In the amphibian auditory system, a subset of hair cells is known to be frequency tuned via electrical resonance. This tuning is thought to contribute to frequency selectivity of the information leaving the auditory periphery via the auditory afferent fibers. At the same time, while most, if not all, afferent fibers are shown to be frequency tuned, electrical resonance has only been experimentally demonstrated in a subset of amphibian auditory hair cells. In this thesis, we validate and use a novel Zap current method to probe the electrical resonance of the bullfrog amphibian papilla hair cells. We uncover the existence of two previously unknown types of electrically resonant auditory hair cells. We then show the existence of resonant hair cells across the length of amphibian papilla, with the range of frequency tuning that is nearly indistinguishable from that previously reported in the of auditory fibers. Therefore, this work further validates amphibian hair cell frequency resonance as the possible mechanism underlying frequency selectivity of the subsequent stages in auditory signal transduction.

No description available.

Spectral domain optical coherence tomography (OCT) is a powerful tool for measuring nanometer-scale displacement responses in the cochlea, as it is capable of volumetric imaging and vibrometry at a depth into a sample. The past decade has seen a wealth of OCT-measured displacement data from structures within the organ of Corti complex (OCC) that had previously been impossible to measure in vivo. These data have revealed surprising features of active intra-OCC motion but have not yet led to a complete understanding of cochlear amplification, the means by which active processes enhance the tuning and gain of the cochlear displacement responses in a level-dependent manner. Certain technical challenges arise from the properties of OCT imaging and vibrometry that obscure the interpretation of intra-OCC displacement measurements. In particular, OCT-measured responses are dependent on the orientation of the system's beam axis. The beam axis is generally chosen based on experimental convenience, and has no inherent relevance to the anatomy of the cochlea. This introduces two problems: 1) OCT-acquired images of the cochlea may be taken at skewed angles relative to the cochlea's naturally endowed anatomical coordinates, and 2) OCT-measured displacement responses are projections of a three-dimensional motion onto the beam axis. This thesis concerns the quantification of these effects on intra-OCC displacement measurements, as well as the development of methods to overcome these complications in vivo. In doing so, previously reported data that appear to disagree can be synthesized. I present a method by which the skew of OCT images relative to cochlear anatomy can be quantified, relating the OCT system's optical coordinates to the cochlear anatomy. With this method, I have shown that OCT images resembling familiar anatomical drawings of longitudinal cross-sections often capture a completely different anatomical slice of the cochlea. This leads to large quantitative shifts in phase responses when measuring displacements along a single beam axis, as opposed to what one would measure if s/he were measuring along an anatomically relevant axis. I have also provided a method by which to account for this phenomenon to capture structures related in some desired anatomical fashion. I then turn to the issue of projection of the three-dimensional cochlear motion onto the OCT beam axis. I have provided a method for reconstructing two- and three-dimensional displacement responses in the relevant anatomical directions by acquiring displacement measurements at multiple locations within the cochlea. In doing so, I have revealed that previously unexplained disagreements between measurements in different experimental preparations can be explained by competing components of motion being projected onto the single axis. I have also shown that motion at the junction between the outer hair cells and Deiters cells follows a lineal pattern, as opposed to non-degenerate elliptical patterns that would be expected of fluid motion in this region. This method requires the acquisition of data at many points within the OCC, making it significantly time-consuming. This makes it vulnerable to sample drift and deterioration, and reduces experimental yield. Certain applications of the method -- such as reconstructing displacement maps over a dense volume -- are thereby intractable. To address this problem, I have developed a compressed sensing method for vibrometry (CSVi). CSVi is a classical optimization method based on a total generalized variation signal prior, which is shown to out-perform methods using total variation and wavelet domain sparsity priors. I have also found that uniform sub-sampling schema offered significant performance benefits over random sub-sampling schema. I found that this CSVi method can reconstruct densely sampled displacement maps in the cochlea in vivo with less than 5% normalized mean square error, using only 10% of samples. While these methods offer new insight into interpretation of OCT displacement measurements, there is still a challenge in measuring the motion of the stereocilia of the hair cells. The stereocilia are too small to be imaged using OCT, and the proxy measurement of differential motion of the reticular lamina and tectorial membrane (between which the stereocilia lie) is not yet achievable in the gerbil base. Stereocilia motion is related to the transduction current through the hair cells, which is critical to understanding cochlear function. These currents lead to neurotransmitter release and active electromotile responses believed to be responsible for cochlear amplification. I present a model for studying another proxy measurement of the stereocilia motion -- the voltage in the cochlea's scala tympani, or cochlear microphonic (CM). This model of CM reveals that to match experimental data 1) stereocilia motion must be more sharply tuned than measured intra-OCC displacement responses, 2) the displacement-current gain of the mechano-electric transducer channels in vivo must be larger than what is measured in vitro by a factor of ~6, and 3) the hair cells at more basal locations of the cochlea must be compromised. These predictions offer insight into aspects of cochlear mechanics that are not easily probed using OCT.

