Search

In mammalian cochlea, sound‐induced vibration is amplified by a three‐row lattice of Y‐shaped microstructures consisting of electromotile outer hair cell and supporting Deiters cell. This highly organized structure is thought to be essential for hearing of low‐level sounds. Prior studies reported differences in geometry and synaptic innervation of the outer hair cells between rows, but how these fine features are achieved at subcellular level still remains unclear. Using serial block‐face electron microscopy, we acquired few‐hundred‐micron‐sized cytoarchitecture of mouse organ of Corti at nanometer resolution. Structural quantifications were performed on the Y‐shapes as well as afferent and efferent projections to outer hair cells (OHCs). Several new features, which support the previously observed inter‐row heterogeneity, are described. Our result provides structural bases for the gradient of mechanical properties and diverse centrifugal regulation of OHC rows., By means of serial block‐face electron microscopy, Wang et al. revealed subcellular structural bases for row‐specific cytoarchitecture and innervation pattern in mouse cochlear amplifier.

In the developing nervous system, axons navigate through complex terrains that change depending on when and where outgrowth begins. For instance, in the developing cochlea, spiral ganglion neurons extend their peripheral processes through a growing and heterogeneous environment en route to their final targets, the hair cells. Although the basic principles of axon guidance are well established, it remains unclear how axons adjust strategies over time and space. Here, we show that neurons with different positions in the spiral ganglion employ different guidance mechanisms, with evidence for both glia-guided growth and fasciculation along a neuronal scaffold. Processes from neurons in the rear of the ganglion are more directed and grow faster than those from neurons at the border of the ganglion. Further, processes at the wavefront grow more efficiently when in contact with glial precursors growing ahead of them. These findings suggest a tiered mechanism for reliable axon guidance., In developing embryos, axons grow through complex and dynamic terrains. Here, the authors show that spiral ganglion neurons in the developing mouse cochlea extend leading axons that interact with a scaffold of glial precursors, with follower axons fasciculating on top.

The auditory system is a complex sensory network with an orchestrated multilayer regulatory program governing its development and maintenance. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) as important regulators in numerous systems, as well as in pathological pathways. However, their function in the auditory system has yet to be explored. Using a set of specific criteria, we selected four lncRNAs expressed in the mouse cochlea, which are conserved in the human transcriptome and are relevant for inner ear function. Bioinformatic characterization demonstrated a lack of coding potential and an absence of evolutionary conservation that represent properties commonly shared by their class members. RNAscope analysis of the spatial and temporal expression profiles revealed specific localization to inner ear cells. Sub-cellular localization analysis presented a distinct pattern for each lncRNA and mouse tissue expression evaluation displayed a large variability in terms of level and location. Our findings establish the expression of specific lncRNAs in different cell types of the auditory system and present a potential pathway by which the lncRNA Gas5 acts in the inner ear. Studying lncRNAs and deciphering their functions may deepen our knowledge of inner ear physiology and morphology and may reveal the basis of as yet unresolved genetic hearing loss-related pathologies. Moreover, our experimental design may be employed as a reference for studying other inner ear-related lncRNAs, as well as lncRNAs expressed in other sensory systems.

Mammalian hearing depends on an amplification process involving prestin, a voltage-sensitive motor protein that enables cochlear outer hair cells (OHCs) to change length and generate force. However, it has been questioned whether this prestin-based somatic electromotility can operate fast enough in vivo to amplify cochlear vibrations at the high frequencies that mammals hear. In this study, we measured sound-evoked vibrations from within the living mouse cochlea and found that the top and bottom of the OHCs move in opposite directions at frequencies exceeding 20 kHz, consistent with fast somatic length changes. These motions are physiologically vulnerable, depend on prestin, and dominate the cochlea's vibratory response to high-frequency sound. This dominance was observed despite mechanisms that clearly low-pass filter the in vivo electromotile response. Low-pass filtering therefore does not critically limit the OHC's ability to move the organ of Corti on a cycle-by-cycle basis. Our data argue that electromotility serves as the primary high-frequency amplifying mechanism within the mammalian cochlea.

Summary This protocol describes how to prepare intact mouse cochleae for serial block-face scanning electron microscopy (SBEM). The detailed workflow includes cochlea fixation, en bloc staining, resin embedding, X-ray microscopy-guided trimming and SBEM data acquisition. This protocol allows large-scale, nanometer-resolution three-dimensional imaging of subcellular structures in a targeted tonotopic range of the cochlea and enables fast volumetric scan at submicron resolution using a compact X-ray microscope. For complete details on the use and execution of this protocol, please refer to Hua et al. (2021)., Graphical abstract, Highlights • En bloc staining and resin embedding of whole mouse cochlea • 3D imaging of cochlea at cellular resolution using compact X-ray microscopy • Large-scale volume electron microscopy imaging of cochlea, This protocol describes how to prepare intact mouse cochleae for serial block-face scanning electron microscopy (SBEM). The detailed workflow includes cochlea fixation, en bloc staining, resin embedding, X-ray microscopy-guided trimming, and SBEM data acquisition. This protocol allows large-scale, nanometer-resolution three-dimensional imaging of subcellular structures in a targeted tonotopic range of the cochlea and enables fast volumetric scan at submicron resolution using a compact X-ray microscope.

