Alfred L. Nuttall - US grants

DESCRIPTION (provided by applicant): A goal of the cochlear physiology laboratory is to understand how sound induced vibration of the tissues in the inner ear results in loss of sensory function and cell pathology. A number of important questions are captured within the scope of the two Aims. Broadly stated they are 1) to learn the mechanisms of the pathophysiology of the sound stimulated lateral wall and 2) to find out how the metabolic load and hypoxia induced by loud sound impacts mitochondria morphology and the redox state of organ of Corti hair cells. Under the first point there are three specific questions about lateral blood flow and stress. We will determine whether there is a local cochlear flow that correlates with the sound tonotopic stimulation and shows the real time metabolic and vascular state of the lateral wall. That metabolic state, we believe, is a functional hypoxia that induces breach of the vascular blood/labyrinth barrier. We study the hypoxia initiated signaling pathway to alter adherens junction proteins of the endothelial cells. We then determine, using molecular biological methods, if hypoxia also initiates lateral wall fibrocyte inflammation. The mutant mouse model (Chd23 mutant or salsa) is employed as a way of inducing altered transduction current, which is a drive for lateral wall metabolism and, possibly, capillary reactivity. Standard techniques are clearly insufficient for answering these questions, so new and innovative methodology will be used. We developed the Optical Micro- Angiography method for the study of lateral wall cochlear blood flow. We also adopt super-resolution imaging of the adherens junction fluorescent labeled proteins to determine how altered junction and cytoskeleton open the trans-cellular permeability gap. The cochlear outer hair cells are subjects of the second question where, in separate experiments using salsa mice and pharmacologic agents, we determine the sub- cellular sources of reactive oxygen species in outer hair cells? We then investigate, using the salsa mouse, if the physical forces of sound (apart from metabolic stress) can produce oxidative stress. Finally, since stress and metabolism directly influence mitochondria population, we study whether OHC mitochondria can dynamically adapt their number under sound stimulation. A GPF- mitochondria transgenic mouse will be used to study the sound altered population of mitochondria in the process called biogenesis.

The objective of this study is to understand the normal and abnormal physiology of the sensory cells and neuronal systems in the mammalian cochlea. The sensory inner and outer hair cells of the organ of Corti are responsible for receiving acoustic information and transducing the basilar membrane mechanical motions into a neural code. Knowledge of how cells functions is essential to other studies which attempt to assess and prevent hair cell loss; the most common denomination of deafness. Hair cell function, in this study, will be measured using intracellular recording techniques. The ac and dc receptor potentials, resting membrane potentials, and sounde-voked cell resistance changes will be recorded for both outer and inner hair cells. A variety of acouostic stimuli will be used to stimulate the cells including pure tones, pure tones in acoustic noise, and two-town combinations. The performance of the hair cells will be characterized for the changes induced by sound and electrical stimulation of the efferent nervous system to the cochlea. These efferent stimulation experiments should help reveal the true purpose of this neural component to organ of Corti. The endocochlear potential will also be manipulated through the administration of the diuretic Furosemide and with direct current, in order to determine if it is required for the high sensitivity and frequency selectivity of hair cells. The sound varying input resistance of inner hair cells also will be measured in order to determine if the extracellular potentials of the cochlea have a role in producing the low-frequency responses of auditory afferent nerve fibers. Recorded hair cells will be positively identified by iontophoretic injection of horseradish peroxidase and subsequent histochemistry and light microscopic inspection of the organ of Corti surface preparations.

The sensory cells of the inner ear are called hair cells, and they are responsible for hearing and balance. With modern tools, there has been a new sophistication in analyzing how these receptors work at the cellular level. This conference will bring together biophysicists, biochemists, pharmacologists, physiol- ogists and anatomists to focus on three major topics: hair cell motility, information processing, and intercellular connections among hair cells and nerve cells. The emphasis will be on reaching some consensus on these three topics, since they have recently emerged as leading issues, as the technology to address them has advanced in the last decade. The impact of this meeting is likely to be a new formulation of the role of interactions between the peripheral sensory system and the central nervous system, and will be important to understanding sensory processing in general, as well as understanding auditory and vestibular function in particular.

