This thesis takes a fresh approach to cochlear mechanics. Over the last quarter of a century, we have learnt that the cochlea is active and highly tuned, observations suggesting that something may be resonating. Rather than accepting the standard traveling wave interpretation, here I investigate whether a resonance theory of some kind can be applied to this remarkable behaviour.¶ A historical survey of resonance theories is first conducted, and advantages and drawbacks examined. A corresponding look at the traveling wave theory includes a listing of its short-comings.¶ A new model of the cochlea is put forward that exhibits inherently high tuning. The surface acoustic wave (SAW) model suggests that the three rows of outer hair cells (OHCs) interact in a similar way to the interdigital transducers of an electronic SAW device. Analytic equations are developed to describe the conjectured interactions between rows of active OHCs in which each cell is treated as a point source of expanding wavefronts. Motion of a cell launches a wave that is sensed by the stereocilia of neighbouring cells, producing positive feedback. Numerical calculations confirm that this arrangement provides sharp tuning when the feedback gain is set just below oscillation threshold.¶ A major requirement of the SAW model is that the waves carrying the feedback have slow speed (5-200 mm/s) and high dispersion. A wave type with the required properties is identified - a symmetric Lloyd-Redwood wave (or squirting wave) - and the physical properties of the organ of Corti are shown to well match those required by theory.¶ The squirting wave mechanism may provide a second filter for a primary traveling wave stimulus, or stand-alone tuning in a pure resonance model. In both, cyclic activity of squirting waves leads to standing waves, and this provides a physical rendering of the cochlear amplifier. In keeping with pure resonance, this thesis proposes that OHCs react to the fast pressure wave rather than to bending of stereocilia induced by a traveling wave. Investigation of literature on OHC ultrastructure reveals anatomical features consistent with them being pressure detectors: they possess a cuticular pore (a small compliant spot in an otherwise rigid cell body) and a spherical body within (Hensens body) that could be compressible. I conclude that OHCs are dual detectors, sensing displacement at high intensities and pressure at low. Thus, the conventional traveling wave could operate at high levels and resonance at levels dominated by the cochlear amplifier. ¶ The latter picture accords with the description due to Gold (1987) that the cochlea is an ‘underwater piano’ - a bank of strings that are highly tuned despite immersion in liquid.¶ An autocorrelation analysis of the distinctive outer hair cell geometry shows trends that support the SAW model. In particular, it explains why maximum distortion occurs at a ratio of the two primaries of about 1.2. This ratio also produces near-integer ratios in certain hair-cell alignments, suggesting that music may have a cochlear basis.¶ The thesis concludes with an evaluation and proposals to experimentally test its validity.

Loss of hair cells due to acoustic trauma results in the loss of hearing. In humans, unlike other vertebrates, the mechanism of hair cell regeneration is not possible. The molecular mechanisms that underlie this regeneration in nonmammalian vertebrates remain elusive. To understand the gene regulation during hair cell regeneration, our previous microarray study on zebrafish inner ears found that growth hormone (GH) was significantly upregulated after noise exposure. In this current study, we utilized Next Generation Sequencing (NGS) to examine the genes and pathways that are significantly regulated in the zebrafish inner ear following sound exposure and GH injection. Four groups of 20 zebrafish each were exposed to a 150 Hz tone at 179 dB re 1μPa RMS for 40 h. Zebrafish were injected with either salmon GH, phosphate buffer or zebrafish GH antagonist following acoustic exposure, and one baseline group received no acoustic stimulus or injection. RNA was extracted from ear tissues at 1 and 2 days post-trauma, and cDNA was synthesized for NGS. The reads from Illumina Pipeline version SCS 2.8.0 were aligned using TopHat and annotated using Cufflinks. The statistically significant differentially expressed transcripts were identified using Cuffdiff for six different pairwise comparisons and were analyzed using Ingenuity Pathway Analysis. I found significant regulation of growth factors such as GH, prolactin and fibroblast growth factor receptor 2, different families of solute carrier molecules, cell adhesion molecules such as CDH17 and CDH23, and other transcription factors such as Fos, FosB, Jun that regulate apoptosis. Analysis of the cell proliferation network in the GH-injected condition compared to buffer-injected day 1 showed significant up-regulation of GH while downregulation of apoptotic transcription factors was found. In contrast, the antagonist-injected condition compared to the GH-injected condition showed an opposite pattern in which up-regulation of apoptotic transcription factors were found while GH was down-regulated. A number of other transcripts (e.g., POMC, SLC6A12, TMEM27, HNF4A, CDH17 and FGFR2) that showed up-regulation in GH-injected condition showed down-regulation in antagonist-injected condition. These results strongly suggest that injection of exogenous GH potentially has a protective role in the zebrafish inner ear following acoustic trauma.

