The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na+,K+-ATPase in the tegmentum vasculosum of the duckling. Na+,K+-ATPase activity was determined by measuring K+-dependent, ouabain-sensitive p-nitrophenyl phosphatase (p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na+,K+-ATPase was identified using K+-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 μmol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 μmol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na+,K+-ATPase-dependent ion transport function related to endolymph synthesis.

No description available.

The cochlea, or inner ear, is a space fully enclosed within the temporal bone of the skull, except for two membrane-covered portals connecting it to the middle ear space. One of these portals is the round window, which is covered by the Round Window Membrane (RWM). A longstanding clinical goal is to gain reliable and precise access to the cochlea with the purpose of delivering therapeutics to treat a plethora of auditory and vestibular disorders, or to aspirate fluids for diagnostic purposes. Standard of care for several difficult-to-treat diseases calls for injection of a therapeutic substance through the tympanic membrane into the middle ear space, after which a portion of the substance diffuses across the RWM into the cochlea. The efficacy of this technique is limited by an inconsistent rate of molecular transport across the RWM. Other solutions for delivery require either traumatic drilling through the bone of the cochlea, or perforating the delicate RWM, which is prone to rupturing with the use of regular surgical tools. For conducting precision diagnostics, even fewer options exist. In our research group, utilizing a newly available technology called Two-Photon Lithography, (2PP) we have developed a suite of ultra-sharp microneedles that are able to create repeatable and reliable perforations in the RWM without tearing. These holes were seen to spontaneously heal within 48 hours, and did not cause any audiological or functional consequences. Furthermore, we have designed needles that can, while inserted into the cochlea, inject or aspirate fluid of microliter quantities, to and from the inner ear, safely. In this thesis, I will discuss the development of these microneedles: their methods, design, use, and modeling. The results show that the microneedles hold great promise to diagnose and treat hearing and balance disorders.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24091号 / 医博第4867号 / 新制||医||1059(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 林 康紀, 教授 髙橋 良輔, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In the mammalian auditory system, specifically in the cochlea of the inner ear, the Basilar Membrane (BM) and hair cells are responsible for transducing incoming acoustic waves into electrical signals. These acoustic signals are carried as traveling waves by the BM and propagate from the base of the cochlea toward its apex where the helicotrema is located. An impressive feature of the mammalian auditory system is to prevent the propagated waves from reflecting which allows mammals to hear sounds without any reflection or overlap. This extraordinary characteristic of the inner ear is the main inspiration for this work. In the present study, the dynamic behavior of a beam structure with one or more attached spring-damper (SD) systems as passive absorbers is studied when excited by a harmonic force. The location of the spring-damper system divides the beam into two dynamic regions: one which exhibits non-reflecting traveling waves and the other with standing waves. In this work, the separation of traveling and standing waves is studied analytically, numerically, and experimentally. To the best of the author's knowledge, this is the first time in the literature that traveling and standing wave separation in a beam is realized experimentally using a single-point excitation and a spring-damper. Experimental results are used to validate the models of the system. Moreover, a parametric study is performed to gain a better understanding of the effect of different parameters on the quality of the generated waves in the structure. Furthermore, the effect of attaching the second spring-damper to the system is presented. Adding the secondary SD system results in increasing the excitation frequency range so that wave separation can be achieved. The results of this work can be used in various applications such as vibration suppression, energy absorption, particle transportation, and in exploring possible explanations for the BM and helicotrema functions in the cochlea. / Doctor of Philosophy / In the inner ear of the mammalian auditory system, the sound waves travel inside the cochlea where they are converted to electrical signals sent to the brain. A fascinating characteristic of the mammalian auditory system is that the sound waves traveling in the cochlea do not reflect when they reach its apex where the helicotrema is located. Therefore, we are able to hear sounds without any reflection or overlap. This work is inspired by the biological behavior of the inner ear and studies the dynamic behavior of a simple structure such as a beam with one (or two) attached spring-damper(s). In this work, the attached spring-damper system(s) prevents the waves traveling from the source to the beam's boundary from reflecting. This is similar to what happens in the inner ear. The location of the spring-damper divides the beam into two dynamic regions, one which exhibits non-reflecting traveling waves and the other with standing waves. The wave separation and parameters affecting the wave quality and its reflective or non-reflective features are studied analytically, numerically, and experimentally. To the best of the author's knowledge, the experiments carried out to generate the aforementioned wave types coexisting with each other on the beam are one of a kind. Furthermore, the results of this study showed a very good agreement between the experimental and theoretical results. The outcomes of this work can potentially be used in exploring possible explanations for the function of the cochlea and helicotrema and various applications such as particle transportation and suppression of unwanted vibrations.

