Ruth Anne Eatock - US grants

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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.

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): 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.

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.

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.