Hearing loss is one of the most prevalent sensory deficits worldwide and can result from the death of mechanosensory hair cells that transduce auditory signals in the cochlea. The mammalian cochlea lacks the capacity to regenerate these hair cells once damaged, and currently there are no biological therapies for hearing loss. Understanding the signaling pathways responsible for hair cell development can inform regenerative strategies and identify targets for treating hearing loss. The canonical Wnt and Notch pathways are critical for cochlear development; they converge on several key molecules, such as Atoh1, to regulate prosensory specification, proliferation, hair cell differentiation, and cellular organization. Much work has focused on Wnt and Notch modulation in the neonatal mouse cochlea, where they can promote hair cell regeneration. However, this regenerative response is limited in the adult cochlea and this might be attributed to age-dependent epigenetic modifications. Indeed, the epigenetic status at key gene loci undergoes dynamic changes during cochlear development, maturation, and aging. Therefore, strategies to improve regenerative success in the adult cochlea might require the modulation of Wnt, Notch, or other pathways, as well as targeted epigenetic modifications to alter the activity of key genes critical for supporting cell proliferation or transdifferentiation., Hearing loss is the most prevalent sensory disorder worldwide. Here we review the progress, challenges, and potential of Wnt and Notch signaling in hearing regeneration. We also discuss ongoing clinical trials and the promise of epigenome editing for the amelioration of hearing loss.

Pathologic mechanisms in cochleae immediately following the onset of noise-induced hearing loss (NIHL) remain unclear. In this study, mice were exposed to 120 dB of octave band noise for 2 h to induce NIHL. Three hours after noise exposure, expression levels of the whole mouse genome in cochleae were analyzed by RNA-seq and DNA microarray. Differentially expressed genes (DEGs) exhibiting >2-fold upregulation or downregulation in noise-exposed cochleae compared to controls without noise exposure were identified. RNA-seq and microarray analyses identified 273 DEGs regulated at 3 h post-noise (51 upregulated and 222 downregulated). Bioinformatic analysis revealed that these DEGs were associated with the functional gene pathway "neuroactive ligand-receptor interaction" and included 28 genes encoding receptors for neurotransmitters such as gamma-aminobutyric acid and glutamate. Other DEGs included 25 genes encoding transcription factors. Downregulation of 4 neurotransmitter receptors (Gabra3, Gabra5, Gabrb1, Grm1) and upregulations of 5 transcription factors (Atf3, Dbp, Helt, Maff, Nr1d1) were validated by RT-PCR. The differentially regulated transcription factor Atf3 immunolocalized to supporting cells and hair cells in the organ of Corti at 12-h post-noise. The present data serve as a basis for further studies aimed at developing medical treatments for acute sensorineural hearing loss.

Laboratory mice have become the dominant animal model for hearing research. The mouse cochlea operates according to standard "mammalian" principles, uses the same cochlear cell types, and exhibits the same types of injury as found in other mammals. The typical mouse lifespan is less than 3 years, yet the age-associated pathologies that may be found are quite similar to longer-lived mammals. All Schuknecht's types of presbycusis have been identified in existing mouse lines, some favoring hair cell loss while others favor strial degeneration. Although noise exposure generally affects the mouse cochlea in a manner similar to other mammals, mice appear more prone to permanent alterations to hair cells or the organ of Corti than to hair cell loss. Therapeutic compounds may be applied systemically or locally through the tympanic membrane or onto (or through) the round window membrane. The thinness of the mouse cochlear capsule and annular ligament may promote drug entry from the middle ear, although an extremely active middle ear lining may quickly remove most drugs. Preclinical testing of any therapeutic will always require tests in multiple animal models. Mice constitute one model providing supporting evidence for any therapeutic, while genetically engineered mice can test hypotheses about mechanisms.

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain-sensing C-fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C-fibers: calcitonin-related polypeptide alpha (Calca/Cgrpα), specific to pain-sensing C-fibers, and tyrosine hydroxylase (Th), specific to low-threshold mechanoreceptive C-fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al, 2016). Whole-mount cochlear preparations from 3-week to 2-month-old CGRPα-EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II-like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co-expression could be observed, most abundantly in the middle cochlear turn.

We have previously shown that in vitro transduction with bovine adeno–associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin–deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. The aims of this study were to manipulate inner ear connexin expression in vivo using BAAV vectors, and to identify the optimal route of vector delivery. Injection of a BAAV vector encoding a bacterial Cre recombinase via canalostomy in adult mice with floxed connexin 26 (Cx26) alleles promoted Cre/LoxP recombination, resulting in decreased Cx26 expression, decreased endocochlear potential, increased hearing thresholds, and extensive loss of outer hair cells. Injection of a BAAV vector encoding GFP-tagged Cx30 via canalostomy in P4 mice lacking connexin 30 (Cx30) promoted formation of Cx30 gap junctions at points of contacts between adjacent non-sensory cells of the cochlear sensory epithelium. Levels of exogenous Cx30 decayed over time, but were still detectable four weeks after canalostomy. Our results suggest that persistence of BAAV-mediated gene replacement in the cochlea is limited by the extensive remodeling of the organ of Corti throughout postnatal development and associated loss of non-sensory cells.

