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1.
Cell ; 141(5): 748-50, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20510920

RESUMO

The actin-filled stereocilia of cochlear hair cells deflect in response to sound by pivoting around rootlets at their insertion points at the sensory cell surface. Kitajiri et al. (2010) now show that the actin-binding protein TRIOBP tightly bundles the actin filaments into rootlets, endowing these stereocilia "pivots" with unique elasticity and robustness.

2.
Proc Natl Acad Sci U S A ; 116(10): 4496-4501, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30782832

RESUMO

Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5' and the other the 3' portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea of Otof-/- mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5' and 3' cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.


Assuntos
Surdez/terapia , Dependovirus/genética , Terapia Genética , Proteínas de Membrana/genética , Animais , Surdez/genética , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
3.
Bioinformatics ; 35(15): 2690-2691, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576403

RESUMO

SUMMARY: Using adequate DNA barcodes is essential to unambiguously identify each DNA library within a multiplexed set of libraries sequenced using next-generation sequencers. We introduce DNABarcodeCompatibility, an R-package that allows one to design single or dual-barcoding multiplex experiments by imposing desired constraints on the barcodes (including sequencer chemistry, barcode pairwise minimal distance and nucleotide content), while optimizing barcode frequency usage, thereby allowing one to both facilitate the demultiplexing step and spare expensive library-preparation kits. The package comes with a user-friendly interface and a web app developed in Java and Shiny (https://dnabarcodecompatibility.pasteur.fr), respectively, with the aim to help bridge the expertise of core facilities with the experimental needs of non-experienced users. AVAILABILITY AND IMPLEMENTATION: DNABarcodeCompatibility can be easily extended to fulfil specific project needs. The source codes of the R-package and its user interfaces are publicly available along with documentation at [https://github.com/comoto-pasteur-fr] under the GPL-2 licence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Software , Sequência de Bases , DNA , Biblioteca Gênica , Análise de Sequência
4.
Proc Natl Acad Sci U S A ; 114(30): 7765-7774, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28705869

RESUMO

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15- and Cdhr23-expressing interneuron precursors in Cdhr23-/- and Cdhr15-/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23- and Cdhr15-expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients.


Assuntos
Córtex Auditivo/embriologia , Proteínas Relacionadas a Caderinas/metabolismo , Caderinas/metabolismo , Interneurônios/fisiologia , Precursores de Proteínas/metabolismo , Animais , Córtex Auditivo/metabolismo , Proteínas Relacionadas a Caderinas/genética , Caderinas/genética , Polaridade Celular , Feminino , Macaca , Masculino , Mecanotransdução Celular , Camundongos , Precursores de Proteínas/genética , Receptores Acoplados a Proteínas G/metabolismo
5.
Proc Natl Acad Sci U S A ; 114(36): 9695-9700, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28835534

RESUMO

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.


Assuntos
Síndromes de Usher/genética , Síndromes de Usher/terapia , Animais , Animais Recém-Nascidos , DNA Complementar/administração & dosagem , DNA Complementar/genética , Dependovirus/genética , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Terapia Genética/métodos , Vetores Genéticos , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Síndromes de Usher/fisiopatologia , Vestíbulo do Labirinto/patologia , Vestíbulo do Labirinto/fisiopatologia
6.
PLoS One ; 19(7): e0305742, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028743

RESUMO

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.


Assuntos
Dependovirus , Orelha Interna , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Dependovirus/genética , Camundongos , Orelha Interna/metabolismo , Orelha Interna/embriologia , Orelha Interna/citologia , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Feminino , Transdução Genética/métodos , Gravidez , Terapia Genética/métodos , Humanos
7.
JCI Insight ; 9(3)2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38194286

RESUMO

Neonatal gene therapy has been shown to prevent inner ear dysfunction in mouse models of Usher syndrome type I (USH1), the most common genetic cause of combined deafness-blindness and vestibular dysfunction. However, hearing onset occurs after birth in mice and in utero in humans, making it questionable how to transpose murine gene therapy outcomes to clinical settings. Here, we sought to extend the therapeutic time window in a mouse model for USH1G to periods corresponding to human neonatal stages, more suitable for intervention in patients. Mice with deletion of Ush1g (Ush1g-/-) were subjected to gene therapy after the hearing onset. The rescue of inner ear hair cell structure was evaluated by confocal imaging and electron microscopy. Hearing and vestibular function were assessed by recordings of the auditory brain stem response and vestibulo-ocular reflex and by locomotor tests. Up to P21, gene therapy significantly restored both the hearing and balance deficits in Ush1g-/- mice. However, beyond this age and up to P30, vestibular function was restored but not hearing. Our data show that effective gene therapy is possible in Ush1g-/- mice well beyond neonatal stages, implying that the therapeutic window for USH1G may be wide enough to be transposable to newborn humans.