No description available.

The mammalian cochlea has been the inspiration to develope contemporary cochlear implants and active dynamic sensors that operate in the sensor's resonance region and possess favorable nonlinear characteristics. In the present work, multi-channel and self-sensing active artificial hair cells (AHCs) made of piezoelectric cantilevers and controlled by a cubic damping feedback controller are developed numerically and experimentally. These novel AHCs function near a Hopf bifurcation and amplify or compress the output by a one-third power-law relationship with the input, analogous to the mammalian cochlear amplifier. The multi-channel AHCs have extended frequency bandwidth to sense over multiple resonant frequencies, unlike conventional single-channel AHCs. Therefore, the adoption of these AHCs reduces the number of required sensors to cover the desired bandwidth of interest in an array format. Furthermore, a novel self-sensing active AHC is created in this study using quadmorph beams for future cochlear implants or sensor design applications. The self-sensing scheme allows miniaturization of the system, embedding AHCs in a limited space, and fabrication of AHC arrays by omitting external sensors from the system for practical implementation. Preliminary research on the extension of this research to MEMS AHCs and arrays of AHCs is also presented. The active AHCs can lead to transformative improvements in the dynamic range, sharpness of the response, and threshold of sound detection in cochlear implants to aid individuals with sensorineural hearing loss. Additionally, they can enhance the dynamic properties of sensors such as fluid flow sensors, microphones, and vibration sensors for various applications. / Doctor of Philosophy / In the mammalian auditory system, the acoustic wave that enters the ear canal is transmitted to the cochlea of the inner ear where it is decomposed into its frequency components. The cochlea then amplifies faint sounds and compresses high-level signals and as these processes stop due to damage, severe hearing loss occurs. Therefore, the present work is focused on developing artificial hair cells (AHCs) that can accurately replicate cochlea's behavior and aid the creation of prostheses for hearing restoration. In this work, the AHC is a beam with piezoelectric layers that is integrated with a control system designed to apply the cochlea-like amplification/compression on the beam. Experimental and simulation results show that the AHC is able to amplify or compress the output based on its input level similar to the mammalian cochlea. In contrast to previous designs of AHCs where each AHC could sense a single frequency, the system developed in this work possesses multiple sensing channels to increase the frequency range of the AHC. Furthermore, the development of a novel self-sensing scheme allows the omission of the external sensor that was required for the AHC operation in previous devices. This advancement in the self-sensing AHC design paves the way for creating fully implantable AHCs to replace the damaged parts of the cochlea. These multi-channel self-sensing AHCs have the potential to be used in the creation of cochlear implants, or sensors such as accelerometers, microphones, and hydrophones with improved dynamic properties. AHCs with different lengths, i.e. different sensing frequencies, can be mounted in an array format to cover the speech frequency range for speech recognition in individuals with hearing loss.