No description available.

En réponse à une stimulation sonore, la cellule ciliée interne libère du glutamate qui va activer des récepteurs distribués sur le bouton post-synaptique. Les courants post-synaptiques vont ensuite dépolariser la terminaison périphérique des neurones auditifs primaires, et initier le déclenchement d'un potentiel d'action. Tandis que la connaissance des mécanismes pré-synaptiques a considérablement progressé ces 10 dernières années, les mécanismes responsables de l'initiation des potentiels d'action sont encore méconnus. Dans cette étude, nous avons déterminé les conductances ioniques nécessaires au déclenchement des potentiels d'action.Les paramètres biophysiques des conductances (Na+ et K+) ont été identifiés (algorithme d'identification trace entière) à partir d'enregistrements de patch clamp acquis sur les corps cellulaires. Un modèle mathématique de nœud de Ranvier a ensuite été développé en faisant l'hypothèse que les canaux présents sur le corps cellulaire et sur un nœud de Ranvier étaient de même nature mais en densité différente. Les paramètres de ce modèle ont été identifiés pour reproduire les potentiels d'action extracellulaire au moyen d'un algorithme de descente du gradient.Nous avons identifié : i) un courant Na+ entrant rapide (GNa activation: V1/2=-33 mV, tau_act< 0.5 ms; inactivation: V1/2=-61 mV, tau_inact < 2 ms) et deux courants K+ sortants, un rectifiant retardé activé à haut seuil (GKH, activation: V1/2=-41 mV; tau_act < 2.5 ms) et un activé à bas seuil (GKL, activation: V1/2=-56 mV; tau_act < 5 ms). Le modèle de nœud de Ranvier génère des potentiels d'action extracellulaire similaires à ceux enregistrés in vivo. La différence de durée du potentiel d'action observée le long de l'axe tonotopique (i.e. 450 µs de durée pic à pic à 1 kHz contre 250 µs à 20 kHz) s'explique parfaitement par un gradient de densité en canaux ioniques le long de la cochlée (GNa ~78 nS, GKL ~9 nS, GKH ~3 nS à 1 kHz contre GNa ~90 nS, GKL ~12 nS, GKH ~6 nS à 20 kHz).Cette étude a permis d'identifier les conductances ioniques et les densités de canaux responsables de l'initiation des potentiels d'action dans les neurones auditifs primaires. Elle suggère que la coopération entre le courant Na+ et des 2 courants K+ est probablement à l'origine de la haute fréquence de décharge de ces neurones. Le modèle de nœud de Ranvier permet en outre de tester de nouvelles stratégies de stimulation électrique dans le contexte de l'implant cochléaire. / In response to sound stimulation, inner hair cell triggers glutamate release onto the dendrite-like processes of primary auditory neurons and drives action potentials, which are convey to the central nervous system. Whereas knowledge of the transfer function at the ribbon synapse has considerably progress, little is known about the voltage-gated ionic channels which shape the action potential. Here, we provide a comprehensive computational model bridging the gap between the voltage-dependent currents measured in vitro on fresh isolated primary auditory neurons and spikes (extracellular action potentials) recorded in vivo from guinea pig auditory nerve fibers.Voltage-dependent currents (Na+ and K+) of SGNs somata patch-clamp recordings were fitted by a Hodgkin-Huxley model with a full trace identification algorithm. Node of Ranvier model was designed from the hypothesis that channel expressed on soma were identical, but differ in density. Simulated spikes were adjusted in order to match in vivo single-unit recordings with gradient-descent algorithm. Computation of the data allows to the identification of: i) one fast inward Na current (GNa activation: V1/2=-33 mV, τact< 0.5 ms; inactivation: V1/2=-61 mV, τinact < 2 ms); and ii) two K conductances, a high voltage-activated delayed-rectifier component (GKH, activation: V1/2=-41 mV; τact < 2.5 ms) and a low voltage-activated component (GKL, activation: V1/2=-56 mV; τact < 5 ms). Node of Ranvier model generate spikes that fit with in vivo recordings. Interestingly, the different spike durations along the tonotopic axis measured in vivo (i.e. 450 µs peak-to-peak duration versus 250 µs for 1 to 20 kHz, respectively) was explain by a gradual change in Na and K channel densities along the cochlea (GNa ~78 nS, GKL ~9 nS, GKH ~3 nS at 1 kHz versus GNa ~90 nS, GKL ~12 nS, GKH ~6 nS at 20 kHz).This study identifies the ionic conductances and densities, which shape the action potential waveform of auditory nerve fibers and suggests that the interplay of fast inward Na+ current and the two K+ enables the auditory nerve fibers to sustain high firing rates. In addition, this node of Ranvier model provides a valuable tool to design new electrical stimulation strategies for cochlear implants.