Assuntos
Síndromes de Usher , Vestíbulo do Labirinto , Humanos , Animais , Camundongos , Síndromes de Usher/genética , Síndromes de Usher/terapia , Audição , Terapia Genética/métodos
8.
Development ; 137(8): 1373-83, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20332152

RESUMO

Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.


Assuntos
Cóclea/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Animais , Cílios/fisiologia , Cílios/ultraestrutura , Cóclea/anatomia & histologia , Cóclea/inervação , Cóclea/ultraestrutura , Orelha Interna/citologia , Cabras , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/ultraestrutura , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Neurônios/citologia , Neurônios/fisiologia , Órgão Espiral/fisiologia , Órgão Espiral/ultraestrutura
9.
iScience ; 25(12): 105628, 2022 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-36483015

RESUMO

Hearing depends on fast and sustained calcium-dependent synaptic vesicle fusion at the ribbon synapses of cochlear inner hair cells (IHCs). The implication of the canonical neuronal SNARE complex in this exocytotic process has so far remained controversial. We investigated the role of SNAP-25, a key component of this complex, in hearing, by generating and analyzing a conditional knockout mouse model allowing a targeted postnatal deletion of Snap-25 in IHCs. Mice subjected to IHC Snap-25 inactivation after hearing onset developed severe to profound deafness because of defective IHC exocytosis followed by ribbon degeneration and IHC loss. Viral transfer of Snap-25 in these mutant mice rescued their hearing function by restoring IHC exocytosis and preventing synapses and hair cells from degeneration. These results demonstrate that SNAP-25 is essential for normal hearing function, most likely by ensuring IHC exocytosis and ribbon synapse maintenance.

10.
Sci Rep ; 10(1): 16430, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009420

RESUMO

The hair bundle of cochlear hair cells is the site of auditory mechanoelectrical transduction. It is formed by three rows of stiff microvilli-like protrusions of graduated heights, the short, middle-sized, and tall stereocilia. In developing and mature sensory hair cells, stereocilia are connected to each other by various types of fibrous links. Two unconventional cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), form the tip-links, whose tension gates the hair cell mechanoelectrical transduction channels. These proteins also form transient lateral links connecting neighboring stereocilia during hair bundle morphogenesis. The proteins involved in anchoring these diverse links to the stereocilia dense actin cytoskeleton remain largely unknown. We show that the long isoform of whirlin (L-whirlin), a PDZ domain-containing submembrane scaffold protein, is present at the tips of the tall stereocilia in mature hair cells, together with PCDH15 isoforms CD1 and CD2; L-whirlin localization to the ankle-link region in developing hair bundles moreover depends on the presence of PCDH15-CD1 also localizing there. We further demonstrate that L-whirlin binds to PCDH15 and CDH23 with moderate-to-high affinities in vitro. From these results, we suggest that L-whirlin is part of the molecular complexes bridging PCDH15-, and possibly CDH23-containing lateral links to the cytoskeleton in immature and mature stereocilia.


Assuntos
Caderinas/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Animais , Proteínas Relacionadas a Caderinas , Diferenciação Celular/fisiologia , Feminino , Masculino , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura/métodos , Isoformas de Proteínas/metabolismo , Estereocílios/metabolismo
11.
J Cell Biol ; 216(6): 1849-1864, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28495838

RESUMO

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal.


Assuntos
Caderinas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Síndromes de Usher/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Caderinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/genética , Larva/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Segmento Externo da Célula Bastonete/ultraestrutura , Síndromes de Usher/genética , Síndromes de Usher/patologia , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genética
12.
Elife ; 62017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29111973

RESUMO

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (OtofAla515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.