The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells' apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca2+-dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca2+-dependent adaptation. The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role.

The mouse has proven to be an essential model system for studying hearing loss. A key advantage of the mouse is the ability to image the sensory cells in the cochlea. Many different protocols exist for the dissection and imaging of the cochlea. Here we describe a method that utilizes confocal imaging of whole-mount preparations followed by 3D analysis using the Imaris software. The 3D analysis of confocal stacks has been successfully used for investigating a number of mouse tissues and developmental processes. We propose that this method is also a valuable tool to analyze the cellular and tissue organization of the sensory hair cells in the cochlea.

Adding rapamycin or acarbose to diet at 9-10 months of age has been shown to significantly increase life span in both male and female UM-HET3 mice. The current study examined cochleae of male and female UM-HET3 mice at 22 months of age to determine if either treatment also influenced age-related loss of cochlear hair cells. A large loss of cochlear outer hair cells was observed at 22 months of age in untreated mice in both apical and basal halves of the cochlear spiral. Addition of acarbose to diet had no significant effect on the amount of outer hair cell loss at 22 months of age or in its pattern, with large loss in both apical and basal halves. The addition of rapamycin to diet, however, significantly reduced outer hair cell loss in the basal half of the cochlea at 22 months of age when compared to untreated mice. There was no significant difference between male and female mice in any of the conditions. Age-related outer hair cell loss in the apical cochlea precedes outer hair cell loss in the base in many mouse strains. The results of the present study suggest that rapamycin but not acarbose treatment can delay age-related loss of outer hair cells at doses at which each drug increases life span.

Medial olivocochlear (MOC) efferent feedback is suggested to protect the ear from acoustic injury and to increase its ability to discriminate sounds against a noisy background. We investigated whether type II spiral ganglion neurons participate in the contralateral suppression of the MOC reflex. The application of ouabain to the round window of the mouse cochlea selectively induced the apoptosis of the type I spiral ganglion neurons, left the peripherin-immunopositive type II spiral ganglion neurons intact, and did not affect outer hairs, as evidenced by the maintenance of the distorted product otoacoustic emissions (DPOAEs). With the ouabain treatment, the threshold of the auditory brainstem response increased significantly and the amplitude of wave I decreased significantly in the ouabain-treated ears, consistent with the loss of type I neurons. Contralateral suppression was measured as reduction in the amplitude of the 2f1−f2 DPOAEs when noise was presented to the opposite ear. Despite the loss of all the type I spiral ganglion neurons, virtually, the amplitude of the contralateral suppression was not significantly different from the control when the suppressor noise was delivered to the treated cochlea. These results are consistent with the type II spiral ganglion neurons providing the sensory input driving contralateral suppression of the MOC reflex.

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell’s stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 µM) or berbamine (≥1.55 µM) protect zebrafish hair cells in vivo from neomycin (6.25 µM, 1 h). Protection of zebrafish hair cells against gentamicin (10 µM, 6 h) was provided by ≥25 µM dTC or ≥12.5 µM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 µM, 48 h) by 20 µM berbamine or 25 µM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 µM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 µM (dTC) and 2.8 µM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell’s basolateral K + current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants.

Conventional microscopy has limitations in viewing the cochlear microstructures due to three-dimensional spiral structure and the overlying bone. But these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM). By using near-infrared lasers for multiphoton excitation, intravital MPM can detect endogenous fluorescence and second harmonic generation of tissues. In this study, we used intravital MPM to visualize various cochlear microstructures without any staining and non-invasively analyze the volume changes of the scala media (SM) without removing the overlying cochlear bone. The intravital MPM images revealed various tissue types, ranging from thin membranes to dense bone, as well as the spiral ganglion beneath the cochlear bone. The two-dimensional, cross-sectional, and serial z-stack intravital MPM images also revealed the spatial dilation of the SM in the temporal bone of pendrin-deficient mice. These findings suggest that intravital MPM might serve as a new method for obtaining microanatomical information regarding the cochlea, similar to standard histopathological analyses in the animal study for the cochlea. Given the capability of intravital MPM for detecting an increase in the volume of the SM in pendrin-deficient mice, it might be a promising new tool for assessing the pathophysiology of hearing loss in the future.