The experiments described in this thesis report were aimed at studying the functions of Sox2 and Serrate1 during the development of the neurosensory elements of the inner ear. First, we have described the expression pattern of Sox2 during inner ear development and compared to that of Sox3 and Serate1. Secondly, we have shown the results of plasmid based in ovo electroporation experiments, designed to manipulate gene expression exogenously, and to study the gain of function of Sox2 and Serrate1. Effects on cell fate and downstream targets were assessed by in situ hybridization immunohistochemistry and quantitative real-time PCR (qRT-PCR).The results show that Sox2 is expressed in the neurosensory domain of the otic epithelium during the neurogenic period of otic development and, later on, during the development of the prosensory patches and sensory organs. As differentiation proceeds, Sox2 is excluded from differentiated neurones and hair cells, but remains expressed in the supporting cells of the sensory organs. Sox3 is co-expressed with Sox2 in the neurogenic domain of the otic cup. But Sox3 is then down-regulated and only Sox2 expression persists in the sensory precursors, where it is co-expressed with the Notch ligand Serrate1. The expression domain of Serrate1 is initially nested within Sox2, however, later in development Sox2 becomes restricted within the boundaries of Serrate1 expression, a process that is concomitant to the formation of the sensory patches. These expression patterns suggest: 1) that Sox2 correlates with neurosensory fate in the otic placode, 2) that neurogenesis is associated with Sox2 and Sox3 and 3) that sensory development is associated with Sox2 and Serrate1.Gain of function studies show that Serrate1 regulates prosensory fate and sensory organ development by maintaining Sox2 expression in restricted domains of the otocyst, without affecting neurogenesis. Serrate1 operates in a Notch-dependent manner, consistently with a mechanism of lateral induction that includes the induction of its own expression and downstream targets of the Notch signalling pathway Hes1, Hey1 and Hey2. Similar studies on Sox2 indicate that it specifies neurosensory fate in the otic epithelium. However, high concentrations of Sox2 suppress sensory fate and promote neuronal fate. Besides, Sox2 prevents cell differentiation though the cooperation with Notch and BMP signalling pathways.We like to propose a model in which an extended neural competence is early established in the otic placode with the early expression of Sox2 and Sox3 genes. The cooperation between Sox2 and Sox3 then provides a high concentration of SoxB1 protein and promote neuronal fate. In parallel, Serrate1 maintains Sox2 expression in restricted domains, after Sox3 down-regulation. These domains retain the neurosensory competence and thereby develop as sensory patches. / Los experimentos descritos en esta tesis tuvieron por objetivo estudiar la función de Sox2 y Serrate1 en el desarrollo de los elementos neurosensoriales del oído. En primer lugar describimos el patrón de expresión de Sox2 durante el desarrollo del oído y lo comparamos con el de Sox3 y Serrate1. En segundo lugar, mostramos los resultados de experimentos de electroporación in ovo, diseñados para manipular exógenamente la expresión génica y estudiar la ganancia de función de Sox2 y Serrate1. Los efectos sobre el destino celular y las dianas moleculares se analizaron mediante hibridación in situ, inmunocitoquímica y real-time PCR (qRT-PCR).Los resultados muestran que Sox2 se expresa en el dominio neurosensoerial del epitelio ótico durante la fase de neurogénesis y, más adelante, durante el desarrollo de los parches prosensoriales y los órganos sensoriales. Con la diferenciación, Sox2 es excluido de las neuronas diferenciadas y las células ciliadas, pero