M. Charles Liberman - US grants

The studies can be divided into two main areas: 1) those intended to elucidate the ways in which the normal patterns of neural activity in the peripheral auditory system are generated, and 2) those intended to understand the mechanisms by which noise exposure can cause temporary and/or permanent hearing deficits. Experiments in both these areas focus on the correlation between structure of the peripheral auditory system (both at the light- and electron-microscopic levels) and the function of the system, as seen in the response of single neurons. The precision of these correlations is greatly enhanced by the application of single-neuron labeling techniques, which allow us to follow single, physiologically identified neurons from their origins to their terminations. The experiments on the normal auditory system are focused on the efferent system. In most vertebrate ears, the sensory cells and/or afferent nerve fibers receive an efferent innervation. This system presumably plays a feedback role in modifying the output of the afferent neurons according to the nature of the acoustic input. We know that electrical stimulation of this efferent system in mammals leads to an increase in the thresholds of afferent neurons. However, we know relatively little of the nature of the sound-evoked activity of single efferent neurons or their cochlear innervation patterns. This type of fundamental information will emerge from the proposed studies of intracellular recording and labeling of single efferent neurons in the cat. The resultant data should form the basis for more realistic speculation about the role of this system in the overall process of auditory perception. The studies on noise-damaged cochleas have two main aims. The first is to infer normal cochlear mechanisms by comparing the changes in auditory-nerve activity with the structural and ultrastructural alterations in the sensory cells which give rise to them. The application of intracellular labeling techniques allow these correlations to be made on the single-cell level. The second main aim is to describe the structural changes underlying permanent vs. temporary threshold shifts and, most importantly, to understand the nature of the acute changes which determine whether the effects of a given exposure will be reversible or irreversible. The combination of physiological and morphological techniques we have in hand put us in a unique position to address this question. If the dynamics of the repair process can be understood, it is possible that post-trauma treatment can be devised to improve the final outcome.

In this program, clinicians and basic scientists study structure and function in the auditory pathway in animals and humans. The projects target clinical and basic-science issues from the middle and inner ear, through brainstem to cortex, in normal and hearing impaired individuals. Engineering, physiological and clinical approaches are combined in Project 1 to study conductive hearing loss in humans. Using measurements in human temporal bones to test models of sound transmission, the efficacies of surgical techniques are predicted and predictions tested against clinical outcomes. Biological and engineering approaches are combined in Project 2 to investigate the role of cochlear supporting cells in maintaining ion balance in normal and high-level sound environments. If our hypotheses are correct, malfunction in this supporting-cell network may lead to cochlear fluid disorders, and understanding their normal function may suggest effective treatments. Another team combines pharmacology, physiology and molecular biology, in Project 3, to prove the molecular mechanisms underlying efferent protection of the inner ear from acoustic injury. Our hypotheses suggest a number of drugs which should enhance protection and also suggest a novel cell-signaling system of general importance to cell biology. The cochlear efferent pathway may also improve auditory performance in noisy environments. The functional role of this feedback pathway is investigated in Project 4, by assaying efferent-reflex strength in human subjects as the auditory task changes to resolve whether up-or down-regulation of this reflex occurs. In Project 5, neurophysiology, psychophysics and neuroanatomy combine to investigate the neural substrate for perceptual phenomena in spatial hearing, such as the improved detectability of masked signals as signal and noise sources are spatially separated. Insight into the underlying physiological mechanisms is important in understanding performance deficits in the hearing impaired, especially in noisy environments. Project 6 studies auditory processing in human subjects via functional magnetic resonance imaging (fMRI) of neuronal activity. Advances in fMRI enable resolution of localized activation throughout the auditory pathway from cochlear nucleus to cortex, allowing this team of basic scientists and clinicians to directly test fundamental and longstanding assumptions concerning the applicability of neurophysiological studies of animal models to human audition, in normal and hearing impaired individuals.