The sharp frequency tuning and exquisite sensitivity of the mammalian cochlea is due to active forces delivered by outer hair cells (OHCs) to the cochlear partition. Force transmission is mediated and modulated by specialized cells, including Deiters’ cells (DCs) and pillar cells (PCs), coupled by gap-junctions composed of connexin 26 (Cx26) and Cx30. We created a mouse with conditional Cx26 knock-out (Cx26 cKO) in DCs and PCs that did not influence sensory transduction, receptor-current-driving-voltage, low-mid-frequency distortion-product-otoacoustic-emissions (DPOAEs), and passive basilar membrane (BM) responses. However, the Cx26 cKO desensitizes mid-high-frequency DPOAEs and active BM responses and sensitizes low-mid-frequency neural excitation. This functional segregation may indicate that the flexible, apical turn cochlear partition facilitates transfer of OHC displacements (isotonic forces) for cochlear amplification and neural excitation. DC and PC Cx26 expression is essential for cochlear amplification in the stiff basal turn, possibly through maintaining cochlear partition mechanical impedance, thereby ensuring effective transfer of OHC isometric forces.

Sensorineural hearing loss (SNHL), which typically originates in the cochlea, is the most common otologic problem caused by aging and noise trauma. The cochlea, a delicate and complex biological mechanosensory transducer in the inner ear, has been extensively studied with the goal of improving diagnosis of SNHL. However, the difficulty associated with accessing the cochlea and resolving the microstructures that facilitate hearing within it in a minimally-invasive way has prevented us from being able to assess the pathology underlying SNHL in humans. To address this problem we investigated the ability of a multimodal optical system that combines optical coherence tomography (OCT) and single photon autofluorescence imaging (AFI) to enable visualization and evaluation of microstructures in the cochlea. A laboratory OCT/AFI imager was built to acquire high resolution OCT and single photon fluorescence images of the cochlea. The imager’s ability to resolve diagnostically-relevant details was evaluated in ears extracted from normal and noise-exposed mice. A prototype endoscopic OCT/AFI imager was developed based on a double-clad fiber approach. Our measurements show that the multimodal OCT/AFI imager can be used to evaluate structural integrity in the mouse cochlea. Therefore, we believe that this technology is promising as a potential clinical evaluation tool, and as a technique for guiding otologic surgeries such as cochlear implant surgery.

Animal models are the only means of assessing the effects of cochlear implantation (CI) at a cellular and molecular level. The range of naturally occurring and genetically-modified mouse strains which mimic human deafness provide excellent opportunities for auditory research. To date, there are very few studies of CI in mice. The main aims of this study were to develop a reproducible and viable technique to enable long term CI in the mouse and to assess the response of the mouse cochlea to implantation as a means of evaluating the success of the procedure. Electrode array implantation via the round window was performed in C57Bl/6 mice aged 3 and 6 months. The contralateral cochlea acted as a control. Auditory brainstem responses (ABR) were recorded prior to and following CI. Analysis showed greater threshold shifts in the implanted ear compared to the control ear post-implantation, but substantial preservation of hearing. There were no cases in which implantation caused a profound hearing loss across all frequencies. Cone beam computerised tomography and light microscopy confirmed correct placement of the electrode array within the scala tympani. Cochleae were prepared for histological examination. Initial analysis revealed encapsulation of the implant in tissue with morphological characteristics suggestive of fibrosis. Our results show that mouse CI via the round window offers a model for exploring tissue responses to implantation.

Sensory perception in the inner ear relies on the hair bundle, the highly polarized brush of movement detectors crowning hair cells. We previously showed that, in the mouse cochlea, the edge of the forming bundle is defined by the 'bare zone', a microvilli-free sub-region of apical membrane specified by the mInsc-LGN-Gαi protein complex. We now report that LGN and Gαi also occupy the very tip of stereocilia that directly abut the bare zone. We demonstrate that LGN and Gαi are both essential to promote the elongation and differential identity of stereocilia across rows. Interestingly, we also uncover that total LGN-Gαi protein amounts are actively balanced between the bare zone and stereocilia tips, suggesting that early planar asymmetry of protein enrichment at the bare zone confers adjacent stereocilia their tallest identity. We propose that LGN and Gαi participate in a long-inferred signal originating outside of the bundle to model its staircase-like architecture, a property essential for direction-sensitivity to mechanical deflection and hearing.

The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells' apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca2+-dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca2+-dependent adaptation. The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role.

The perception of complex acoustic stimuli begins with the deconstruction of sound into its frequency components. This spectral processing occurs first and foremost in the inner ear. In vertebrates, two very different strategies of frequency analysis have evolved. In nonmammalian vertebrates, the sensory hair cells of the inner ear are intrinsically electrically tuned to a narrow band of acoustic frequencies. This electrical tuning relies on the interplay between BK channels and voltage-gated calcium channels. Systematic variations in BK channel density and kinetics establish a gradient in electrical resonance that enables the coding of a broad range of acoustic frequencies. In contrast, mammalian hair cells are extrinsically tuned by mechanical properties of the cochlear duct. Even so, mammalian hair cells also express BK channels. These BK channels play critical roles in various aspects of mammalian auditory signaling, from developmental maturation to protection against acoustic trauma. This review summarizes the anatomical localization, biophysical properties, and functional contributions of BK channels in vertebrate inner ears. Areas of future research, based on an updated understanding of the biology of both BK channels and the inner ear, are also highlighted. Investigation of BK channels in the inner ear continues to provide fertile research grounds for examining both BK channel biophysics and the molecular mechanisms underlying signal processing in the auditory periphery.