Sound stimulation of the cochlea leads to mechanoelectric activity in the organ of Corti. Outer hair cells play a central but little understood role in the normal cochlea response to sound. Furthermore, outer hair cell (OHC) activity is controlled in an almost completely unknown way by a complex innervation from the olivocochlear efferent fibers. This proposal seeks to add to our understanding of organ of Corti function by examining its mechanoelectric responses to sound and to efferent nerve electric stimulation. Organ of Corti responses will be measured as gross cochlear potentials, ear canal otoacoustic responses, receptor potentials of hair cells, and most importantly as velocity and displacement responses measured from various locations in the or an of Corti. Each of these measurements provides a different viewpoint on the performance of inner and OHCs acting together in vivo. This proposal applies a dramatic new technology for the study of cellular vibration in the inner ear. Laser feedback interferometry (LFI) is a method with sufficient sensitivity to register the vibration of nearly transparent cellular elements. LFI applied through a microscope allows one to focus a laser beam onto the different cellular structures in the organ of Corti. A detailed study of the traveling wave will provide an important empirical base for theoretical studies of organ of Corti function by providing information on mechanical displacements of its cellular structures. This understanding is necessary in order to determine how mechanical energy stimulates the inner and OHCs. LFI microscopy can provide the cellular displacement measurements needed to determine how OHCs serve as motile elements in this system. The proposal will also study the physiology of the olivocochlear efferent system and determine whether activation of the efferent nerves changes the mechanical properties of OHCs. The physiology of the descending efferent system from the inferior colliculus is included as a way to cause activation of both the medial and the lateral efferent systems in a topographic manned Finally, with this proposal we are beginning the study of the physiology of the supporting cells of the organ of Corti. Until recently supporting cells were thought to be largely passive elements only lending structural support to the system.There is mounting evidence now that Deiters' cells are motile and dynamic structures. We will measure the changes in organ of Corti vibration following types of stimulation that lead to changes in Deiters' cell morphology. Taken together the studies of this proposal will provide an understanding of: how OHCs generate high frequency selectivity and sensitivity in the normal inner ear; the function of the olivocochlear efferent system and the structural dynamics of supporting cells in the organ of Corti.

DESCRIPTION: (Adapted from the Applicants' Abstract.) This proposal seeks to add to our knowledge concerning organ of Corti function by examining its mechanoelectric responses to sound using four distinct, but linked measurement approaches; gross cochlear potentials, ear canal otoacoustic responses, intracellular hair cell receptor potentials, and most importantly, basilar membrane motion. Each of these provides a different viewpoint on the performance of inner and outer hair cells. The detailed motion pattern of the basilar membrane will be studied first, using a new method for this purpose. A new and detailed view of the traveling wave pattern will provide an important empirical base for future theoretical models of the organ of Corti function and establish a point of experimental departure to measure olivocochlear efferent activity. Natural, sound evoked, activation of efferents as well as electrical activation will be studied. Ear canal otoacoustic changes during efferent activation will be directly compared to basilar membrane motion changes. This comparison will reveal some mechanical aspects of neurally controlled outer hair cell performance. The role of the efferent system to protect OHCs from the damaging effects of loud sound will be studied, as will how efferent activity can improve our ability to detect and code sounds in the presence of masking noises. The investigators will also study the physiology of the higher brain stem levels of the descending efferent pathways. Electric current stimulation of areas of the inferior colliculus will be used to activate this system, leading not only to new information on the descending pathways proper but perhaps also on the unknown physiology of the "lateral" efferent fibers which terminate on the peripheral processes of afferent neurons.