Introdução: Sabe-se que muitas pessoas sofrem de hipertensão arterial sistêmica (HAS) e indivíduos cada vez mais jovens estão apresentando este quadro. A HAS pode causar várias complicações na saúde geral do indivíduo, em virtude do comprometimento da circulação sanguínea, o que pode afetar até mesmo o sistema auditivo. Porém, os estudos encontrados na literatura, até o momento, são bastante controversos quanto à influência ou não da HAS no sistema auditivo periférico. Objetivos: Verificar se existe influência da hipertensão arterial no sistema auditivo periférico, por meio dos achados na audiometria de altas frequências, nas emissões otoacústicas transientes e produto de distorção, comparando grupos de indivíduos normoouvintes pela audiometria convencional, com e sem HAS; e identificar se alguma das variáveis referentes às avaliações realizadas pode ser utilizada para discriminar os indivíduos nos grupos com ou sem HAS. Método: Participaram deste estudo 40 sujeitos de 30 a 50 anos, divididos em grupos com e sem hipertensão arterial. Todos os indivíduos foram submetidos à meatoscopia, imitanciometria, audiometria tonal nas frequências de 250 Hz a 16000 Hz, logoaudiometria, emissões otoacústicas evocadas transientes e emissões otoacústicas produto de distorção. Resultados: Não foi observada diferença estatisticamente significante entre os grupos com e sem HAS na audiometria convencional e em altas frequências. Para as emissões otoacústicas evocados por estímulo transiente, houve tendência à diferença estatisticamente significante, com as respostas do grupo com HAS piores que do grupo sem HAS. Já as emissões otoacústicas produto de distorção, nas frequências de 1501, 2002 e 3003 Hz, apresentaram diferenças estatisticamente significantes, sendo que o grupo com HAS apresentou amplitudes menores. A análise discriminante mostrou que as variáveis das emissões otoacústicas produto de distorção foram as que melhor discriminaram os indivíduos com e sem HAS. Conclusão: Nossos achados sugerem presença de disfunção coclear em indivíduos com hipertensão arterial, uma vez que as respostas das emissões otoacústicas foram piores no grupo com HAS / Introduction: It is widely known that many people have systemic arterial hypertension (SAH) and, currently, the number of young people with such disease is increasing. SAH may cause a wide range of complications in the general health of an individual due to the involvement of blood circulation, which may affect the hearing system. Nevertheless, the reports found in the scientific field are still controversial regarding the influence of SAH on the peripheral auditory system. Purpose: To verify if there is any influence of SAH on the peripheral auditory system, through data from high frequency audiometry, transient-evoked otoacustic emissions and distortion-product otoacoustic emissions and compare such results to groups of typical hearing individuals, with and without SAH, who underwent the pure-tone audiometry test; and also investigate if any variable regarding the hearing assessment may be used to differentiate individuals with and without SAH. Methods: This study counted on 40 individuals aged between 30 and 50 years old, divided into groups with and without SAH. All individuals underwent the following procedures: otoscopy, imitanciometry, pure-tone audiometry in frequencies from 250Hz to 16000 Hz, logoaudiometry, transient-evoked otoacoustic emissions and distortion-product otoacoustic emissions. Results: There was found no significant statistically difference between the groups with and without SAH neither in pure-tone nor high-frequency audiometry. Regarding transient-evoked otoacustic emissions there was a tendency to a significant statistically difference once the SAH group showed lower results. Concerning distortion-product otoacustic emissions there was found significant statistically difference in the following frequencies: 1501, 2002 e 2003 Hz in which the SAH group showed lower results. Discriminant analysis pointed that variables of distortion-product otoacustic emissions best distinguished individuals with and without SAH. Conclusion: Data from this study suggest cochlear dysfunction in individuals with SAH since their results in otoacoustic emissions were lower than in the group with SAH

O epitélio pulmonar é formado por uma grande diversidade celular, que incluí: células secretoras, ciliadas, basais e neuroendócrinas (NE). A distribuição balanceada destes tipos celulares é crucial para a função pulmonar e pode ser dramaticamente alterada em doenças como a asma. Neste trabalho, estudamos o papel de Notch no pulmão em desenvolvimento ao inativar condicionalmente Rbpjk ou Pofut1, componentes críticos da sinalização Notch. Pulmões mutantes apresentaram-se superpopulados por células ciliadas e NE, além da ausência de células de Clara. Nossos dados sugeriram que Notch suprime os programas de diferenciação de células ciliadas e NE para permitir a diferenciação de células de Clara, através de um mecanismo de inibição lateral. Identificamos também genes associados com a diferenciação de células secretoras e ciliadas através de microarrays. A heterogeneidade no padrão de expressão gênica sugeriu que a via de sinalização Notch estabelece múltiplos subtipos de células ciliadas e secretoras no epitélio pulmonar em desenvolvimento. / The airway epithelium comprises a diverse population of secretory, ciliated, basal and neuroendocrine cells (NE). The proper balance of these cell types is critical for normal lung function and can be altered dramatically in conditions, such as asthma. We studied the role of Notch in airway progenitor cell fate by conditionally inactivating Rbpjk or Pofut1, two critical Notch pathway components in mouse mutants. This resulted in airways overpopulated with ciliated and NE cells and absence of secretory Clara cells. We found that Notch suppresses the ciliated and the NE cell programs to allow secretory cell differentiation through a lateral inhibition mechanism. We also identified genes associated with the differentiation of secretory and ciliated cells through a microarray gene profiling experiment. The great heterogeneity of gene expression patterns suggested that Notch plays a role in establishing multiple subsets of secretory and ciliated cells in the developing lung.