computer system hardware; image processing; biomedical equipment purchase;

DESCRIPTION: The mammalian ear is connected to the brain via four types of nerve fibers: two afferent types carry sensory input to the brain, while two efferent types carry feedback control from the brain. The overall goal of our research effort is to understand the role(s) of each fiber type in audition: our current understanding of these roles is rudimentary in many areas. The present application includes one aim directed at each of the four fiber types. 1) Type-I afferents are subdivided into spontaneous-rate (SR) groups, which differ in threshold sensitivity. We will use intracellular labeling to study the central projections of these fibers, testing the hypothesis that an SR-based spatial organization within the cochlear nucleus (CN) is superimposed on its frequency-based organization, thereby allowing CN cell-types (and the higher centers to which they project) to sample activity from different SR groups according to the nature of the decoding operations performed. 2) Response properties of type-11 afferents from outer hair cells (OHCS) have never been studied, both because their axons are small and because they are few in number. We will use a drug (carboplatin) to selectively eliminate the large type-I population and then apply electrode technology for recording from unmyelinated fibers. The resultant type-II recordings will allow a test of the hypothesis that type-11's mediate the sensation of auditory pain. 3) Olivocochlear (OC) efferent fibers to OHCs comprise a sound-evoked reflex, and chronic de-efferentation greatly increases vulnerability to permanent noise-induced hearing loss (NIHL). Interestingly, the strength of the OC reflex varies among normal individuals, and so does the vulnerability to NIHL. We will test the hypothesis that "tough" ears are those with the most active OC reflex and that "toughening" of the ear seen with long-term, moderate-level acoustic exposure arises via an amplification of the OC reflex. Since OC reflex strength can be measured non-invasively, a NIHL vulnerability screen for humans could be devised based on our findings. 4) The OC fibers to the inner hair cell area are unmyelinated, and their response properties and functional effects are completely unknown, largely because their axons are difficult to excite electrically. Our preliminary results suggest that this part of the OC pathway may be excitable via electric activation of the inferior colliculus. We will pursue this approach to elucidate the peripheral effects these OC efferents. Such information is fundamental to an understanding of the functional role of the entire OC system.

DESCRIPTION (provided by applicant): Three Research Cores are proposed to facilitate interdisciplinary research into hearing and deafness at the Massachusetts Eye and Ear Infirmary (MEEI). The Research Center comprises 19 NIDCD-funded principal investigators, all affiliated with the Eaton-Peabody Laboratory (EPL). They include clinicians and basic scientists, with academic ties to graduate programs and departments at Harvard Medical School and MIT. The Research Base covers a wide range of basic and applied research projects from peripheral mechanics to cortical processing, from in vitro systems to human patients, from animal models to neural nets. The EPL research group has a long history of fruitful collaboration based on sharing of equipment, resources and scientific expertise via a system of research cores supported by a sunsetting program project grant. The current proposal builds on the present core structure and personnel to maintain existing, and facilitate further, interdisciplinary research efforts into hearing and deafness. Each of the Cores will support highly experienced personnel to 1) staff, stock, maintain and upgrade existing shared research facilities; 2) train users and/or render expert technical services in these facilities; and 3) provide the necessary expertise to enhance research productivity and facilitate the fusion of different research approaches across the many disciplines represented in the Research Center. The three Cores and the major aims of each include 1) an Engineering Core to design, build and maintain data-acquisition systems, custom acoustical devices, stimulus generation systems, a distributed multi-platform system for computational infrastructure and the local area network to link it all together 2) a Surgery/Histology Core to maintain existing shared facilities and assist research teams in animal surgery and histological preparation for both light and electron microscopy; and 3) an Imaging Core to support the growing needs of Center investigators for digital image acquisition and analysis, including confocal microscopy, computer-aided anatomical reconstruction, automation of morphometry, 3-D reconstruction/rendering, and analysis of functional imaging data.