Assuntos
Células Ciliadas Auditivas/fisiologia , Fusão de Membrana , Proteínas de Membrana/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Sinapses/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Cálcio/metabolismo , Técnicas de Introdução de Genes , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Receptores de Detecção de Cálcio/genética
13.
J Cell Biol ; 212(2): 231-44, 2016 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-26754646

RESUMO

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.


Assuntos
Células Ciliadas Auditivas/fisiologia , Microvilosidades/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo III/fisiologia , Estereocílios/fisiologia , Sequência de Aminoácidos , Animais , Padronização Corporal , Surdez/genética , Células HEK293 , Células Ciliadas Auditivas/ultraestrutura , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/ultraestrutura , Dados de Sequência Molecular , Estereocílios/ultraestrutura
14.
J Neurosci ; 22(22): 9850-7, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12427841

RESUMO

The vibration of the hearing organ that occurs during sound stimulation is based on mechanical interactions between different cellular structures inside the organ of Corti. The exact nature of these interactions is unclear and subject to debate. In this study, dynamic structural changes were produced by stepwise alterations of scala tympani pressure in an in vitro preparation of the guinea pig temporal bone. Confocal images were acquired at each level of pressure. In this way, the motion of several structures could be observed simultaneously with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical flow estimation algorithm. Under these conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. Despite being enclosed in several types of supporting cells, the inner hair cells, together with the adjacent inner pillar cells, moved in a manner signifying high compliance. The outer hair cells displayed radial motion indicative of cellular bending. Together, these results show that shearing motion occurs between several parts of the organ, and that structural relationships within the organ change dynamically during displacement of the basilar membrane.


Assuntos
Membrana Basilar/fisiologia , Rampa do Tímpano/fisiologia , Animais , Membrana Basilar/citologia , Cobaias , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/fisiologia , Técnicas In Vitro , Microscopia Confocal , Movimento (Física) , Órgão Espiral/citologia , Órgão Espiral/fisiologia , Perfusão , Pressão , Rampa do Tímpano/citologia , Estresse Mecânico
15.
J Mech Mater Struct ; 4(4): 755-778, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20485573

RESUMO

The organ of Corti is the sensory epithelium in the cochlea of the inner ear. It is modeled as a shell-of-revolution structure with continuous and discrete components. Our recent work has been on the inclusion of the viscous fluid. Measurements from various laboratories provide the opportunity to refocus on the elastic properties. The current detailed model for the organ of Corti is reasonably consistent with diverse measurements. Most components have little stiffness in the propagation direction. However, the isotropic stiffness of the pillar heads is found to offer an explanation for the difference in point load and pressure measurements. The individual rows of inner hair cell stereocilia with tip links and the Hensen stripe are included, since these details are important for the determination of the neural excitation. The results for low frequency show a phase of tip link tension similar to auditory nerve measurements. The nonlinearity of fluid in the small gaps is considered. A result is that as amplitude increases, because of the near contact with the Hensen stripe, the excitation changes polarity, similar to the peak-splitting neural behavior sometimes observed.

16.
Biophys J ; 93(9): 3279-84, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17604314

RESUMO

The hearing organ contains sensory hair cells, which convert sound-evoked vibration into action potentials in the auditory nerve. This process is greatly enhanced by molecular motors that reside within the outer hair cells, but the performance also depends on passive mechanical properties, such as the stiffness, mass, and friction of the structures within the organ of Corti. We used resampled confocal imaging to study the mechanical properties of the low-frequency regions of the cochlea. The data allowed us to estimate an important mechanical parameter, the radial strain, which was found to be 0.1% near the inner hair cells and 0.3% near the third row of outer hair cells during moderate-level sound stimulation. The strain was caused by differences in the motion trajectories of inner and outer hair cells. Motion perpendicular to the reticular lamina was greater at the outer hair cells, but inner hair cells showed greater radial vibration. These differences led to deformation of the reticular lamina, which connects the apex of the outer and inner hair cells. These results are important for understanding how the molecular motors of the outer hair cells can so profoundly affect auditory sensitivity.