Mammalian hair cells possess only a limited ability to repair damage after trauma. In contrast, sea anemones show a marked capability to repair damaged hair bundles by means of secreted repair proteins (RPs). Previously, it was found that recovery of traumatized hair cells in blind cavefish was enhanced by anemone-derived RPs; therefore, the ability of anemone RPs to assist recovery of damaged hair cells in mammals was tested here. After a 1 h incubation in RP-enriched culture media, uptake of FM1-43 by experimentally traumatized murine cochlear hair cells was restored to levels comparable to those exhibited by healthy controls. In addition, RP-treated explants had significantly more normally structured hair bundles than time-matched traumatized control explants. Collectively, these results indicate that anemone-derived RPs assist in restoring normal function and structure of experimentally traumatized hair cells of the mouse cochlea.

The complex anatomy of the mammalian cochlea is most readily understood by representation in three-dimensions. However, the cochlea is often sectioned to minimize the effects of its anatomic complexity and optical properties on image acquisition by light microscopy. We have found that optical aberrations present in the decalcified cochlea can be greatly reduced by dehydration through graded ethanols followed by clearing with a mixture of five parts methyl salicylate and three parts benzyl benzoate (MSBB). Clearing the cochlea with MSBB enables acquisition of high-resolution images with multiple fluorescent labels, through the full volume of the cochlea by laser scanning confocal microscopy. The resulting images are readily applicable to three-dimensional morphometric analysis and volumetric visualizations. This method promises to be particularly useful for three-dimensional characterization of anatomy, innervation and expression of genes or proteins in the many new animal models of hearing and balance generated by genetic manipulation. Furthermore, the MSBB is compatible with most non-protein fluorophores used for histological labeling, and may be removed with traditional transitional solvents to allow subsequent epoxy embedding for sectioning.

Nuclear factor kappa B(NF-κB) is one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli, including inflammatory cytokines, phorbol esters, growth factors, and bacterial and viral products. The aim of this study is to demonstrate NF-κB expression in the mouse cochlea and its enhancement in response to lipopolysaccharides(LPS) and kanamycin(KA) treatment. Methods KA treatment consisted of subcutaneous KA injections at 700 mg/kg twice a day with an eight-hour interval between the two injections for 3 or 7 days. For animals in the LPS treatment group, a single dose of 0.3 mg LPS dissolved in 0.2 ml sterile saline were injected into both bullae through the tympanic membrane and kept there for 3 hours. Animals in the control group received subcutaneous saline injection for 7 days. Following immmunohistochemichal processing with rabbit polyclonal anti-NF-κB p65 antibodies, cryosections of the cochlea were examined for expression of NF-κB p65 in various structures in the cochlea. Results NF-κB p65 expression, identified by presence of brown reaction products characteristic of DAB immunohistochemistry, was visible in the spiral ligament, spiral prominence, tectorial membrane(TM), spiral ganglion and nerve fibers. Relatively weak NF-κB p65 expression was also visualized in the organ of Corti. Within the organ of Corti, the inner hair cells (IHC), outer hair cells (OHC), inner pillar cells (IP), outer pillar cells (OP), Deiter’s cells(DC), and Boettcher’s cells exhibited stronger staining than the inner sulcus cells, Hensen’s cells(HC) and Claudius’cells. No NF-κB p65 expression was seen in the nucleus of the IHC and OHC. NF-κB p65 expression was increased in animals exposed to LPS or KA, demonstrating significant differences in the staining between control animals and LPS/KA-treated animals. NF-κB p65 expression was not significantly different between LPS treated and KA treated animals or between 3 and 7 days in KA-treated animals. Conclusion LPS and KA exposure increases expression of NF-κB p65 in the mouse cochlea.

A multi-scale finite element (FE) model of the mouse cochlea, based on its anatomy and material properties is presented. The important feature in the model is a lattice of 400 Y-shaped structures in the longitudinal direction, each formed by Deiters cells, phalangeal processes and outer hair cells (OHC). OHC somatic motility is modeled by an expansion force proportional to the shear on the stereocilia, which in turn is proportional to the pressure difference between the scala vestibule and scala tympani. Basilar membrane (BM) and reticular lamina (RL) velocity compare qualitatively very well with recent in vivo measurements in guinea pig [2]. Compared to the BM, the RL is shown to have higher amplification and a shift to higher frequencies. This comes naturally from the realistic Y-shaped cell organization without tectorial membrane tuning.