[unreadable] DESCRIPTION (provided by applicant): Three Research Cores are proposed to facilitate interdisciplinary research into hearing and deafness at the Massachusetts Eye and Ear Infirmary. The Research Center comprises 23 investigators, all affiliated with the Eaton-Peabody Laboratory (EPL). They include clinicians and basic scientists, with academic ties to graduate programs and departments at Harvard Medical School and MIT. The Research Base covers a wide range of basic and applied research projects from peripheral mechanics to cortical processing, from in vitro systems to human patients, from animal models to neural nets. The EPL research group has a long history of fruitful collaboration based on sharing of equipment, resources and scientific expertise via a system of research cores supported by a program project grant in the 80's and 90's, and by this P30 for the last 4 years. Each of the Cores supports highly experienced personnel to 1) staff, stock, maintain and upgrade existing shared research facilities; 2) train users and/or render expert technical services in these facilities; and 3) provide the necessary technical expertise to enhance research productivity and facilitate the fusion of different research approaches across the many disciplines represented in the Research Center. The three Cores and the major aims of each include 1) an Engineering Core to design, build, program and maintain data-acquisition systems, custom acoustical devices, and stimulus generation systems, and to provide a precision machining service to build custom mechanical devices for a wide variety of research applications; 2) an Imaging Core to support the needs of Center investigators for digital image-acquisition and analysis, including confocal microscopy, transmission electron microscopy, computer-aided anatomical reconstruction, automation of morphometry, 3-D reconstruction/rendering, and analysis of functional imaging data; and 3) a Histology/Surgery Core to maintain existing shared facilities and assist research teams in animal surgery and histological preparation for both light and electron microscopy; and [unreadable] [unreadable] Core Center Administration [unreadable] Program Director: Charles Liberman, Ph.D. [unreadable] [unreadable] [unreadable]

Administrative Structure Program Director, M. Charles Liberman, has 30 years of experience as an auditory neuroscientist, 25 years of continuous RO1 funding from the NIDCD, 8 years experience as PI of a PO1 grant entitled "Basic and Clinical Studies of the Auditory System" which supported six collaborative projects among investigators at the Center, and 5 years experience as PI of this P30 Research Center. He was PI on one Shared Instrumentation grant (and co-Pi on another) which funded the acquisition of much of the major equipment powering the Imaging Core. He has been Director of the Eaton-Peabody Laboratory for 8 years and has co-authored collaborative research papers with 12 of the 23 participating Investigators in this Research Center over the last 5 years (Core Progress Report Summary, p. 112). Thus, he is familiar with all the research projects underway. The Program Director will be responsible for interacting with all Center Investigators and Core technical personnel to facilitate smooth operation of each Core. There will continue to be regular meetings of each Core group with the Program Director, and relevant Core Pl(s) to discuss past progress, overall operations, as well as to set future goals and priorities. For the Engineering Core, these meetings will continue to held on a monthly basis. For the Imaging and Histology/Surgery Cores, meetings will be held quarterly. These Core meetings will be open to all participating Investigators. Additional informal meetings will occur on an as needed basis. This type of overall structure has worked well over the past five years, as documented in the individual Core Progress Reports. Experience has shown that meetings of the Engineering Core need to be held more often than for the other two Cores, because there are more Core staffers in Engineering, thus there are more projects underway simultaneously. All Core technical personnel in all three Cores are required to track their time and effort allocations in Excel spreadsheet format. These spreadsheets are reviewed on at least a quarterly basis by the Program Director and individual Core Pis, to assess which Center Investigators are receiving investigator-specific services, and how the time allocation for investigator-specific requests are balanced by time spent on Core-wide projects. An overview of the balance of activities over the past five years in each of the Cores is given in the relevant Core Progress Reports. The Program Director will be assisted in the day-to day operations and in integration of the three Cores by an administrator, Dianna Sands. She has over 15 years experience working with the Program Director and as well as most of the participating investigators. She will be responsible for interacting with MEEI research administration to track grant expenditures for consumables, to allocate salary expenditures among appropriate cost centers, to track incoming orders to make sure they are appropriately routed, and to arrange meetings as needed. Administrator Sands, in turn, will be aided by an office assistant, who is salary is paid by the MEEI research administration budget. As a team, Ms. Sands and her assistant are also responsible for many aspects of pre-award and post-award grant administration for all other grants held by Center Investigators.