No description available.

Les cellules ciliées internes (CCI) sont les cellules sensorielles de l'organe de l'audition, elles transforment les ondes sonores en messages nerveux. Avant l’entrée en fonction de la cochlée, les CCI émettent spontanément des potentiels d’action (PA) calciques, ce qui active la voie auditive ascendante et assure le développement de l’axe tonotopique, à savoir la représentation du codage en fréquence, dans chaque relais de la voie auditive. Le profil et les mécanismes à l’origine des PA des CCI sont fortement débattus. Nous nous sommes donc attachés à étudier l’empreinte développementale des cellules sensorielles, c'est à dire déterminer le profil et les mécanismes à l’origine de leur activité.Après avoir incubé l’épithélium neuro-sensoriel avec la sonde calcique Fura2-AM, nous avons observé des vagues calciques se propageant le long des cellules de soutien et des cellules sensorielles. Plus précisément, l’activité des cellules ciliées se caractérisait par des élévations transitoires de calcium (pics calciques) à intervalles de temps réguliers. Nous avons ensuite démontré que les pics calciques des CCI correspondaient bien à des bouffées de PA en mesurant simultanément les oscillations calciques et l’émission de PA en patch-clamp. La fréquence, la durée et la distribution temporelle des pics calciques des CCI étaient en grande partie invariantes le long de l’axe base-apex de la cochlée. Enfin, les cellules voisines montraient une activité fortement synchrone à l’inverse des cellules spatialement éloignées. Ces résultats indiquent donc que l’activité des CCI est majoritairement identique le long de l’axe tonotopique de la cochlée.Nous nous sommes ensuite intéressés au mécanisme responsable de l’activité spontanée, la dépendance à l’ATP. L’incubation d’apyrase, une ecto-nucléotidase, entraine une diminution de l’activité des cellules de soutien, à savoir une réduction de l’aire et de la vitesse de propagation des vagues calciques. En revanche, l'activité des CCI n'est pas altérée par la déplétion d’ATP. Ces résultats suggèrent 2 mécanismes distincts, le premier ATP-dépendant et le second ATP-indépendant dans les cellules de soutien et sensorielles, respectivement.L’ensemble de ces résultats indique que la maturation des centres supérieurs serait déterminée par l’activation synchrone d’un nombre limité de cellules sensorielles. / During development, the sensory cells of the cochlea, the inner hair cells (IHCs), fire spontaneous calcium action potentials. This spontaneous spiking activity at the pre-hearing stage allows the IHCs to automatically stimulate the auditory nerve fibers and hence, ensures the proper shaping of the tonotopic organization along the ascending auditory pathway. Spontaneous spiking patterns may depend on the IHCs position on the cochlea (the tonotopic axis). Those patterns may also rely on ATP secretion from neighboring supporting cells. In this study, we used calcium imaging in the immature neuro-sensory epithelium of the cochlea, the Kölliker´s organ, to gain insights in the IHCs spiking activity. After loading the Kölliker´s organ with the calcium dye fura-2 AM, propagation of spontaneous calcium waves was readily observed across supporting and sensory cells. Both basal and apical IHCs were characterized by similar spontaneous calcium transients interspaced with silent periods, reminiscent of bursts of action potential recorded in patch-clamp. In addition, neighboring cells show a strong degree of synchronous activity. Incubation with apyrase, which hydrolyzes ATP, prevents the spontaneous calcium increase that propagates across the supporting cells within the Kölliker's organ. However, it leaves the spontaneous calcium transients in IHCs mostly unaffected. All these results show that the tonotopic map refinement in higher auditory centers comes from a coordinated activity of neighboring sensory cells, whose activity seems to be independent of ATP