The migraine related inner ear symptoms for phonopobia , tinnitus, hearing fluctuation, hearing loss, and increased noise sensitivity provide evidence for a possible neurological substrate connecting basilar artery migraine and cochlear pathophysiological mechanisms. Recently we have identified a previously unreported sensory innervation of the cochlear blood vessels originating from the trigeminal ganglia. We have shown that this sensory innervation has a significant effect on cochlear blood flow (CBF) in both normal and pathological conditions (e.g., in the animal model of endolymphatic hydrops, one of the symptoms of Meniere's disease). This proposal seeks to further define the anatomical basis and mechanisms of the trigemino-sensory network around the vertebrovasilar and cochlear vascular system. The proposal offers the hypothesis that the trigemino-sensory system and its related neuropeptide system are important factors contributing to basilar migraine and vascular homeostasis of the cochlea. The study has three specific aims. Aim 1. To establish if there is a physiological basis for the cochlear symptoms in basilar artery migraine headache. Positive results will confirm a common functional basis for basilar migraine and cochlear symptoms, the basis could be neurogenic inflammation. Aim 2. To demonstrate if vanilloid receptor (VR1) and substance P (SP) are co-localized around cochlear blood vessels, the basilar artery and its related branches. Positive immunocytochemical results will demonstrate: (a) network of the VR1 and (b) SP co-labeled primary sensory neurons around the basilar artery; anterior inferior cerebellar artery (AICA), spiral modiolar artery (SMA) and radial artery; (c), Capsaicin will cause a significant reduction in the density of labeled sensory fibers. Aim 3. To determine the vasoregulatory disturbance of the trigemino-sensory neurons in endolymphatic hydrops. In this study positive results will demonstrate that endolymphatic hydrops causes a reduction in the stimulated trigeminal ganglion induced CBF change. The studies of the proposal will help clarify how trigemino-sensory neurons regulate the vertebro-basilar vascular system and cochlear fluid balance under normal and pathological conditions.

The Oregon Hearing Core (OHCC) Bioengineering Core (Core 1) will support six of the OHCC users and four collaborating investigators. The purpose of the Bioengineering core is to provide engineering and software expertise and to provide electrophysiological characterization of certain animals needed in projects of the users. In engineer will design and build custom electronic and mechanical devices that solve experimental and data acquisition problems. A computer programmer will write computer software (e.g. a Lab View virtual instrument) to address the experimental needs of the users. Projects undertaken by engineer and programmer will extend the funded work of the core users and stimulate collaborative studies between users. The programmer will help create common data bases that would allow sharing of experimental data among users. In addition, an electrophysiology technician will be available to measure (in mice, guinea pigs or gerbils) auditory brainstem responses, cochlear compound action potentials, distortion product otoacoustic emissions, and endocochlear potentials. An important activity of the technician will be to characterize the functional status of the cochlea in mutant mice produced by the OHCC Mouse Molecular Genetics Core. As for the engineer and programmer, the technician will work with Core users to extend work on supported R01s and enhance collaborative studies.

DESCRIPTION (provided by applicant): The major goal in this proposal is to develop an instrument to measure the vibration of the organ of Corti in the living guinea pig. The instrument will use the principles of optical coherence tomography (OCT) and low optical coherence interferometry to visualize the basilar membrane and the internal structures of the organ and to measure their vibratory displacements induced by sound stimulation of the ear, respectively. The OCT interferometer will utilize light from two different super luminescent diodes coupled into a common optical fiber, allowing simultaneous measurement of the vibratory displacement of the basilar membrane and the reticular lamina components of the organ of Corti. The first two aims of the proposal are 1) to develop a dual source (two channels) OCT interferometer for measurements at a single location in the organ and 2) to develop a scanning OCT interferometer to measure organ of Corti motion in three dimensions. OCT interferometry will have the advantage of not requiring any reflective objects (such as mirrors or beads) to be placed on the ear tissues and will have high optical spatial resolution (particularly in the 'z' direction along the optical axis) allowing accurate magnitude and phase measurements of organ of Corti tissue vibration. The third aim is to use the OCT instrument to measure the three-dimensional vibration pattern of the basilar membrane and reticular lamina in the guinea pig. The data will give critical new insight into the micro mechanical motion of the organ of Corti and on the mechanism of the cochlear amplification process whereby outer hair cells are thought to be actively enhancing organ of Corti motion.