DESCRIPTION: Three Research Cores are proposed to facilitate interdisciplinary research into hearing and deafness at the Massachusetts Eye and Ear Infirmary. The Research Center comprises 23 senior investigators and 9 junior investigators, all affiliated with the Eaton-Peabody Laboratory (EPL). They include clinicians and basic scientists, with academic ties to graduate programs and departments at Harvard Medical School and MIT. The Research Base covers a wide range of basic and applied research projects from peripheral mechanics to cortical processing, from in vitro systems to human patients, from animal models to neural nets. The three Cores and the major aims of each include 1) an Engineering Core to design, build, program and maintain data-acquisition systems, custom acoustical devices, and stimulus generation systems, to provide a precision machining service to build custom mechanical devices for a wide variety of research applications, and to share these software and hardware advances with the greater scientific community; 2) a Histology-Surgery Core to maintain existing shared facilities, to assist research teams in animal surgery and histological preparation for both light and electron microscopy and to continually enhance the quality of both histological and surgical preparations throughout the center; and 3) an Imaging Core to support digital image-acquisition and analysis, including confocal microscopy, transmission electron microscopy, computer-aided anatomical reconstruction, automation of morphometry, 3-D reconstruction/rendering, analysis of functional imaging data, and to share virtual teaching models with the greater scientific community.

Section II - Administrative Shell A. Abstract and Key Personnel Three Research Cores are proposed to facilitate interdisciplinary research into hearing and deafness at the Massachusetts Eye and Ear Infirmary. The three Cores and the major aims of each include 1) an Engineering Core to design, build, program and maintain data-acquisition systems, custom acoustical devices, and stimulus generation systems, and to provide a precision machining service to build custom mechanical devices for a wide variety of research applications; 2) an Imaging Core to support the needs of Center investigators for digital image-acquisition and analysis, including confocal microscopy, transmission electron microscopy, computer-aided anatomical reconstruction, automation of morphometry, 3-D reconstruction/rendering, and analysis of functional imaging data; and 3) a Histology-Surgery Core to maintain existing shared facilities and assist research teams in animal surgery and histological preparation for both light and electron microscopy. The Administrative Shell for this P30 includes the Program Director and a Grants Manager. Only fractional additional effort level is required to effectively administer this P30, because 1) the Center Investigators, Core Personnel, Program Director and Grants Manager have a long history of productive collaboration, 2) the Program Director is also Co-PI on both Imaging and Histology-Surgery Cores and is an active user of the Engineering Core, and 3) the Grants Manager also handles many pre-award and post-award functions for the numerous R-grants of almost all Center Investigators and is thus thoroughly conversant with all the components of this Center.