permanece expresado en las células de soporte. Sox3 se coexpresa con Sox2 en el dominio neurogénico de la copa ótica. Pero entonces, la expresión de Sox3 se reduce y sólo Sox2 persiste en los precursores sensoriales, en donde se co-expresa con el ligando de Notch Serrate1. El dominio de expresión de Serrate1 está inicialmente contenido en el de Sox2, sin embargo, más adelante, Sox2 se restringe dentro de los límites de Serrate1, un proceso que es concomitante con la formación de los parches sensoriales. Estos experimentos sugieren que : 1) Sox2 se correlaciona con el destino neurosensorial de la placoda ótica, 2) la neurogénesis está asociada con Sox2 y Sox3, y 3) el desarrollo sensorial está asociado a la expresión de Sox2 y Serrate1Los estudios de ganancia de función muestran que Serrate1 regula el destino prosensorial y el desarrollo de los órganos sensoriales mediante el mantenimiento de la expresión de Sox2 en dominios restringidos del otocisto, sin afectar a la neurogénesis. Serrate1 opera en un modo dependiente de Notch, consistente con un mecanismo de inducción lateral que comprende la inducción de su propia expresión y la de las dianas de Notch Hes1, Hey1 and Hey2. Estudios similares sobre Sox2 indican que Sox2 especifica el destino neurosensorial en el epitelio ótico. Sin embargo, las concentraciones altas de Sox2 suprimen el destino sensorial y promueven el destino neuronal. Además, Sox2 previene la diferencoiación celular mediante la cooperación con Notch y Bmp. Se propone un modelo en el cual la competencia neural se establece tempranamente en la placoda ótica mediante la expresión temprana de Sox2 y Sox3. La cooperación entre Sox2 y Sox3 provee una alta concentración de factores SoxB1 que promueven el destino neuronal de los progenitores. En paralelo, Serrate1 mantiene la expresión de Sox2 en dominios restringidos tras la supresión de Sox3. Estos dominios, retienen el potencial neurosensorial y, más adelante, se desarrollan como parches sensoriales.

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Many speech enhancement algorithms suffer from musical noise - an estimation residue noise consisting of music-like varying tones. To reduce this annoying noise, some speech enhancement algorithms require post-processing. However, a lack of auditory perception theories about musical noise limits the effectiveness of musical noise reduction methods. Scientists now have some understanding of the human auditory system, thanks to the advances in hearing research across multiple disciplines - anatomy, physiology, psychology, and neurophysiology. Auditory models, such as the gammatone filter bank and the Meddis inner hair cell model, have been developed to simulate the acoustic to neuron transduction process. The auditory models generate the neuron firing signals called the cochleagram. Cochleagram analysis is a powerful tool to investigate musical noise. We use auditory perception theories in our musical noise investigations. Some auditory perception theories (e.g., volley theory and auditory scene analysis theories) suggest that speech perception is an auditory grouping process. Temporal properties of neuron firing signals, such as period and rhythm, play important roles in the grouping process. The grouping process generates a foreground speech stream, a background noise stream, and possibly additional streams. We assume that musical noise is the result of grouping to the background stream the neuron firing signals whose temporal properties are different from the ones grouped to the foreground stream. Based on this hypothesis, we believe that a musical noise reduction method should increase the probability of grouping the enhanced neuron firing signals to the foreground speech stream, or decrease the probability of grouping them into the background stream. We propose a post-processing musical noise reduction method for the auditory Wiener filter speech enhancement method, in which we employ a proposed complex gammatone filter bank for the cochlear decomposition. The results of a subjective listening test of our speech enhancement system show that the proposed musical noise reduction method is effective. / Thesis (Master, Electrical & Computer Engineering) -- Queen's University, 2008-05-28 16:11:28.374