DESCRIPTION (provided by applicant): The Ninth Mechanics of Hearing Workshop - Auditory Mechanisms: Processes and Models - The Benson Hotel, Portland Oregon, July 23-28, 2005 - This proposal requests support for a workshop to bring together active senior experimentalists and theoreticians working in hearing mechanics in diverse species, along with students and junior investigators entering this field of research. The purpose of the workshop is to discuss, at length and in depth, the most recent theoretical and experimental work in the field, together with the currently important issues and controversies. One primary goal of the workshop is to advance the understanding of the peripheral mechanisms of hearing. An increased understanding of auditory mechanics will aid in our understanding of, and future treatment of hearing impairment. The primary objective is to promote strong interaction and connections between theoretical and experimental scientists. To ensure involvement of all attendees in each interest area, the number of orally presented papers will be limited to approximately 50 to avoid parallel sessions. This number will allow ample representation of different opinions while still being conducive to free discussion. Poster sessions will permit individuals who are not selected for oral presentations to present their data and ideas. Written copies of the papers will be circulated to registrants of the meeting a month before the meeting and the proceedings of the meeting will be published immediately afterward. This rapid publication time scale will be accomplished by requiring authors to submit manuscripts electronically at the time of registration or at the meeting itself. The secondary goal is to introduce mechanics-of-hearing scientists to the idea and feasibility of science outreach. To this end, the workshop will have a scientific outreach component where one of the plenary speakers will provide a lecture for high school students. In addition, attendees will receive information and experience in how to be proactive in the area of public education for hearing science.

[unreadable] DESCRIPTION (provided by applicant): This proposal is concerned with the mechanical and electrical properties of cells in the organ of Corti and how those properties influence the ability of the organ to process sounds. In particular, the experiments are designed to determine how the outer hair cells (OHCs) function in vivo to control the vibration of the basilar membrane and cellular structure of the organ. It is thought that OHCs use their inherent motile ability to amplify sound-evoked vibration of the neurosensory structures in the cochlea. This process is called cochlear amplification. However, it is not known how OHCs accomplish this amplification, a property critical to normal hearing because it provides both for the great sensitivity of the ear and for the acuity needed to understand speech in a noisy environment. The amplifier mechanism must work within a system of mechanical and electrical filters. In Aim 1 we test the hypothesis that the stereocillia of OHCs act as a critical mechanical filter that has not yet been studied. The amplifier must also generate power and it is clear OHCs have cellular or subcellular motor activity that may provide the power. In Aim 2 we will determine whether cochlear amplification requires forces produced by the stereocilia (via so called fast adaptation) of OHCs or prestin motor molecules in the baso-lateral membrane of the OHC or both. The manipulation of endolymphatic calcium is key to the Aim 1 and 2 experiments. Finally, cochlear amplification can only come about with the proper (but as yet unknown) micromechanical motions of the system of cells that make up the organ of Corti. In Aim 3 we study the micro or cellular mechanics of the organ of Corti (in yivo) using custom designed measurement instrumentation based on low coherence optical interferometry. We investigate the mechanical processing of sound that occurs with activity of the OHCs and the resulting stimulation of the stereocilia of inner hair cells. Knowledge of OHCs and organ of Corti mechanics is essential to our understanding of the normal and pathophysiologial function of the cochlea. Data derived in these studies are critically needed for appropriate mathematical modeling of the inner ear and in turn such models provide the only way to completely understand its complex mechanics. [unreadable] [unreadable] [unreadable]

The Bioengineering Core will support all P30 investigators and collaborating investigators. The purpose of this Core is to provide engineering and software expertise needed in the projects of the users and to maintain the OHRC sound-exposure and auditory-brainstem-response (ABR) facilities. The P30 engineer will design and build custom electronic and mechanical devices that solve experimental and data acquisition problems. The P30 computer programmer will write computer software (e.g., a Lab- View virtual instruments) to address the experimental needs of the users. Projects undertaken by the engineer and programmer will extend the funded work of the Core users and stimulate collaborative studies between users. The P30 staff will help create common databases that would allow sharing of experimental data among users and enable more secure data backup. Core staff also advise faculty on specification and purchase of instrumentation and software.