Retrograde degeneration of spiral ganglion cells in the cochlea following hair cell loss is similar to dying back in pathology. The EFR3A gene has recently been discovered to be involved in the pathogenesis of dying back. The relationship of EFR3A and spiral ganglion degeneration, however, was rarely investigated. In this study, we destroyed the hair cells of the mouse cochlea by co-administration of kanamycin and furosemide and then investigated the EFR3A expression during the induced spiral ganglion cell degeneration. Our results revealed that co-administration of kanamycin and furosemide quickly induced hair cell loss in the C57BL/6J mice and then resulted in progressive degeneration of the spiral ganglion beginning at day 5 following drug administration. The number of the spiral ganglion cells began to decrease at day 15. The expression of EFR3A increased remarkably in the spiral ganglion at day 5 and then decreased to near normal level within the next 10 days. Our study suggested that the change of EFR3A expression in the spiral ganglion was coincident with the time of the spiral ganglion degeneration, which implied that high expression of EFR3A may be important to prompt initiation of spiral ganglion degeneration following hair cell loss.

We recorded K+ currents in inner (IHCs) and outer (OHCs) hair cells from mice at embryonic days 16 and 18 and on the day of birth (PO) to characterize their early physiological differentiation. In both cell types, outward currents increased in size during late embryonic development, in cells situated in both the apical and basal coils of the cochlea. Currents increased up to six-fold, with current density increasing four-fold. Currents in basal cells were generally larger than in the apex, and currents in IHCs were larger than in OHCs at any given stage. In OHCs, they were initially non-inactivating but gained the partial inactivation characteristic of the K+ current of neonatal mouse cochlear hair cells, IK,neo, by day 18 in the base and by P0 in the apex. In IHCs, there was little change, other than in amplitude, with partial inactivation already evident in the base by embryonic day 16. These results suggest that changes in the channel complement of OHCs occur within a few days of terminal mitosis, whereas in IHCs any such development would occur earlier. The progressive development of K+ currents correlates with a developmental delay of around 2 days from the base to the apex of the cochlea.

The snail-shell-shaped cochlea of the inner ear contains some 15,000 hair cells that are needed for humans to hear. Audiologist dogma holds that once these cells die off, they never grow back, leading to hearing loss. But a new study suggests that hair cell death may not be as immutable as it seems. Scientists from Harvard, MIT, Brigham & Women’s Hospital, and Massachusetts Eye & Ear Infirmary have discovered a mixture of molecules that can dramatically reverse hair cell loss in the cochleas of mice (Cell Rep. 2017, DOI: 10.1016/j.celrep.2017.01.066). Hair cells are surrounded by a population of so-called supporting cells that studies suggest could behave like stem cells, meaning they can multiply and mature into other forms. So Albert S. B. Edge of Massachusetts Eye & Ear Infirmary and colleagues tested mixtures of small molecules on mouse cochlea cells growing in a dish until they found combinations that coaxed

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca2+-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca2+ concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca2+, intracellular Ca2+ buffering, and membrane potential, by their common effect on intracellular free Ca2+.\ud \ud Hearing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3⇓–5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca2+ entry through the channel itself (6⇓⇓⇓⇓⇓⇓–13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14⇓–16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17, 18) indicates that Ca2+, to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca2+ entry is required for fast adaptation, and instead proposed an as-yet-undefined mechanism involving a Ca2+-independent reduction in the viscoelastic force of elements in series with the MET channels (19). In the present study, we further investigated the role of Ca2+ in MET channel adaptation in mouse cochlear hair cells by deflecting their hair bundles using a piezo-driven fluid jet, which is believed to produce a more uniform deflection of the hair bundles (20⇓⇓–23) compared with the piezo-driven glass rod (19, 24).

We demonstrate imaging of mouse cochlea using two-photon excited autofluorescence for the diagnosis of pathologic changes. We also show the potential of phase-conjugation through multimode fiber as an endoscopy-based solution for in vivo diagnosis.

Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a(6J) and Myo7a(4626SB). on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction. The hair bundles need to be displaced beyond their physiological operating range for mechanotransducer channels to open. Transducer currents also adapt more strongly than normal to excitatory stimuli. We conclude that myosin VIIA participates in anchoring and holding membrane-bound elements to the actin core of the stereocilium. Myosin VIIA is therefore required for the normal gating of transducer channels.

Prolonged exposure to noise results in loss of ribbon synapses, composed in part by superclusters of neurotransmitter vesicles in the sensory hair cells of the cochlea. Expression of a nerve growth factor in mouse cochlea mitigates noise-induced damage and hearing loss.

Hearing over a wide range of sound intensities is thought to require complementary coding by functionally diverse spiral ganglion neurons (SGNs), each changing activity only over a subrange. The foundations of SGN diversity are not well understood but likely include differences among their inputs: the presynaptic active zones (AZs) of inner hair cells (IHCs). Here we studied one candidate mechanism for causing SGN diversity—heterogeneity of Ca(2+) influx among the AZs of IHCs—during postnatal development of the mouse cochlea. Ca(2+) imaging revealed a change from regenerative to graded synaptic Ca(2+) signaling after the onset of hearing, when in vivo SGN spike timing changed from patterned to Poissonian. Furthermore, we detected the concurrent emergence of stronger synaptic Ca(2+) signals in IHCs and higher spontaneous spike rates in SGNs. The strengthening of Ca(2+) signaling at a subset of AZs primarily reflected a gain of Ca(2+) channels. We hypothesize that the number of Ca(2+) channels at each IHC AZ critically determines the firing properties of its corresponding SGN and propose that AZ heterogeneity enables IHCs to decompose auditory information into functionally diverse SGNs.