Avant d'être libéré dans la fente synaptique, le glutamate est accumulé dans les vésicules présynaptiques par les transporteurs vésiculaires du glutamate (VGLUTs). Il existe 3 types de VGLUTs. VGLUT3 possède une distribution anatomique et des fonctions atypiques. Au sein du système nerveux central, VGLUT3 est exprimé dans des neurones glutamatergiques mais aussi non glutamatergiques, dans lesquels il assure les fonctions de co-transmission ou de synergie vésiculaire. On le retrouve notamment dans certains neurones sérotoninergiques du raphé. Au sein de l'oreille interne, VGLUT3 est l'unique VGLUT décrit dans les cellules ciliées internes (CCI). La sérotonine joue un rôle essentiel dans le contrôle respiratoire néonatal. En étudiant la respiration de souriceaux n'exprimant plus le VGLUT3, nous avons démontré le rôle de VGLUT3 dans l'adaptation au stress hypoxique néonatal. Une mutation de VGLUT3 a été mise en évidence dans une surdité humaine très proche cliniquement de la presbyacousie et appelée DFNA25. En étudiant le phénotype auditif de souris exprimant cette mutation, nous avons prouvé l'implication de cette mutation dans l'atteinte des CCI à l'origine de la surdité DFNA25. L'étude des processus biochimiques mis en jeu nous a permis d'envisager un rôle indirect de VGLUT3 dans l'activation de la mort autophagique, via la protéine Becline 1 et une possible interaction au sein de la voie de la Culline 3. L'ensemble de ce travail nous a permis de mettre en évidence un rôle de VGLUT3 dans l'adaptation aux conditions extrêmes telles que le développement néonatal ou le processus de vieillissement. Il ouvre de nouvelles perspectives sur les diverses fonctions des VGLUTs. / Before its release into synaptic cleft, glutamate is accumulated in presynaptic vesicles by vesicular glutamate transporters (VGLUTs). There are 3 types of VGLUTs. VGLUT3 presents atypical functions and anatomical distribution. In the central nervous system, VGLUT3 is expressed in glutamatergic and non glutamatergic neurons, in which it performs the co-transmission and the vesicular synergy. Particularly, we can observe VGLUT3 in serotoninergic neurons of raphe. In the inner ear, VGLUT3 is the unique VGLUT described in the inner hair cells (IHCs). Serotonin plays a key role in the neonatal respiratory control. By exploring the respiration of VGLUT3 knock out mice pumps, we have demonstrated the role of VGLUT3 in the response to neonatal hypoxic stress. One VGLUT3 mutation has been described in a human deafness clinically very close to presbycusis, the DFNA25 deafness. By studying the auditory phenotype of mice expressing this VGLUT3 mutation, we have proved the implication of this mutation in the IHCs impairment at the origin of DFNA25 deafness. Biochemical analysis have helped us to consider an indirect role of VGLUT3 in the autophagic death. Beclin 1 and a possible interaction between VGLUT3 and the Cullin3 pathways could be implicated. All of our results allowed us to highlight a role of VGLUT3 in the adaptation to extreme conditions like neonatal development or aging process. They open new perspectives on the various functions of VGLUTs.

No description available.

In the inner and outer hair cells (OHCs) of the inner ear, an unconventional myosin 15a localizes at the tips of mechanosensory stereocilia and plays an important role in forming and maintaining their normal structure. A missense mutation makes the motor domain of myosin 15a dysfunctional and is responsible for the congenital deafness DFNB3 in humans and deafness and vestibular defects in Shaker-2 (Sh2) mouse model. All hair cells of homozygous Shaker-2 mice (Myo15sh2/sh2) have abnormally short stereocilia, but, only stereocilia of Myo15sh2/sh2OHCs start to degenerate after the first few days of postnatal development and lose filamentous tip links between stereocilia that are crucial for mechanotransduction. The exact mechanisms of this degeneration are unknown even though they may underlie DFNB3 deafness in humans. We hypothesize that structural abnormalities in Myo15sh2/sh2 OHCs may alter the mechanical forces applied to the mechano-electrical transduction (MET) channels resulting in abnormal ionic homeostasis, which may lead to eventual degeneration of Myo15sh2/sh2 OHCs. Therefore, we investigated the ionic conductances and integrity of mechanotransduction apparatus in Myo15sh2/sh2 OHCs. Surprisingly, we found that myosin 15a-deficiency is associated not only with structural abnormalities of OHC stereocilia but also with alterations of voltage-gated ion conductances.