC emphasizes the diverse ways in which auditory and related information is encoded, broadly interpreted so as to include new results in areas from genetics to central nervous system physiology and behavior. Topics include genetic regulation of inner ear epithelia, hair bundles, and birdsong; hair cell transduction and synaptic transmission; noise trauma at cochlear and systems levels; and neural encoding of sound location, speech, and auditory scenes. We have learned from experience to spread different model systems and technologies throughout the sessions, so that each session has something for everyone - encouraging unusually high attendance throughout the meeting and cross-fertilization across disciplines. The clinical importance of auditory research will be highlighted with presentations that consider the mechanisms underlying human disorders (e.g. noise trauma and aging) and translational efforts to fix them (e.g., hair cell regeneration). For the Auditory GRS, talks are selected from registrants' submitted abstracts and anchored by a keynote address by an invited mentor-participant who is an assistant professor. Mentoring is an active component of the GRS - in addition to the keynote speaker, the GRC organizers (more senior investigators) are available for discussion, and a mentoring session featuring a representative from NIDCD and other grant agencies is planned.

PROJECT SUMMARY/ABSTRACT Vestibular disorders affect as many as 35% of adults past age 40. Studies of the vestibular inner ear have yielded important insights into how we process and compensate for head motion including the existence of parallel channels of information in the afferent nerve. In macular organs, for example, two populations of hair cells adopt opposite planar orientations of their hair bundles and thus opposite responses to head movements. This highly conserved bidirectional organization was first described in neuromasts, the lateral line organs sensing water movements in fish, but the genetic program implementing this reversal during development is only starting to be deciphered. Consequently, ablation studies to reveal the importance of reversal for vestibular function have not been possible until recently. Here we propose to address this question by investigating the consequences of inactivating an orphan G protein coupled receptor (GPCRx), implicated by our preliminary data in orientation reversal in mouse hair cell epithelia. Based on our preliminary data, we suggest that mouse GPCRx functions downstream of the transcription factor EMX2 and upstream of the heterotrimeric G protein G?i to reverse a ground state of polarity established by planar cell polarity proteins. We will test this hypothesis and also use the GPCRx mutant as an animal model to pinpoint how polarity reversal shapes macular organ responses and downstream effects on vestibular behaviors. To reach these goals, we will: 1) Use genetics to determine how GPCRx instructs reversal at the molecular level, solving its epistatic relationship to EMX2, G?i and planar cell polarity proteins in mice, and use zebrafish to test whether GPCRx-G?i is a conserved effector pathway for reversal. 2) Use molecular markers, electrophysiology and calcium imaging to resolve hair cell maturation and function in absence of polarity reversal. 3) Determine how polarity reversal affects afferents' organization and function, with afferent recordings, as well as overall vestibular function using behavioral tests. Our coherent body of preliminary evidence ensures the feasibility and the high interest of the project, and our focus on a virtually unstudied receptor protein guarantees innovation. The multi-PI team is ideally suited to address complementary questions in both the mouse and zebrafish acoustico-lateralis systems. We anticipate that this collaborative effort will be decisive towards solving the mechanism of hair cell orientation reversal, its conservation across vertebrates and its significance for mammalian vestibular physiology. Thorough understanding of polarity reversal will help interpret and design treatments for vestibular dysfunctions.