La neuropathie auditive est une forme de surdité caractérisée par une atteinte des cellules ciliées internes (qui détectent les ondes sonores et les transforment en message nerveux) et/ou des neurones afférents primaires (qui véhiculent les messages nerveux jusqu'au noyau cochléaire), associée à une activité normale des cellules ciliées externes (qui amplifient les ondes sonores). AUNA1 est la première neuropathie auditive héréditaire à avoir été décrite. Elle est causée par une mutation ponctuelle située dans le promoteur du gène DIAPH3, résultant en une surexpression de DIAPH3. La protéine DIAPH3, un membre de la famille des formines, est connue pour promouvoir la nucléation et l’élongation des filaments d’actine ainsi que la stabilisation des microtubules. Nous avons étudié les mécanismes d’AUNA1 à partir d’un modèle murin transgénique surexprimant le gène diap3, l’orthologue murin de DIAPH3. Les souris transgéniques développent une surdité dont les caractéristiques sont semblables à celles d’AUNA1. Cette surdité est due à une perte d’activité des cellules ciliées internes. L’activité synaptique et les courants potassiques de ces cellules ne sont pas altérés. En revanche, la microscopie électronique révèle une fusion des stéréocils (expansions cytoplasmiques qui permettent la détection des ondes sonores) et une déformation de la plaque cuticulaire (plateforme qui assure l’ancrage des stéréocils). Par la technique d’immunomarquage, nous avons mis en évidence une invasion de la plaque cuticulaire par des microtubules. Enfin, nous avons démontré que la protéine Diap3 est localisée dans la plaque cuticulaire des cellules ciliées internes, suggérant ainsi que la surexpression de diap3 provoque un remodelage du réseau de microtubule des cellules ciliées internes, à l’origine de la surdité AUNA1. / Auditory neuropathy is a type of deafness characterized by an alteration of the inner hair cells (which detect the acoustic waves and transform them into neural messages) and/or of the primary afferent neurons (which conduct the neural messages to the cochlear nucleus), associated with a normal activity of the outer hair cells (which amplify the acoustic waves).AUNA1 is the first hereditary auditory neuropathy which has been described. It is caused by a point mutation in the promoter of the DIAPH3 gene, resulting in an overexpression of DIAPH3. The DIAPH3 protein, a formin family member, is known to promote the actin filament nucleation and elongation and to stabilize the microtubules.We studied the AUNA1 mechanisms using a transgenic mouse model which overexpresses the diap3 gene, the mouse homologue of DIAPH3. Transgenic mice develop a deafness whose characteristics are similar to the ones of AUNA1. The hearing loss is due to a defect in the inner hair cell activity. The synaptic activity and the potassium currents of these cells are not altered. However, electron microscopy reveals a fusion of the stereocilia (cytoplasmic expansions which detect the acoustic waves) and a disruption of the cuticular plate (plateform which maintains stereocilia). By immunolabeling, we showed an invasion of the cuticular plate by microtubules. Eventually, we demonstrated that Diap3 is located in the inner hair cell cuticular plate, suggesting that the overexpression of diap3 provokes a remodeling of the inner hair cell microtubule network, underlying the AUNA1 deafness.