Assuntos
Estimulação Acústica , Audição/fisiologia , Órgão Espiral/fisiologia , Som , Animais , Cobaias , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Técnicas de Cultura de Órgãos
17.
Neuroimage ; 35(4): 1393-400, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17382563

RESUMO

Confocal laser scanning microscopy permits detailed visualization of structures deep within thick fluorescently labeled specimen. This makes it possible to investigate living cells inside intact tissue without prior chemical sample fixation and sectioning. Isolated guinea pig temporal bones have previously been used for confocal experiments in vitro, but tissue deterioration limits their use to a few hours after the death of the animal. In order to preserve the cochlea in an optimal functional and physiological condition, we have developed an in vivo model based on a confocal microscopy approach. Using a ventral surgical approach, the inner ear is exposed in deeply anaesthetized, tracheotomized, living guinea pigs. To label the inner ear structures, scala tympani is perfused via an opening in the basal turn, delivering tissue culture medium with fluorescent vital dyes (RH 795 and calcein AM). An apical opening is made in the bony shell of cochlea to enable visualization using a custom-built objective lens. Intravital confocal microscopy, with preserved blood and nerve supply, may offer an important tool for studying auditory physiology and the pathology of hearing loss. After acoustic overstimulation, shortening and swelling of the sensory hair cells were observed.


Assuntos
Orelha Interna/anatomia & histologia , Estimulação Acústica , Animais , Cóclea/anatomia & histologia , Orelha Interna/fisiologia , Cobaias , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/fisiologia , Células Ciliadas Auditivas Externas/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Ruído/efeitos adversos , Rampa do Tímpano/anatomia & histologia , Rampa do Tímpano/fisiologia
18.
Proc Natl Acad Sci U S A ; 103(6): 1918-23, 2006 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-16446441

RESUMO

The cochlea contains two types of sensory cells, the inner and outer hair cells. Sound-evoked deflection of outer hair cell stereocilia leads to fast force production that will enhance auditory sensitivity up to 1,000-fold. In contrast, inner hair cells are thought to have a purely receptive function. Deflection of their stereocilia produces receptor potentials, transmitter release, and action potentials in the auditory nerve. Here, we describe a method for rapid confocal imaging. The method was used to image stereocilia during simultaneous sound stimulation in an in vitro preparation of the guinea pig cochlea. We show that inner hair cell stereocilia move because they interact with the fluid surrounding the hair bundles, but stereocilia deflection occurs at a different phase of the stimulus than is generally expected. In outer hair cells, stereocilia deflections were approximately 1/3 of the reticular lamina displacement. Smaller deflections were found in inner hair cells. The ratio between stereocilia deflection and reticular lamina displacement is important for auditory function, because it determines the stimulus applied to transduction channels. The low ratio measured here suggests that amplification of hair-bundle movements may be necessary in vivo to preserve transduction fidelity at low stimulus levels. In the case of the inner hair cells, this finding would represent a departure from traditional views on their function.


Assuntos
Cílios/fisiologia , Células Ciliadas Auditivas/fisiologia , Mecanotransdução Celular/fisiologia , Som , Animais , Cobaias , Microscopia Confocal , Fatores de Tempo
19.
Biophys J ; 86(1 Pt 1): 535-43, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14695298

RESUMO

A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modifications to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.


Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Movimento/fisiologia , Órgão Espiral/citologia , Órgão Espiral/fisiologia , Estimulação Acústica/métodos , Animais , Técnicas de Cultura , Desenho de Equipamento , Análise de Falha de Equipamento , Cobaias , Audição/fisiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vibração
20.
Audiol Neurootol ; 7(1): 27-30, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11914522

RESUMO

To obtain a more integrated view of the cellular behaviour of the cochlea it is essential to observe not only wider regions of the exposed turns but also to visualize structures below the reticular lamina. Using confocal microscopy and in vitro preparations of guinea pig and mouse inner ears, cellular structures within the intact organ of Corti can be visualized at high resolution. The approach thus offers a means to investigate detailed cellular events, e.g. structural reorganization following acoustic overstimulation. Confocal microscope images, although sharper than images acquired using regular light microscopy, are still subject to problems related to light scattering within the optical system and low signal-to-noise ratio. Significant image restoration can, however, be obtained by applying a combination of wavelet denoising techniques and deconvolution algorithms. Future work will focus both on more dynamical cellular events and on new in vivo models where the inner ear is visualized at a better functional state.


Assuntos
Audição/fisiologia , Órgão Espiral/fisiologia , Transdução de Sinais/fisiologia , Animais , Cobaias , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Confocal , Fibras Nervosas/ultraestrutura , Transmissão Sináptica/fisiologia
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