The three-dimensional fibrillar arrangement of the basilar membrane in the mouse cochlea was studied using scanning electron microscopy. Fibrils of the basilar membrane were exposed by removing cellular elements of the cochlea using a sodium hydroxide maceration technique. The arrangement of fibrils in the basilar membrane was different between the pars arcuata and pars pectinata. In the pars arcuata, fibrils were arranged in radial and spiral direction, showing a woven pattern. In the pars pectinata, most of the fibrils ran in the radial direction. These findings suggest that the vibration pattern of the pars arcuata and pars pectinata is different when the basilar membrane vibrates.

A “Y”-shaped structure of cells formed by the basally tilted force generating outer hair cells (OHC) and apically tilted passive phalangeal process (PhP) connected to a Deiter cell (DC) has been hypothesized to be an essential organ of Corti (OoC) building block for cochlear amplification (Yoon et al., 2011). We developed a COMSOL finite-element model of the mouse cochlea, taking into account the spatial arrangement of Y-shaped elements and the 3-D fluid-structure interaction. The model was validated by comparison with previously reported basilar membrane (BM) displacement for both passive and active cases (Lee et al., 2015). The baseline model can reproduce an increase of 40 dB gain of the BM (re stapes velocity) for low level sounds. However, the absence of the PhP results in an unstable response of the BM for active cases. The model with the roles of the PhP and OHC switched, could not reproduce the active gain. Finally, the model with reversed titling orientation of OHC and PhP cells generated signifi...

The motor protein prestin in the outer hair cells is a prime candidate for the molecular amplifier that ensures the sensitivity, frequency tuning and dynamic range of the mammalian cochlea. Absence of prestin results in a 40-60 dB reduction in cochlear neural sensitivity. Here we show that sound-evoked basilar membrane (BM) vibrations in the basal cochleae of prestin(-/-) mice are as sensitive as those of their prestin(+/+) siblings. BM vibrations in prestin(-/-) mice are, however, broadly tuned to a frequency similar to a half octave below the characteristic frequency (CF) of similar BM locations in prestin(+/+) mice. The peak sensitivity of prestin(+/+) BM tuning curves matches the neural thresholds, while prestin(-/-) BM tuning curves at the best frequency are > 50 dB more sensitive than the neural responses. We conclude that prestin influences properties of the cochlear partition that are crucial for BM frequency tuning and for converting its vibrations into neural excitation.

This prospective randomized study investigates the possible toxic effects of interferon (IFN) α2A on the mouse cochlea. Thirty-six albino Swiss mice that were randomly assigned to 3 groups underwent baseline auditory brain stem response testing bilaterally to objectively assess baseline hearing levels. The first group received a single dose of 50,000 units, and the second group received 100,000 units of IFN-α2A intraperitoneally, whereas the third group was given no medication. Repeat auditory brain stem response testing revealed a significant rise in mean baseline peak equivalent sound pressure level thresholds in the groups that received IFN (P < 0.001). Histologicallly, the cochleae of mice that received IFN had a decreased number of fibroblasts in the spiral limbus, as well as prominent cytoplasmic vacuolation of these cells, compared with control animals. Loss of hair cells was not observed. It is possible that reversible biochemical and metabolic changes in the cochlea, rather than morphologic abnormalities, manifest IFN ototoxicity. (Otolaryngol Head Neck Surg 2001;124:107-10.)

The present study was designed to clarify the chronological developmental process of monovalent ions (Na+, K+, Cl–) in the endolymph of the mouse in relation to the development of the endocochlear potential (EP). The EP and ionic concentrations were measured simultaneously with the ion-sensitive double-barreled microelectrodes from the scala media of the basal turn. The EP increased abruptly 7 days after birth (DAB) and reached approximately 80 mV 14 DAB. In the earliest postnatal days, the endolymphatic Na+ concentration was significantly higher than that in adult mice, however, the K+ and the Cl– concentrations were lower. The concentrations of all the monovalent ions in endolymph reached adult levels at 7 DAB when the EP was still under 20 mV. These data strongly suggest the presence of a different mechanism between the production of monovalent ions, especially of high K+ in the endolymph and that of EP.