Les cellules sensorielles auditives, les cellules ciliées internes (CCI), transforment les ondes sonores en message nerveux. Les synapses des CCI se distinguent de celles du système nerveux par leur anatomie. En effet, les synapses des CCI sont dotées d'un organite appelé ruban synaptique. Ce dernier a pour fonction de concentrer les vésicules synaptiques à proximité des zones actives. Il est important de souligner qu'un déficit de la libération synaptique à la première synapse auditive est à l'origine de surdités chez l'homme. Si la physiologie des synapses à rubans des cellules ciliées a été intensivement étudiée, la composition moléculaire des ces synapses reste en grande partie inconnue. L'objectif de cette thèse était donc d'isoler les protéines clefs de la machinerie synaptique. Pour ce faire, nous avons utilisé la technique du double hybride à partir d'une banque d'ADN complémentaire de cochlée et de la protéine Ribeye, composant majeur des rubans, comme appât. La difficulté majeure de notre étude provient de la structure de Ribeye, qui est constitué par deux domaines A et B. Tandis que le domaine A est dirigé vers le cœur du ruban synaptique et aurait une fonction structurale, le domaine B est fortement homologue au facteur de transcription Ctbp2. Ainsi, nous avons identifié plusieurs candidats comme étant des facteurs de transcription. Ces derniers interagissent probablement avec Ctbp2 dans le noyau. Nos résultats obtenus soulignent la difficulté d'identifier des protéines d'interactions, inhérente à l'utilisation de Ribeye comme appât. Parmi les autres candidats, nous avons isolés des composants du système de l'ubiquitine, suggérant une régulation ubiquitine-dépendante de l'activité ou de la structure des rubans synaptiques. / Inner hair cells (IHCs) are the sensory cells of the cochlea, the organ of hearing. IHCs transduce sound stimulation into the release of glutamate onto the afferent auditory nerve fibers. To achieve this task, IHCs harbor at their presynaptic side a large organelle, the so-called synaptic ribbon, surrounded by a monolayer of glutamate-filled synaptic vesicles. Exocytosis of glutamate at the hair cell ribbon synapse seems to be unconventional as the synaptic machinery, depicted so far, differs from most of the nervous system synapses. The goal of this work was to identify new members of the synaptic machinery of the hair cell ribbon synapse. To do so, we took advantage of the yeast two-hybrid system using a cochlea cDNA library as the prey and Ribeye (the major ribbon component) as the bait. Transcription factors were highly represented in our screening assay, most probably because Ribeye is highly homologous to the transcription factor Ctbp2. They probably interact with Ctbp2 in the nucleus. Our results underlined the difficulty to identify protein interactions because of the nature of Ribeye itself. However, we found ubiquitin system components among the other candidates, suggesting an ubiquitin-dependent regulation of the activity and/or structure of synaptic ribbons.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23094号 / 医博第4721号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 井上 治久, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Recent advances in the field of audiology have indicated that there has been a growing concern regarding the potential damage to the hearing mechanism induced by recreational noise exposure from personal music devices (PMD). Regular PMD use may have a long-term damaging effect on the outer- and inner hair cells of the cochlea which may result in a progressive hearing loss. As PMDs have advanced to a stage where the memory of the devices are able to contain hours of listening content, the environments where these devices are being used are rapidly expanding. Many young adults tend to use their PMDs whilst exercising. Exercise in itself induces physiological and metabolic changes such as increased blood flow and oxygen levels within the structures of the cochlea. The purpose of this study was to determine the differential impact and short-term effects of simultaneous cardiovascular workout and personal music device (PMD) use on the otoacoustic emissions of young adults. Seven female and five male subjects completed three testing conditions: (i) one hour exposure to PMD use in isolation, (ii) one hour exposure to cardiovascular workout in isolation, and (iii) one hour simultaneous exposure to PMD use and cardiovascular workout. Distortion product otoacoustic emissions (DPOAEs) were conducted prior to, as well as directly following each testing condition, as primary indicator of cochlear responses emitted through a preset stimulus frequency sequence measuring the 2f₁ - f₂ (75 – 70 dB SPL) and constructing a plot of DPOAE levels as a function of frequency. While each of the testing conditions on its own did not result in statistically significant changes of the DPOAE response, a highly significant different profile in the DPOAE response level increase/decrease for the higher frequencies (6-8 kHz) was obtained when comparing the different sessions to each other. Where exposure to cardiovascular workout showed a clear trend of an increased DPOAE response level between the pre-exposure and post-exposure testing from 2 kHz to 8 kHz with a maximum increase at 6 kHz, both the music only condition and the combined condition where the cardiovascular workout was combined with music resulted in a significant different profile. During combined exposure a clear trend of decreased DPOAE response amplitudes between the pre-exposure and post-exposure testing were seen for the higher frequencies. These findings may support the notion of a clear effect of cardiovascular workout on the otoacoustic emissions at higher test frequencies, measured by DPOAEs when performed with and without music exposure. / Dissertation (MLOG)--University of Pretoria, 2014. / tm2015 / Speech-Language Pathology and Audiology / MLOG / Unrestricted

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