DESCRIPTION (provided by applicant): Auditory neuropathy (AN) is a type of deafness characterized by absence of auditory brainstem responses despite robust otoacoustic emissions, suggesting that outer hair cells are intact while inner hair cells (IHCs) and/or afferen neurons are dysfunctional. The AN phenotype can be either hereditary or acquired. Prematurity is an important risk factor for acquired AN in human populations, and, in animal models, AN can also be produced by the ototoxic anti-cancer drug, carboplatin. Two recent observations from our laboratory suggest that thiamine deficiency may be a key to the genesis of selective IHC loss, and therefore to acquired AN, in both prematurity and carboplatin therapy: 1) there is a striking prevalence of selective IHC loss in premature infants, and 2) IHCs have a unique vulnerability to thiamine (vitamin B1) deficiency in a mouse lacking a key transporter gene. Using animal models and human temporal bones obtained at autopsy, we will test the following hypotheses: 1) that the unique vulnerability of neonatal IHCs arises because of late-onset of expression of the key thiamine transporter genes in the inner ear, 2) that thiamine deficiency during late gestation or early post-natal life can cause selective IHC loss in otherwise healthy mice, and 3) that thiamine supplementation during carboplatin treatment can rescue the IHC loss that produces AN. If these novel hypothesized links are validated, simple therapies based on thiamine supplementation could be safe and effective in reducing the prevalence of hearing loss among infants in the neonatal intensive care unit and among children or adults undergoing anti-cancer therapies with platinum-based drugs. PUBLIC HEALTH RELEVANCE: Recent work has generated the novel hypothesis that a particular type of acquired deafness known as auditory neuropathy arises from the special dependence of one type of inner ear sensory cell, the inner hair cell, on thiamine, also known as vitamin B1. If the experiments proposed here to test this hypothesis are successful, the results should lead to a clinical trial of the efficacy of thiamine supplementation in the prevention of auditory neuropathy among newborns.

Project 2 Summary - Abstract In acquired sensorineural hearing loss, the dogma has been that hair cells, as primary targets, are the first to degenerate, and that cochlear nerve fibers die only after the hair cells disappear. Recent animal work in the Kujawa and Liberman laboratories has shown, in both noise-induced and age-related hearing loss, that the most vulnerable elements are actually the synaptic connections between hair cells and cochlear neurons, and that this cochlear synaptopathy can be widespread (> 50%) even in ears with no threshold elevation and no hair cell degeneration. Synaptic loss silences the affected neurons, absent a cochlear implant, however the slow death of the cell bodies and central projections offers a long therapeutic window in which neurotrophin-related therapies could potentially reverse the damage. We hypothesize that partial de-afferentation of surviving inner hair cells is widespread in acquired sensorineural hearing loss and is a major cause of difficulties understanding speech in a noisy environment, regardless of the degree of hair cell damage, as measured by the audiogram. A recent pilot study from the Liberman lab showed that the same immunostaining techniques we developed to quantify cochlear synaptopathy in mouse, rat, guinea pig, rhesus and other mammals can be applied to human post-mortem material. An analysis of a small number of ears without explicit otologic disease revealed significant cochlear synaptopathy in aged ears, despite no significant loss of hair cells. Here we propose to build on these preliminary results to quantify, as broadly as possible, the prevalence of cochlear de- afferentation in a wide range of hearing loss etiologies, using newly acquired human temporal bones as well as archival sections from the Mass. Eye and Ear collection. Specifically we will, quantify cochlear afferent and efferent innervation in age-graded ?normal-hearing? subjects (Aim 1) and characterize cochlear synaptopathy in subjects with sensorineural hearing loss (Aim 2), with etiologies including noise damage, aminoglycoside antibiotics, and cisplatin-based chemotherapy. Completion of these foundational studies will reveal how widespread the problem of primary neural degeneration is across the spectrum of sensorineural hearing loss in human ears.

Abstract The present proposal seeks to transfer the University of Pittsburgh temporal bone collection to Mass Eye and Ear. The University of Pittsburg has entrusted Mass Eye and Ear and the National Temporal Bone Registry to house and preserve the Sando Temporal Bone collection. This collection is one of the largest collections in the world and is of significant scientific value in the study of human hearing and vestibular disorders. It includes 880 trays of stained and a similar amount of unstained sections. There are 265 temporal bones in celloidin with accompanying histories, autopsy reports and audiograms. The case also includes 21 cases of unique coronal sections that include the entire Eustachian tube. We will make this collection accessible to investigators by incorporating the collection into the National Temporal Bone Registry database. Housing the collection at Mass Eye and Ear will also facilitate the digitalization of the histological slides for remote access.