This study examined the utility of a methacrylate-based embedding medium and microwave staining for light microscopic quantification of hair cells and spiral ganglion cells in the mouse cochlea. The most important phase of the preparation process involved slowing down the polymerization process. The tissue molecules so locked within the plastic matrix produced excellent preservation of the organ of Corti and adjacent structures including the spiral ganglion, as well as tissue ionic charges. Excitable by microwaves, these ionic charges accelerated the movement of the basic dye (hematoxylin) into the tissue, reducing the time for this segment of the staining process from approximately 45 minutes to 1-2 minutes. When embedded in glycol methacrylate (GMA), acidic dyes show less stain-cell affinity so that staining intensity and time cannot be improved significantly. However, addition of color extenders to the counterstain eosin produced distinguished staining of all tissue constituents. Thus, a combination of GMA embedding medium, use of the microwave for staining and addition of color extenders to the counterstain generated excellent structural resolution and contrast. This made both hair cell and spiral ganglion cell counts possible from within the same specimen and provided an opportunity for qualitative evaluation as well.

Resin replicas of the microvasculature of the cochlear lateral wall of the mice were studied by scanning electron microscopy. The vascular system of the mouse cochlea showed a similar architecture to that of the guinea pig, cat, and rat. Several thin connecting branches were found between the vessels of the osseous spiral lamina and the cochlear lateral wall at the apical end. The apical turn and the basal part of the lower basal turn were innervated by several radiating arterioles originating from a single stem arteriole. The hook portion and the apical end had a particular vascular meshwork different from other parts of the cochlea.

ObjectiveTo identify presence of inflammasome activated in mouse cochlea with sensorineural hearing loss (SNHL) caused by cytomegalovirus (CMV) infection.MethodMCMV was injected into the right cerebral hemisphere in neonatal BALB/c mice at 2000 pfu virus titers. Auditory brainstem responses (ABRs) were tested to evaluate hearing at 21 days. Histopathological studies were conducted to confirm localizations of MCMV infected cells in the inner ear. Expression of inflammasome related factors was assessed by immunofluorescence, Quantitative real-time PCR and Western blotting.ResultsIn the mouse model of CMV induced SNHL, inflammasome related kinase Caspase-1 and downstream inflammatory factor IL-1β and IL-18 were found increased and activated after CMV infection in the cochlea. These factors could further up-regulate expression of IL-6 and TNF-α. These inflammatory factors are neurotoxicity and may contribute to hearing impairment. Furthermore, we also detected significantly increased AIM2 protein that accumulated in the SGN of cochleae with CMV infection.SignificanceWe have shown that inflammasome as a novel inherent immunity mechanism may contribute to hearing impairment.ConclusionOur data indicate that imflammasome assemble in mouse inner ear in response to CMV infection. We have revealed a novel pathology event in CMV induced SNHL involving activation of inflammasome in mouse cochlea. Additionally, we have shown that inflammasome may be a novel target for prevention and treatment of CMV related SNHL.

Genetic loss of VGLUT3 in cochlear inner hair cells results in profound deafness. In this issue of Neuron, Akil et al. (2012) show that AAV-mediated introduction of wild-type VGLUT3 in the genetically deaf mouse cochlea results in significantly improved hearing.

Histologic development of the mammalian cochlea has been studied by many investigators. In 1978, Li reported on a preliminary scanning electron microscopic study of hair cell development in the embryonic mouse cochlea. The present study further examines the surface morphology of the embryonic cochlea.

A technique is described that can nontraumatically assess cochlear microphonic (CM) and cochlear action potential (AP) thresholds to pure tones in the intact mouse. This method was applied to the normal hearing CBA/J, and to the C57BL/6J, which has early-onset hearing loss. CM thresholds are most sensitive in both mice by 23 days of age, and the C57BL/6J CM responses degenerate rapidly thereafter, especially at the high-frequency end. The CMs of the CBA/J continue to change past 23 days, developing a threshold dip at 24 kHz, and becoming slightly less sensitive at 32 kHz. The AP of the C57BL/6J is also most sensitive at 23 days, and becomes gradually less sensitive to 32- and 64-kHz tones over the next 36 days. The CBA/J AP thresholds are not maximally sensitive until 41 days of age.

Cochleas of mice homozygous for the Bronx waltzer gene (symbol bv ) were investigated using scanning (SEM) and transmission (TEM) electron microscopy. An age-related study was done from birth to postnatal day 100. With SEM, the arrangement of hair cells confirmed the unique feature of the bv/bv cochlea: the inner hair cells (IHCs) were either absent or abnormally haired but the outer hair cells (OHCs) appeared normal. No significant difference was observed with age. Using TEM, the remaining (20–25%) IHCs could be divided into two groups: normal-looking IHCs but with an abnormal synaptic pole, and abnormal, abortive-like IHCs. Very little if any sign of degeneration was observed whatever the age. OHCs displayed an almost normal cytology and pattern of innervation. The neurons of the spiral ganglion were very rare, even at birth. These findings suggest that the bv mutation should rather be classified in another group, than ‘degenerative’. The persistence of normal structures at OHC level is discussed in light of the cochlear physiology of the bv/bv : it again raises the question of the real role of OHCs in the peripheral auditory mechanisms.

Combined exposure to noise and salicylates was found to produce greater acoustic trauma induced audiogenic seizure risk than exposure to the noise alone. The result suggests that salicylates could make the mouse cochlea more vulnerable to the traumatic action of noise.