DESCRIPTION (provided by applicant): Low optical coherence tomography (OCT) has been used to image biological tissue and is the theoretical basis of microscopes that are commercially available to image the lens and cellular structures of the human eye. Interferometers based on OCT have not been produced but have unique properties useful for vibration measurements of the tissues and cells of the inner ear. We propose to develop an OCT interferometer that has the ability to both image the living organ of Corti and measure its cellular motion in 3-dimensions down to a vibration as small as 0.1 nm. The basic concept of OCT interferometry has already proven usefulness for the micromechanics of the organ of Corti. This proposal implements technical advances that permit needed higher resolution that enables determination of the direction and phase of the organ displacement vector at the cellular level. The instrument will have an imaging and vibration resolution of about 3 cubic micrometers through the use of a femtosecond pulsed laser. This is accomplished by incorporation of a novel phase-sensitive OCT approach allowing the instrument to be used to test the hypothesis that the tectorial membrane is mechanically resonant in the lateral (radial) direction. Knowledge of the in vivo mechanics of the tectorial membrane, including resonance, will set to rest a quarter century of conjecture on how the organ achieves the efficient mechanical stimulation of the inner ear hair cell stereocilia and the subsequent remarkable sensitivity of mammalian hearing. PUBLIC HEALTH RELEVANCE: The discoveries of cochlear mechanics that this optical coherence tomography (OCT) instrument will allow are critical to the understanding of normal hearing, the mechanisms of the otoacoustic emissions that are used as clinical audiometric tests and the defects in hearing caused by loud sound and by deafness genes. The OCT method we will develop also has other applications for human health in Otolaryngology: OCT could image the vibration of the middle ear structures as an audiometric method, measure blood flow in the human inner ear to classify which patients with sudden deafness have deficient flow or measure and map blood flow in a microvascular skin flap to improve the viability of flaps.

? DESCRIPTION (provided by applicant): A goal of the cochlear physiology laboratory is to understand how the components of the organ of Corti enhance the sound induced vibration of the basilar membrane, a process known as cochlear amplification (CA). Two questions of broad interest are to be studied; how do the outer hair cells transmit force to activate the CA and what is the mechanical role of the tectorial membrane in CA? Standard techniques are clearly insufficient for answering these questions, so new and innovative methodology will be used. The Fourier Domain Optical (Low) Coherence Tomography (FDOCT) system that we developed gives unique possibilities for studying CA. Using this method; we propose to determine how the complex motion of the organ of the Corti, powered by OHC forces, results in mechanical stimulation of the inner hair cell stereocilia. This is the critical first step in hearing, the stiulation of inner hair cells. We also propose to determine whether the tectorial membrane has a mechanical resonance relevant to organ of Corti function. This is fundamental to understanding how the displacement of the stereocilia hair bundles on outer hair cells can have the proper timing to make effective any force that they actively produce. The distribution of outer hair cell activity is fundamental to knowing how the traveling wave propagates. The spatial distribution of the CA is studied with FDOCT and mathematical modeling.

Project Summary A goal of the cochlear physiology laboratory is to understand how the components of the organ of Corti tune the sound induced vibration of the organ of Corti. A process known as cochlear amplification (CA), now the subject of intense work around the world, has critical components not yet studied. Two questions of broad interest that this proposal address are; 1) how does the fundamental hydrodynamic viscosity contribute to the unique frequency analysis capacity of the cochlear apex where speech frequencies are processed and 2) does the tectorial membrane in have a central and biochemical role in regulating the calcium ion concentration that is so critical to hair cell function. There are three Aims. New and innovative experimental approaches are needed to address these questions. For the measurement of output variables, we continue to use the optical coherence tomography (OCT) method, that we pioneered, to record inner ear tissue vibration. We use state of the art confocal imaging methods applied to whole organ explant systems and measure calcium ion concentrations in quiescent and stimulated inner ears. In Aim 1, about question 1, we also propose to determine if perilymph macroscopic viscosity is a crucial parameter of apical frequency tuning. As well as whether the tuning is dependent upon the process of cochlear amplification within the traveling wave as it propagates to the apex. To manipulate viscosity, normal perilymph is replaced with altered viscosity perilymph via a real time perfusion system. Aims 2 and 3 are about question 2 where we seek to understand how and with what consequence is calcium stored by the tectorial membrane. Involved is the use of mutant mural models of defective tectorial membrane structural proteins and quantitative fluorescent determination of calcium concentrations in endolymph and tectorial membrane. Additionally, in Aim 3, we explore how age might factor into the tectorial membrane calcium sequestration via two models that manipulate the physiology of the stria vascularis a known target of age degeneration. Model 1 is the chronic application of furosemide, an agent to suppress endocochlear potential. Model 2 is the genetically targeted chemical alteration of stria vascularis blood flow). Taken together the work will significantly advance not only fundamental knowledge of organ of Corti function but open a path to pharmacological interventions to treat tectorial membrane calcium pathology.